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Demonstration of (Ca2+-Mg2+-ATPase activity of the neural cell adhesion molecule
Abstract
In this study a possible association between (Ca(2+)-Mg2+)-ATPase activity and the neural cell adhesion molecule, NCAM, was investigated. The effects of various detergents on ATPase activity were evaluated, and it was found that solubilization of rat brain microsomes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, CHAPS, released a major fraction of the (Ca(2+)-Mg2+)-ATPase activity together with NCAM. Using different types of solid phase immunoadsorption it was shown that NCAM antibodies selectively isolated ATPase activity. Furthermore, agarose gel immunoelectrophoresis of solubilized brain microsomes followed by ATPase assay directly in the gel revealed ATPase activity associated with the NCAM immunoprecipitate. The NCAM-associated enzyme activity had a broad nucleoside triphosphate specificity and no strict selectivity for divalent cations, indicating that the enzyme probably is an ecto-ATPase. This raises a series of intriguing questions in relation to NCAM adhesive functions.
... To check the possibility that the observed ATP hydrolysis was the result of secreted soluble enzymes, as observed for other parasites [16], a reaction mixture was prepared with cells that were incubated in the absence of ATP. The suspension was centrifuged to remove the cells, and the supernatant was checked for ATPase activity. ...
In this work, we have described the expression of ecto-ATPDase on the external surface of Leishmania donovani. This enzyme has the ability to hydrolyze extracellular ATP. There is a low level of ATP hydrolysis in the absence of divalent cation 2.5 ± 0.51 nM Pi 10(7) cells/h which shows the divalent cation-dependent activity of this enzyme in the intact parasite. However, MgCl2 stimulated the ATP hydrolysis to a greater extent compared with CaCl2 and ZnCl2. This activity was also observed when replaced by MnCl2. The Mg-dependent ecto-ATPase activity was 46.58 ± 6.248 nM Pi 10(7) cells/h. The apparent K m for ATP was 5.76 mM. Since Leishmania also possesses acid phosphatase activity and to discard the possibility that the observed ATP hydrolysis was due to acid phosphatase, the effect of pH was examined. In the pH range 6.0-9.0, in which the cells were viable, the phosphatase activity decreased while ATPase activity increased. To show that the observed ATP hydrolysis was not due to phosphatase or nucleotidase activity, certain inhibitors for these enzymes were tested. Vandate and NaF inhibited the phosphatase activity; Ammonium molybdate inhibited 5'-nucleotidase activity, but these inhibitors did not inhibit the observed ATP hydrolysis. However, when ADP was used as a substrate, there was no inhibition of ATP hydrolysis showing the possibility of ATP diphosphohydrolase activity. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, 4,4'-diisothiocyanostilbene 2,-2'-disulfonic acid, as well as suramin, an antagonist of P2-purinoceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The presence of L. donovani E-NTPDase activity was demonstrated using antibodies against NTPDase by Western blotting and flow cytometry. The presence of Mg(2+)-dependent ATP diphosphohydrolase activity on the surface of L. donovani modulates the nucleotide concentration and protects the parasite from the lytic effects of the nucleotides mainly ATP. Ecto-ATPDase from L. donovani may be further characterized as a good antigen and as a target for immunodiagnosis and drug development, respectively.
... Immunoprecipitates of NCAM have been demonstrated to possess Ca 2+Mg 2+-dependent ATP hydrolysing activity, indicating that NCAM is tightly associated with ATPase ( Dzhandzhugazyan and Bock, 1993). It has also been demonstrated that ATP can bind to NCAM directly (Dzhandzhugazyan and Bock, 1997). ...
Väitekirja elektroonilisest versioonist puuduvad publikatsioonide täistekstid. Recent hypothesis of the pathogenesis of depression links the development of this disease with reduced brain plasticity. In the central nervous system, plasticity and connectivity in the brain are mediated by the neural cell adhesion molecule (NCAM) and its polysialylated form PSA-NCAM. Therefore, according to the plasticity theory of depression, NCAM may have a crucial role in the development of this condition. NCAM is able to bind a series of counter-receptors including fibroblast growth factor receptor (FGFR), and through the binding, activate the downstream signalling pathways, which all lead to the activation of cyclic-AMP-response element binding protein (CREB). The aim of this study was to investigate whether mice with the constitutive deficiency in NCAM exhibited dysfunctional neuronal plasticity and whether it results in the depressive-like phenotype. By using NCAM-deficient mice, NCAM interaction partners and downstream signalling pathways were studied in detail. Also was investigated, whether a synthetic peptide FGL, which mimics the actions of NCAM, is able to reverse emerged disturbances and restore the activation of intracellular signalling cascades in these animals. The results showed that NCAM deficient mice do have depression-like phenotype accompanied with reduced adult hippocampal neurogenesis. Also was seen the reduction in the activation of NCAM interaction partner FGFR and in downstream signalling pathways like calcium-calmodulin dependent kinases II and IV (CaMKII and IV) and CREB in NCAM-deficient mice. FGL peptide was able to ameliorate the signs of depressive-like behaviour, increase the neurogenesis and furthermore, FGL peptide restored the activation levels of FGFR, CaMKII, CaMKIV and CREB. The ability of FGL to modulate the levels of phosphorylated interaction partners and intracellular signalling pathways might partly explain antidepressant-like properties of the peptide. Üks viimaseid hüpoteese depressiooni patogeneesis seob selle haiguse tekkemehhanismid vähenenud aju plastilisusega. Kesknärvisüsteemis on peamisteks aju plastilisuse kujundajateks närviraku adhesioonimolekul (NCAM) ja tema polüsiaalhappega seotud vorm (PSA-NCAM). Sellest lähtuvalt võiks olla NCAM seotud depressiooni tekkemehhanismidega. Eelnevalt on näidatud, et NCAM on võimeline seostuma ka paljude rakupinna molekulidega ja sealtkaudu vahendama rakusisest signaaliülekannet. Üheks olulisemaks interaktsioonipartneriks NCAM’le on fibroblastide kasvufaktori retseptor (FGFR). Omavahelise seostumise kaudu käivitavad nad mitmeid signaalradu, mis kõik viivad transkriptsioonifaktor CREB aktiveerumiseni. Antud töö eesmärkideks oli selgitada, kas hiirtel, kellel puuduvad kõik NCAM isovormid, võiks esineda depressiooni-sarnane fenotüüp, muutused täiskasvanuea neurogeneesis ja häireid NCAM interaktsioonipartnerite ja NCAM-vahendatud rakusiseste signaalradade aktivatsioonis. Samuti hinnata FGL peptiidi toimet esinevatele muutustele. Töö tulemused näitasid, et NCAM-defitsiitsetel hiirtel esineb depressiooni-sarnane fenotüüp, millega kaasneb ka langenud täiskasvanuea hipokampaalne neurogenees. Samuti esinesid muutused FGFR, kaltsium-kalmoduliin sõltuvate kinaaside II ja IV (CaMKII ja IV) ning CREB aktivatsioonis nendel loomadel. FGL peptiid oli võimeline kõrvaldama kõiki esinevaid muutusi. Kuna FGL peptiid on võimeline aktiveerima NCAM interaktsioonipartnerit, FGFR’i ja alanevaid signaalradu, võiks see osaliselt seletada ka tema antidepressiivset efekti.
... Ecto-ATPases have been purified from a range of tissues (see Ziganshin et at., 1994) and in some cases cDNA sequences isolated. The ecto-ATPases were found to be identical to previously identified cell adhesion molecules (Lin & Guidotti, 1989;Culic et al., 1992;Dzhandzhugazyan & Bock, 1993;Edlund et al., 1993;McCuaig et al., 1993;Najjar et al., 1993). These glycoproteins are largely extracellular and the region which encodes ecto-ATPase activity is found in the extracellular portion. ...
Field stimulation of the sympathetic nerves of the guinea‐pig isolated vas deferens with trains of pulses for 20 s at 1–8 Hz produced characteristic biphasic contractions. The effect of the novel ecto‐ATPase inhibitor, 6‐N,N‐diethyl‐D‐β,γ‐dibromomethyleneATP (ARL 67156, formerly known as FPL 67156), on the magnitude of the initial, predominantly purinergic peak of this response was studied in order to determine the influence of enzymatic degradation of adenosine 5′‐triphosphate (ATP) on its action as a neurotransmitter.
The peak magnitude of the response to nerve stimulation was significantly increased in a concentration‐dependent manner by ARL 67156 (5–100 μ m ) and the size of the neurogenic response at 4 Hz was approximately doubled in the presence of ARL 67156 (100 μ m ).
ARL 67156 (100 μ m ) has a rapid onset of action. The enhancing effect on neurogenic contractions was maximal after 10 min, was well maintained for at least 30 min and was rapidly reversed, with responses returning to control levels 10 min after washout.
The neurogenic contraction in the presence of prazosin (0.1 μ m ) was purely purinergic, as it was abolished by the P 2 ‐purinoceptor antagonist, PPADS (100 μ m ). ARL 67156 (100 μ m ) produced a similar degree of enhancement of neurogenic responses in the absence and presence of prazosin, supporting the view that the enhancing effects of ARL 67156 on neurogenic contractions result from potentiation of the action of ATP.
Exogenous ATP and α,β‐methyleneATP produced rapid transient contractions. Responses to ATP were increased in magnitude and duration in the presence of ARL 67156 (100 μ m ), whereas those to the stable analogue, α,β‐methyleneATP were not significantly affected.
Contractions to exogenous noradrenaline (10 μ m ) and KCl (40 mM) were significantly enhanced by ARL 67156 (100 μ m ), but this potentiation was abolished by PPADS (100 μ m ). Therefore, this effect of the ecto‐ATPase inhibitor may be due to a build up of endogenous ATP, increasing the sensitivity of the smooth muscle to other agonists.
It is concluded that ARL 67156 potentiates the action of ATP, and that when ATP acts as a neurotransmitter its postjunctional actions are greatly attenuated by enzymatic degradation.
... The enzyme activity is stimulated by a number of divalent cations, of which Ca 2 ϩ and Mg 2 ϩ are the most relevant ones (Plesner 1995; Nagy et al. 1997). Ecto-ATPases are related to several different proteins (Dzhandzhugazyan and Bock 1993,1997; Handa and Guidotti 1996; Vasconcelos et al. 1996). The ...
The aim of this study was to investigate the distribution pattern of Ca2+- and Mg2+-dependent ecto-ATPases on the surface of rat brain capillary endothelial cells (ECs) in control and lipopolysaccharide (LPS)-treated animals. Ecto-ATPases in the membrane of vascular endothelial cells are suggested to play a crucial role in thromboregulation. Loss of this enzyme activity after oxidative stress and upregulation of the enzyme chain hydrolyzing extracellular ATP after transient forebrain ischemia have also been reported. We used histochemistry to localize the activities of this enzyme on ECs and found pH- and cation-dependent changes in the localization of enzyme activity both in control and in LPS-treated animals. These findings suggest the presence of more than one ecto-ATPase enzyme on the surface of rat capillary ECs. The different behavior of ECs after LPS treatment is the target of further investigations. The increased ecto-nucleotidase activity might play a role in nucleotide-mediated cellular responses after bacterial infections.
The publication of a report on the molecular cloning of the rat liver ecto-ATPase1 in 1989 was considered a major break-through in ecto-ATPase research, especially since no other ecto-ATPase had been purified at that time. The extensive homology of the cDNA sequence of the rat liver ecto-ATPase with human biliary glycoprotein I (BGPI) also gave hope that the function of the ecto-ATPases might soon be revealed since functional studies of BGPI and related proteins in the carcinoembryonic antigen (CEA) gene family had begun and a cell adhesion function had been suggested2, 3. Subsequent reports on amino acid sequence similarity of the rat liver ecto-ATPase with a rat liver cell adhesion molecule (cell-CAM 105)4, cross-reactivity of the ecto-ATPase and eell-CAM105 with antibodies generated against the other protein5, and functional assays4–6 unambiguously established that the BGP-like cDNA codes for a cell adhesion molecule. However, the important question of the relationship of ATPase activity and cell adhesion function was not addressed. In later reports where consequences of manipulating the cDNA on cell aggregation were described6–8, there was no concomitant evaluation of the ATPase activity of the mutants.
Extracellularly released ATP has a variety of functions, such as activating different purinoreceptors1, affecting cell adhesion via certain adhesion molecules2 and serving as a substrate for numerous ATP-hydrolysing enzymes. Ecto-ATPases are proposed to terminate the activation of purinoreceptors, participate in the salvage of purines, and to modulate cell adhesion3,4. Thus, an enzyme of the cascade of ATP hydrolysis, 5′-nucleotidase, or CD73, has recently been recognized as an adhesion molecule5.
Extracellular ATP is involved in cell-cell communication in many species ranging from invertebrates to man (1). It modulates neuronal functions via a set of purinoreceptors and ATP-utilizing ecto-enzymes (1,2). Some of the proposed functions of ecto-ATPases in the nervous system are termination of neurotransmission via purinoreceptors, salvage of purines, signal transduction and regulation of cell adhesion (3,4). This variety of functions indicates the existence of a variety of ecto-ATPases differing in structure and enzymatic characteristics. In this article we evaluated a series of inhibitors of different ecto-ATPases in order to provide tools for studies of the functions of these enzymes and for the identification of the individual proteins.
The plasma membrane Ca2+/Mg2+ ecto-ATPase is an acidic glycoprotein, which hydrolyzes different nucleoside triphosphates and is activated by millimolar concentrations of various divalent cations. Unlike transport ATPases, it does not require Mg-ATP as a substrate and is different from the mitochondrial, myofibrillar, and sarcoplasmic reticulum ATPases. This enzyme is present in all tissues of the body including liver, brain, heart, kidney, blood, platelets, endothelium, and smooth muscles. The Ca2+/Mg2+ ecto-ATPase is considered to play diverse physiological roles such as termination of purinergic transmission, regulation of extracellular ATP concentration, gating mechanism for Ca2+ and Mg2+ fluxes, ATP-driven proton pump, cell-to-cell communication as well as cellular differentiation and transformation in a tissue specific manner. The activity of Ca2+/Mg2+ ecto-ATPase is altered by a wide variety of physiological, pharmacological, and pathological interventions which change membrane fluidity and its composition with respect to cholesterol and phospholipid contents. The molecular weight of this enzyme varies from tissue to tissue in the range of 180–240 kDa with subunits of 90, 80, 67, 20, and 10 kDa. The cDNA sequence for the plasma membrane Ca2+/Mg2+ ecto-ATPase from different tissues show homology with different adhesion molecules including CD36, CD39, and CD70. The evidence in the existing literature suggests that the Ca2+/Mg2+ ecto-ATPase is a multifunctional adhesion molecule which exists in different isoforms in various tissues.
Strong evidence has recently been provided that ATP can act as a transmitter not only in smooth muscle but also in peripheral ganglia and brain [1,2]. ATP can act through ligand-gated ion channels or through receptors coupled to trimeric G proteins. At neurones from cultured or intact mammalian celiac ganglia and in slices of the medial habenula, ATP can function as a fast excitatory neurotransmitter [3]. Ca2+-dependent neurogenic release of ATP can be directly measured. This was shown for fractions of synaptosomes isolated from the electric-ray electric organ and mammalian brain, and for peripheral tissue preparations such as the taenia coli, bladder, vas deferens, and rat neuromuscular junction [4,5]. ATP is costored with acetylcholine in brain and at the neuromuscular junction, with noradrenaline in sympathetic nerves and possibly also with other neurotransmitter substances [6]. In either case the vesicular nucleotide is outnumbered by its cotransmitter. Intravesicular concentrations of ATP are in the millimolar range. Molecular ratios of released acetylcholine and ATP close to those of synaptic vesicle storage have been reported [7]. K+-induced synaptosomal ATP release was suggested to be quantal in nature [8]. However, release of ATP was not found to be directly related to its cotransmitter in all cases [9,10]. ATP can be released from cellular sites apparently lacking vesicular storage organelles. This includes the postsynaptic target cell [11,12]. In this case ATP may be released by a carrier or channel similar to the product of the multidrug resistance gene that has recently been implicated as an ATP channel [13].
During the past several years, convincing evidence has accumulated that ATP is stored within vesicles and co-released with various neurotransmitters upon depolarization of the nerve endings1–5. When released into neural junctions, ATP can fulfill multiple roles. As a co-transmitter, ATP modulates the release and effectivity of other neurotransmitters1, 2, 6–8 either by acting through its own, postjunctionally localized receptors9, or by altering the sensitivity of cholinoreceptors10 and adrenoreceptors 11. ATP has been well- established as a neurotransmitter in the peripheral nervous system4,9. The same role has also been suggested for ATP in the central nervous system12, which was supported by evidence showing that ATP could act as a fast excitatory transmitter in the brain13–15. In this role, ATP is assumed to act through a P2x-like purinoreceptor16–17 which is directly coupled to a non-voltage-dependent, high Ca2+-permeability ion channel18,19. On the other hand, ATP represents a major source of adenosine, a powerful neurosupressant4,23, which was shown to inhibit the release of excitatory neurotransmitters, such as acetylcholine24,25, dynorphin and glutamate8. Extracellular ATP can also serve as a substrate for ecto-protein kinases 20 and is thus involved in phosphorylation of various proteins on the neuronal cell surface21. In addition to its action in neurons, extracellular ATP is also assumed to be a signal molecule in neuronal-astrocytic interactions22.
Ecto-nucleotidases are cell surface-located enzymes which catalyze extra-cellular nucleotide hydrolysis. An extracellular hydrolysis pathway for nucleotides has been detected in essentially all tissues and also in a large variety of cell culture systems. Its general features include the following:
1.
Nucleoside 5′-triphosphates are sequentially metabolized to the nucleoside with nucleoside 5′-diphosphate and nucleoside 5′-monophosphate appearing as intermediate products. The nucleoside may then be further deaminated to inosine by adenosine deaminase (Franco et al. 1997).
2.
Not only ATP, ADP, and AMP but essentially all physiologically occurring purine and pyrimidine nucleotides are hydrolyzed.
3.
Extracellular hydrolysis of nucleotides is not inhibited by known inhibitors of intracellular ATPases such as P-type, F-type, and V-type ATPases.
4.
Nucleotide hydrolysis depends on divalent cations, generally millimolar concentrations of either Ca2+ or Mg2+.
5.
Nucleotide hydrolysis has an alkaline pH optimum.
6.
A major function of the extracellular enzyme chain appears to be the termination of the physiological action of nucleotides released from cells.
To date no information is available as to whether the hydrolysis of ATP is used to drive energy-dependent processes (for reviews of the earlier work see Arch and Newsholme 1978; Fox 1978; Pearson 1985; Dhalla and Zhao 1988; Ziganshin et al. 1994a; Plesner 1995; Sarkis et al. 1995; Beaudoin et al. 1996; Zimmermann 1996a,b; Plesner et al. 1997; Zimmermann and Pearson 1998).
The ecto-ATPase from chicken smooth muscle was solubilized, purified, and characterized. Mono- and polyclonal antibodies were raised and the tissue distribution based on Western analysis was determined. N-terminal and internal protein sequences were determined and used to design degenerate oligonucleotide probes to screen a chicken muscle cDNA library. Two overlapping partial clones encoding most of the ecto-ATPase were isolated and sequenced. A unified theory as to the mechanism by which many varied types of molecules modulate ecto-ATPase activity was developed, and the theory was supported by cross-linking data.
This chapter focuses on the members of the immunoglobulin (Ig) superfamily of cell-recognition molecules that are predominantly or exclusively expressed in the vertebrate nervous system. The chapter discusses the neural cell-adhesion molecule, NCAM. Cell-recognition molecules of the Ig-superfamily frequently express structural feature, repetitive elements homologous to the fibronectin type III (FnIII) domain. These repeats are generally expressed in the extracellular portion of the molecule between the Ig-like domains and the plasma membrane. The prototype of this domain was found to be repeated up to 16 times in the extracellular matrix protein, fibronectin. Structural data suggest that the FnIII domain and related structural units might be composed of two β-sheets with three and four antiparallel β-strands, thus exhibiting a remarkable resemblance to the C-type structure. The Ig superfamily recognition molecules so far characterized in the nervous system may be grouped into three subfamilies based on their structural features: (1) molecules containing Ig-like domain(s), but lacking FnIII-like repeats, (2) molecules combining Ig- and FnIII-type units, and (3) molecules that combine Ig-domains with structural units other than FnIII-like domains.
Monoclonal antibody RB13-6 recognizes a subset of rat brain glial precursor cells that are highly susceptible to malignant conversion by the carcinogen N-ethyl- N-nitrosourea. The corresponding cell surface antigen was identified as a membrane glycoprotein (gp130RB13-6) and purified by immunoaffinity chromatography from the tumorigenic neuroectodermal rat cell line BT4Ca. Sequencing of 5 endoproteinase-generated peptides of the purified antigen permitted the specific amplification of a cDNA fragment by reverse transcription-polymerase chain reaction and subsequent isolation of the complete coding sequence from a fetal rat brain cDNA library. The derived amino acid sequence indicates that the RB13-6 antigen is related to the human and murine plasma cell membrane protein PC-1, a nucleotide pyrophosphatase/alkaline phosphodiesterase and ectoprotein kinase. Similarly, purified gp130RB13-6possesses 5′-nucleotidase activity that can be inhibited with EDTA. Different from PC-1, gp130RB13-6isolated from BT4Ca cells is not a disulfide-linked dimer and contains an RGD-tripeptide sequence which, together with other structural features, suggests a possible function in cell adhesion and its subversion in malignant phenotypes.
A variety of nucleotides and the nucleoside adenosine can act as extracellular signaling substances. Their function is terminated by extracellular degradation via surface-located enzymes. The breakdown products may be recycled. This review discusses recent developments in the cellular and molecular biology of enzymes involved in extracellular purine metabolism, including diadenosine polyphosphate hydrolase, ATP-diphosphohydrolase (apyrase), nucleotide pyrophosphatase, 5′-nucleotidase, alkaline phosphatase, NAD-glycohydrolase, and adenosine deaminase. The potential of the surface-located enzymes for ADP-ribosylation and phosphorylation of extracellular proteins is also briefly discussed. Drug Dev. Res. 39:337–352, 1996. © 1997 Wiley-Liss, Inc.
The neural cell adhesion molecule (NCAM) is a member of the immunoglobulin superfamily and involved in the development and regeneration of the nervous system. NCAM is expressed in three major isoforms. Two of them have large intracellular domains of different lengths and are named according to their apparent molecular weight as NCAM140 or NCAM180. NCAM140 is known to promote neurite outgrowth, whereas NCAM180 stabilizes cell-cell contacts. Beside its role in neurite outgrowth, NCAM also shows ATPase activity on its extracellular domain. Here, we quantified neurite outgrowth of neuroblastoma cells, which were transfected with either NCAM140 or NCAM180 in the absence or presence of ATP. We could confirm that NCAM140 strongly promotes neurite outgrowth, whereas NCAM180 stimulates neurite outgrowth only to a minor extent. Furthermore, application of ATP reduced the NCAM-induced neurite outgrowth to background levels in a concentration-dependent and isoform-dependent manner.
In this review the hypothesis is developed, that high concentrations of adenosine are anti-inflammatory and therefore beneficial in inflammatory disease like rheumatoid arthritis. The anti-inflammatory effect of adenosine is mediated mainly via specific adenosine receptors on immune cells that are involved in the disease process. Unfortunately, at least three mechanisms support a decrease in the local concentration of adenosine at the site of inflammation: 1) A decrease in sympathetic innervation, which leads to decreased local release of the cotransmitter ATP at the site of inflammation and therefore to a decreased level of its degradation product adenosine. 2) High activities of the adenosine degrading enzyme adenosine deaminase, which leads to increased degradation of adenosine to inosine and therefore decreases the concentration of adenosine 3) Lower activity of the ATP degrading enzyme 5'-Ecto-nucleotidase, which leads to a slower degradation of ATP to adenosine, also resulting in lower local levels of adenosine present. Therefore, it has to be considered that the optimal treatment for rheumatoid arthritis has to include drugs that specifically target adenosine receptors or the adenosine metabolism to increase local adenosine concentrations. In this respect the mechanism of action of low-dose methotrexate therapy is discussed, which is thought to involve the inhibition of adenosine degrading enzymes rather than antiproliferative effects.
The role of ecto-ATPase in modulating the purinergic component of neurotransmission in the guinea-pig vas deferens has been investigated using ARL 67156, a recently developed inhibitor of ecto-ATPase. ARL 67156 rapidly and reversibly potentiated neurogenic contractions in a concentration-dependent manner. ARL 67156 also potentiated contractions evoked by exogenous ATP, but had no effect on those to the stable analogue α,β-methyleneATP or on those to noradrenaline and KCl in the presence of the P2-purinoceptor antagonist PPADS. These results are consistent with an inhibitory action of ARL 67156 on ecto-ATPase and suggest that ecto-ATPase modulates purinergic neurotransmission in the guinea-pig vas deferens.
Cell adhesion molecules (CAMs) are proteins mediating cell-cell or cell-extracellular matrix (ECM) interactions. CAMs are
traditionally divided into four groups, the cadherins, the selectins, the integrins and CAMs belonging to the immunoglobulin
superfamily (IgSF). The present chapter describes CAMs belonging to IgSF, that exclusively or in part, are expressed in the
nervous system. The chapter includes descriptions of myelin protein zero (P0), integrin-associated protein (CD47), neuroplastin,
activated leukocyte-cell adhesion molecule (ALCAM), melanoma cell adhesion molecule (MCAM), myelin-associated glycoprotein
(MAG), the neural cell adhesion molecules 1 and 2 (NCAM, NCAM2), Down Syndrome cell adhesion molecule (DSCAM) and Down Syndrome
cell adhesion molecule-like-1 (DSCAML1), sidekick 1 and 2 (SDK1, SDK2), signal-regulatory proteins (SIRPs), nectins, nectin-like
proteins (necls), and members of the CTX, IgLON, Robo, contactin, and L1 families. The individual descriptions include sections
describing the expression, isoforms, structure, posttranslational modifications, homo- and heterophilic binding partners,
signaling and functions of the individual CAMs. The chapter demonstrates CAMs to be more than simple regulators of adhesion.
Many CAMs are important mediators of intracellular signal transduction, and CAMs are involved in many biological phenomena
including migration, proliferation, and differentiation of cells, as well as axonal guidance, neurite outgrowth, and synaptic
plasticity and maturation.
In this review, the structural biology of interaction between the neural cell adhesion molecule (NCAM) and the fibroblast
growth factor (FGF) receptor is described and a possible mechanism of the FGF-receptor activation by NCAM is discussed. Most
of the FGF-receptor molecules are thought to be constantly involved in a transient interaction with NCAM. However, the FGF-receptor
becomes activated only when NCAM is involved in the trans-homophilic binding (mediating cell-cell adhesion). The trans-homophilic binding between the NCAM molecules is believed to result in the formation of either one- or two-dimensional “zipper”-like
arrays of the NCAM molecules, which leads to NCAM clustering and as a result to clustering of the FGF-receptor, which in turn
may lead to its activation through a direct receptor-receptor dimerization (and thus activation) due to an increase in the
local concentration of the receptor.
KeywordsNeural cell adhesion molecule-NCAM-Fibroblast growth factor receptor-FGFR
NCAM-type proteins modulate multiple neuronal functions, including the outgrowth and guidance of neurites, the formation,
maturation, and plasticity of synapses, and the induction of both long-term potentiation and long-term depression. The ectodomains
of NCAM proteins have a basic structure of five amino-terminal immunoglobulin (Ig), followed by two fibronectin type III (FnIII)
modules. As a result of alternative splicing, many NCAM-type proteins exist in several isoforms, including both transmembrane
and glycosylphosphatidylinositol (GPI)-anchored versions. Extracellularly, NCAM proteins mediate cell–cell adhesion through
homophilic interactions and bind to growth factors, growth factor receptors, glutamate receptors, other CAMs, and components
of the extracellular matrix. Intracellularly, NCAM-type proteins interact with various cytoskeletal proteins and regulators
of intracellular signal transduction. A central feature of the synaptic function of NCAM proteins is the regulation of their
extracellular interactions by adhesion-modulating glycoepitopes, their removal from the cell surface by endocytosis, and the
elimination of their adhesion-mediating interactions by the proteolytic cleavage of their ectodomains. Although specific aspects
of NCAM proteins have changed through evolution, core structural and functional features are conserved between NCAM-type proteins
in vertebrates and invertebrates, demonstrating that the functions of this class of adhesive proteins are of general importance
during nervous system formation.
KeywordsapCAM-ATP-Fasciclin II-Immunoglobulin-NCAM-PSA
Cell-cell adhesion molecules (CAMs) comprise a broad range of membrane-associated glycoproteins characterized by affinities for constituents of the extracellular matrix and cell-surface molecules expressed by other cells. Based on structural similarities and related functions, CAMs can be grouped into several families: the immunoglobulin (Ig) superfamily (IgSF), the cadherin superfamily, the integrin family and the selectin family (1). The neural cell adhesion molecule, NCAM, initially described as a synaptic membrane protein termed D2 (2), is the most extensively studied CAM. NCAM, mediating cell-cell as well as cell-substratum interaction and promoting neurite outgrowth, plays a key role in nervous system development, regeneration and learning (1,3).
Extracellular ATP is an abundant signaling molecule that has a number of functions in the nervous system. It is released by
both neurons and glial cells, activates purinergic receptors, and acts as a trophic factor as well as a neurotransmitter.
In this review, we summarize the evidence for a direct ATP-NCAM interaction and discuss its functional implications. The ectodomain
of NCAM contains the ATP binding Walker motif A and has intrinsic ATPase activity, which could modulate NCAM-dependent signaling
processes. NCAM interacts directly with and signals through FGFR. The NCAM binding site to ATP overlaps with the site of NCAM-FGFR
interaction, and ATP is capable of disrupting NCAM-FGFR binding. This implies that NCAM signaling through FGFR can be regulated
by ATP, which is supported by the observation that ATP can abrogate NCAM-induced neurite outgrowth. Finally, ATP can induce
NCAM ectodomain shedding, possibly affecting the structural plasticity associated with learning and memory.
KeywordsNCAM-ATP-Ecto-ATPase-Extracellular proteolysis-FGFR-Neural cell differentiation
The neuronal protein of cell adhesion belongs to the immunoglobulin superfamily of cell adhesion proteins. It consists of
an extracellular domain providing homo-and heterophilic interactions with surrounding molecules that are located on the cell
surface or are components of the extracellular matrix, a transmembrane part, and intracellular domains (NCAM140 and NCAM180).
In addition to its role in cell adhesion, NCAMs act as a signal receptor molecule. Adhesion and initiation of signal cascades
induced by binding to the NCAM extracellular domains occur interdependently but influence each other. The homo-and heterophilic
binding of NCAM can activate a number of intracellular signal cascades resulting in neurite growth, axone guidance, axone
myelinization, and formation of nerve fibers. It has been established that the intracellular signal is initiated by the interactions
between NCAMs and fibroblast growth factor receptors (FGFR), non-receptor tyrosine kinases (Fyn and FAK), glia-derived neurotrophic
factor (GDNF), ATP, prion proteins, and several other molecules. The review discusses possible mechanisms of functioning of
these signal cascades.
Ecto-nucleotidases play a pivotal role in purinergic signal transmission. They hydrolyze extracellular nucleotides and thus can control their availability at purinergic P2 receptors. They generate extracellular nucleosides for cellular reuptake and salvage via nucleoside transporters of the plasma membrane. The extracellular adenosine formed acts as an agonist of purinergic P1 receptors. They also can produce and hydrolyze extracellular inorganic pyrophosphate that is of major relevance in the control of bone mineralization. This review discusses and compares four major groups of ecto-nucleotidases: the ecto-nucleoside triphosphate diphosphohydrolases, ecto-5'-nucleotidase, ecto-nucleotide pyrophosphatase/phosphodiesterases, and alkaline phosphatases. Only recently and based on crystal structures, detailed information regarding the spatial structures and catalytic mechanisms has become available for members of these four ecto-nucleotidase families. This permits detailed predictions of their catalytic mechanisms and a comparison between the individual enzyme groups. The review focuses on the principal biochemical, cell biological, catalytic, and structural properties of the enzymes and provides brief reference to tissue distribution, and physiological and pathophysiological functions.
Cell adhesion molecules (CAMs) constitute a large class of plasma membrane-anchored proteins that mediate attachment between
neighboring cells and between cells and the surrounding extracellular matrix (ECM). However, CAMs are more than simple mediators
of cell adhesion. The neural cell adhesion molecule (NCAM) is a well characterized, ubiquitously expressed CAM that is highly
expressed in the nervous system. In addition to mediating cell adhesion, NCAM participates in a multitude of cellular events,
including survival, migration, and differentiation of cells, outgrowth of neurites, and formation and plasticity of synapses.
NCAM shares an overall sequence identity of ∼44% with the neural cell adhesion molecule 2 (NCAM2), a protein also known as
olfactory cell adhesion molecule (OCAM) and Rb-8 neural cell adhesion molecule (RNCAM), and the region-for-region sequence
homology between the two proteins suggests that they are transcribed from paralogous genes. However, very little is known
about the function of NCAM2, although it originally was described more than 20 years ago. In this review, we summarize the
known properties and functions of NCAM2 and describe some of the differences and similarities between NCAM and NCAM2.
Dado que las glicoproteínas denominadas moléculas de adhesión celular neural (NCAM, su forma polisializada, PSA-NCAM, y LI) desempeñan un papel importante en la consolidación de la memoria, se estudió su expresión en diferentes regiones cerebrales, en ratas entrenadas en el paradigma conductual del condicionamiento del miedo al contexto, bajo diferentes niveles de estrés intrínseco (ligado a la tarea de aprendizaje), y a diferentes tiempos post-entremamiento (12 y 24 horas). También se estudiaron los posibles efectos del estrés crónico sobre la expresión de dichas moléculas de adhesión celular neural en ratas sometidas a inmovilización y/o entrenadas en el condicionamiento del miedo al contexto. Los resultados obtenidos mostraron una diferencial implicación de la polisilaización de NCAM hipocampal en el almacenamiento de información relativa a experiencias que implican diferentes grados de estrés. En conjunto, los resultados mostrados en esta Tesis, apoyan la idea de una modulación de las moléculas de adhesion celular en situaciones de estrés, tanto agudo, como crónico, y sugieren la posibilidad de que los cambios producidos en los niveles de dichas moléculas como consecuencia del estrés participen en los mecanismos neurales implicados en diversas funciones cognitivas. Asimismo, los datos neuroendocrinos y conductuales refuerzan las hipoteizadas acciones de los glucocorticoides sobre los procesos de formación de la memoria, incluidas las memorias traumáticas. Por otro lado, el estrés crónico indujo un descenso de los niveles de NCAM hipocampales, que podría estar implicado en los procesos deletéreos que a nivel sináptico se han descrito en otros estudios a nivel de dicha estructura. Por el contrario, incrementó los niveles de PSA-NCAM y de Ll, moléculas implicadas en el remodelamiento estructural de tipo compensatorio ante el daño neural inducido por el estrés crónico. Además, el estrés crónico también indujo alteraciones en el contenido de dichas moléculas de adhesión en otras regiones cerebrales
Närvikoele on iseloomulik võime reageerida erinevatele organismi sisekeskkonnast või väliskeskkonnast tulevatele stiimulitele kohaste adaptatiivsete muutustega, eeltoodud omadust nimetatakse aju plastilisuseks. Käesolev töö uurib täiskasvanuea struktuurset plastilisust, mis hõlmab erinevaid ajukoe morfoloogilisi muutusi, keskendudes neurogeneesi (uute närvirakkude teke) fenomenile ja raku adhesioonisüsteemide rollile aju plastilisuses. Töö eesmärgid: Töö eesmärgiks oli uurida plii varajase sünnijärgse manustamise mõju täiskasvanud roti mälule ja õppimisvõimele, samuti neurogeneesile ja närviraku adhesioonimolekuli ekspressiooni tasemele hipokampuse hammastuumas. Samuti oli eesmärgiks uurida amfetamiini erinevate manustamisviiside toimet hiirte käitumisele ja närviraku adhesioonimolekuli ekspressiooni tasemele aju erinevates piirkondades. Töö tulemused: Madalalävine plii manustamine varajasel sünnijärgsel perioodil põhjustas katseloomadel täiskasvanueas ärevuse tõusu ja mäluhäireid, neurogeneesi languse ning tekkinud rakkude diferentseerumise häire, samas suurendas PSA-NCAM- positiivsete rakkude arvu hipokampuses. Eeltoodud muutused võivad osaleda plii ekspositsiooni kaugtagajärjena tekkivate kognitiivsete häirete kujunemisel. Akuutne amfetamiini manustamine langetas selektiivselt ja ajast sõltuvalt 180–200 kD molekulmassiga PSA-NCAM-i isovormi hipokampuses nii keskkonnaga assotsieeritud (Paired) kui ka keskkonnaga assotsieerimata (Unpaired) loomade grupis, mis võib olla oluline amfetamiini manustamise kaugtagajärjena tekkiva kognitiivse düsfunkstiooni tekkes. Krooniline amfetamiini manustamine kutsus esile märkimisväärse käitumusliku sensitisatsiooni, kuid ei mõjutanud PSA-NCAM ekspressiooni aju erinevates struktuurides, millest võib järeldada, et PSA-NCAM ei osale sensitisatsiooni välja kujunemisel. Brain plasticity refers to the ability of brain tissue to adequately react and adapt to continuous endogenous and environmental changes. The present study mainly explores the field of adult structural plasticity focusing on cell adhesion systems and on the phenomenon of neurogenesis. Aims of the study: To assess whether low-level lead exposure during early postnatal period induces emotional and cognitive dysfunction, alterations in neurogenesis and cell maturation as well as affects the expression levels of PSA-NCAM expressing cells and their phenotype in adult rat. To study the effects of different amphetamine administration regimens on the expression levels of neural cell adhesion molecule and its polysialylated form in various brain regions of adult mouse. Results: Early post-natal low-level lead exposure induced persistent increase in the level of anxiety and inhibited contextual fear conditioning, reduced the generation of new cells in the dentate gyrus of adult animal and altered the pattern of differentiation of BrdU-positive cells. Lead exposure induced significant increase in the total number of PSA-NCAM expressing cells and did not alter the proportion of cells co-expressing PSA-NCAM with glial or neuronal markers, which suggests perturbations in the differentiation process. Observed alteration could, at least partly, account for the cognitive impairments in adulthood following developmental lead exposure. Chronic amphetamine administration produced robust behavioral sensitization. The immunoblotting analysis demonstrated that acute administration of amphetamine selectively and time-dependently decreases the expression of 180–200 kDa isoform of PSA-NCAM in hippocampus in both context associated (the Paired) as well as context non-associated (the Unpaired) groups, which could be relevant in different synaptic-plasticity associated processes, however, according to our results, PSA-NCAM is not involved in the amphetamine-induced associated learning mechanism.
Strong evidence has been provided that ATP can act as a transmitter not only in smooth muscle but also in peripheral ganglia and in brain. The cloning and molecular identification of two putative ATP receptors supports the previously established pharmacological receptor classifications. This review places into perspective the evidence for ATP as a neural signalling substance by examining sites of storage, release and hydrolysis, as well as potential actions and targets. The action of ATP is related to that of the nucleoside adenosine, and the potential of additional nucleotides to function as neural messenger is examined briefly.
Ecto-ATPases are ubiquitous in eukaryotic cells. They hydrolyze extracellular nucleoside tri- and/or diphosphates, and, when isolated, they exhibit E-type ATPase activity, (that is, the activity is dependent on Ca2+ or Mg2+, and it is insensitive to specific inhibitors of P-type, F-type, and V-type ATPases; in addition, several nucleotide tri- and/or diphosphates are hydrolysed, but nucleoside monophosphates and nonnucleoside phosphates are not substrates). Ecto-ATPases are glycoproteins; they do not form a phosphorylated intermediate during the catalytic cycle; they seem to have an extremely high turnover number; and they present specific experimental problems during solubilization and purification. The T-tubule Mg2+-ATPase belongs to this group of enzymes, which may serve at least two major roles: they terminate ATP/ADP-induced signal transduction and participate in adenosine recycling. Several other functions have been discussed and identity to certain cell adhesion molecules and the bile acid transport protein was suggested on the basis of cDNA clone isolation and immunological work.
Monoclonal antibody RB13-6 recognizes a subset of rat brain glial precursor cells that are highly susceptible to malignant conversion by the carcinogen N-ethyl-N-nitrosourea. The corresponding cell surface antigen was identified as a membrane glycoprotein (gp130RB13-6) and purified by immunoaffinity chromatography from the tumorigenic neuroectodermal rat cell line BT4Ca. Sequencing of 5 endoproteinase-generated peptides of the purified antigen permitted the specific amplification of a cDNA fragment by reverse transcription-polymerase chain reaction and subsequent isolation of the complete coding sequence from a fetal rat brain cDNA library. The derived amino acid sequence indicates that the RB13-6 antigen is related to the human and murine plasma cell membrane protein PC-1, a nucleotide pyrophosphatase/alkaline phosphodiesterase and ectoprotein kinase. Similarly, purified gp130RB13-6 possesses 5'-nucleotidase activity that can be inhibited with EDTA. Different from PC-1, gp130RB13-6 isolated from BT4Ca cells is not a disulfide-linked dimer and contains an RGD-tripeptide sequence which, together with other structural features, suggests a possible function in cell adhesion and its subversion in malignant phenotypes.
The chicken gizzard smooth muscle extracellular ATPase (ecto-ATPase) is a low abundance, high specific activity, divalent cation-dependent, nonspecific nucleotide triphosphatase (NTPase). The ATPase is a 66-kDa glycoprotein with a protein core of 53 kDa (Stout, J.G. and Kirley, T.L. (1994) J. Biochem. Biophys. Methods 29, 61-75). In this study we evaluated the characteristics of a bank of monoclonal antibodies raised against a partially purified chicken gizzard ecto-ATPase. 18 monoclonal antibodies identified by an ATPase capture assay were tested for effects on ATPase activity as well as for their Western blot and immunoprecipitation potential. The five most promising monoclonal antibodies were used to immunopurify the ecto-ATPase. The one-step immunoaffinity purification of solubilized chicken gizzard membranes with all five of these monoclonal antibodies isolated a 66-kDa protein whose identity was confirmed by N-terminal sequence analysis to be the ecto-ATPase. Several of these monoclonal antibodies stimulated ecto-ATPase activity similar to that observed previously with lectins. Western blot analysis revealed that three of the five monoclonal antibodies recognized a major immunoreactive band at 66 kDa (53-kDa core protein), consistent with previous purification results. The other two antibodies recognized proteins of approximately 90 and 160 kDa on Western blots. The 90-kDa co-immunopurifying (and presumably associated or related) protein was identified by N-terminal analysis as LEP100, a glycoprotein that shuttles between the plasma and lysosomal membranes. The approximately 160-kDa co-immunopurifying protein was identified by N-terminal analysis as integrin, a protein involved in extracellular contacts with adhesion molecules. Extended N-terminal sequence analysis of the immunopurified 66-kDa ecto-ATPase revealed some sequence homology with mouse lysosomal associated membrane protein. Tissue distribution of the ecto-ATPase showed that the highest levels of protein were expressed in muscle tissues (cardiac, skeletal, and smooth) and brain.
The extracellular ATPase (ecto-ATPase) is a divalent cation-dependent nucleoside triphosphatase with an unusually high specific activity. Monoclonal antibodies, described previously [Stout, J. G., Strobel, R. S., & Kirley, T. L. (1995) J. Biol. Chem. 270, 11845-11850], and newly generated polyclonal antibodies, both raised against the chicken gizzard ecto-ATPase, were evaluated for their ability to cross-react with mammalian ecto-ATPases and were used as specific immunochemical probes to identify non-cross-linked and cross-linked ecto-ATPase. Unlike previous results obtained with the rabbit skeletal muscle ecto-ATPase enzyme, cross-linking the chicken gizzard smooth muscle ecto-ATPase with 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) and dithiobis(succinimidylpropionate) (DSP) increased the activity of the enzyme which corresponded to an increase in a approximately 130 kDa immunoreactive band, proposed to be a ecto-ATPase homodimer, and a concomitant decrease in a approximately 66 kDa immunoreactive band, the ecto-ATPase monomer. Ecto-ATPase was immunochemically identified in chicken, rat, mouse, rabbit, and pig. Interestingly, under nonreducing conditions, the ecto-ATPase activity in rat and pig (unlike chicken and rabbit) was evident on Western blots as an immunoreactive band at approximately 200 kDa, proposed to be an intermolecularly disulfide-linked ecto-ATPase homotrimer. Nonreducing Western blot analysis of various rat tissues with three different monoclonal antibodies that recognize the 66 kDa chicken gizzard ecto-ATPase monomer strengthened the hypothesis that this 200 kDa band indeed represents the trimeric ecto-ATPase. After reduction, ecto-ATPase monomers were found to be approximately 66 kDa in all species examined. The differences in ecto-ATPase quaternary structure stability may account for the observed species differences in ecto-ATPase enzymatic properties. Intermolecular disulfide bonds appear to be one of the species-specific ways to stabilize the native, active ecto-ATPase quaternary structure (the homotrimer). Based on the data obtained, as well as previous data from this and other laboratories, a hypothesis was developed to explain the modulation of ecto-ATPase activity by a variety of agents, including detergents, chemical cross-linkers, lectins, antibodies, and small molecule inhibitors. It is proposed that agents and conditions stabilizing ecto-ATPase oligomers stimulate enzyme activity, whereas agents and conditions destabilizing ecto-ATPase homooligomers would inhibit the ecto-ATPase.
There has been controversy for some time as to whether a posttraumatic influx of calcium ions occurs in stretch/nondisruptively injured axons within the central nervous system in both human diffuse axonal injury and a variety of models of such injury. We have used the oxalate/pyroantimonate technique to provide cytochemical evidence in support of such an ionic influx after focal axonal injury to normoxic guinea pig optic nerve axons, a model for human diffuse axonal injury. We present evidence for morphological changes within 15 min of injury where aggregates of pyroantimonate precipitate occur in nodal blebs at nodes of Ranvier, in focal swellings within axonal mitochondria, and at localized sites of separation of myelin lamellae. In parallel with these studies, we have used cytochemical techniques for localization of membrane pump Ca(2+)-ATPase and ecto-Ca-ATPase activity. There is loss of labelling for membrane pump Ca(2+)-ATPase activity on the nodal axolemma, together with loss of ecto-Ca-ATPase from the external aspect of the myelin sheath at sites of focal separation of myelin lamellae. Disruption of myelin lamellae and loss of ecto-Ca-ATPase activity becomes widespread between 1 and 4 h after injury. This is correlated with both infolding and retraction of the axolemma from the internal aspect of the myelin sheath to form periaxonal spaces which are characterized by aggregates of pyroantimonate precipitate, and the development of myelin intrusions into invaginations of the axolemma such that the regular profile of the axon is lost. There is novel labelling of membrane pump Ca(2+)-ATPase on the cytoplasmic aspect of the internodal axolemma between 1 and 4 h after injury. There is loss of an organized axonal cytoskeleton in a proportion of nerve fibres by 4-6 h after injury. We suggest that these changes demonstrate a progressive pathology linked to calcium ion influx after stretch (non-disruptive) axonal injury to optic nerve myelinated fibres. We posit that calcium influx, linked to or correlated with changes in Ca(2+)-ATPase activities, results in dissolution of the axonal cytoskeleton and axotomy between 4 and 6 h after the initial insult to axons.
A calcium-adenosine triphosphatase (Ca(2+)-ATPase) activity expressed by CNS nerve fibres has been examined during demyelination and remyelination in rats, 21-26 days after an intraspinal injection of ethidium bromide. The Ca(2+)-ATPase distribution was determined cytochemically, using a technique believed primarily to reflect the presence of ecto-ATPases. We confirm that in normal nerve fibres Ca(2+)-ATPase activity was present on the external surface of the myelin sheath, and on the axolemma at the nodes of Ranvier. Labelling of the internodal axolemma was restricted to small, scattered, punctate regions. However, following demyelination the Ca(2+)-ATPase activity was expressed continuously along both the exposed, previously internodal axolemma of entirely naked axons, and it was particularly prominent at sites of contact between axons and glial-cell processes. During remyelination (which in this lesion is accomplished predominantly by Schwann cells) the proportion of the axonal surface exhibiting Ca(2+)-ATPase activity decreased in concert with the progressive thickening of the new myelin sheath. The non-myelin forming plasmalemma of Schwann cells was positive for the Ca(2+)-ATPase activity, but activity was abruptly lost at the site of compaction between the inner and outer leaflets of the forming myelin sheath. Ecto-ATPase activity is a property of some cell adhesion molecules, and it follows that the changes observed in the distribution of ATPase activity in this study may reflect changes in the axolemma which are important for the successful repair of the lesion by remyelination. The ATPase activity may, for example, reflect the changing distribution of molecules important in aiding axo-glial recognition and the establishment of axo-glial contacts.
The cutaneous sensory nerve formations belong to the structures which are studied intensely by the enzyme activity histochemistry since the early history of this technique. The histochemical localization of the activities of nonspecific cholinesterase, alkaline phosphatases, acid phosphatase, adenosine tri- and diphosphatases, adenylate cyclase, and dipeptidylpeptidase-IV in the cutaneous sensory nerve formations, mainly sensory corpuscles, is reviewed. The histochemical approach has brought new knowledge of both morphological building of these unique structures and their biochemical constituents. Taken together, the present results of enzyme histochemistry provide insight into the function of enzymes, and disclose new relationships between the sensory terminals and auxiliary structures in the cutaneous sensory nerve formations.
Nucleotides such as ATP, ADP, UTP or the diadenosine polyphosphates and possibly even NAD+ are extracellular signaling substances in the brain and in other tissues. Enzymes located on the cell surface catalyze the hydrolysis of these compounds and thus limit their spatio-temporal activity. As a final hydrolysis product they generate the nucleoside and phosphate. The paper discusses the biochemical properties, cellular localization and functional properties of surface-located enzymes that hydrolyse nucleotides released from nervous tissue. This is preceded by a brief discussion of nucleotide receptors, cellular storage and mechanisms of nucleotide release. In nervous tissue nucleoside 5'-triphosphates are hydrolysed by ecto-ATP-diphosphohydrolase and possibly in addition also by ecto-nucleoside triphosphatase and ecto-nucleoside diphosphatase. The molecular identity of the ATP-diphosphohydrolase has now been revealed. The hydrolysis of nucleoside 5'-monophosphates is catalysed by 5'-nucleotidase whose biochemical properties and molecular structure have been studied in detail. Little is known about the molecular properties of the diadenosine polyphosphatases. Surface located enzymes for the extracellular hydrolysis of NAD+ and also ecto-protein kinases are discussed briefly. The cellular localization of the ecto-nucleotidases is only partly defined. Whereas in adult mammalian brain activity for hydrolysis of ATP and ADP may be associated with nerve cells or glial cells 5'-nucleotidase appears to have a preferential glial allocation in the adult mammal. The extracellular hydrolysis of the nucleotides is of functional importance not only during synaptic transmission where it functions in signal elimination. It plays a crucial role also for the survival and differentiation of neural cells in vitro and presumably during neuronal development in vivo.
The ecto-ATPase from chicken gizzard (smooth muscle) was solubilized, and the 66-kDa cell membrane ecto-ATPase protein was purified. The protein was then subjected to both enzymatic and chemical cleavage, and the resultant peptides were purified by reverse phase high pressure liquid chromatography and sequenced. Several of these internal peptide sequences were used to design oligonucleotides to screen a chicken muscle library to identify the cDNA encoding the ecto-ATPase. Two overlapping partial clones were sequenced, yielding the complete coding region and a long 3'-untranslated sequence. The deduced amino acid sequence is in agreement with the N-terminal and peptide sequences obtained from the purified protein. The chicken muscle ecto-ATPase is a slightly basic (predicted pI = 7.93) 494-amino acid protein (54.4 kDa), containing a single transmembrane domain at each end of the protein. The majority of the protein is predicted to be extracellular, making it a Type Ia plasma membrane protein. There are four putative N-glycosylation sites, a single potential cAMP/cGMP-dependent protein kinase phosphorylation site, as well as a single putative tyrosine kinase phosphorylation site. Analysis of the sequence using the BLAST programs demonstrated homology with other ecto-ATPases and ecto-apyrases, including those from the parasitic protozoan Toxoplasma gondii, potato tubers, and garden pea, as well as a guanosine diphosphohydrolase from yeast. However, the most striking homology observed was to the human and mouse lymphoid cell activation antigen 39 (CD39), a molecule now known to have apyrase activity. The chicken ecto-ATPase showed considerable amino acid sequence homology with CD39 over the entire length of the sequence, excluding about 30-40 amino acids at the extreme ends of the protein (which include the two membrane-spanning helices). The sequence homology between the gizzard ecto-ATPase and CD39 was confirmed by Western blots demonstrating immunocross-reactivity between mono- and polyclonal antibodies raised against the chicken ecto-ATPase and two commercially available monoclonal antibodies against the human CD39 protein. The results suggest that the muscle ecto-ATPase may be involved in cell adhesion, since the highly homologous CD39 protein is involved in homotypic adhesion of activated B lymphocytes.
Extracellular nucleotides acting as signaling molecules are inactivated by hydrolysis catalyzed by ecto-nucleotidases. ATP is sequentially degraded via ADP and AMP to adenosine. Enzymes that can be involved in the extracellular hydrolysis chain are ecto-ATP diphosphohydrolase (ecto-apyrase), ecto-ATPase, ecto-ADPase and 5'-nucleotidase. Mammalian ecto-ATP diphosphohydrolase is a member of a family of apyrases sharing four "apyrase conserved regions" that presumably participate in the formation of the catalytic site. We report the presence of ecto-ATP diphosphohydrolase in rat brain and the primary structure of a new mammalian member of the apyrase family. Expression in CHO cells shows that it represents an ecto-ATPase. As revealed by Northern analysis of rat tissues, the ecto-ATPase is co-expressed with ecto-ATP diphosphohydrolase in heart, kidney, spleen, thymus, lung, skeletal muscle and brain. Signals for both ecto-nucleotidases are very weak in liver. mRNAs for both proteins are present in PC12 cells, suggesting that the two nucleotidases may be co-expressed in the same neural cell. Using computer-aided sequence analysis, primary structure and membrane topography are compared with those of other members of the apyrase family.
A minor fraction of the total ecto-type (E-type) ATPase activity of rat synaptosomes has been detected in immunoprecipitates of the neural cell adhesion molecule, NCAM, indicating that this either is an intrinsic enzymatic activity of NCAM or of an ATPase tightly associated to NCAM [Dzhandzhugazyan & Bock (1993) FEBS Lett. 336, 279-283]. We here demonstrate ATPase activity in preparations of the lipid-anchored as well as the transmembrane NCAM isoforms immunoisolated from transfected L-cells. A fraction of the E-type ATPase activity is spontaneously released from synaptosomes. Released material was fractionated by various chromatographic procedures and an extracellular fragment of NCAM was shown to co-elute with the major part of the enzymatic activity. Furthermore, it was shown that agarose-coupled NCAM-antibodies retained 85% of the ATPase activity released from synaptosomes after treatment with phosphatidylinositol-specific phospholipase C. These findings restricted the association or expression of the enzymatic activity to the extracellular part of NCAM. An affinity reagent, 5'-p-fluorosulfonylbenzoyl adenosine, FSBA, was shown to inhibit ATPase activity of immunoisolated NCAM, and incorporation of FSBA was detected in all three major NCAM isoforms (A, B, and C). An excess of ATP prevented both inactivation of the enzyme and affinity labeling of NCAM. Thus, NCAM contains an ATP-binding site, and this site is localized extracellularly and probably has the catalytic function. Binding of the substrate or FSBA protected a proteolytic cleavage site in NCAM localized close to the membrane presumably by induction of a local conformational change in NCAM, indicating a mechanism by which ATP may regulate NCAM adhesion and adhesion-triggered processes. A possible role of this mechanism in synaptic plasticity and memory consolidation is proposed.
NCAM is a member of the Ig superfamily that is highly expressed in the nervous system. NCAM mediates cell-cell adhesion and adhesion of cells to the extracellular matrix. In addition to influencing the adhesive behaviour of cells, NCAM binding triggers the activation of intracellular signalling pathways. It is well established that NCAM is important for the formation of proper neuronal connections in the developing nervous system. Recently, plasticity of neuronal connections in the mature brain has also been shown to be dependent on NCAM function.
An extracellular ATPase (E-type ATPase) clone was isolated from a human brain cDNA library and sequenced. The transcript shows similarity to the previously published chicken smooth muscle and rat brain ecto-ATPase cDNAs, human CD39L1 cDNA (putative human ecto-ATPase), and mammalian CD39 (lymphoid cell activation antigen, ecto-apyrase, ATPDase, ATP-diphosphohydrolase) cDNAs. The full-length human brain cDNA encodes a 529 amino acid glycoprotein with a putative membrane spanning region near each terminus, with the majority of the protein found extracellularly. Expression of this clone in mammalian COS-1 cells yielded NaN3-sensitive ATPase and ADPase activity detectable both on intact cells and cell membrane preparations. The nucleotide hydrolysis ratio of the expressed protein is approx. 2.75:1 (ATPase:ADPase activity), classifying it as an ecto-apyrase. However, this hydrolysis ratio is intermediate between that observed for the ecto-ATPases and the CD39 ecto-apyrases (L. Plesner, Int. Rev. Cytol. 158 (1995) 141-214). Quantitative analyses of amino acid identities and similarities between this ecto-apyrase and other vertebrate E-type ATPases suggest that this human brain enzyme is nearly equally related to the ecto-ATPases and the CD39s, and phylogenetic analysis suggests that it could be an ancestral enzyme from which both ecto-ATPases and CD39 ecto-apyrases are derived.
Rat cardiac sarcolemmal Ca2+/Mg2+ ectoATPase (Myoglein), a membrane-bound enzyme requiring millimolar concentrations of Ca2+ or Mg2+ for maximal hydrolysis of ATP, has been purified to apparent homogeneity. Tryptic digestion and amino acid sequencing was used to design an oligonucleotide probe for screening a rat heart cDNA library; this produced a partial cDNA clone (pND2.1), and sequencing of a 400 base pair portion revealed a 100% homology to human platelet CD36. Northern blotting with pND2.1 detected a 3.1 kb transcript in rat heart but not in other tissues. Interspecies expression analysis (cardiac tissue total RNA blot probed with pND2.1) detected a approximately 2.0 kb transcript in canine, rabbit and porcine heart, whereas transcripts of a 4.1 kb, approximately 3.0 kb and 2.1 kb were observed in human cardiac tissue. A rat genomic DNA Southern blot, probed with pND2.1, indicated that there was a single copy of the gene in the rat genome. Expression of the pND2.1 cDNA in E. coli produced an 89 kDa polypeptide recognized by anti-human CD36 antibody but not by anti-rat Ca2+/Mg2+ ectoATPase antibody. It is concluded that rat cardiac Ca2+/Mg2+ ectoATPase is tightly associated with a protein highly homologous to the adhesion molecule CD36.
It has become clear that members of both the E-NTPase and PDNPase protein family can act as ectoenzymes or exoenzymes, or both, or may even have an intracellular localization. The functional characterization of recently cloned members of the ecto-NTPase family is expected to widen the range of catalytic properties and cellular locations even further. The molecular diversity of ectonucleotidases now appears to be comparable with that of receptors for nucleotides. It seems likely that additional family members including splice variants remain to be discovered. The enormous molecular diversity and overlapping tissue distribution of the members of the E-NTPase and PDNP family make it difficult to assign specific functions to enzymes in individual tissues. However, first examples have been elaborated. The definition of their physiological substrates remains a major challenge. It is expected that the generation of heterozygous and homozygous knockout mice for each subtype followed by double-knockouts obtained by crossbreeding of single knockout mice will help to elucidate the functional roles of ectonucleotidases. These novel insights, together with the development of specific enzyme inhibitors or recombinant enzymes, or both, promise to offer important tools for basic research and therapy, and may provide new understanding of the control of cellular function at the local level.
The neural cell adhesion molecule (NCAM) plays an important role in synaptic plasticity in embryonic and adult brain. Recently, it has been demonstrated that NCAM is capable of binding and hydrolyzing extracellular ATP. The purpose of the present study was to evaluate the role of extracellular ATP in NCAM-mediated cellular adhesion and neurite outgrowth. We here show that extracellularly added adenosine triphosphate (ATP) and its structural analogues, adenosine-5'-O-(3-thiothiophosphate), beta, gamma-methylenadenosine-5'-triphosphate, beta, gamma-imidoadenosine-5-triphosphate, and UTP, in varying degrees inhibited aggregation of hippocampal neurons. Rat glial BT4Cn cells are unable to aggregate when grown on agar but acquire this capacity when transfected with NCAM. However, addition of extracellular ATP to NCAM-transfected BT4Cn cells inhibited aggregation. Furthermore, neurite outgrowth from hippocampal neurons in cultures allowing NCAM-homophilic interactions was inhibited by addition of extracellular nucleotides. These findings indicate that NCAM-mediated adhesion may be modulated by extracellular ATP. Moreover, extracellularly added ATP stimulated neurite outgrowth from hippocampal neurons under conditions non-permissive for NCAM-homophilic interactions, and neurite outgrowth stimulated by extracellular ATP could be inhibited by a synthetic peptide corresponding to the so-called cell adhesion molecule homology domain (CHD) of the fibroblast growth factor receptor (FGFR) and by FGFR antibodies binding to this domain. Antibodies against the fibronectin type-III homology modules of NCAM, in which a putative site for ATP binding and hydrolysis is located, also abolished the neurite outgrowth-promoting effect of ATP. The non-hydrolyzable analogues of ATP all strongly inhibited neurite outgrowth. Our results indicate that extracellular ATP may be involved in synaptic plasticity through a modulation of NCAM-mediated adhesion and neurite outgrowth.
Diethyl pyrocarbonate (DEPC) in conditions that favour carbethoxylation of histidyl residues strongly inactivated E-type ATPase activity of a rat lung membrane preparation, as well as ecto-ATPase activity of rat vessels and human Epstein-Barr virus-transformed B lymphocytes. Inactivation of the enzyme (up to 70%) achieved at concentrations of DEPC below 0.5 mM could be fully reversed by 200 mM hydroxylamine at pH 7.5, thus confirming histidine-selective modification. UTP effectively protected the enzyme activity from DEPC inactivation. This was taken to indicate that the conformation adopted by the enzyme molecule upon substrate binding was not compatible with DEPC reaching and/or modifying the relevant histidyl residue. Substrate activation curves were interpreted to show the enzyme molecule to be inactive, at all substrate concentrations tested, when the target histidyl residue had been modified by DEPC. Comparison of known sequences of CD39-like ecto-ATP(D)ases with the results on inactivation by DEPC revealed His-59 and His-251 (according to the human CD39 sequence) as equally possible targets of the inactivating DEPC modification. Potato apyrase lacks a homologue for the former residue, while the latter is preserved in the enzyme sequence. Therefore, this enzyme was exposed to DEPC, and since hydrolysis of ATP and ADP by potato apyrase was insensitive to modification with DEPC, it was concluded that His-59 is the essential residue in CD39 that is affected by DEPC modification in a way that causes inactivation of the enzyme.
In this work, we describe the ability of living hemocytes from an insect (Manduca sexta, Lepidoptera) to hydrolyze extracellular ATP. In these intact cells, there was a low level of ATP hydrolysis in the absence of any divalent metal (8.24 +/- 0.94 nmol of Pi/h x 10(6) cells). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 15.93 +/- 1.74 nmol of Pi/h x 10(6) cells. Both activities were linear with cell density and with time for at least 90 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.33 mM MgCl2. This stimulatory activity was not observed when Ca2+ replaced Mg2+. The apparent Km values for ATP-4 and Mg-ATP2- were 0.059 and 0.097 mM, respectively. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.6 and 7.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced by an increase of pH. These ecto-ATPase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, sodium fluoride, tartrate, and levamizole. To confirm the observed hydrolytic activities as those of an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), as well as suramin, an antagonist of P2-purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents. Interestingly, lipopolysaccharide, a component of cell walls of gram-negative bacteria that increase hemocyte aggregation and phagocytosis, increased the Mg2+-dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg2+-independent ecto-ATPase activity.
The adhesion molecule on glia, AMOG, an integral cell surface glycoprotein highly expressed by cerebellar astrocytes and involved in neuron to astrocyte adhesion and granule neuron migration (Antonicek, H., Persohn, E., and Schachner, M. (1987) J. Cell Biol. 104, 1587-1595) has been identified as a beta2 subunit isoform of the mouse sodium pump (Gloor, S., Antonicek, H., Sweadner, K. J., Pagliusi, S., Frank, R., Moos, M., and Schachner, M. (1990) J. Cell Biol. 110, 165-174). Here we demonstrate that AMOG/beta2 expressed by cRNA injection in Xenopus oocytes is capable of combining with endogenous Xenopus alpha1 subunits or coexpressed Torpedo alpha1 subunits to yield a functional alpha1/AMOG sodium pump isozyme. Determinations of the number of ouabain binding sites and ouabain-sensitive Rb-86+ uptake suggest that the alpha1/AMOG isozyme has slightly lower maximum transport rate and apparent affinity for external K+ than the alpha1/beta1 isozyme. Immunoprecipitation of alpha1/AMOG complexes from digitonin extracts of [S-35]methionine-labeled oocytes with a monoclonal anti-AMOG antibody provides direct evidence for a stable association between AMOG and the alpha1 subunits of Xenopus and Torpedo.
Recombinant plant plasma membrane H(+)-ATPase has been produced in a yeast expression system comprising a multicopy plasmid and the strong promoter of the yeast PMA1 gene. Western blotting with a specific monoclonal antibody showed that the plant ATPase is one of the major membrane proteins made by the transformed cells, accounting for about 1% of total yeast protein. The plant ATPase synthesized in yeast is fully active. It hydrolyzes ATP, pumps protons, and the reaction cycle involves a phosphorylated intermediate. Phosphorylation is possible from both ATP and Pi. Unlike the situation in plants, however, most of the plant ATPase is not expressed in the yeast plasma membrane. Rather, the enzyme appears to remain trapped at a very early stage of secretory pathway: insertion into the endoplasmic reticulum. This organelle was observed to proliferate in the form of stacked membranes surrounding the yeast nucleus in order to accommodate the large amount of plant ATPase produced. In this location, the plant ATPase can be purified with high yield (70 mg from 1 kg of yeast) from membranes devoid of endogenous yeast plasma membrane H(+)-ATPase. This convenient expression system could be useful for other eukaryotic membrane proteins and ATPases.
AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.
The surface distribution of the plasma membrane Ca2+ (Mg2+)-ATPase (ecto-ATPase) in rat hepatocytes was determined by several methods. 1) Two polyclonal antibodies specific for the ecto-ATPase were used to examine the distribution of the enzyme in frozen sections of rat liver by immunofluorescence. Fluorescent staining was observed at the bile canalicular region of hepatocytes. 2) Plasma membranes were isolated from the canalicular and sinusoidal regions of rat liver. The specific activity of ecto-ATPase in the canalicular membranes was 22 times higher than that of sinusoidal membranes. The enrichment of the ecto-ATPase activity in the canalicular membrane is closely parallel to that of two other canalicular membrane markers, gamma-glutamyltranspeptidase and leucine aminopeptidase. 3) By immunoblots with polyclonal antibodies against the ecto-ATPase and the Na+,K+-ATPase, it was found that the ecto-ATPase protein was only detected in canalicular membranes and not in sinusoidal membranes, while the Na+,K+-ATPase protein was only detected in sinusoidal membranes and not in canalicular membranes. These results indicate that the ecto-ATPase is enriched in the canalicular membranes of rat hepatocytes.
The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.
Plasma membranes of many mammalian cells contain a Mg2+-dependent ATPase activity which is easily inactivated by detergents. This activity is the combined expression of at least two ATP-hydrolyzing enzymes (Knowles, A.F., Isler, R.E., and Reece, J.F. (1983) Biochim. Biophys. Acta 731, 88-96). We have purified one of these enzymes from the plasma membranes of a human oat cell carcinoma xenograft. The enzyme was extracted from the membranes by 0.5% digitonin and purified on a DE52 column. The purified enzyme contained a major protein band of Mr = 30,000 when dissociated by sodium dodecyl sulfate. It hydrolyzed all nucleoside triphosphates in the presence of Mg2+ or Ca2+, but showed little activity toward nucleoside diphosphates. The enzyme was inhibited by p-chloromercuriphenyl sulfonate, slowly inactivated by p-fluorosulfonylbenzoyl-5'-adenosine and dithiothreitol at room temperature, and lost activity readily in solutions containing low concentrations of several detergents. This knowledge of the macromolecular structure of the Mg2+(Ca2+)-ATPase and its catalytic properties is important in determining the orientation of the enzyme in the membrane and its physiological function.
The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B and a 115,000 Mr polypeptide C, whereas neurons expressed a 200,000 Mr polypeptide A as well as polypeptide B. Skeletal muscle cells produced polypeptide B. The polypeptides synthesized by the three cell types were immunochemically identical. The membrane association of polypeptide C was investigated with methods that distinguish peripheral and integral membrane proteins. Polypeptide C was found to be a peripheral membrane protein, whereas polypeptides A and B were integral membrane proteins with cytoplasmic domains of approximately 50,000 and approximately 25,000 Mr, respectively. The affinity of the membrane binding of polypeptide C increased during postnatal development. The posttranslational modifications of polypeptide C were investigated in glial cell cultures, and it was found to be N-linked glycosylated and sulfated.
We have previously shown that the neural adhesion molecules L1 and NCAM interact with each other to form a complex which binds more avidly to L1 than L1 to L1 alone (Kadmon, G., A. Kowitz, P. Altevogt, and M. Schachner. 1990a. J. Cell Biol. 110:193-208). This cis-association between L1 and NCAM is carbohydrate-dependent (Kadmon, G., A. Kowitz, P. Altevogt, and M. Schachner. 1990b. J. Cell Biol. 110:209-218). In the present study, we report that L1 and NCAM bind to each other via oligomannosidic carbohydrates expressed by L1, but not by NCAM, as shown in several experiments: (a) complex formation between L1 and NCAM is inhibited by a mAb to oligomannosidic carbohydrates and by the oligosaccharides themselves; (b) NCAM binds to oligomannosidic carbohydrates; (c) within the L1/NCAM complex, the oligomannosidic carbohydrates are hidden from accessibility to a mAb against oligomannosidic carbohydrates; (d) the recombinant protein fragment of NCAM containing the immunoglobulin-like domains and not the fragment containing the fibronectin type III homologous repeats binds to oligomannosidic glycans. Furthermore, the fourth immunoglobulin-like domain of NCAM shows sequence homology with carbohydrate recognition domains of animal C-type lectins and, surprisingly, also with plant lectins. A peptide comprising part of the C-type lectin consensus sequence in the fourth immunoglobulin-like domain of NCAM interferes with the association between L1 and NCAM. The functional importance of oligomannosidic glycans at the cell surface was shown for neurite outgrowth in vitro. When neurons from early postnatal mouse cerebellum were maintained on laminin or poly-L-lysine, neurite outgrowth was inhibited by oligomannosidic glycans, by glycopeptides, glycoproteins, or neoglycolipids containing oligomannosidic glycans, but not by nonrelated oligosaccharides or oligosaccharide derivates. Neurite outgrowth was also inhibited by the peptide comprising part of the C-type lectin consensus sequence in the fourth immunoglobulin-like domain of NCAM. The combined results suggest that carbohydrate-mediated cis-associations between adhesion molecules at the cell surface modulate their functional properties.
A new procedure is described for the isolation of synaptosomes from various parts of mammalian brain. This method utilizes an isoosmotic Percoll/sucrose discontinuous gradient and has some advantages over the traditionally used synaptosomal isolation techniques: (1) it is possible to prepare suitable gradients while retaining isoosmolarity; (2) the time of the preparation is remarkably short (approximately 1 h); (3) if necessary, the gradient material can be easily removed from the samples. Intact synaptosomes were recovered from the 10%/16% (vol/vol) Percoll interphase. The fractions were identified and characterized by electron microscopy and by several biochemical markers for synaptosomes and other subcellular organelles. The homogeneity of the preparations is comparable to or better than that of synaptosomes prepared by the conventional methods. This procedure has been successfully used for the isolation of synaptosomes from very small tissue samples of various experimental animals and human brain.
A microsomal fraction from canine brain gray matter has been extracted with the detergent sodium dodecyl sulfate to partially purify the membrane-bound (Na+ + K+)-stimulated adenosine triphosphatase. Phospholipid, glycolipid, and a family of other glycoproteins are also enriched by the procedure; it is proposed that the product is an intrinsic membrane protein fraction. 6--8-fold purification of (Na+ + K+)-ATPase is obtained without solubilizing the enzyme and without irreversibly altering its turnover number. Final specific activities are 350--400 mumol of ATP hydrolyzed/h per mg protein. The stimulation and reversible inactivation of the (Na+ + K+)-ATPase by dodecyl sulfate were examined for information relevant to the mechanism of action of the detergent.
When isolated rat mesenteric small arteries were submitted to 2 s of sonication, a nucleoside triphosphatase activity was released to the medium, mainly from the plasma membrane of the vascular smooth muscle cells. The activity was kinetically characterized: It hydrolysed ATP, UTP and GTP with the same substrate affinity and the same specific activity. CaATP, as well as MgATP were substrates for the enzyme with an apparent Km in the micromolar range. ATPase inhibitors: ouabain, vanadate, AlF4-, oligomycin and N-ethylmaleimide were without effect on the hydrolytic activity. Among other modifiers tested only N,N'-dicyclohexylcarbodiimide caused significant (greater than 30%) inhibition. In the presence of micromolecular concentrations of Ca2+ and Mg2+, small (less than 20 mM) concentrations of Na+, K+, Rb+, Cs+ and choline+, irrespective of the nature of the anion, activated the hydrolysis with an equilibrium ordered pattern, but concentrations of monovalent cation salts above 20 mM decreased the hydrolysis rate. No activation by monovalent cation salts was seen at millimolar concentrations of divalent cations and substrate. On the basis of the results a standard mixture is proposed, which allows a sensitive assay of the specific enzyme activity.
One of the cell-adhesion molecules (CAMs) responsible for rat hepatocyte aggregation has been described as a glycoprotein having an Mr of 105,000 (cell-CAM105). The Mr and localization of cell-CAM105 in liver membranes are very similar to those of liver ecto-ATPase, an ATPase with its nucleotide-hydrolysing site localized on the outside of the cell membrane. The protein sequence of the ecto-ATPase has been deduced from cDNA cloning. Structural analysis of the sequence indicates that the ecto-ATPase has immunoglobulin-like domains and is a member of the immunoglobulin superfamily. Since a group of proteins in the immunoglobulin superfamily has been shown to have functions related to cell adhesion, the structural characteristics of the ecto-ATPase further led to the possibility that the ecto-ATPase may have functions related to cell adhesion. In this paper, using the cDNA for the ecto-ATPase, the anti-peptide antibodies produced against peptides derived from the ecto-ATPase cDNA sequence and monoclonal antibodies against the cell-CAM105, we present evidence of identity between cell-CAM105 and ecto-ATPase. First, in Western immunoblots, two anti-cell-CAM105 monoclonal antibodies cross-reacted with the purified ecto-ATPase. Secondly, in immunodepletion experiments, antibodies against the ecto-ATPase depleted the same protein recognized by the anti-cell-CAM105 antibodies. Thirdly, in two-dimensional gel-electrophoretic analysis, anti-peptide antibodies generated against an extracellular N-terminal peptide and the intracellular C-terminal peptides of the ecto-ATPase immunoprecipitated proteins of similar isoelectric points and Mr values to those of the cell-CAM105. Fourthly, proteins immunoprecipitated by anti-ecto-ATPase antibodies and anti-cell-CAM105 antibodies have similar V8-proteinase-digest peptide maps. Finally, monoclonal antibodies against the cell-CAM105 specifically recognized the protein expressed in COS cells transfected with the ecto-ATPase cDNA. These results indicate that the ecto-ATPase cDNA codes for a protein that is identical with the cell-CAM105. Since the ecto-ATPase has structural features of immunoglobulin domains, the identity of cell-CAM105 with ecto-ATPase leads to the conclusion that this liver CAM, similarly to neuronal CAM, is also a member of the immunoglobulin supergene family. Furthermore, immunological studies indicate that the cell-CAM105/ecto-ATPase is composed of two isoforms of different C-terminal sequences. The association of ATPase activity with cell-CAM105 raises the possibility that extracellular nucleotides may play important roles in regulating cell adhesion.
Cell recognition and adhesion, being of prime importance for the formation and integrity of tissues, are mediated by cell adhesion molecules, which can be divided into several distinct protein superfamilies. The cell adhesion molecule C-CAM (cell-CAM 105) belongs to the immunoglobulin superfamily, and more specifically is a member of the carcinoembryonic antigen (CEA) gene family. C-CAM can mediate adhesion between hepatocytes in vitro in a homophilic, calcium-independent binding reaction. The molecule, which occurs in various isoforms, is expressed in liver, several epithelia, vessel endothelia, platelets and granulocytes and its expression is dynamically regulated under various physiological and pathological conditions. It is proposed that C-CAM in different cells and tissues plays different functional roles, where the common denominator is membrane-membrane binding.
Cell-CAM 105 (C-CAM), a cell adhesion molecule in rat hepatocytes, was digested with trypsin, and peptides were isolated and sequenced by Edman degradation. The sequences of 4 peptides agreed with different regions of rat liver ecto-ATPase. Detailed biochemical analyses confirmed the identity between C-CAM and the ecto-ATPase. C-CAM/ecto-ATPase is a transmembrane protein having 4 immunoglobulin-like domains in the extracellular portion, demonstrating membership of the immunoglobulin superfamily. The ATPase activity suggests that ATP might influence cell adhesion, which would explain the inhibitory effect of exogenously added ATP on adhesion of several cell types.
Na+,K(+)-ATPase has distinctly different distributions in mesenchymal cells, where it has an unrestricted distribution over the entire cell surface, compared with polarized epithelial cells, where it is restricted to the basal-lateral membrane domain. The generation of this restricted distribution is important in mesenchyme to epithelia conversion in development and the function of transporting epithelia, but the mechanisms involved are unknown. Here we show that expression of the epithelial CAM uvomorulin in transfected fibroblasts is sufficient to induce a redistribution of Na+,K(+)-ATPase to sites of uvomorulin-mediated cell-cell contacts, similar to that in polarized epithelial cells. This restricted distribution of Na+,K(+)-ATPase occurs in the absence of tight junctions but coincides with the reorganization of the membrane cytoskeleton. The results indicate a direct role for CAMs as inducers of cell surface polarity of selective cytoplasmic and membrane proteins.
An individual catalytic component of calmodulin-independent adenylate cyclase has been isolated from bovine brain cortex. Affinity chromatography on an immunosorbent was used. The amino acid sequence of adenylate cyclase as well as the corresponding nucleotide sequence of the cDNA has been determined. cDNA of adenylate cyclase encodes a protein consisting of 834 amino acid residues and the signal peptide (19 amino acid residues). A series of adenylate cyclase isoforms has been found. A homology between adenylate cyclases from bovine brain, E. coli and Bordetella pertussis has been revealed.
The original lead-trapping method for demonstrating Na+--K+-ATPase activity was discredited because of the effect that lead ions can have on the substrate and on the enzyme. Current methods, that measure this activity by the related K+-dependent phosphatase activity, do not appear to measure activity that is known, from microchemistry, to occur in proximal convoluted tubules. The disadvantages of using lead appear to have been overcome by the use of a new reagent in which the lead is complexed with ammonium citrate ions; phosphate, liberated enzymatically, successfully competes with these ions. The activities of total ATPase and of the ouabain sensitive Na+--K+-ATPase have been measured in three regions of the nephron in the guinea-pig and in the rat. The relative activities found, by this method, in the different regions of the latter, appear to be comparable with results found by others, using microchemical methods applied to isolated regions of the nephron.
D2 is a nervous-specific membrane protein enriched in fractions of synaptosomal membranes from rat brain. Recently, an immunochemical relationship between D2 and the chick cell adhesion molecule (CAM) has been demonstrated. There is reason to believe that D2 is involved in adhesion phenomena between neurites. The purpose of the present study was to purify and further characterize the D2 protein from rat brain. In the developed purification procedure synaptosomal membranes from rat brains were prepared and solubilized by means of non-ionic detergent. The subsequent purification steps were hydroxylapatite chromatography, wheat germ lectin affinity chromatography, gel filtration, and lysine affinity chromatography. The purified D2 was found to be enriched 240 times compared with the starting brain homogenate and 120 times compared with the synaptosomal membrane fraction. The recovery of D2 was 26% when the amount of D2 in the synaptosomal membrane fraction was set to 100%. The purified D2 antigen was used for production of monospecific rabbit antisera, and it was found to be composed of two polypeptides of apparent molecular weights 130,000 and 150,000, respectively.
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