Niggli Verena

Universität Bern | UniBe · Institute of Pathology

Flotillin-1 (Flot1) is an evolutionary conserved, ubiquitously expressed lipid raft-associated scaffolding protein. Migration of Flot1-deficient neutrophils is impaired because of a decrease in myosin II-mediated contractility. Flot1 also accumulates in the uropod of polarized T cells, suggesting an analogous role in T cell migration. In this study...

Flotillin-1 (Flot1) is a highly conserved, ubiquitously expressed lipid raft-associated scaffolding protein. Migration of Flot1-deficient neutrophils is impaired due to a decrease in myosin II - mediated contractility. Flot1 also accumulates in the uropod of polarized T cells, suggesting an analogous role in T cell migration. Here, we analyzed morp...

Plasma membrane Ca²⁺ ATPases (PMCA pumps) are key regulators of cytosolic Ca²⁺ in eukaryotes. They extrude Ca²⁺ from the cytosol, using the energy of ATP hydrolysis and operate as Ca²⁺ -H⁺ exchangers. They are activated by the Ca²⁺ -binding protein calmodulin, by acidic phospholipids and by other mechanisms, among them kinase-mediated phosphorylati...

This review is focused on mechanisms of chemokine-induced polarization of T-lymphocytes. Polarization involves, starting from spherical cells, formation of a morphologically and functionally different rear (uropod) and front (leading edge). This polarization is required for efficient random and directed T-cell migration. The addressed topics concer...

We have shown previously that the raft-associated proteins flotillin-1 and -2 are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Other proteins such as the adhesion receptor PSGL-1, the actin-membrane linker proteins ezrin/radixin/moesin (ERM) and the signaling enz...

We have previously identified phosphatidylinositol-4-phosphate 5-kinase type I (PIPKI)γ90 as a T cell uropod component. However, the molecular determinants and functional consequences of its localization remain unknown. In this report, we seek to better understand the mechanisms involved in PIPKIγ90 uropod targeting and the role that PIPKIγ90 plays...

Determinants of uropod targeting of PIPKIγ90 in murine T cells Representative immunofluorescence pictures showing the localization of GFP-tagged wild type and mutant PIPKIγ (green) and phospho-ERM (red) in murine D10 T cells randomly migrating on ICAM-1. Scale bar represents 5 µm. Images are representative of 30 cells from 3 independent experiments...

T cell uropods are enriched in specific proteins including adhesion receptors such as P-selectin glycoprotein ligand-1 (PSGL-1), lipid raft-associated proteins such as flotillins and ezrin/radixin/moesin (ERM) proteins which associate with cholesterol-rich raft domains and anchor adhesion receptors to the actin cytoskeleton. Using dominant mutants...

Random migration of human T cells expressing constitutively active T567D ezrin-EGFP. T cells were transfected with T567D ezrin-EGFP and placed 4 h later on an Il-1-beta-stimulated human endothelial cell layer in the absence of added chemokine. Random migration of the transfected T cells was assessed for 10 min by time-lapse videomicroscopy at 37°C...

We have shown previously that endogenous flotillin-1 and -2, closely related proteins implicated in scaffolding of membrane microdomains, are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells, and are involved in cell polarization. Coexpressed flotillin-1 and -2, but not singly expressed proteins, are also...

Coomassie-stained immunoblot of protein lysates from tumor samples. 40 µg protein extract per sample were separated using SDS-PAGE (7.5%) under reducing conditions and blotted onto a polyvinylidene difluoride (PVDF)-membrane. The Coomassie-stained blot confirms equal loading and blotting of proteins across the samples. (JPG)

Inhibition assay of Mannan-binding lectin (MBL) and meprin-α and meprin-β. There was no evidence for an inhibitory activity of Mannan-binding lectin towards recombinant human, mouse and rat meprins, and human meprins in transfected cells and in purified human intestinal brush border membrane. (PDF)

Meprin-α is a metalloprotease overexpressed in cancer cells, leading to the accumulation of this protease in a subset of colorectal tumors. The impact of increased meprin-α levels on tumor progression is not known. We investigated the effect of this protease on cell migration and angiogenesis in vitro and studied the expression of meprin-α mRNA, pr...

Different types of membrane microdomains (rafts) have been postulated to be present in the rear and front of polarized migrating T-lymphocytes. Disruption of rafts by cholesterol sequestration prevents T-cell polarization and migration. Reggie/flotillin-1 and -2 are two highly homologous proteins that are thought to shape membrane microdomains. We...

Rho family proteins are constitutively activated in the highly invasive human fibrosarcoma HT1080 cells. We now investigated the specific roles of Rac1 and Rac2 in regulating morphology, F-actin organization, adhesion, migration, and chemotaxis of HT1080 cells. Downregulation of Rac1 using specific siRNA probes resulted in cell rounding, markedly d...

Background: Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that...

Cation-transporting P-type ATPases show a high degree of structural and functional homology. Nevertheless, for many members of this large family, the molecular mechanism of transport is unclear; namely, whether transport is electrogenic or not and if countertransport is involved remains to be established. In a few well-studied cases such as the Na(...

Ezrin, radixin and moesin (ERM) proteins are widely distributed proteins located in the cellular cortex, in microvilli and adherens junctions. They feature an N-terminal membrane binding domain linked by an alpha-helical domain to the C-terminal actin-binding domain. In the dormant state, binding sites in the N-terminal domain are masked by interac...

Rat Walker 256 carcinosarcoma cells spontaneously develop front-tail polarity and migrate in the absence of added stimuli. Constitutive activation of phosphatidylinositol-3 kinase (PI 3-kinase), Rac, Rho and Rho kinase are essential for these processes. Ezrin and moesin are putative targets of these signaling pathways leading to spontaneous migrati...

At least 18 different cytoskeletal proteins have been shown to interact in vitro with, and in some cases are regulated by, specific membrane lipids, mostly phosphoinositides, whose synthesis is themselves regulated by extracellular signals. These lipid interactions are mediated by structurally diverse specific binding sites. Lipid interactions can...

Myelosuppression is the most common unwanted side effect associated with the administration of anticancer drugs, and infections remain a common cause of death in chemotherapy-treated patients. Several mechanisms of the cytotoxicity of these drugs have been proposed and may synergistically operate in a given cell. Survivin expression has been associ...

We present evidence for differential roles of Rho-kinase and myosin light chain kinase (MLCK) in regulating shape, adhesion, migration, and chemotaxis of human fibrosarcoma HT1080 cells on laminin-coated surfaces. Pharmacological inhibition of Rho-kinase by Y-27632 or inhibition of MLCK by W-7 or ML-7 resulted in significant attenuation of constitu...

In a mendelian (sr 3 ) and an uniparental (sr 35 ) streptomycin resistant mutant of Chlamydomonas reinhardi the influence of streptomycin on protein synthesis on the chloroplast and cytoplasmic ribosomes was investigated in vitro . Hetero‐, mixo‐ and phototrophic agar cultures and heterotrophic liquid cultures were used. Protein synthesis on the cy...

As previously shown, constitutive activation of the small GTPase Rho and its downstream target Rho-kinase is crucial for spontaneous migration of Walker carcinosarcoma cells. We now show that after treatment of cells with either the Rho inhibitor C3 exoenzyme or the Rho-kinase inhibitor Y-27632, constitutive myosin light chain (MLC) phosphorylation...

Phosphoinositide phosphates (PIPs) correspond to phosphorylated derivatives of phosphatidylinositol (PI). Despite their relatively low abundance in the plasma membrane, PIPs play a crucial role as precursors of second messengers and are themselves important signaling and targeting molecules. Indeed, modulation of levels of PIPs affects, for example...

Electrophilic alkylation of colchicine at C(4) was accomplished by a multicomponent aromatic electrophilic substitution reaction with electrophilic aldehydes and carboxylic acids or amides in H2SO4. A series of new derivatives were obtained and evaluated for their antiproliferative effect towards various tumor cell lines, and their stimulatory effe...

Stimulation of neutrophils with chemotactic peptide induces actin reorganization, formation of actin-rich protrusions, and development of polarity. Shape changes and actin polymerization can also be induced by phorbol ester-mediated direct activation of protein kinase C (PKC). We have investigated the role of cholesterol in stimulus-dependent motil...

Stimulation of neutrophils with chemotactic peptide induces actin reorganization, formation of actin-rich protrusions, and development of polarity. Shape changes and actin polymerization can also be induced by phorbol ester-mediated direct activation of protein kinase C (PKC). We have investigated the role of cholesterol in stimulus-dependent motil...

Neutrophils, a major type of blood leukocytes, are indispensable for host defense of bacterial infections. Directed migration in a gradient of chemotactic stimuli enables these cells to rapidly find the site of infection and destroy the invading pathogens. Chemotactic factors bind to seven-transmembrane-domain receptors and activate heterotrimeric...

We studied the role of Rho kinase and extracellular signal-regulated kinase (ERK)-2 in the polarization and migration of T lymphocytes in response to the CCR7 ligands EBI1 ligand chemokine (ELC; CCL19) and secondary lymphoid-tissue chemokine (SLC; CCL21). Both Rho kinase protein isoforms are expressed in T lymphocytes. Inhibition of the Rho kinases...

Neutrophil granulocytes rely on a functional actin network for directed migration. Microtubule disassembly does not impair receptor-linked chemotaxis, instead it induces development of polarity and chemokinesis in neutrophils concomitant with polarized distribution of alpha-actinin and F-actin. Cells stimulated with colchicine, which disassembles m...

We have carried out a detailed comparison of the motile properties of differentiated HL-60 cells and human peripheral blood neutrophils. We compared the effects of chemotactic stimuli and of inhibitors of signalling proteins on morphology, chemokinesis and chemotaxis of neutrophils and differentiated HL-60 cells using videomicroscopy and a filter a...

Several cytoskeletal proteins have been shown to interact in vitro with, and in some cases are regulated by, specific membrane lipids. In some cases, evidence for in situ interactions has been provided. The molecular basis for such interactions is now being unravelled. At least five structurally distinct types of lipid-binding sites in cytoskeletal...

Signal transduction pathways controlling spontaneous locomotion of Walker carcinosarcoma cells are not well understood. We have therefore investigated the role of signalling proteins in development of polarity and locomotion of these cells. Treatment of the cells with 100 ng/ml pertussis toxin had no significant effect on the percentage of polarize...

The cytoskeleton-membrane linker protein ezrin has been shown to associate with phosphatidyl-inositol 4,5-bisphosphate (PIP(2))-containing liposomes via its NH(2)-terminal domain. Using internal deletions and COOH-terminal truncations, determinants of PIP(2) binding were located to amino acids 12-115 and 233-310. Both regions contain a KK(X)(n)K/RK...

The role of protein kinase C (PKC) isoforms in the regulation of cell shape [switch between fibroblast-like and crescent shape (CS)] and of locomotion of human fibrosarcoma HT1080 cells has been investigated. The PKC activator phorbol myristate acetate (PMA) induced the transition of elongated fibroblast-like cells into CS cells and stimulated loco...

Activity of phosphatidylinositol (PI) 3-kinase is required for optimal migration of human neutrophils [Niggli and Keller (1999) Eur. J. Pharmacol. 335, 43-52]. We have tested the direct effect of a product of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), on neutrophil migration. To this end, a membrane-permeant ester of PIP(3), di...

We report a selective, differential stimulus-dependent enrichment of the actin-associated protein alpha-actinin and of isoforms of the signaling enzyme protein kinase C (PKC) in the neutrophil cytoskeleton. Chemotactic peptide, activators of PKC, and cell adhesion all induce a significant increase in the amount of cytoskeletal alpha-actinin and act...

Treatment with low (nanomolar) concentrations of phorbol-12-myristate-13-acetate (PMA) for 5 to 30 min suppresses locomotion of Walker 256 carcinosarcoma cells, suggesting that activation of protein kinase C (PKC) is a stop signal for tumor cell locomotion. We have compared the effects of PMA on cell shape and motility with down-regulation of speci...

The role of a Rho-associated coiled-coil forming kinase in migration of neutrophils has been investigated. Rho-associated coiled-coil forming kinase I was expressed in human neutrophils. Chemotactic peptide led to a Rho-associated coiled-coil forming kinase-dependent increase in phosphorylation of myosin light chain. This was determined with the he...

The tail domain of vinculin (Vt) contains a salt-insensitive binding site for acidic phospholipids which is masked by the intramolecular head-tail interaction in native vinculin [Johnson, R. P., and Craig, S. W. (1995) Biochem. Biophys. Res. Commun. 210, 159-164]. To characterize further this phospholipid binding site, we have used hydrophobic phot...

Rapid and significant progress has been made in understanding lipid/protein interactions involving cytoskeletal components and the plasma membrane. Covalent and noncovalent lipid modifications of cytoskeletal proteins mediate their interaction with lipid bilayers. The application of biophysical techniques such as differential scanning colorimetry,...

We have studied activation-induced dephosphorylation of proteins in human neutrophils loaded with [32P]orthophosphate using two-dimensional gel electrophoresis and autoradiography. A major phosphoprotein of 20 kDa in resting neutrophils was markedly dephosphorylated upon activation of cells with chemotactic peptide or phorbol 12-myristate 13-acetat...

To define a possible role of the enzyme phosphatidylinositol 3-kinase (PI 3-kinase) in motile functions of neutrophils, we have used a potent inhibitor of this enzyme, [1S-(1alpha,6b alpha,9a beta,11alpha,11bbeta)]-1-(acetyloxy)-1,6b,7,8,9a,10,11 ,11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3,2-de]indeno [4,5-h]-2-benzopyran-3,6,9-tri...

The phosphatase inhibitor okadaic acid exerted a biphasic effect on the shape of spontaneously polarized Walker carcinosarcoma cells. At lower concentrations, the drug suppressed cell polarity (IC50 = 0.14 microM) and the cells reverted to a spherical shape. At higher concentrations (> 0.25 microM), cells developed large blebs (IC50 = 0.4 microM)....

We have investigated the binding of PI, PIP and PIP2 to talin and the effect of phosphoinositides and adenosinenucleotides on talin-induced actin polymerization. At physiological salt concentrations, talin coprecipitates with liposomes when containing phosphoinositides but not when containing PI. The nucleating effect of talin as reflected by a two...

Signal transduction pathways controlling tumor cell locomotion are not yet well understood. We have studied the role of protein kinase C (PKC)-dependent protein phosphorylation associated with changes in cell shape and locomotor activity of Walker carcinosarcoma cells in culture. We show that the inhibitory effect of phorbol-12-myristate-13-acetate...

Cytoskeleton-membrane linkage is important for organelle transport, adhesion of cells to the substrate or to other cells, shapes changes, extension of pseudopods, and cell migration. These cellular processes are involved in physiological events, such as embryonal development, immune defense, and wound healing. An understanding of the mechanism and...

Purified human recombinant ezrin cosediments with large liposomes containing phosphatidylserine (PS). This interaction is optimal at low ionic strength. At physiological ionic strength (130 mM KCl) ezrin interacts strongly with liposomes containing > or = 5% phosphatidylinositol-4,5-bisphosphate (PIP2), the residual being phosphatidylcholine (PC)....

We report that cytochalasin D (CD) is not a reliable tool to inhibit all forms of cell motility and actin polymerization in neutrophil granulocytes. In addition to the well-established effects of CD such as altered localization of F-actin, inhibition of surface ruffling, fluid pinocytosis and actin polymerization in agonist-stimulated cells, we fin...

Using two newly synthesized inhibitors, Ro 31-8220 and CGP 41,251, of protein kinase C (PKC), we analysed: (1) how distinct PMN functions (shape changes, locomotion, pinocytosis) are regulated, and (2) the role of protein phosphorylation and PKC in this process. We were able to transform: (1) resting PMNs into locomoting cells using fNLPNTL, (2) lo...

The interaction of the actin-binding protein filamin with mixtures of zwitterionic and anionic phospholipids (DMPC, DMPG, PC, PS) was studied in reconstituted lipid monolayers and bilayers. Protein-lipid interactions were investigated by differential scanning calorimetry, the film balance technique, and hydrophobic photoradiolabeling. For calorimet...

The cytoskeletal protein talin potentially plays a key role in actin–membrane linkage. It is able to nucleate actin filament growth in vitro while binding simultaneously to lipid bilayers. Thrombin digestion of human platelet talin yields two polypeptide domains of 200 kDa and 47 kDa. We have purified these fragments and analyzed their functional p...

The PKC-inhibitor Ro 31-8220 inhibits stimulated fluid pinocytosis of human PMNs induced by the PKC-activators phorbol myristate acetate (PMA, IC50 = 1.35 x 10(-6) M) or diacylglycerols (OAG, diC8) by 95%, whereas Ro 31-8220 has no effect on D2O- or fNLPNTL-induced pinocytosis and enhances cytochalasin D- induced pinocytosis. Also formation of F-ac...

The cytoskeletal component vinculin has been demonstrated by hydrophobic photoradiolabelling, to insert into bilayers containing acidic phospholipids and trace amounts of a photoactivatable analogue of lecithin. It is shown in this study that the higher-molecular-mass variant metavinculin and alpha-actinin, also share this property. alpha-Actinin a...

Several protein kinase inhibitors of the staurosporine type displaying different enzyme specificity were used to study the role of protein kinase C (PKC) in motile neutrophil functions. Effects on protein phosphorylation, F-actin localization, morphology, and locomotion were determined. Only staurosporine, but not another inhibitor more specific fo...

Colchicine-induced stimulation of polymorphonuclear leukocyte (PMN) locomotion is an interesting model because extension of blebs at the front occurs at a rate (about 2.4 microns/s) which is far above that reported for growth of actin filaments. The following cytoskeletal changes were observed in colchicine-treated PMNs: (1) a small increase in cyt...

The phosphatase inhibitors okadaic acid and calyculin A were found to elicit or to modify several neutrophil responses, suggesting that dephosphorylation plays a regulatory role. The concentrations of okadaic acid (> or = 1 mumol/L) that were effective on neutrophil functions (shape changes and marginal stimulation of pinocytosis) were shown to sti...

The phosphatase inhibitors okadaic acid and calyculin A were found to elicit or to modify several neutrophil responses, suggesting that dephosphorylation plays a regulatory role. The concentrations of okadaic acid (> or = 1 mumol/L) that were effective on neutrophil functions (shape changes and marginal stimulation of pinocytosis) were shown to sti...

Talin purified from human platelets and chicken gizzard smooth muscle is an actin and lipid binding protein. Here, we have investigated the effect of vinculin on (a) talin-nucleated actin polymerization and (b) insertion of talin into lipid bilayers. Calorimetric data show ternary complex formation between talin, vinculin, and actin. Actin-talin, a...

We have studied the reorganization of vinculin and α-actinin during the process of adhesion in human neutrophils using immunofluorescence microscopy and interference reflection microscopy (IRM). Neutrophils in contact with uncoated glass formed black IRM areas in the cell periphery, indicative of very close contact with the substratum. Eight to twe...

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents...

ForeignmRNA was expressedin Xenopus laevis oocytes.Newly expressedioncurrentslocalized indefinedplasma membrane areaswere measured usingthetwo-electrodevoltageclamp techniquein combinationwitha speciallydesignedchamber, that exposed onlypartofthesurfaceon theoocytesto channelagonistsor inhibitors .Newly expressedcur- rentswere found tobe unequallyd...

We have investigated the effect of staurosporine-type protein kinase inhibitors, displaying different enzyme specificity, on the association of actin with the neutrophil cytoskeleton. In resting cells, nanomolar concentrations of staurosporine induced a rapid increase in cytoskeleton-associated actin. Other inhibitors, more specific for protein kin...

Neutrophil granulocytes are multifunctional cells, capable of locomotion, Chemotaxis, adhesion, pinocytosis, phagocytosis, intracellular killing or degradation and exocytosis. Several of the functions require generation of force and may thus be associated with different forms of motility. In the circulating blood of healthy individuals neutrophils...

The present study demonstrates new properties of H-7. The protein kinase inhibitor H-7 is a potent activator of several neutrophil functions. Stimulation of initially spherical nonmotile neutrophils elicits vigorous shape changes within a few seconds, increases in cytoskeletal actin, altered F-actin distribution, increased adhesiveness and a relati...

The cytoskeletal protein vinculin, a putative actin–plasma-membrane linker, has been shown by hydrophobic photo-labeling to interact in vitro directly with bilayers of acidic phospholipids [Niggli et al. (1986) J. Biol. Chem. 261, 6912–6918]. In order to demonstrate that such an interaction occurs also in intact cells, chicken embryo fibroblasts we...

The results available so far allow to distinguish between three major groups of responses on the basis of the type of shape changes, functional activity and distribution of F-actin. 1. Agents capable of polarizing cells, such as cheraotactic peptides, microtubule disassembling agents and the protein kinase inhibitor H-7 elicit, at appropriate conce...

We show that diacylglycerols, like phorbol myristate acetate (PMA), suppress cell polarity and locomotor activity of Walker carcinosarcoma cells in a dose-dependent fashion in vitro. OAG and diC8 show significant activity at concentrations above 3 x 10(-5) M. The inhibitory effect on locomotion is due to a reduction in the proportion of locomoting...

The neutrophil cytoskeleton, especially the actin network, is thought to play a crucial role in neutrophil migration. However, little is known on the modulation of this network by actin-associated proteins. We have demonstrated the presence of immuno-reactive forms of alpha-actinin (an actin cross-linking and bundling protein) and vinculin (a putat...

Shape changes have been determined in human blood lymphocytes stimulated with OAG, diC8, PMA, colchicine or the hexapeptide fNLPNTL in short-term assays (30 min). Distinct types of shape-change responses were observed. Colchicine was active in generating a relatively small proportion of polarized lymphocytes (front-tail polarity). OAG, diC8 and PMA...

The widely distributed cytoskeletal protein vinculin has been implicated to participate in actin-membrane linkage. This hypothesis is mainly based on the highly specific location of vinculin in areas where this linkage occurs. However, conclusive evidence confirming this hypothesis is yet lacking, and the molecular mechanism of the proposed linkage...

This chapter discusses the procedure of Ca2+ pumping ATPase of plasma membrane to yield relatively large quantities of homogeneous enzyme from human erythrocytes, permitting detailed chemical and enzymological studies using techniques developed in laboratory. A model for the trypsin-catalyzed breakdown of the purified ATPase is described. This mode...

The cytoskeletal component vinculin has been proposed to act as an actin-plasma membrane linker. In order to demonstrate a possible direct interaction of vinculin with bilayers, photolabeling with a phospholipid generating a highly reactive carbene was used. This phosphatidylcholine analogue (1-palmitoyl-2-[10-[4-[(trifluoromethyl)diazirinyl]phenyl...

We have been using a preparation of permeabilised human blood platelets to probe the response of amine storage granule and lysosomal secretion to variation in Ca2+ concentration and the nature of various factors which may modulate these responses. Permeabil-isation is achieved by exposure of a suspension of platelets to a series of intense electric...

The systems responsible for phosphorylating tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, were investigated in situ in adrenal medullary cells made permeable to solutes of up to 1,000 dalton by exposure to brief intense electric fields. Two different phosphorylation systems were found. One is dependent on Ca2+, the o...

Addition of thrombin enhances secretion of both [ ¹⁴ C]serotonin and β‐ N ‐acetylglucosaminidase induced by Ca ²⁺ in human platelets rendered permeable by exposure to intense electric fields. Enhancement of β‐ N ‐acetylglucosaminidase secretion by thrombin results from an increase in the maximal extent of the response with no significant change in...

A variety of presumed anti-calmodulin (anti-CaM) drugs was tested for their potential inhibitory effects on the isolated, purified and reconstituted Ca2+-pump ATPase of human red blood cell membranes. Anti-CaM drugs inhibited the Ca2+-pump ATPase both in the absence and presence of added CaM. Qualitatively similar inhibition was observed in two dif...

The Ca2+-pumping ATPase from human erythrocyte membranes, purified by the method previously reported [Niggli, V., Penniston, J. T., & Carafoli, E. (1979) J. Biol. Chem. 254, 9955-9958], was freed of minor impurities by extensive washing while bound to the calmodulin-Sepharose column. The pure enzyme showed a single band of Mr 138000, which containe...

The sickle cell (Hb SS) membrane-bound Ca2+-ATPase was found to have a Vmax in a range of 30-100% of the Vmax of the normal enzyme. In all sickle cell preparations, the Ca2+-ATPase could be stimulated at least 4-fold by calmodulin, but the stimulation factor varied considerably (4-26 fold) in the different preparations. The affinity of the ghost si...

The purified Ca2+-pumping ATpase of human erythrocyte membranes transports Ca2+ after reconstitution in asolectin liposomes, and the ATPase activity is stimulated 9-to 10-fold by A 23187. This indicates tight coupling between ATP hydrolysis and Ca2+ transport. In this system 'extra' protons (i.e. in excess of those produced during the hydrolysis of...

The Ca 2+-ATPase of erythrocytes is the first Ca 2+-pumping enzyme ever identified in a plasma membrane. In the 13 years that have elapsed since its discovery, it has become obvious that Ca 2+-pumping ATPase are in all likelihood a constant feature of the plasma membranes, and constitute a family of enzymes in which subtle differences are superimpo...

The purified Ca2+-pumping ATPase of human erythrocyte membranes (Niggli, V., Adunyah, E. S., Penniston, J. T., and Carafoli, E. (1981) J. Biol. Chem. 256, 395-401) can be stimulated, in the absence of calmodulin, by other treatments. 1. A variety of acidic phospholipids (phosphatidylserine, cardiolipin, phosphatidylinositol, and phosphatidic acid)...

The Ca2+-pumping ATPase has been purified in a functional form from human erythrocytes by calmodulin affinity chromatography. The purified enzyme has a specific activity at least 300-fold higher than the membrane bound enzyme. It consists of one major protein band of 140000 Dalton, and after reconstitution in liposomes it transports Ca2+ with an ef...

The Ca2+-pumping ATPase from human erythrocyte membranes, purified nearly to homogeneity (Niggli, V., Penniston, J. T., and Carafoli, E. (1979) J. Biol. Chem. 254, 9955-9958), can be reconstituted into phospholipid vesicles. The purified and the reconstituted forms of the enzyme displayed the properties expected of the intact Ca2+ pump; they had an...

The stimulation of the (Ca2+ + Mg2+)ATPase of erythrocyte ghosts by calmodulin was observed not only in intact ghosts, but also in the solubilized (Triton X-100) and partially purified, reconstituted (phosphatidylserine liposomes) forms. Since the solubilized form of the enzyme migrated on Sepharose 6B at a position corresponding to a molecular wei...

The (Ca2+-Mg2+)-ATPase from human erythrocyte membranes has been solubilized in Triton X-100 and purified on a calmodulin affinity chromatography column in the presence of phosphatidylserine, to limit the inactivation of the enzyme. The enzyme was purified at least 150 times when compared with the original ghosts and showed a specific activity of 3...

During storage of whole blood at 4°C the red cell Na-K pump is slowed to a negligible rate (white cells may be disregarded). The cells gain Na and lose K through leak channels under these circumstances (these movements are not across the arrested pump because they are not sensitive to ouabain).

Inner membrane vesicles have been prepared by cholate treatment of rat liver mitoplasts. The vesicles can actively accumulate Ca2+ in the absence or presence of inorganic phosphate. The uptake is inhibited by ruthenium red and uncouplers of oxidative phosphorylation. Like in intact mitochondria the driving force for the uptake reaction seems to be...