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A Study on the Effect of Lysolecithin and Phospholipase A on Membrane-Bound Galactosyltransferase
Abstract
Addition of lysolecithin caused very marked activation of UDP-galactose:glycoprotein galactosyltransferase in rat liver microsomes and in Golgi-rich membranes. Lysolecithin activated galactosyltransferase when the enzyme was assayed both with endogenous acceptor and with exogenous proteins or monosaccharides as acceptors. Lactose synthetase activity in presence of α-lactalbumin was also stimulated by lysolecithin. Lecithin, lysophosphatidylethanolamine, lysophosphatidic acid, and glycerophosphorylcholine did not activate the enzyme, suggesting that both fatty acyl and phosphorylcholine groups of the lysolecithin molecule are required for the observed activation. The degree of activation was about the same when myristoyl-, palmitoyl-, oleoyl-, or stearoyllysolecithin were tested. The activation by lysolecithin was observed well within the physiological concentration of the lipid in the liver cell. Saturating amounts of Triton masked the effect of lysolecithin.Brief preincubation with phospholipase A activated the enzyme and generated lysolecithin in the membranes. Triton and lysolecithin activated the enzyme without any lag time, whereas phospholipase A activation was dependent on preincubation and also on an alkaline pH favorable for the hydrolysis of phospholipid. EDTA blocked the activation effect of phospholipase A but had no effect on activation by lysolecithin. Albumin and cholesterol opposed the effects of lysolecithin and phospholipase A on the enzyme. Two successive incubations of the microsomes with lysolecithin caused considerable release of the enzyme into the soluble fraction. The role of lysolecithin in the activation of the enzyme is probably related to the solubilization of the membrane and consequent enhanced interaction of the enzyme with substrate. Lysolecithin also activated N-acetylglucosaminyl- and sialyltransferase activities in microsomes. A possible role of lysolecithin is indicated in the regulation of glycosylation reactions in mammalian system.
... Recent investigations have demonstrated that mammalian cell glycosyltransferase enzymes that are cell surface associated in their natural state may require the presence of a phospholipid for maximum catalytic activity (1,15,23,31,32,40). However, there are few reports of bacterial glycosyltransferases that respond to the presence of phospholipids (8,35,36). ...
Lysophosphatidylcholine (LPC) and other phosphoglycerides stimulated water-insoluble and water-soluble glucan production by the Streptococcus mutans 6715 dextransucrase (EC 2.4.1.5). LPC stimulated crude extracellular dextransucrase 1.7-fold, the water-insoluble glucan-producing alpha form of the enzyme 6.5-fold, the water-soluble glucan-producing beta form of the enzyme 2.1-fold, and the cell-associated dextransucrase 2.0-fold. Kinetic studies demonstrated that LPC did not change the K(m) for sucrose of alpha or beta but increased the maximum velocity of the enzymes. The K(m) for LPC of the alpha enzyme was 10(-5) M. LPC from various sources and synthetic preparations of lauroyl-LPC, myristoyl-LPC, and palmitoyl-LPC all stimulated glucan formation. Portions of phosphoglyceride molecules including fatty acids, phosphatidic acid, glycerophosphoric acid, glycerophos-phorylcholine, and choline, when tested individually or in combinations, did not enhance dextransucrase activity. The increased rates of glucan production caused by LPC and primer dextran were additive. Enzyme incubated with LPC before addition of sucrose was stimulated by dextran primer, and, conversely, enzyme treated with dextran was stimulated by addition of LPC with the sucrose substrate. Thus, dextransucrase can be activated by binding of intact phosphoglyceride molecules to a site on the enzyme that is distinct from either the glucosyl donor or glucosyl acceptor (primer) binding sites. Interactions between the S. mutans dextransucrase and amphipathic phosphoglycerides may explain properties of this enzyme which contribute to the cariogenicity of S. mutans.
The isolated perfused cat hearts incorporated radioactive arachidonic (20:4) and stearic (18:0) acids in the rapidly metabolizing pool of membrane phospholipids. Stereospecific analysis of the phospholipid fractions studied revealed that all of the 20:4 was acylated at the sn-2 position and about 80% of 18:0 was acylated at the sn-1 position of the phospholipid fractions studied. Comparison of the radioactivities of 20:4 at 2-position to 18:0 at 1-position of individual phospholipid fractions obtained from ischemic and non-ischemic zones of the same heart after 40 min of LAD occlusion showed a significant decrease in the ratio (20:4/18:0) of radioactivities. This decrease (19–50%) in the ratio for phosphatidylcholine and phosphatidylethanolamine in the ischemic myocardium suggests that phospholipase A2 activity in comparison to A1 is accelerated due to ischemia. The decrease (16%) in the ratio for phosphatidylinositol (PI) probably suggests increased turnover due to stimulated PI-specific phospholipase C activity.
Phospholipids are the major form of lipid in all cell membranes and their fixed composition and disposition within membranes are genetically predetermined. In combination with other lipids and proteins, phospholipids are responsible for both structural characteristics and functional properties of the biological membranes such as fluidity (1, 2), ionic permeability (3, 4), transport of material across cell membranes (5), activities of a large number of membrane-bound enzymes (6) and receptor-mediated cell responses (7, 8). Phospholipases are the enzymatic systems responsible for the catabolism of membrane phospholipids, and the interplay between these catabolic enzymes and those of anabolic systems determines the turnover rate and maintains the biological properties and fixed composition of these lipids in membranes. Various physiological stimuli (e.g. certain hormone-receptor interactions, increase in intracellular Ca2+, fat absorption, etc.) enhance phospholipid turnover in membranes (8–11). In some disease conditions, membrane phospholipid composition and metabolism are progressively altered resulting in the functional abnormalities of the cell (12–15).
Since the early work of Bergenhem and Fahraeus on the hemolytic activity of naturally occurring 2-lysophosphatidylcholine (lysolecithin) (LPC) [12] this substance has off and on been considered as a biologically active compound. It is present as a minor phospholipid in the plasma (8–12%) [23] and cellular membranes (≥ 3%) [21, 27, for review 79]. It is highly surface-active (44.3 dyn/cm) [4] and, therefore, potentially cytotoxic if incubated with cells in serum — or plasma free —media [4, 30, 31, 33, 44, 72, 76]. Addition in sublytic amounts, however, stimulates phagocytosis [16, 20, 80], changes the surface properties of erythrocytes [36], increases the Concanavalin-A (Con-A)-induced agglutination of erythrocytes [75], and may be used as a cell-fusing agent [58]. Furthermore, it has been claimed to be involved in hypersensitivity [38, 70] and inflammatory reactions [17].
Concentrations of GPC in the caput epididymidis are similar to those in the cauda epididymidis in rats (Riar et al., 1973; Brooks et al., 1974; Brown-Woodman et al., 1976) and rabbits (Holtz and Foote, 1978), others estimate slightly more in the cauda (Dawson and Rowlands, 1959; Dacheux et al., 1970; Setty et al., 1979). Epithelial cells from the caput contain more GPC than those from the corpus or cauda (Hoffmann and Killian, 1981) and the principal cells contain more than basal cells (Killian and Chapman, 1980; Hoffmann and Killian, 1981). Within the epididymal lumen there is more GPC in fluid than sperm cells (Brooks, et al., 1974). The concentration in epididymal fluid is high in the caput and is maintained distally (Table 15) whereas concentrations in spermatozoa increase as they mature (Quinn and White, 1967).
Since the discovery that sialic acid was the receptor site for influenza virus on the red blood cell (Gottschalk, 1960), recognition of the biological importance of glycoproteins and glycolipids has been increasing. Oligosaccharide moieties of these complex carbohydrates function as the primary antigenic determinants of blood-group substances, and may be involved in such diverse biological phenomena as contact inhibition and cell-cell adhesion of cultured cells, gamete recognition, transplant rejection, and recognition of specific receptor sites for hormones, viruses, and agglutinins. In addition, a large and increasing number of pathological conditions have been shown to be related to complex carbohydrates, including the glycosphingolipid storage diseases (see Chapter 11), cholera (Holmgren et al., 1973), herpes (Nahmias and Roizman, 1973), neoplasia (Burger and Martin, 1972), diabetes (Spiro and Spiro, 1971) hepatic cirrhosis (Marshall et al., 1974), and hemostasis (Barber and Jamieson, 1971). A better understanding of the chemistry and the mechanisms of biosynthesis of these complex molecules may provide information leading to the control of these diseases.
Cells contain a variety of hydrolytic enzymes that play important rules in regulation and turnover. Suitable subtrates for these enzymes are found within the cell itself, including essential structural and informational macromolecules, but a variety of mechanisms exist which prevent the cell's hydrolytic enzymes from prolonged attack on its own components. This situation makes it possible to envisage a class of cytolytic mechanisms in which hypothetical toxins could circumvent these controls allowing one or more of the cell's hydrolytic enzymes to attack its own essential structures, destroying them and leading to cell death. Of the hydrolytic enzymes found in cells, phospholipases, nucleases and proteases show the greatest promise for forming the basis of a cytotoxic mechanism of this type. Among these, phospholipases C and A2 are the most promising, because there are numerous examples of exogenous phospholipase C and A2 enzymes which function as effective toxins. The mechanisms by which these two enzymes lead to destruction of cell membranes are only partially understood, but it is reasonable to expect that prolonged activation of the corresponding endogenous enzyme would also lead to membrane destruction by a similar mechanism. Endogenous phospholipases C and A2 have been identified in mammalian cells, where they are believed to play important roles in regulating the synthesis of prostaglandins and other arachidonic acid metabolites, and possibly to play a role in the regulation of other membrane-related phenomena by direct effects on membrane properties. In the case of phospholipase A2 the products of its action on the major membrane-forming lipid in mammalian cells are two natural detergent lipids (lysolecithin and free fatty acids) which can alter membrane properties and the activities of membrane-associated enzymes. To date no toxin which activates an endogenous phospholipase C has been identified, but a series of toxins which activate endogenous phospholipase A2 have been. Partial characterization of these enzymes has provided evidence for multiple, independently activatable phospholipases in a cloned mammalian cell line.
It is established that buprenorphine (0.3 mg/kg) induces considerable alterations in the phospholipid composition of hepatocyte
plasma membranes as a result of phosphatidylserine accumulation and a considerable loss of sphingomyelin and lysophosphatidylserine.
When administered in a dose of 0.03 mg/kg, buprenorphine facilitates normalization of the phosphoinositol turnover in hepatocyte
plasma membranes.
Triacylglycerol ester hydrolase was isolated from bat adipose tissue and characterized. The partially purified enzyme had
pH optimum of 8.6 and a Km value of 0.6 mM. The enzyme was denaturated upon freezing and thawing, which was prevented by 25% glycerol. The enzyme was
activated by EDTA and NaCl, while it was inhibited by serum and bovine serum albumin. Heparin, sodium fluoride and diisopropyl
fluorophosphate had no effect on triacylglycerol ester hydrolase activity. It hydrolyzed triglycerides partially. Triacylglycerol
ester hydrolase lost its activity during delipidation but it was reactivated by endogenous lipids and phospholipids, viz.
phosphatidyl ethanolamine, phosphatidyl choline and sphingomyelin. The enzyme shows kinetic properties altogether different
from lipoprotein lipase and hormone sensitive lipase
The in vitro effects of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, sphingomyelin, lysophosphatidyl choline, lysophosphatidyl ethanolamine, lysophosphatidyl serine, glycerophosphoryl choline, and phosphatidic acid on rubidium uptake and amino acid transport of rat lens are presented. None of these lipids affected the rubidium uptake mechanism; however, a large increase in the apparent leak-out of rubidium was observed with lysophosphatidyl choline. Lysophosphatidyl choline also inhibited all of the amino acid transport systems (A, ASC, L, Gly, Ly and β) as well as the uptake of inositol.
The guanylate cyclase activity [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] in membrane preparations from 3T3 mouse fibroblasts is stimulated approximately 5-fold by lysolecithin at concentrations of 100 mug/ml and above.
Recent biochemical studies have demonstrated profound effects of phospholipases on cellular enzyme systems. In the present study selected enzymes were demonstrated in canine kidney after incubation (1) in buffer alone, (2) in 500 μgm/ml of phospholipase C in buffer, and (3) in 50 μgm/ml of phospholipase A in buffer. Results indicate a dramatic decrease in all enzymes in tissue sections subjected to preincubation in phospholipase A. Phospholipase C had no discernible effect on succinate dehydrogenase, α-glycerophosphate dehydrogenase, or alkaline phosphatase but produced an activation of acid phosphatase and an inactivation of β-OH butyrate dehydrogenase. Results are discussed in relation to human disease.
1. Galactosyltransferase activities in postnuclear supernatants and Golgi fractions from rat liver were assayed with two improved and simplified methods, using high‐ and low‐molecular‐weight acceptors. Transfer to N ‐acetylglucosamine was measured after the separation of the reaction product N ‐acetyllactosamine from all other radioactive molecules (including galactose) on an ion‐exchange column partially converted to the borate form. To determine the transfer of galactose to a glycoprotein acceptor we used ovomucoid, which accepts galactose without any previous chemical or enzymic modification.
2. Both enzymic activities were enriched 60–80‐fold (compared with the post‐nuclear supernatant) in Golgi fractions, which were isolated on two subsequent sucrose gradients and identified morphologically by their high contents of stacked Golgi elements. The two activities could not be resolved by isolation of the Golgi fractions or by detergent solubilization. Each acceptor inhibited the galactose transfer to the other one (up to 95%), presumably because both compete for the same enzyme.
3. The transferase activities were enhanced by the nonionic detergent Triton X‐100. The degree of activation depended directly on the amount of Triton bound to the membrane, i.e. the Triton/phospholipid ratio and not the w/v concentration of the detergent in the assay medium. This relationship persisted, regardless of the purity of the Golgi preparation: Half‐maximal activation occurred at the same Triton/phospholipid ratio in postnuclear supernatants as well as in isolated Golgi fractions. The activation could not be explained by complete solubilization, because 50% of the fully activated enzyme could still be sedimented (1 h, 100000 × g ).
4. Galactose transfer to the high‐molecular‐weight acceptor required a higher Triton/phospholipid ratio for half‐maximal activation than did the transfer to the monosaccharide N ‐acetylglucosamine (1 mg/mg compared with 0.5 mg/mg). The degree of activation maximally achieved was much higher with the protein acceptor (400%) than with the sugar (150%). Because both activities are probably due to the same enzyme, it is suggested that these differences in activation reflect properties of the membrane rather than the enzyme, e.g . the presence of a tight diffusion barrier for ovomucoid and the breakdown of this barrier by the detergent.
Abstract— Total rat cerebral homogenate, with nuclei removed, yielded sialyltransferase activity peaks that were distinct from the protein distribution profile in a continuous sucrose density gradient. Marker enzyme studies and electron microscopic examinations on the gradient fractions suggested that most of the sialyltransferase activities were not associated with the synaptosomes.
The sialyltransferases appeared to be localized in the smooth microsomal membranes and the Golgi complex derivatives. The sialyltransferase activities were stimulated by non-ionic detergent mixture, Triton CF-54/Tween 80 (2/1, w/w), the effect being much more pronounced with exogenous substrates. The stimulatory effect was dependent on detergent concentration. With 1 mg detergent mixture per mg enzyme protein, the percent increases in enzyme activities with the different substrates were: endogenous glycolipids, 100; endogenous glycoproteins, 50; exogenous GM1a, 700; exogenous DS-fetuin, 230. The action of the nonionic detergents appears to be on a hydrophobic segment of the enzyme molecule, bearing the active site, which is buried in the membrane lipid bilayer. This was substantiated by the partial trypsin resistance of the sialyltransferase activities and the abolition of that resistance when trypsiniza-tion was performed in the presence of nonionic detergents. Furthermore, the sialyltransferase activities were markedly inhibited by organic solvents; and these inhibitory effects were inversely proportional to the solvent dielectric constants.
1-Palmitoyl-sn-glycerol-3-phosphocholine and 3-palmitoyl-sn-glycerol-1-phosphocholine have been found to be equipotent in the stimulation of membrane-bound glycosyltransferases in microsomes of rat intestinal villus cells. This indicates that the stimulatory effect of lysophosphatidylcholine is not stereospecific, but that it may be related to a specific detergent property dependent upon the peculiar balance of hydrophilic and hydrophobic components in the molecule.
Exponential-phase cells of Neisseria gonorrhaeae 2686 were examined for phospholipid composition and for membrane-associated phospholipase A activity. When cells were harvested by centrifugation, washed, and lyophilized before extraction, approximately 74% of the total phospholipid was phosphatidylethanolamine, 18% was phosphatidylglycerol, 2% was cardiolipin, and 10% was lysophosphatidylethanolamine. However, when cells still suspended in growth medium were extracted, the amount of lysophosphatidylethanolamine decreased to approximately 1% of the phospholipid composition. This suggests that a gonococcal phospholipase A may be activated by conditions encountered during centrifugation and/or lyophilization of cells preceding extraction. Phospholipase A activity associated with cell membranes was assayed by measuring the conversion of tritiated phosphatidylethanolamine to lysophosphatidylethanolamine. Optimal activity was demonstrated in 10% methanol at pH 8.0 to 8.5, in the presence of calcium ions. The activity was both detergent sensitive and thermolabile. Comparisons of gonococcal colony types 1 and 4 showed no significant differences between the two types with respect to either phospholipid content or phospholipase A activity.
1. Rat liver microsomal preparations incubated with 200mM-NaCl at either 0 or 30 degrees C released about 20-30% of the membrane-bound UDP-galactose-glycoprotein galactosyl-transferase (EC 2.4.1.22) into a 'high-speed' supernatant. The 'high-speed' supernatant was designated the 'saline wash' and the galactosyltransferase released into this fraction required Triton X-100 for activation. It was purified sixfold by chromatography on Sephadex G-200, and appeared to have a higher molecular weight than the soluble serum enzyme. 2. Rat serum galactosyltransferase was purified 6000-7000-fold by an affinity-chromatographic technique using a column of activated Sepharose 4B coupled with alpha-lactalbumin. The purified enzyme ran as a single broad band on polacrylamide gels and contained no sialytransferase, N-acetylglucosaminyltransferase and UDP-galactose pyrophosphatase activities. 3. The highly purified enzyme had properties similar to those of both soluble and membrane-bound galactosyltransferase. It required 0.1% Triton X-100 for stabilization, but lost activity on freezing. The enzyme had an absolute requirement for Mn2+, not replaceable by Ca2+, Mg2+, Zn2+ or Co2+. It was active over a wide pH range (6-8) and had a pH optimum of 6.8. The apparent Km for UDP-galactose was 12.5 x 10(-6) M. Alpha-Lactalbumin had no appreciable effect on UDP-galactose-glycoprotein galactosyltransferase, but it increased the specificity for glucose rather than for N-acetylglucosamine, thus modifying the enzyme to a lactose synthetase. 4. The possibility of a conversion of higher-molecular-weight liver enzyme into soluble serum enzyme is discussed, especially in relation to the elevated activities of this and other glycosyltransferases in patients with liver diseases.
1. 1. A number of detergents were used to dissolve calf thymus plasma membranes rich in alkaline phosphatase (orthophosporic-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) activity. 2. 2. The Stokes' radius (r) of alkaline phosphatase in each detergent was measured by gel filtration. The size of the solubilized enzyme varied from r = 6.2 nm in sodium cholate to r = 8.3 nm in Berol EMU-043. With N-alkylsulphates, the apparent size increased with alkyl chain length, with r = 6.4 nm (C9) and r = 7.3 nm (C12). Tween 20 failed to solubilise the enzyme. 3. 3. The effect of each detergent on the catalytic activity of alkaline phosphatase was determined. The non-ionic detergents Triton X-100, Nonidet P-40, Berol EMU-043, Tween 20 and the zwitterionic detergent Empigen BB increased V by 10-50% without substantially altering the Km for p-nitrophenylphosphate. The bile salts sodium deoxycholate and sodium cholate decreased V and increased the apparent affinity of the enzyme for nitrophenylphosphate. Inhibition was concentration-dependent up to the critical micellar concentration, above which it remained constant (deoxycholate, 33%; cholate, 76%). Alkylsulphates (C8-2) had no significant inhibitory effect during 24 h at 23°C. 4. 4. Exchanging one detergent for another altered alkaline phosphatase activity to a state characteristic for the second detergent, e.g. the activity of cholateinhibited alkaline phosphatase was restored to normal levels by excess of Triton X-100 and vice versa. The inhibitory effect of deoxycholate and cholate therefore result primarily from interactions between detergent and alkaline phosphate, rather than from selective removal of lipids from the enzyme. 5. 5. Pure lecithin, lysolecithin and an ether-deoxylysolecithin each reactivated cholate-inhibited alkaline phosphatase in a concentration-dependent fashion. Cholesterol had no effect.
1. Phospholipids activated the enzyme, lactosylceramide: UDP-galactose alpha-galactosyltransferase in hamster cells (NIL 2E clone 8) when assayed in the presence of neutral detergents. 2. Phosphatidylserine and phosphatidylinositol were the most effective phospholipids in activating the enzyme. Other phospholipids were also effective, but sphingomyelin and lysophosphatidylcholine were inhibitory. 3. Considerable enzyme activity was obtained in the absence of any detergent. Most of this activity was due to glycosylation of endogenous acceptors. 4. There was a complex effect of detergents on the enzyme activity. Very low concentrations were sharply inhibitory, but higher concentrations, above the critical micelle concentration for detergent, caused regeneration of activity. 5. The phospholipids, in the absence of a detergent, are required to maintain the lipid substrate, lactosylceramide, in a suitable dispersion where it can be acted upon by the enzyme. In the presence of detergents, it is proposed that the phospholipids also act by affecting the state of the lipid substrate.
Phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4, 5-bisphosphate (PIP2), 1, 2-diglyceride (DG), lysophosphatidylcholine (LPC), and free fatty acids (FFA) contents, as well as their fatty acid composition, were measured in transient global cerebral ischemia. ATP and CTP were also studied. Male Wistar rats were subjected to 1, 5, and 30 min of ischemia and 10, 30, and 60 min of recirculation following 30 min of ischemia. In addition, for the quantification of PI, PIP, and PIP2, rats were also subjected to 30 and 60 min of recirculation following 5 min of ischemia. PIP2 and PIP decreased rapidly during 5 min of ischemia and recovered completely after recirculation. DG increased almost at the same rate during ischemia and returned to normal after recirculation. PI showed almost no changes throughout entire course. LPC increased during 5 min of ischemia and returned to normal after recirculation. Stearic acid and arachidonic acid contained in DG increased during 5 min of ischemia, whereas saturated fatty acids increased in LPC. Among the FFA accumulated during ischemia, stearic acid and arachidonic acid increased rapidly and were followed by increases of other FFA. From these results, the pathways for the increase of FFA during ischemia and the fate of FFA after recirculation are discussed. In addition, the importance of the changes of PIP, PIP2, and LPC is also discussed.
Sonication of lysophosphatidylcholine (lysoPC; 20 mumol/mL) and cholesterol (chol) in aqueous medium produces lamellar structures over a wide range of concentrations. From 25 to 47 mol % cholesterol, electron microscopy (EM) after negative staining showed extended stacklike lamellae about 40 A thick. From 50 to 60 mol % chol, freeze-fracture EM showed homogeneous populations of small unilamellar vesicles averaging 260-310 A in diameter. Phosphorus-31 nuclear magnetic resonance was used to characterize the stacklike lamellae and to measure the distribution of the lysophospholipid between the outer and inner leaflet of the vesicles as a function of sterol concentration. We found that in lysoPC/chol dispersions containing less than equimolar amounts of cholesterol (25-47 mol %), the entire phosphorus signal (40.5 ppm) was shifted downfield by 10.5 ppm upon addition of Pr3+ (2.4 mM), consistent with the stacklike lamellar structures in which all lysoPC head groups are accessible to the ions. By contrast, addition of Pr3+ to lysoPC/chol vesicles containing equimolar or higher amounts of cholesterol (up to 60 mol %) gave rise to two phosphorus peaks. The more intense downfield signal (51.0 ppm) responsive to paramagnetic ions was assigned to lysoPC located in the outer vesicle leaflet. The upfield signal (40.5 ppm), which was not affected by the ions, was assigned to inside lysoPC. For lysoPC/chol (1:1) vesicles, an outside to inside lysophospholipid ratio (Ro/i) of 6.5 was determined. Essentially the same Ro/i value (6.7) was obtained on lysoPC/chol (1:1) vesicles which after dialysis contained only entrapped Pr3+.(ABSTRACT TRUNCATED AT 250 WORDS)
Sialyltransferase was measured in serum of normal and hepatoma Mc-29 bearing chickens. By preparative isoelectric focusing the multiple forms of sialyltransferase from both kind of serums was studied as well. By using influenza virus neuraminidase an attempt was made for partial structural characterization of the sialylation sites in asialofetuin applied as exogenous acceptor for sialyltransferase determination. It was established an elevated serum sialyltransferase activity in tumor bearing chickens with tumor an enzyme form was detected with pI-4.99 identical with an enzyme form described previously in solubilized plasma membrane preparations from hepatoma Mc-29. Monitoring of multiple forms of serum glycosyltransferases may be of value in answering the problem concerning the tissue origin of serum enzymes.
A galactosyltransferase activity is located in the cell-sap of aortic intima-media cells. This enzymatic system calatyzes [14C]galactose transfer from UDP-[14C]galactose into endogenous and exogenous proteinic acceptors. Labelled products are isolated from the proteinic fraction obtained in 20% trichloroacetic acid pellet or from organic solvent extractions. Maximal [14C]galactose incorporation occurs at pH 7.8 in Tris-HCl buffer in the presence of 0.1 mM MnCl2 at 30 degrees C. The enzymatic activity is modified by phospholipids, particularly by phosphatidic acid and lysophosphatidylcholine, which behave as mixed inhibitors, while L-alpha-phosphatidylserine interacts as a competitive inhibitor. The effect of phospholipids is not stereospecific but appeared to be closely related to their polar headgroups, especially the acidic headgroups of phosphatidylcholine and phosphatidic acid. The chain length and the unsaturation degree of fatty acids involved in phospholipid structures are not a main factor of regulation. The lysophosphatidylcholine effect could be explained by its solubilization properties, as non-ionic detergents interact in the same way with galactosyltransferase activity. Exogenous phospholipids probably interact with the enzymatic environment by their own molecular arrangement and so could exert a control on galactosyltransferase activity or lead to a conformation change of this enzyme.
Insulin does not influence either the yield of the liver Golgi membrane-enriched fraction or the activity of its UDP-Gal→ G1cNAc transferase activity. Morphological studies show that the hormone slightly stimulates the secretory action of the Golgi apparatus in normal rat liver and prevents the formation of rounded cisternae of this organelle in the streptozotocin-diabetic rat liver. Other morphological alterations of the liver Golgi apparatus in streptozotocin-diabetic rats are not improved by insulin treatment.
Phospholipids interact on the membrane-bound and solubilized mannosyltransferase activity. The biosynthesis of Dol-P-Man is strongly inhibited by phosphatidic acid and lysophosphatidylcholine. The effect of phospholipids is not related to stereospecificity. Chemical properties of phospholipids (ester or ether bond, length of fatty acids and polarity of head groups) are not an essential factor for inhibition. The different parameters involved in enzymatic reaction of glycosylation are not modified by phospholipids, in particular the integrity of GDP-[14C]mannose. The inhibitory effect of lysophosphatidylcholine and phosphatidic acid on mannosyltransferase activities is related to their possible formation of micellar structures which definitely induce a conformation change of this enzyme.
Asialomucin-sialyltransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) activity was solubilized from mouse liver microsomes by sonication. The catalytic activity was markedly inhibited by a series of lysophosphatidylcholines, particularly 1-palmitoyl-sn-glycero-3-phosphorylcholine. This lysophospholipid did not alter optimal conditions for enzyme activity. In contrast, it was found that affinities for binding of Mn2+, desialylated mucin and CMP-sialic acid were decreased by adding the lipid. A reasonable interpretation of these data is that the presence of phospholipid modifies the enzyme conformation.
In rats fed orotic acid, the incorporation in liver subcellular fractions of sugars injected intraperitonealy is altered only for mannose, but not for fucose or galactose. Direct determinations of several glycosyltransferases are done in smooth and rough microsomes: fucosyl-, glactosyl-, N-acetylglucosaminyltransferase activities are at quite similar levels in normal and fatty livers. By contrast, sialyltransferase activity is increased (+50%) in smooth microsomes of fatty livers, while mannosyltransferase activity is inhibited by 30%. These alterations are not caused by interfering reactions (pyrophosphatases or proteases). For the mannosyltransferase activity, the inhibition is found in the dolichylphosphorylmannose intermediates. Kinetic studies suggest that there is deficiency of both enzyme and endogenous dolichyl phosphate.
This study was performed to determine the early and delayed metabolic effects of myocardial ischemia on the major membrane phospholipids and to reassess the potential role of lysophospholipids in the genesis of malignant dysrhythmias induced by ischemia. Samples taken from in situ hearts before ant at various intervals up to 40 minutes after abrupt ligation of LAD were extracted by the classical Folch technique with modifications to avoid artifactual lysophospholipid production and losses. Following thin layer chromatography of lipid extracts, phospholipid fractions were quantified by phosphorus estimation and lysophospholipids by a more sensitive method employing gas liquid chromatography. The total phospholipid content with the exception of lysophospholipids remained essentially constant throughout the early phases of acute ischemia, but fell by 6 and 14% after 8 and 24 ours, respectively. At 8 minutes, lysophospholipid levels n ischemic myocardium were significantly increased by 60% compared to pre-occlusion controls in the ischemic zone and by 25% in post-occlusion controls. They changed little thereafter. The molecular species of lysophospholipids remained unchanged throughout the period of ischemia studied. The mole fraction of other phospholipids as well as their fatty acyl and aldehyde profiles also were unchanged. Despite significant elevations in lysophospholipids levels, their absolute quantities were very small (0.6% of total phospholipid P) and 15-fold smaller than that reported in vitro to simulate electrophysiological manifestation of ischemia. However, such small amounts in vivo, if produced in the microenvironment of certain membrane-bound enzymes along with acidosis, hypoxia, and fatty acids, could be potentially deleterious to cell functions.
CMP-NAcNeu:GM3 ganglioside sialytransferase (GD3 synthase) was concentrated 80-100-fold, relative to total homogenates, in Golgi apparatus fractions from rat liver. Ultrasound treatment of Golgi apparatus in a low salt medium extracted 40-60% of the original protein but did not dissociate the transferase from membranes. The acivity was greatest in the presence of certain detergents, had a pH optimum of 6.2, was stimulated by mg2+ and diacylphospholipids and was inhibited by lysophospholipids. Apparent Km values for CMP-NAcNeu and GM3 were about 0.8 and 0.2 mM, respectively. On chromatographic separation, virtually all the reaction product migrated as GD3. GD3 synthase appeared to be a glycoprotein since the activity bound to concanavalin A-Sepharose and was eluted, with increased specific activity, by alpha-methyl mannoside.
Comparative effects of the partial opiate agonists buprenorphine and butorphanol on the phospholipid composition of liver cell plasma membranes were investigated in cats under conditions of hemorrhagic shock. Buprenorphine administration (0.03 mg/kg) normalized the level of phosphatidylinositol. The higher dose (0.3 mg/kg) induced additional disturbances in the phospholipid composition of liver cell plasma membranes by increasing the content of phosphatidylserine and by decreasing the contents of sphingomyelin and lysophosphatidylcholine. The protective effect of butorphanol on liver cells was more pronounced than that of buprenorphine.
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