Cholinesterase solubilizing factor from Cytophaga sp. is a phosphatidylinositol-specific phospholipase C

In the culture supernatant of Cytophaga sp. we detected an enzyme that converted glycosylphosphatidyl-inositol-anchored acetylcholinesterase to the hydrophilic form. This enzyme had a cleavage specificity of a phospholipase C. It hydrolyzed phosphatidylinositol but did not act on phosphatidylcholine. On gel filtration the enzyme migrated with an apparent molecular mass of about 17 kDa. It displayed maximal activity between pH 6-6.5 and did not require cofactors for the expression of catalytic activity. Mercurials and zinc ions inhibited the enzyme and its activity also decreased with increasing ionic strength in the assay. With acetylcholinesterase as substrate optimal activity was obtained in pure micelles of Triton X-100, whereas in mixed micelles containing Triton X-100 and phosphatidylcholine the activity was reduced. The enzyme from Cytophaga sp. showed little activity towards acetylcholinesterase embedded in intact membranes where more than 1000-times higher concentrations of phosphatidylinositol-specific phospholipase C was necessary to solubilize acetylcholinesterase as compared to acetylcholinesterase in detergent micelles.     

High sensibility to reactivation by acidic lipids of the recombinant human plasma membrane Ca2+-ATPase isoform 4xb purified from Saccharomyces cerevisiae

The human plasma membrane Ca2+ pump (isoform 4xb) was expressed in Saccharomyces cerevisiae and purified by calmodulin-affinity chromatography. Under optimal conditions the recombinant enzyme (yPMCA) hydrolyzed ATP in a Ca2+ dependent manner at a rate of 15 micromol/mg/min. The properties of yPMCA were compared to those of the PMCA purified from human red cells (ePMCA). The mobility of yPMCA in SDS-PAGE was the expected for the hPMCA4xb protein but slightly lower than that of ePMCA. Both enzymes achieved maximal activity when supplemented with acidic phospholipids. However, while ePMCA in mixed micelles of phosphatidylcholine-detergent had 30% of its maximal activity, the yPMCA enzyme was nearly inactive. Increasing the phosphatidylcholine content of the micelles did not increase the activity of yPMCA but the activity in the presence of phosphatidylcholine improved by partially removing the detergent. The reactivation of the detergent solubilized yPMCA required specifically acidic lipids and, as judged by the increase in the level of phosphoenzyme, it involved the increase in the amount of active enzyme. These results indicate that the function of yPMCA is highly sensitive to delipidation and the restitution of acidic lipids is needed for a functional enzyme.     

Studies on the interactions between phospholipids and membrane-bound enzymes in microsomes. Effects of phospholipases C on the glucose-6-phosphatase system of rat liver microsomes

The role of phospholipids in the glucose-6-phosphatase system, including glucose-6-P phosphohydrolase and glucose-6-P translocase, was studied in rat liver microsomes by using phospholipases C and detergents. In the time course experiments on detergent exposure, the maximal activation of glucose-6-P phosphohydrolase varied according to the nature of the detergent used. On treatment of microsomes with phospholipase C of C. perfringens, the activity of glucose-6-P phosphohydrolase without detergent (i.e. without rupture of translocase activity) was gradually decreased with the progressive hydrolysis of phosphatidylcholine and phosphatidylethanolamine on the microsomal membrane, and was restored by incubation of these microsomes with egg yolk phospholipids. The extent of decrease in this phosphohydrolase activity in the detergent-exposed microsomes (with rupture of translocase activity) also varied depending on the detergent used (Triton X-114 or taurocholate). When 66% of the phosphatidylinositol on the membrane was hydrolyzed by phosphatidylinositol-specific phospholipase C of B. thuringiensis, the inhibition of glucose-6-P phosphohydrolase activity without detergent was very small. Although the inhibition of enzyme activity with detergent was apparently greater than that without detergent, the enzyme activity was stimulated by the breakdown of phosphatidylinositol when the enzyme activity was measured at lower concentration (0.5 mM) of substrate, glucose-6-P. The latency of mannose-6-P phosphohydrolase, a plausible index of microsomal integrity, remained above 70% after the hydrolysis of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol. The results show that the glucose-6-phosphatase system requires microsomal phospholipids for its integrity, suggesting that there exists a close relation between phosphatidylinositol and glucose-6-P translocase.     

Effects of temperature and bovine serum albumin on lysis of erythrocytes induced by dilauroylglycerophosphocholine and didecanoylglycerophosphocholine.

The effects of the incubation temperature and bovine serum albumin on hemolysis induced by short-chain phosphatidylcholine were examined. The rate of hemolysis of human, monkey, rabbit, and rat erythrocytes by dilauroylglycerophosphocholine showed biphasic temperature-dependence: hemolysis was rapid at 5-10 degrees C and above 40 degrees C, but slow at around 25 degrees C. In contrast, the rate of lysis of cow, calf, sheep, pig, cat, and dog erythrocytes did not show biphasic temperature-dependence, but increased progressively with increase in the incubation temperature. Bovine serum albumin increased the hemolysis of human erythrocytes induced by dilauroylglycerophosphocholine or didecanoylglycerophosphocholine: it shortened the lag time of lysis and reduced the amount of phosphatidylcholine required for lysis. A shift-down of the incubation temperature from 40 to below 10 degrees C also shortened the lag time of lysis of human erythrocytes induced by dilauroylglycerophosphocholine and reduced the amount of phosphatidylcholine required for lysis.     

Stereochemical and positional specificity of the lipase/acyltransferase produced by Aeromonas hydrophila.

Aeromonas species secrete a glycerophospholipid-cholesterol acyltransferase (GCAT) which shares many properties with mammalian plasma lecithin-cholesterol acetyltransferase (LCAT). We have studied the stereochemical and positional specificity of GCAT against a variety of lipid substrates using NMR spectroscopy as well as other assay methods. The results show that both the primary and secondary acyl ester bonds of L-phosphatidylcholine can be hydrolyzed but only the sn-2 fatty acid can be transferred to cholesterol. The enzyme has an absolute requirement for the L configuration at the sn-2 position of phosphatidylcholine. The secondary ester bond of D-phosphatidylcholine cannot be hydrolyzed, and this lipid is not a substrate for acyl transfer. In contrast to the phospholipases, but similar to LCAT, the enzyme does not interact stereochemically with the phosphorus of phosphatidylcholine. In fact, the phosphorus is not required for enzyme activity, as GCAT will also hydrolyze monolayers of diglyceride, although at much lower rates.     

Hydrolysis of phosphatidylcholine during LDL oxidation is mediated by platelet-activating factor acetylhydrolase

Degradation of phosphatidylcholine to lysophosphatidylcholine occurs during oxidative modification of low density lipoproteins (LDL). In this study, we have shown that this phospholipid hydrolysis is brought about by an LDL-associated phospholipase A2 that can hydrolyze oxidized but not intact LDL phosphatidylcholine. The chemical nature of the oxidized phospholipids that can act as substrates for this enzyme was not fully characterized, but we hypothesized that the specificity of the enzyme for oxidized LDL phosphatidylcholine might be explained by fragmentation of polyunsaturated sn-2 fatty acyl groups in LDL phosphatidylcholine during oxidation. To facilitate characterization of this enzyme, we therefore selected a fluorescent phosphatidylcholine substrate that had a short-chain, polar residue in the sn-2 position: 1-palmitoyl 2-(6-[7-nitrobenzoxadiazolyl]amino) caproyl phosphatidylcholine, (C6NBD PC). This substrate was efficiently hydrolyzed by LDL, but the dodecanoyl analogue of C6NBD PC, which differed only in that a 12-carbon rather than a 6-carbon acyl derivative was present in the sn-2 position, was not hydrolyzed. The phospholipase activity was heat-stable, calcium-independent, and was inhibited by the serine esterase inhibitors phenylmethylsulfonyl-fluoride and diisopropylfluorophosphate, but was resistant to p-bromophenacylbromide and dithiobisnitrobenzoic acid. The phospholipid hydrolysis could not be attributed to the action of lecithin:cholesterol acyltransferase or lipoprotein lipase. Nearly all of the activity in EDTA-anticoagulated normal plasma was physically associated with apoB-containing lipoproteins, but this apoprotein was not essential as enzyme activity was present in plasma from abetalipoproteinemic patients. These properties are very similar to those recently reported for human plasma platelet-activating factor (PAF) acetylhydrolase. In the present study, we found that acylhydrolase activity against C6NBD PC, PAF, and oxidized phosphatidylcholine copurfied through gel filtration and ion-exchange chromatography. Substrate competition was demonstrated between C6NBD PC, PAF, and oxidized 2-arachidonyl phosphatidylcholine, suggesting that a single enzyme was active against all three substrates. The enzyme had an apparent molecular weight of 40,000-45,000 by high pressure gel exclusion chromatography. Inhibition of this activity with disopropyfluorophosphate prior to oxidative modification of LDL prevented phospholipid hydrolysis but did not affect the production of thiobarbituric acid reactive compounds or the change in electrophoretic mobility. In addition, this inhibition of phospholipase did not prevent the rapid degradati     

Studies on phospholipase C from Pseudomonas aureofaciens. I. Purification and some properties of phospholipase C.

Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Pseudomonas aureofaciens was purified 3600-fold from the culture filtrate with a recovery of 1.6%. Purification was performed with the useof (NH4)2SO4 precipitation, Sephadex G-100 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50. The purified enzyme appeared to be homogeneous as revealed by polyacrylamide disc gel electrophoresis at pH 9.3. The molecular weight was estimated to be 35 000 by gel filtration on Sephadex G-75. Under our experimental conditions, phosphatidylethanolamine was more rapidly hydrolysed than phosphatidylcholine. Lyso forms of these two phosphatides were poor substrates. Phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cardiolipin and sphingomyelin were not hydrolysed. The enzyme activity with phosphatidylcholine as substrate was slightly stimulated by Ca2+, Mg2+, and Mn2+. However, these cations inhibited the activity with phosphatidylethanolamine as substrate. An anionic detergent, sodium deoxycholate, slightly enhanced the activity when phosphatidylcholine and phosphatidylethanolamine were used as substrates. A cationic detergent, cetyltrimethylammonium bromide, inhibited enzyme activity. EDTA and o-henanthroline inhibited the activity of the enzyme to a marked degree.     

Reconstitution of the purified calmodulin-dependent (Ca2+ + Mg2+)-ATPase from smooth muscle

The purified calmodulin dependent (Ca2+ + Mg2+)-ATPase (CaMg ATPase) from porcine antral smooth muscle transports Ca2+ after reconstitution in lipid vesicles indicating that this enzyme is indeed a Ca2+-transport ATPase. For CaMg ATPase reconstituted in asolectin vesicles a good correlation was found between the time course of Ca2+ accumulation and the corresponding changes in CaMg ATPase activity. The ATPase activity was stimulated 8-fold by A23187, which further indicates a tight coupling between ATP hydrolysis and Ca2+ transport. Asolectin vesicles with incorporated enzyme accumulated Ca2+ with a ratio approaching one Ca2+ ion transported for each ATP hydrolyzed. For CaMg ATPase reconstituted in phosphatidylcholine vesicles on the other hand, Ca2+ transport and CaMg ATPase were poorly coupled as is shown by the approximately 3.5 fold stimulation by A23187. The activity of the CaMg ATPase when reconstituted in asolectin vesicles was stimulated 1.25 fold by calmodulin while in phosphatidylcholine a value of 4.25 was obtained. The CaMg ATPase activity of the enzyme reconstituted either in asolectin or phosphatidylcholine was, after its stimulation by A23187, still further stimulated by detergent by a factor of 5.     

Mechanism of human erythrocyte hemolysis induced by short-chain phosphatidylcholines and lysophosphatidylcholine

The incorporation and accumulation of a certain amount of short-chain phosphatidylcholine or lysophosphatidylcholine into lipid bilayers of erythrocyte membranes is the first step causing membrane perturbation in the process of hemolysis. Accumulation of dilauroylglycerophosphocholine into membranes makes human erythrocytes "permeable cells"; Ions such as Na+ or K+ can permeate through the membrane, though large molecules such as hemoglobin can not. The "pore" formation was partially reproduced in liposomes prepared from lipids extracted from human erythrocyte membranes; C12:0PC induced the release of glucose from liposomes but did not significantly induce the release of dextran. It was suggested that the phase boundary between dilauroylglycerophosphocholine and the host membrane bilayer or dilauroylglycerophosphocholine rich domain itself behaves as "pores." Erythrocytes could expand to 1.5 times the original cell volume without any appreciable hemolysis when incubated with C12:0PC at 37 degrees C. The capacity of the erythrocytes to expand was temperature dependent. The capacity may play an important role in the resistance of the cells against lysis. The "permeable cell" stage could be hardly observed when erythrocytes were treated with didecanoylglycerophosphocholine and lysophosphatidylcholine. Perturbation induced by accumulation of didecanoylglycerophosphocholine or lysophosphatidylcholine may cause non specific destruction of membranes rather than formation of a kind of "pore."     

Functional reconstitution of sphingomyelin synthase in Chinese hamster ovary cell membranes.

Sphingomyelin synthase (phosphatidylcholine:ceramide phosphocholinetransferase) activity in the membranes of Chinese hamster ovary cells was found to be detectable with a fluorescent ceramide analog, containing a short acyl chain, as a substrate. We developed a method for the functional reconstitution of sphingomyelin synthase in detergent-treated membranes. Treatment of membranes with 1.5% octyl glucoside in the absence of exogenous phosphatidylcholine resulted in almost complete loss of sphingomyelin synthase activity, even after removal of the detergent by dialysis. In contrast, membranes treated with the detergent in the presence of exogenous phosphatidylcholine showed partial activity and, after dialysis of this mixture, enzyme activity was restored to almost the same level as the activity in dialyzed intact membranes. The effects of various lipids on enzyme activity in this reconstitution system suggested that L-alpha-phosphatidylcholine was the environmental lipid essential for the functional reconstitution of the enzyme. Furthermore, diacylglycerol was suggested to serve as an inhibitory regulator of sphingomyelin synthesis.     

Hydrolysis of sphingomyelin and phosphatidylcholine by midgut homogenates of the stable fly

Qualitative and quantitative analyses were made to characterize the enzymatic degradation of sphingomyelin and phosphatidylcholine by midgut homogenates of the adult stable fly, Stomoxys calcitrans (L.). The results indicated that sphingomyelin was hydrolyzed by an enzyme with sphingomyelinase-like properties, and that phosphatidylcholine was hydrolyzed by an enzyme with properties similar to phospholipase C. The optimum pH for the sphingomyelinase was 7.6, and the rate of hydrolysis of sphingomyelin at that pH was linear from 1 to 4 nmol of substrate and 5 to 25 micrograms of enzyme preparation. Dialysis of the homogenates against Tris-HCl and imidazole buffers resulted in a decrease of sphingomyelinase activity by 59% and 98%, respectively, and the original activity was not restored with the addition of Ca++, Mg++, or Mn++.     

The effects of phospholipids on the properties of hepatic 5'-nucleotidase

Arrhenius plots of 5'-nucleotidase activity in microsomes or plasma membranes from rat liver exhibited transitions at approximately 35 degrees C. The enzyme was purified from homogenates after solubilization in 2% Triton X-100 and 1% sodium deoxycholate. After the initial steps of the purification, the enzyme was recovered in membranes, as judged by both thin section and freeze-fracture electron microscopy, which contained sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. The purest fractions of 5'-nucleotidase were enriched approximate 3,000-fold, consisted of similar membranes, but only contained sphingomyelin. Thermal transitions were detected in Arrhenius plots of 5'-nucleotidase after detergent solubilization, in the membranes which contained the three phospholipids, but not in the purified fraction which contained only sphingomyelin; transitions were also detected after reassociation of the purified enzyme with microsomal or plasma membrane lipids and phosphatidylcholine but not with phosphatidylethanolamine. Phosphatidylcholines containing specific fatty acids all affected the energy of activation of 5'-nucleotidase, and the detergent Sarkosyl, which has been shown to dissociate phospholipids from 5'-nucleotidase (Evans, W. H., and Gurd, J. W. (1973) Biochem. J. 133, 189-199), caused a marked decrease in the stability of the enzyme to heating. Inhibition of 5'-nucleotidase by concanavalin A followed by reactivation with alpha-methyl-D-mannoside resulted in linear Arrhenius plots of 5'-nucleotidase activity in membrane fractions, and in lower transition temperatures for the detergent, solubilized enzyme. It is concluded that in situ, 5'-nucleotidase interacts with both sphingomyelin and phosphatidylcholine; the first apparently influences the stability of the enzyme and the second, the energy of activation. In addition, the lipid environment of the enzyme seems to be altered as a result of lectin binding.     

Reactivation of lipid-depleted Ca2+-ATPase by a nonionic detergent.

The Ca2+-ATPase of sarcoplasmic reticulum can be reversibly delipidated by precipitation with polyethyleneglycol in the presence of deoxycholate and glycerol to as low as 4 mol of phospholipid/mol of enzyme polypeptide and can then be reactivated to 90% of its original ATPase activity by the addition of phosphatidylcholine. Furthermore, the preparation exhibits nearly the same activity if the nonionic detergent dodecyl octaoxyethyleneglycol monoether is substituted for the added phospholipid. The delipidated ATPase is soluble in the detergent and retains activity for several days. This is the first report of the Ca2+-ATPase retaining high activity with less than about 30 mol of phospholipid bound per mol of polypeptide.     

Purification and characterization of a novel monomeric glutathione peroxidase from rat liver

A novel glutathione peroxidase, which is active toward hydroperoxides of phospholipid in the presence of a detergent, has been purified to homogeneity from a rat liver postmicrosomal supernatant fraction by ammonium sulfate fractionation and three different column chromatographies. From a DE52 column, glutathione peroxidase active toward phosphatidylcholine dilinoleoyl hydroperoxides was eluted in one major and two minor peaks. The enzyme in the major peak was found to be separated from the "classic" glutathione peroxidase and glutathione S-transferases and further purified by Sephacryl S-200 and Mono Q column chromatographies. The purified enzyme was found to be homogeneous on polyacrylamide gel electrophoresis under nondenaturing conditions as well as that in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 22,000, and that by gel filtration was comparable, indicating that the enzyme protein is a single polypeptide. The purified enzyme was found to catalyze the reduction of phosphatidylcholine dilinoleoyl hydroperoxides to the corresponding hydroxy derivatives. The isoelectric point of the enzyme was found at pH 6.2, and the optimum pH for the enzyme activity was 8.0. The enzyme was active toward cumene hydroperoxide, H2O2, and 1-monolinolein hydroperoxides in the absence of a detergent. The enzyme activity toward phospholipid hydroperoxides was minute in the absence of a detergent but was remarkably enhanced by the addition of a detergent. From these results, the presently purified enzyme is obviously different from the classic glutathione peroxidase and also from phospholipid hydroperoxide glutathione peroxidase purified from pig heart (Ursini, F., Maiorino, M., and Gregolin, C. (1985) Biochim. Biophys. Acta 839, 62-70), though considerably similar to the latter.     

Comparison of the phospholipase activity of bovine milk lipoprotein lipase against rat plasma very low density and high density lipoprotein.

The hydrolytic activity of a lipoprotein lipase from bovine milk against triacylglycerol and phosphatidylcholine of rat plasma very low density lipoprotein was determined and compared to that against phosphatidylcholine of high density lipoprotein. 85--90% of the triacylglycerol in very low density lipoprotein were hydrolyzed to fatty acids and 25--35% of the phosphatidylcholine to lysophosphatidylcholine. High density lipoprotein phosphatidylcholine was only minimally susceptible to the enzyme. Even with high amounts of enzyme and prolonged incubation periods, lysophosphatidylcholine generation did not exceed 2--4% of the original amounts of labeled phosphatidylcholine in the high density lipoprotein. We conclude that phospholipids in high density lipoprotein are not substrates for the phospholipase activity of this lipoprotein lipase. These observations suggest that factors other than the presence of apolipoprotein C-II and of glycerophosphatides are of importance for the activity of lipoprotein lipases.     

DNA-Breaking Action of 2-tert-Butyl-p-quinone

Phospholipase B from baker’s yeast (Saccharomyces cerevisiae) was purified by acid treatment of the crude extract, ammonium sulfate fractionation, and column chromatographies on DEAE-Sepharose CL-6B, Sepharose 4B, and Bio-Gel HTP. The purified preparation had lysophospholipase activity and phospholipase B activity in a ratio of 16:1. The optimum pH of both activities was 3.5 ~ 4.0. The enzyme was a glycoprotein and its molecular size was somewhat heterogeneous, ranged from about 280,000 to 420,000 by gel filtration. Phospholipase B activity was strongly stimulated by 0.1 % DOC, but lysophospholipase activity was completely inhibited by the detergent. Neither activity was stimulated by Ca²⁺ and both were inhibited by SDS, Triton X-100, and Fe³⁺. The enzyme hydrolyzed the acyl ester bonds of phosphatidylcholine sequentially, first the 2-acyl and then the 1-acyl groups. The Km values for phosphatidylcholine and lysophosphatidylcholine were 0.63 mm and 0.05 mm, respectively.     

Phosphatidylinositol synthase from Saccharomyces cerevisiae. Reconstitution, characterization, and regulation of activity

Purified membrane-associated phosphatidylinositol synthase (CDP diacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) from Saccharomyces cerevisiae was reconstituted into unilamellar phospholipid vesicles. Reconstitution of the enzyme was performed by removing detergent from an octylglucoside/phospholipid/Triton X-100/enzyme mixed micelle mixture by Sephadex G-50 superfine column chromatography. The average diameter of the vesicles was 40 nm and chymotrypsin treatment of intact vesicles indicated that over 90% of the reconstituted enzyme had its active site facing outward. The enzymological properties and reaction mechanism of reconstituted phosphatidylinositol synthase were determined in the absence of detergent. The reconstituted enzyme was used as a model system to study the regulation of activity. Phosphatidylinositol synthase was constitutive in wild type cells grown in the presence of water-soluble phospholipid precursors as determined by enzyme activity and immunoblotting. Reconstituted enzyme was not effected by water-soluble phospholipid precursors or nucleotides. Maximum activity was found when the enzyme was reconstituted into phosphatidylcholine: phosphatidylethanolamine: phosphatidylinositol: phosphatidylserine vesicles. Phosphatidylserine stimulated reconstituted activity, suggesting that the local phospholipid environment may regulate phosphatidylinositol synthase activity.     

Enzyme and phospholipid asymmetry in liver microsomal membranes

The transverse distribution of enzyme proteins and phospholipids within microsomal membranes was studied by analyzing membrane composition after treatment with proteases and phospholipases. Upon trypsin treatment of closed microsomal vesicles, NADH- and NADPH-cytochrome c reductases as well as cytochrome b5 were solubilized or inactivated, while cytochrome P-450 was partially inactivated. When microsomes were exposed to a concentration of deoxycholate which makes them permeable to macromolecules but does not disrupt the membrane, the detergent alone was sufficient to release four enzymes: nucleoside diphosphatase, esterase, beta-glucuronidase, and a portion of the DT-diaphorase. Introduction of trypsin into the vesicle lumen inactivated glucose-6-phosphatase completely and cytochrome P-450 partially. The rest of this cytochrome, ATPase, AMPase, UDP-glucuronyltransferase, and the remaining 50% of DT-diaphorase activity were not affected by proteolysis from either side of the membrane. Phospholipase A treatment of intact microsomes in the presence of albumin hydrolyzed all of the phosphatidylethanolamine, phosphatidylserine, and 55% of the phosphatidylcholine. From this observation, it was concluded that these lipids are localized in the outer half of the bilayer of the microsomal membrane; Phosphatidylinositol, 45% of the phosphatidylcholine, and sphingomyelin are tentatively assigned to the inner half of this bilayer. It appears that the various enzyme proteins and phospholipids of the microsomal membrane display an asymmetric distribution in the transverse plane.     

Purification of a phospholipase A2 from human monocytic leukemic U937 cells. Calcium-dependent activation and membrane association

The existence of an intracellular phospholipase A2 (PLA2) involved in the production of 1-O-alkyl-sn-glycero-3-phosphocholine and free arachidonic acid has been repeatedly postulated. Using 1-O-hexadecyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine as a substrate and a series of conventional and high-pressure liquid chromatographic techniques, we have purified a PLA2 from the soluble fraction of differentiated human monocytic U937 cells. The enzyme has been purified nearly 2000-fold to homogeneity. The purified enzyme has a molecular mass of 56 kDa, under reducing conditions, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The enzyme activity has a pH optimum of 8.0 and is calcium concentration-dependent. The EC50 for the activation of the enzyme activity by calcium is 300 nM. When the cells were homogenized in the presence of the calcium chelator EGTA (0.2 mM), the enzyme was found to be soluble (more than 90% of the activity in the 100,000 x g supernatant). However, when Ca2+ concentration was controlled from 10 nM to 100 microM in Ca2(+)-EGTA buffers, increasing amounts of the activity were found in the particulate fraction (100,000 x g pellet). This suggests that membrane translocation and activation of the soluble PLA2 may be regulated by physiological intracellular levels of Ca2+. The purified enzyme hydrolyzed different phosphatidylcholine substrates presented in either vesicular or Triton X-100 mix micellar forms. In both situations, the enzyme showed a high degree of specificity for arachidonic acid on the sn-2 position of the substrate. Substitution of palmitic or oleic on the sn-2 position substantially reduced the hydrolytic activity of the enzyme. When vesicles of arachidonic acid-containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol were presented to the purified enzyme, all of them were hydrolyzed with comparable efficiency. However, only phosphatidylcholine and phosphatidylinositol were hydrolyzed when presented in Triton X-100 mixed micelles.     

Chemical modification of rat liver microsomal glutathione transferase defines residues of importance for catalytic function.

Amino acid residues that are essential for the activity of rat liver microsomal glutathione transferase have been identified using chemical modification with various group-selective reagents. The enzyme reconstituted into phosphatidylcholine liposomes does not require stabilization with glutathione for activity (in contrast with the purified enzyme in detergent) and can thus be used for modification of active-site residues. Protection by the product analogue and inhibitor S-hexylglutathione was used as a criterion for specificity. It was shown that the histidine-selective reagent diethylpyrocarbonate inactivated the enzyme and that S-hexylglutathione partially protected against this inactivation. All three histidine residues in microsomal glutathione transferase could be modified, albeit at different rates. Inactivation of 90% of enzyme activity was achieved within the time period required for modification of the most reactive histidine, indicating the functional importance of this residue in catalysis. The arginine-selective reagents phenylglyoxal and 2,3-butanedione inhibited the enzyme, but the latter with very low efficiency; therefore no definitive assignment of arginine as essential for the activity of microsomal glutathione transferase can be made. The amino-group-selective reagents 2,4,6-trinitrobenzenesulphonate and pyridoxal 5'-phosphate inactivated the enzyme. Thus histidine residues and amino groups are suggested to be present in the active site of the microsomal glutathione transferase.