Purification and biochemical characterization of cytosolic glutathione-S-transferase from filarial worms Setaria cervi

The present study reports the purification and characterization of GST from cytosolic fraction of Setaria cervi. GST activity was determined in various subcellular fractions of bovine filarial worms S. cervi (Bubalus bubalis Linn.) and was found to be localized mainly in the cytosolic and microsomal fractions. The soluble enzyme from S. cervi was purified to homogeneity using a combination of salt precipitation, centrifugation, cation exchange and GSH-Sepharose affinity chromatography followed by ultrafiltration. SDS-PAGE analysis revealed a single band and activity staining was also detected on PAGE gels. Gel filtration and MALDI-TOF studies revealed that the native enzyme is a homodimer with a subunit molecular mass of 24.6 kDa. Comparison of kinetic properties of the parasitic and mammalian enzymes revealed significant differences between them. The substrate specificity and inhibitor profile of cytosolic GST from S. cervi appeared to be different from GST from mammalian sources.     

MALDI mass sequencing and characterization of filarialglutathione-S-transferase

Glutathione-S-transferase has been detected in the somatic extract and excretory-secretory products of different life stages of Setaria cervi, a bovine filarial parasite. The enzyme was subjected to MALDI-TOF followed by mass spectrometry and the nearest match found was Pleuronectes platessa GST. Molecular mass of the purified enzyme was approximately 26 kDa as determined by SDS-PAGE and MALDI-TOF. Setaria cervi GST exhibited high activity towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene and glutathione as substrate revealed a K(m) of 2.22 mM and 0.61 mM, respectively. The activity was inhibited significantly by Cibacron blue and alpha-tocopherol.     

Effect of ferulic acid from Hibiscus mutabilis on filarial parasite Setaria cervi: Molecular and biochemical approaches

In the reported work the in vitro activity of a methanolic extract of leaves of Hibiscus mutabilis (Malvaceae) against bovine Setaria cervi worms has been investigated. Bioassay-guided fractionation led to isolation of ferulic acid from ethyl acetate fraction. The crude extract and ferulic acid, the active molecule, showed significant microfilaricidal as well as macrofilaricidal activities against the microfilaria (L(1)) and adult of S. cervi by both a worm motility and MTT reduction assay. The findings thus provide a new lead for development of a filaricidal drug from natural products. To examine the possible mechanism of action of ferulic acid, the involvement of apoptosis in adult worms of S. cervi was investigated. We found extreme cellular disturbances in ferulic acid-treated adult worms characterized by chromatin condensation, in situ DNA fragmentation and nucleosomal DNA laddering. In this work we are reporting for the first time that ferulic acid exerts its antifilarial effect through induction of apoptosis and by downregulating and altering the level of some key antioxidants (GSH, GST and SOD) of the filarial nematode S. cervi. Our results have provided experimental evidence supporting that ferulic acid causes an increased proapoptotic gene expression and decreased expression of anti-apoptotic genes simultaneously with an elevated level of ROS and gradual dose dependent decline of parasitic GSH level. We also observed a gradual dose dependent elevation of GST and SOD activity in the ferulic acid treated worms.     

Characterization of Ca(2+)-ATPase of Setaria cervi (Nematoda: Filarioidea): effect of phenothiazines and anthelmintics.

1. A Mg2+ independent, Ca(2+)-ATPase requiring high concentrations of Ca2+ (5 mM) for the activation, equally distributed in cuticle-muscular-hypodermis, genital organs and gastrointestinal tissues and mainly localized in 10,000 g pellet fraction, was identified in Setaria cervi, a bovine filarial parasite. 2. Filarial enzyme showed Km value of 3.33 mM for ATP as computed from the double reciprocal Lineweaver-Burk plot. 3. The enzyme could be completely solubilized by sonication with about 4-fold increase in specific activity of the enzyme. 4. The enzyme showed about 2-fold activation by the calmodulin fractions isolated from S. cervi and rat brain homogenates. 5. The enzyme was highly sensitive to inhibition with some phenothiazine derivatives. Trifluoperazine was observed to be the most potent inhibitor followed by promethazine and chlorpromazine. 6. Some anthelmintics viz. diethycarbamazine and centperazine were found to be highly potent inhibitors of the enzyme, significant inhibition of filarial Ca(2+)-ATPase was also observed with levamisole and suramine. 7. Studies indicate Ca(2+)-ATPase of S. cervi as a potential chemotherapeutic target.     

Setaria cervi: kinetic studies of filarial glutathione synthetase by high performance liquid chromatography

The bovine filarial worm Setaria cervi was found to have abundance of glutathione synthetase (GS; EC 6.3.2.3) activity, the enzyme being involved in catalysing the final step of glutathione (GSH) biosynthesis. A RP-HPLC method involving precolumn derivatization with o-phthalaldehyde has been followed for the estimation of GS activity in crude filarial preparations. Subcellular fractionation of the enzyme was undertaken and it was confirmed to be a soluble protein residing mainly in cytosolic fraction. Attempts to determine the Km value for L-gamma-glutamyl-L-cysteine gave a distinctly nonlinear double-reciprocal plot in which data obtained at relatively high dipeptide concentrations (>1 mM) extrapolate to a Km value of about 400 microM whereas data obtained at lower concentrations (<0.1 mM) extrapolate to a value of about 33 microM. Km was determined to be around 950 and 410 microM for ATP and glycine, respectively. The effect of various amino acids was studied on enzyme activity at 1mM concentration. L-cystine caused a significant enzyme inhibition of 11%. Preincubation with N-ethylmaleimide also resulted in significant inhibition of GS activity.     

Identification and characterization of a selenium-dependent glutathione peroxidase in Setaria cervi

Setaria cervi a bovine filarial parasite secretes selenium glutathione peroxidase during in vitro cultivation. A significant amount of enzyme activity was detected in the somatic extract of different developmental stages of the parasite. Among different stages, microfilariae showed a higher level of selenium glutathione peroxidase activity followed by males then females. However, when the activity was compared in excretory secretory products of these stages males showed higher activity than microfilariae and female worms. The enzyme was purified from female somatic extract using a combination of glutathione agarose and gel filtration chromatography, which migrated as a single band of molecular mass approximately = 20 kDa. Selenium content of purified enzyme was estimated by atomic absorption spectroscopy and found to be 3.5 ng selenium/microg of protein. Further, inhibition of enzyme activity by potassium cyanide suggested the presence of selenium at the active site of enzyme. This is the first report of identification of selenium glutathione peroxidase from any filarial parasite.     

Setaria cervi: immunoprophylactic potential of glutathione-S-transferase against filarial parasite Brugia malayi

Glutathione-S-transferase (GST) has been detected in the adult female Setaria cervi, a bovine filarial parasite. The role of S. cervi GST antigen in inducing immunity in the host against Brugia malayi microfilariae and infective larvae was studied by in vitro antibody dependent cell mediated reaction as well as in situ inoculation of filarial parasites within a microchamber in Mastomys. The immune sera from glutathione-S-transferase immunized Mastomys promoted the adherence of peritoneal exudate cells to B. malayi microfilariae and infective larvae in vitro inducing 80.7 and 77.6% cytotoxicity, respectively in 72 h. In the microchambers implanted in the immunized Mastomys host cells could migrate and adhere to the microfilariae and infective larvae and induced 77.8 and 75% cytotoxicity to B. malayi microfilariae and infective larvae in 72 h, respectively. These results suggest that native GST from S. cervi is effective in inducing protection against heterologous B. malayi filarial parasite and thus has potential in immunoprophylaxis.     

The in vitro metabolism and bioactivation of 1,2-dibromoethane (ethylene dibromide) by human liver

The nematocide, grain fumigant, and gasoline additive 1,2-dibromoethane (DBE) is both a cellular and a genetic toxin that is metabolically activated in rats and mice by mixed function oxidases (MFO) as well as glutathione 5-transferases (GST). The purpose of this study was to determine whether DBE is similarly metabolized and bioactivated by human liver in vitro. Human liver microsomal and cytosolic metabolism of DBE was monitored by the production of aqueous-soluble metabolites from [14-C]-DBE. Reactive intermediates were detected as irreversibly bound adducts to protein or DNA. 1,2-Dibromoethane was metabolized by human liver cytosolic GST, microsomal GST, and microsomal MFO. Cytosolic GST activity (9 +/- 2 nmol/20 min/mg protein) was about four times greater than the other two activities. Only MFO activity resulted in adducts irreversibly bound to protein (1.5 +/- .4 nmol/20 min/mg protein) and was inhibited by the presence of glutathione. Both MFO and GST activity resulted in irreversibly bound adducts to DNA. Microsomal and cytosolic GST activity each produced about twice as many DNA adducts as microsomal MFO activity. These results suggest that human liver, like rat and mouse liver, metabolizes DBE to aqueous-soluble metabolites by both MFO and GST activity. Furthermore, each of these activities produces reactive metabolites that can irreversibly bind to cellular macromolecules.     

A novel, endogenous inhibitor of 7-ethoxyresorufin-O-deethylase activity isolated from liver cytosolic fractions of bream (Abramis brama L.)

The cytosolic supernatant of bream (Abramis brama L.) liver homogenates inhibits the 7-ethoxyresorufin-O-deethylase (EROD) activity of pike (Esox lucius) microsomal fractions. The inhibitor shows no activity against 7-ethoxycoumarin-O-deethylase and benzo(a)pyrene hydroxylase indicating a high isoenzyme specificity. The inhibiting component is a heat-sensitive substance (56 degrees C for 5') which is not self regenerating after subsequent cooling. It can be isolated from the cytosolic fraction using two combined steps of ion exchange chromatography. The purification factor is 500-fold with a recovery rate of 70%. SDS-PAGE of the purified fractions indicate that electrophoretic purity was not achieved. However, a prominent band at about 97 kDa was present in all fractions in a close intensity activity relationship. The molecular weight of the native form of the purified protein was determined to be 175 +/- 35 kDa using gel filtration on a Sephacryl S 300 HR column. So far the inhibitor can be characterized as a protein. It shows strong tendencies to aggregate due to lipophilic interactions. These interactions can be repressed by the addition of 1% sodium cholate. The inhibitor has an optimum activity at 25 degrees C and pH 8.0. The inhibitor does not correspond to any of the known cytosolic, endogenous inhibitors of EROD activities in fish, including proteases, cytosolic phosphatases, kinases and resorufin reductase (e.g. DT-diaphorase), although a non-dicoumarol (10 microM)-inhibited menadione oxidoreductase activity of up to 46.7 +/- 0.4 nmol/min per mg inhibitory protein was measured. Kinetic studies using Michaelis-Menten kinetics with purified inhibitor fractions prove a non-competitive mode of inhibition. As this kind of inhibitor is not described yet it is named CERODIP (cytosolic, EROD-inhibiting protein).     

Biochemical Characterization and Distribution of Glutathione S-Transferases in Leaping Mullet (Liza saliens)

In this study, feral leaping mullet (Liza saliens) liver cytosolic glutathione S-transferases (GSTs) were investigated and characterized using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as substrates. The average GST activities towards CDNB and EA were found to be 1365 +/- 41 and 140 +/- 20 nmol/min per mg protein, respectively. The effects of cytosolic protein amount and temperature ranging from 4 to 70 degrees C on enzyme activities were examined. While both activities towards CDNB and EA showed similar dependence on protein amount, temperature optima were found as 37 and 42 degrees C, respectively. In addition, the effects of pH on GST-CDNB and -EA activities were studied and different pH activity profiles were observed. For both substrates, GST activities were found to obey Michaelis-Menten kinetics with apparent V(max) and K(m) values of 1661 nmol/min per mg protein and 0.24 mM and 157 nmol/min per mg protein and 0.056 mM for CDNB and EA, respectively. Distribution of GST in Liza saliens tissues was investigated and compared with other fish species. Very high GST activities were measured in tissues from Liza saliens such as liver, kidney, testis, proximal intestine, and gills. Moreover, our results suggested that GST activities from Liza saliens would be a valuable biomarker for aquatic pollution.     

Androgen concentration and partial characterization of 5alpha reductase in the epididymis of the rhesus monkey.

The 5alpha reductase activity ofthe monkey epididymis was studied. The enzyme was found in particulate subcellular fractions, its distribution closely resembling that of the microsomal marker enzyme NADPH: cytochrome c reductase, suggesting an association of 5alpha reductase with membranes of the endoplasmic reticulum. Maximal enzyme activity was found at pH 5.4 and at 32--37 C. The crude nuclear preparation had a Km: 0.315 x 10(-6)M and Vmax: 168 pmoles/mg protein/h. The microsomal enzyme had a Km: 0.243 x 10(-6)M and Vmax: 828 pmoles/mg protein/h. Neither enzyme preparation was affected by addition to the incubation media of dihydrotestosterone (DHT) or 5alpha-androstane-3alpha,17beta-diol. The endogenous androgen concentration in the epididymides of 2 different monkeys, in ng/g wet weight was: DHT 20.81 +/- 1.98; T: 9.0L +/- 2.83; diol: 3.03 +/- 0.41.     

Hexose metabolism in pancreatic islets: compartmentation of hexokinase in islet cells

Hexokinase activity was found in both soluble (cytosolic) and particulate subcellular fractions prepared from rat pancreatic islet homogenates. The bound enzyme was associated with mitochondria rather than secretory granules. Relative to the total hexokinase activity, the amount of bound enzyme was higher in islet homogenates prepared at pH 6.0 (72 +/- 7%) than in islets homogenized at pH 7.4 (38 +/- 1%). The affinity of hexokinase for equilibrated D-glucose was not different in the cytosolic and mitochondrial fractions. In both fractions, hexokinase displayed a greater affinity for alpha- than beta-D-glucose, but a higher maximal velocity with the beta- than alpha-anomer. Glucose 6-phosphate inhibited to a greater extent cytosolic than mitochondrial hexokinase. A high Km glucokinase-like enzymic activity was also present in both subcellular fractions. It is proposed that the ambiguity of hexokinase plays a propitious role in the glucose-sensing function of pancreatic islet cells.     

Some properties of the fatty alcohol oxidation system and reconstitution of microsomal oxidation activity in intestinal mucosa

This paper describes the metabolism of fatty alcohols by microsomal and cytosolic fractions from intestinal mucosa. Microsomes of rabbit intestinal mucosa had a high activity of [1-14C]dodecanol oxidation as did those of liver. The intestinal cytosolic fraction also exhibited oxidation activity to a lesser extent than the microsomes did. The reaction product was determined as lauric acid using thin-layer chromatography. Laurylaldehyde was detected as another product, when semicarbazide was added to the incubation system. Cyclodextrins exhibited a stimulation effect similarly to bovine serum albumin on the microsomal activity. We have compared the stimulatory effects of dimethyl-beta-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and alpha-cyclodextrin, which decrease in that order. Effects of NAD+ and dodecanol concentrations, pH and pyrazole on microsomal activity were compared with those on cytosolic activity. Dodecanol oxidation activity was solubilized and reconstituted with a fatty alcohol dehydrogenase and a fatty aldehyde dehydrogenase separated from the intestinal microsomes. These findings indicate that both the dehydrogenases participate in microsomal oxidation of fatty alcohols to fatty acids with fatty aldehydes as intermediates in the reaction.     

Biochemical composition and metabolic pathways of filarial worms Setaria cervi: search for new antifilarial agents

The main problem regarding the chemotherapy of filariasis is that no safe and effective drug is available yet to combat the adult human filarial worms. Setaria cervi, the causal organism of setariasis and lumbar paralysis in cattle, is routinely employed as a model organism for conducting biochemical and enzymatic studies on filarial parasites. In view of the practical difficulties in procuring human strains of Wuchereria bancrofti and Brugia malayi for drug screening, the bovine filarial parasite S. cervi, resembling the human species in having microfilarial periodicity and chemotherapeutic response to known antifilarial agents, is widely used as a model in such studies. For a rational approach to antifilarial chemotherapy, knowledge of the biochemical composition and metabolic pathways of this helminth parasite may be of paramount importance, so that more potent antifilarial agents based on specific drug targets can be identified in drug discovery programmes. The present review provides an update on the biochemistry of the important metabolic pathways functioning within this potentially important bovine parasite, that have so far been studied, and on those that need to be investigated further so as to identify novel drug targets that can be exploited for designing new antifilarial drugs.     

Synthesis and biological evaluation of potential modulators of malarial glutathione-S-transferase(s)

Glutathione-S-transferase(s) (E.C.2.5.1.18, GSTs) have been investigated in parasitic protozoans with respect to their biochemistry and they have been identified as potential vaccine candidates in protozoan parasites and as a target in the synthesis of new antiparasitic agents. In a search towards the identification of novel biochemical targets for antimalarial drug design, the area of Plasmodium glutathione metabolism provides a number of promising chemotherapeutic targets. GST activity was determined in various subcellular fractions of malarial parasites Plasmodium yoelii and was found to be localized mainly in the cytosolic fraction (specific activity, c. 0.058 +/- 0.016 micromol/min/mg protein). Hemin, a known inhibitor of mammalian GST(s), maximally inhibited this enzyme from P. yoelii to nearly 86%. In a search towards synthetic modulators of malarial GST(s), 575 compounds belonging to various chemical classes were screened for their effect on crude GST from P. yoelii and 92 compounds belonging to various chemical classes were studied on recombinant GST from P. falciparum. Among all the compounds screened, 83 compounds inhibited/stimulated the enzyme from P. yoelii/P. falciparum to the extent of 40% or more.     

Arylamine activation following chronic ethanol ingestion by rats: studies on the liver S9, microsomal and cytosolic fractions and comparison with Aroclor 1254 pretreatment

That enzyme fractions derived from animals chronically fed alcohol can alter the metabolism of carcinogenic xenobiotic compounds has been documented. To further understand this relationship the mutagenicity of 3 aromatic amines was determined in the Ames test, employing activation systems derived from rats maintained on an alcohol-containing liquid diet, an isocaloric control liquid diet or Aroclor 1254-pretreated animals fed standard laboratory chow. Depending upon protein and substrate concentrations, S9 from ethanol-fed rats was 30-50% less efficient than S9 from pair-fed rats in activating arylamines (2-aminofluorene, 2-aminoanthracene and 2-acetylaminofluorene) to mutagens in Salmonella typhimurium TA98 and TA100. Cytosolic fractions from ethanol-fed animals always resulted in greater arylamine activation than that of controls whereas the opposite was true of the microsomal compartment in which the ethanol-treated group was consistently less active than the controls. The cytosolic N-acetyltransferase activities with respect to 2 different substrates, isoniazid and 2-aminofluorene, were unaffected by ethanol consumption, indicating that this activity probably does not account for the different activation profiles exhibited by the ethanol and pair-fed cytosolic systems. Both the cytosolic and microsomal compartments are required for maximal expression of the mutagenicity of each arylamine however, each compartment can activate arylamines independently of the other. Reconstituting cytosol with microsomes from ethanol- and pair-fed rats, but not Aroclor-pretreated rats, resulted in a synergistic activation of the aromatic amines and displayed an effect similar to that of S9. Compared to Aroclor pretreatment and pair-fed controls, microsomes from ethanol-fed rats displayed the least capacity for activating any of the arylamines to mutagens. Microsomes from Aroclor-pretreated rats accounted for at least 80% of the S9-mediated activation of each of the arylamines to mutagenic metabolites which was in marked contrast to the contribution of the microsomal fractions to the S9 activity in the ethanol- (5-20% of S9 activity) and pair-fed systems (22-30% of S9 activity). The data indicate that 2 opposing reactions occur in S9, a cytosolic activity that augments and a microsomal activity that attenuates the mutagenicity of arylamines. Both activities are modified by ethanol consumption and Aroclor pretreatment.     

Prostaglandin biosynthesis by midgut tissue isolated from the tobacco hornworm, Manduca sexta

We describe prostaglandin (PG) biosynthesis by isolated midgut preparations from tobacco hornworms, Manduca sexta. Microsomal-enriched midgut preparations yielded four PGs, PGA/B(2), PGD(2), PGE(2) and PGF(2alpha), all of which were confirmed by analysis on gas chromatography--mass spectrometry (GC--MS). PGA and PGB are double bond isomers which do not resolve on TLC but do resolve by GC; for convenience, we use the single term PGA(2) for this product. PGA(2) was the major product under most conditions. The midgut preparations were sensitive to reaction conditions, including radioactive substrate, protein concentration (optimal at 1mg/reaction), reaction time (optimal at 0.5 min), temperature (optimal at 22 degrees C), buffer pH (highest at pH 6), and the presence of a co-factor cocktail composed of reduced glutathione, hydroquinine and hemoglobin. In vitro PG biosynthesis was inhibited by two cyclooxygenase inhibitors, indomethacin and naproxen. Subcellular localization of PG biosynthetic activity in midgut preparations, determined by ultracentrifugation, revealed the presence of PG biosynthetic activity in the cytosolic and microsomal fractions, although most activity was found in the cytosolic fractions. This is similar to other invertebrates, and different from mammalian preparations, in which the activity is exclusively associated with the microsomal fractions. Midgut preparations from M. sexta pupae, adult cockroach, Periplaneta americana, and corn ear worms, Helicoverpa zea, also produced the same four major PG products. We infer that insect midguts are competent to biosynthesize PGs, and speculate they exert important, albeit unrevealed, actions in midgut physiology.     

Characterization and modulation by drugs of sheep liver microsomal flavin monooxygenase activity

The flavin monooxygenases (FMO) catalyse the NADPH and oxygen-dependent oxidation of a wide range of nucleophilic nitrogen-, sulfur-, phosphorus-, and selenium heteroatom-containing chemicals, drugs, and agricultural agents. In the present study, sheep liver microsomal FMO activity was determined by measuring the S-oxidation rate of methimazole and the average specific activity obtained from different microsomal preparations was found to be 3.8 +/- 1.5 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 7). The presence of 0.1% Triton X-100 in the reaction mixture caused an increase of specific sheep liver microsomal FMO activity towards methimazole to 6.1 +/- 1.4 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 6). Metabolism of imipramine and chlorpromazine was measured by following the oxidation of cofactor NADPH spectrophotometrically at 340 nm. Sheep liver microsomal FMO activity towards imipramine and chlorpromazine was found to be 10.7 and 12.3 nmol NADPH oxidized min(-1) mg(-1) microsomal protein, respectively. Characterization of sheep liver enzyme was carried out using methimazole as substrate and the maximum FMO enzyme activity was detected at 37 degrees C and at pH 8.0. The apparent K(m) value of sheep liver microsomal FMO for methimazole was 0.118 mM. Effects of the detergents Triton X-100, Cholate, and Emulgen 913, on FMO activity were determined and FMO activity was found to increase with the addition of detergents to the reaction medium. Sheep liver microsomal FMO-catalysed methimazole oxidation was inhibited by imipramine and chlorpromazine when these drugs were used at high concentrations. Western blot-immunochemical analysis revealed the presence of FMO3 in sheep liver microsomes.     

Subcellular localization of the phospholipases A of rat heart: evidence for a cytosolic phospholipase A1

During myocardial ischemia increased levels of lysoglycerophospholipids have been reported which may be deleterious to myocardial function. Phospholipases are presumed to be important in the regulation of this process. To further quantify and characterize the activity of heart phospholipases, we carried out a systematic analysis of phospholipase A activity in rat heart subcellular fractions isolated by the method of Palmer et al. (J. Biol. Chem. 1972. 262: 8731-8739). Neutral phospholipase A was recovered predominately in the cytosolic (soluble) fraction which represented 46% of recovered activity, while the microsomal and subsarcolemmal mitochondrial fractions represented 15% and 12% of the total recovered activity, respectively. Cytosolic phospholipase A differed from the two principal membrane-bound phospholipases A in its pH dependence and apparent Km for substrate. The cytosolic enzyme had a Km (apparent) for dioleoylphosphatidylcholine of 0.07 mM versus 0.28-0.33 mM for the membrane-associated phospholipases A. Acid phospholipase A activity had a subcellular distribution consistent with a lysosomal localization. Lysophospholipase was found principally in the cytosolic, microsomal, and the subsarcolemmal and interfibrillar mitochondrial fractions where it represented 46, 17, 6.3, and 6.9% of the recovered activity, respectively. The positional specificity of the respective phospholipases was assessed. This analysis was complicated by the fact that in heart, lysophospholipase has an observed Vmax 3.6- to 4.5-fold greater than that of phospholipase A in the various subcellular fractions. Equations were derived to obtain corrected values for the activity of phospholipases A1 and A2. Using this method we found that the cytosolic and lysosomal fractions contained phospholipase A1, while the mitochondrial fractions contained primarily phospholipase A2. In heart microsomes, the positional specificity of phospholipase A could not be determined because lysophospholipase activity was very high and lysophosphatidylcholine did not accumulate.