Phosphatide Acyl-hydrolase Research Articles

Phospholipase C and diacylglycerol lipase activities associated with plasma membranes of chromaffin cells isolated from bovine adrenal medulla

The plasma membranes of bovine adrenal chromaffin cells were isolated and the activities of enzymes involved in arachidonic acid liberation were investigated. Only a minute activity of phospholipase A 2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) could be detected using externally added phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as substrate. When membranes were treated with exogenous phospholipase C (orthophosphoric acid diester phosphohydrolase, EC 3.1.4.1) there was a liberation of free fatty acids from the sn-2 position of PC. The enzyme responsible for this effect could be demonstrated to be a diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) localized in the plasma membrane. Using phosphatidylinositol (PI) as a substrate, it was found that an endogenous phospholipase C exists which co-purifies with the membrane preparation. The produced diacylglycerol is subsequently hydrolyzed by diacylglycerol lipase liberating arachidonic acid. The two enzymes, phospholipase C and diacylglycerol lipase were characterized. Phospholipase C was found to be calcium dependent and PI specific, showing an activity of 60 pmol/μg protein per h (1.2 mM Ca 2+), whereas the diacylglycerol lipase was calcium independent hydrolyzing diacylglycerol at a rate of 7.2 pmol/μg protein per h. The lipase but not the phospholipase C was inhibited 50% by 1.7 mM para-bromophenacylbromide.

Inhibition of human platelet phospholipase A2 activity by unsaturated fatty acids.

Phospholipase A2 (PLA2; phosphatide 2-acylhydrolase, EC 3.1.1.4) activity from human platelets increases significantly when the enzyme is separated from an endogenous inhibitor(s). The inhibitor, associated mainly with a particulate fraction, has now been identified as a mixture of unsaturated fatty acids. Treatment of the inhibitor with trypsin, RNase, DNase, or heat did not diminish its inhibitory activity, which was extractable by organic solvents. Incubation of PLA2 with phospholipids or various neutral lipids, including saturated fatty acids, had little or no effect on enzymatic activity. In contrast, unsaturated fatty acids such as palmitoleic acid (16:1), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4), all of which were detected in the particulate fraction, or longer chained unsaturated fatty acids inhibited PLA2 activity by 50% at approximately equal to 5 X 10(-7) M. The level of unsaturated fatty acids in the inhibitor fraction was equivalent to approximately equal to 10(-4) M, apparently sufficient to effectively inhibit PLA2 activity. Methylation of unsaturated fatty acids caused a complete loss of inhibitory activity, and subsequent demethylation restored the activity, suggesting that a free carboxyl group was necessary. Inhibition of PLA2 by unsaturated fatty acids appeared to be noncompetitive. PLA2 absolutely required Ca2+ for activity; the inhibition by unsaturated fatty acids was not reversed by Ca2+. The finding that unsaturated fatty acids are potent inhibitors of PLA2 would explain its generally low activity in human platelet extracts and its marked increase of activity during the course of enzyme purification.

Marked increase of human platelet phospholipase A2 activity in vitro and demonstration of an endogenous inhibitor.

When soluble and particulate fractions of human platelet homogenate were chromatographed on DEAE-cellulose and hydroxylapatite columns, total phospholipase A2 (PLA2; phosphatide 2-acylhydrolase, EC 3.1.1.4) activity increased sharply. PLA2 activity detected in these partially purified preparations was approximately 12 times greater than that associated with the original homogenate. Chromatography of the particulate enzyme on a DEAE-cellulose column yielded one activity peak. Fractions eluted near the activity peak showed a trace of PLA2 activity but inhibited purified PLA2 stoichiometrically, suggesting the presence of an endogenous "inhibitor" in the homogenate. The inhibitor activity was heat-stable, trypsin insensitive, and extractable by chloroform/methanol, and thus appears to be associated with a lipid(s). PLA2 activity was Ca2+ dependent. The crude enzyme was variably stimulated by calmodulin, whereas the purified enzyme was not, suggesting that the effect of calmodulin on the crude enzyme is indirect. Our results suggest that human platelet PLA2 activity reported in the literature may have been underestimated, apparently due to the presence of an endogenous inhibitor.

The complete primary structure of phospholipase A 2 from human pancreas

The complete amino acid sequence of phospholipase A 2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from human pancreas was determined. The protein consists of a single polypeptide chain of 125 amino acids and has a molecular weight of 14 003. The chain is cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digestion of the reduced and thialaminated derivative of the protein with clostripain, yielding three fragments. The largest fragment (residues 7–100) was further degraded both with staphylococcal proteinase and chymotrypsin. The sequence was determined by automated Edman degradation of the intact protein and of several large peptide fragments. Phospholipase A 2 from human pancreas contains the same number of amino acids (125) as the enzyme from horse, while the enzymes from pig and ox contain 124 and 123 residues, respectively. The enzymes show a high degree of homology; human phospholipase differs from the other enzymes by substitutions of 26 (porcine), 28 (bovine) and 32 (equine) residues, respectively.

Biphasic modulation of platelet phospholipase A 2 activity and platelet aggregation by mepacrine (quinacrine)

The modulation of rat platelet phospholipase A 2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) and rat platelet aggregation by mepacrine was investigated. The 2-acyl specificity of phospholipase A 2 activity was confirmed by using 1-[ 14C]palmitoyl-2-[ 3H]arachidonylphosphatidylcholine as substrate. Under optimal pH, phospholipase A 2 activity was not affected by aspirin but was inhibited by indomethacin. Contrary to previous reports, a biphasic modulatory role of mepacrine on phospholipase A 2 activity and platelet aggregation was demonstrated. The data suggest that platelet aggregation is mediated via phospholipase A 2.

Biochemical signal transmitted by Fc gamma receptors: phospholipase A2 activity of Fc gamma 2b receptor of murine macrophage cell line P388D1.

The detergent lysate of the P388D1 macrophage cell line was subjected to affinity chromatography on two different media, Sepharose coupled to heat-aggregated human IgG (IgG-Sepharose) and Sepharose coupled to the phosphatidylcholine analog rac-1-(9-carboxyl)nonyl-2-hexadecylglycero-3-phosphocholine (PC-Sepharose). Both IgG- and phosphatidylcholine-binding proteins were further purified by Sephadex G-100 gel filtration and isoelectric focusing in the presence of 6 M urea. The isolated IgG-binding proteins specifically bound to IgG2a, but not to IgG2b, whereas the isolated phosphatidylcholine-binding proteins specifically bound to IgG2b but not to IgG2a. Phosphatidylcholine-binding proteins possessed a typical phospholipase A2 activity (phosphatide 2-acylhydrolase, EC 3.1.1.4), which was maximal (10 mumol/min per mg of protein) at pH 9.5, depended on Ca2+, and was specific for cleavage of fatty acid from the C-2 position of the glycerol backbone of phosphatidylcholine. The noted enzymatic activity was augmented 4-fold by preincubating phosphatidylcholine-binding proteins with heat-aggregated murine IgG2b but not with IgG2a. IgG-binding proteins, on the other hand, are devoid of any detectable phospholipase A2 activity. Thus, the functional significance of Fc gamma 2b receptor of P388D1 macrophage cell line would be the generation of phospholipase A2 activity at the cell surface upon specific binding to Fc gamma 2b fragment.

Phospholipid methylation and phospholipase A2 activation in cytotoxicity by human natural killer cells.

The role of phospholipid methylation and phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) in natural killer (NK) function by human peripheral blood mononuclear cells was studied. Pretreatment of effector cells with a methyltransferase inhibitor, 3-deazaadenosine, in the presence of homocysteine thiolactone, reduced cytotoxicity in a dose-dependent fashion. This effect was closely associated with inhibition of methylation of lipids but not of nucleic acids or proteins. The suggestion for a role of phospholipid methylation was supported by the observation that the interaction between NK-susceptible tumor targets and peripheral blood mononuclear cells caused increased phospholipid methylation only when susceptible target cells were used. Phospholipase A2 was also implicated in human NK activity. Inhibitors of the enzyme such as tetracaine, mepacrine, Rosenthal's inhibitor, and corticosteroids impaired NK function. Rosenthal's inhibitor was also shown to exert an inhibitory effect on a purified NK-cell population obtained by the isolation of large granular lymphocytes on Percoll gradients. Peripheral blood mononuclear cells were also directly shown to display phospholipase A2-like activity, as measured by the decrease in radioactive arachidonate from prelabeled phospholipids, specifically phosphatidylcholine, in effector cells. These data suggest that enhanced phospholipid methylation occurs during the recognition function of NK cells. Consequent activation of phospholipase A2 might be involved in the mechanisms leading to lytic events within the target cell.

Localization and partial purification of a neutral-active phospholipase A 2 from BCG-induced rabbit alveolar macrophages

The localization and partial purification of a Ca 2+-dependent, membrane-associated phospholipase A 2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from BCG-induced rabbit alveolar macrophages is described. Phospholipase A activity was determined using autoclaved Escherichia coli, the phospholipids of which were labelled in the 2-acyl position with [1- 14C]oleate. Sonicated macrophages or granule preparations exhibited maximal phospholipase A 2, activity at pH 7.0, with 5 mM Ca 2+. Activity was quantitatively recovered in the pellet after centrifugation of homogenates at 100 000 × g, indicating that the enzyme is membrane-associated. At least two populations of macrophage granules were separated that contained phospholipase A 2 activity. Plasma membranes enriched 15-fold with respect to alkaline phosphodiesterase I were devoid of phospholipase activity. The enzyme was purified 1278-fold in a yield of 34%, was active over a broad pH range, and was extremely sensitive to low concentrations of Ca 2+, Mg 2+ and Mn 2+ would not substitute for Ca 2+, 1 mM EDTA completely inhibited enzymatic activity. Absolute specificity for the 2-position was demonstrated using 1-[1- 14C]stearyl-2-acyl 3-sn- glycerophosphorylethanolamine as substrate. Phospholipase A 2 activity was inhibited by the nonsteroidal anti-inflammatory agent indomethacin; the amount of drug required for 50% inhibition was 5·10 −4 M .

79] Isolation of phospholipase A 2 from red cell membranes of sheep : EC 3.1.1.4 Phosphatide 2-acylhydrolase

79] Isolation of phospholipase A 2 from red cell membranes of sheep : EC 3.1.1.4 Phosphatide 2-acylhydrolase

Mitochondrial calcium transport and calcium-activated phospholipase in porcine malignant hyperthermia

The interaction of Ca 2+ with mitochondria isolated from longissismus dorsi, a predominantly white skeletal muscle, of normal and malignant hyperthermia pigs was investigated using tightly-coupled preparations. Arrhenius plots of mitochondrial Ca 2+-stimulated respiration for succinate oxidation of malignant hyperthermia pigs showed a transition temperature ( T t) of 26.31 ± 0.80° C ( n = 5), which was decreased by spermine to 15.41 ± 0.69° C ( n = 3), a value very similar to that for normal pigs. No difference in either the T t or in the activation energy ( E a) was observed between the two types of pigs when ADP was used instead of Ca 2+. Mitochondria of malignant hyperthermia pigs were uncoupled at 40°C by exogenous Ca 2+ at 1221 ± 301 ( n = 9) nmol Ca 2+ per mg protein during succinate oxidation and the uncoupled mitochondria showed large amplitude swelling. Both the Ca 2+-induced uncoupling and swelling were prevented by bovine serum albumin and by the phospholipase inhibitors, spermine and tetracaine. In contrast, mitochondria of normal pigs were still tightly coupled even after a total addition of 2313 ± 287 ( n = 5) nmol Ca 2+ per mg protein and retained the original condensed configuration in the presence or absence of spermine and tetracaine. Mitochondria of malignant hyperthermia pigs contained significantly ( P < 0.001) higher quantities of endogenous Ca 2+ and showed a significantly ( P < 0.001) faster FCCP-induced endogenous Ca 2+ efflux rate than normal when monitored spectroscopically with murexide. No significant difference was observed in either the rate of exogenous Ca 2+ uptake or in the extent of Ca 2+ accumulated in the aerobic steady state during succinate oxidation between the two types of pigs. The rate of mitochondrial Ca 2+ efflux of malignant hyperthermia pigs during anaerobiosis was about twice that of normal. Experimental evidence suggests that mitochondria from musculi longissimus dorsi of malignant hyperthermia pigs contained a Ca 2+-stimulated phospholipase A 2 (EC 3.1.1.4, phosphatide 2-acylhydrolase), and that this enzyme if present in mitochondria of normal pigs is either latent or in very low concentration. The significance of the Ca 2+-stimulated phospholipase A 2 and its association with the enhanced rate of glycolysis in porcine malignant hyperthermia syndrome and in the post-mortem formation of the pale, soft and exudative conditions observed in white skeletal muscles of malignant hyperthermia pigs is discussed.

80] Phospholipase A 2 from bee venom : EC 3.1.1.4 Phosphatide acylhydrolase

80] Phospholipase A 2 from bee venom : EC 3.1.1.4 Phosphatide acylhydrolase

A phospholipase A2 inhibitory protein in rabbit neutrophils induced by glucocorticoids.

When rabbit peritoneal neutrophils were treated with glucocorticoids, their chemotactic response to stimulation by the chemoattractant fMet-Leu-Phe was markedly reduced. Preincubation of cells with glucocorticoids also decreased phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) activity in situ as measured by the release of [1-14C]arachidonic acid previously incorporated into phospholipids. The inhibitory potencies of glucocorticoids on phospholipase A2 activity correlated well with their anti-inflammatory activities and their abilities to bind to glucocorticoid receptors. Inhibitors of RNA and protein synthesis suppressed the inhibitory effect of glucocorticoids on phospholipase A2 activity. Digestion of the glucocorticoid-treated cells by Pronase overcame the inhibitory activity. Phospholipase A2 activity induced by Ca2+ ionophore A23187 was not affected by Pronase treatment. Gel filtration of proteins from neutrophil membranes labeled with [3H]lysine showed an induction of protein(s) (about 40,000 daltons) after glucocorticoid treatment. This protein inhibited a partially purified pancreatic phospholipase A2 and reduced the peptide-initiated chemotactic response of neutrophils.

Mitochondria and sarcoplasmic reticulum as model targets for neurotoxic and myotoxic phospholipases A2.

Certain neurotoxins and myotoxins from snake venoms have phospholipase A(2) activity (phosphatide 2-acylhydrolase, EC 3.1.1.4), which appears to be necessary for their toxicity. Several of these toxins inhibit the net uptake of Ca(2+) into sarcoplasmic reticulum vesicles and brain mitochondria. We have obtained evidence that the ability to inhibit this Ca(2+) uptake is a mechanistically relevant correlate of the toxicity of these proteins rather than being just a nonspecific consequence of their phospholipase A(2) activity. Two of the toxins, beta-bungarotoxin and notexin, had 5% and 50%, respectively, of the phospholipase A(2) activity of IVa phospholipase A(2)(a nontoxic enzyme), but beta-bungarotoxin was as effective as IVa in inhibiting Ca(2+) uptake into brain mitochondria and notexin was more effective. Each of the myotoxic enzymes substantially inhibited Ca(2+) uptake into sarcoplasmic reticulum, notexin being the most effective in this regard. This ability correlated better with their myotoxic potency than with their phospholipase A(2) activity. beta-Bungarotoxin lost its toxicity but not its measurable phospholipase A(2) activity after modification with ethoxyformic anhydride in the presence of dihexanoylphosphatidylcholine. The modified toxin also lost most of its ability to inhibit Ca(2+) uptake into sarcoplasmic reticulum and brain mitochondria. Sarcoplasmic reticulum vesicles reconstituted from solubilized sarcoplasmic reticulum retained their sensitivity to notexin.

Mepacrine blocks beta-adrenergic agonist-induced desensitization in astrocytoma cells.

C6 astrocytoma cells contain beta-adrenergic receptors coupled to adenylate cyclase. A 2-hr exposure to l-isoproterenol results in an 80% decrease in cyclic AMP production in response to a subsequent challenge by l-isoproterenol (desensitization). This loss in responsiveness is paralleled by a 20-30% decrease in the apparent number of beta-adrenergic receptors and by increased release of arachidonic aciid into the medium. The increased release of arachidonic acid is caused by the action of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) and corresponds to increased turnover of methylated phospholipids. Mepacrine and tetracaine, both inhibitors of this phospholipase A2, are able to block l-isoproterenol-induced desensitization of cyclic AMP production and the decrease in beta-adrenergic receptors. Mellitin and phorbol ester, two activators of phospholipase A2, when preincubated with the cells cause a decreased cyclic AMP response of the cells to l-isoproterenol. These results suggest that the activation of phospholipase A2 in the local domain of the beta-adrenergic receptor may be involved in desensitization.

Perfusion-dependent induction of de novo synthesis of renal phosphatide acyl hydrolase in ureter-obstructed rabbit kidney.

Perfusion of rabbit kidney ex vivo, removed 72 h after ureter obstruction, results in a dose-dependent increase in bioassayable prostaglandin Ez (PGE2). In addition, there are progressive increases in PGEz release with time and by 5 h of perfusion, there is a markedly enhanced PGEz release in response to bradykinin. Previous work has demonstrated that this increased basal and hormone-stimulated PGEz biosynthesis is in part dependent on de novo synthesis of cyclooxygenase. Measurement of arachidonic acid released into the venous effluent as a marker of renal phosphatide acyl hydrolase activity following bradykinin stimulation of the perfused kidneys showed dose-dependent release of arachidonic acid that was markedly enhanced by 5 h of perfusion. Kidneys removed from normal rabbits exhibited dose-dependent release of arachidonic acid in response to bradykinin with no enhancement by 5 h of perfusion. The ureter-obstructed kidney exhibited a time-dependent enhanced arachidonic acid release reflecting increase phosphatide acyl hydrolase activation, which was inhibitable by cycloheximide, thus suggesting that the enhanced arachidonate release was dependent on de novo synthesis of phosphatide acyl hydrolase. Thus, perfusion of the hydronephrotic kidney apparently results in the simultaneous induction of new protein synthesis of phosphatide acyl hydrolase, cyclooxygenase, and thromboxane synthetase. Earlier studies have demonstrated enhanced prostaglandin biosynthesis in response to peptide stimulation in the isolated perfused kidney of the rabbit perfused ex vivo 72 h after unilateral ureteral ligation in vivo (1). Furthermore, ureteral ligation unmasked the biosynthesis of thromboxane A2 (2,3). Inhibition of the endogenous cyclooxygenase (in vivo) with acetylsalicylic acid (aspirin), which irreversibly acetylates this regulatory enzyme in the cascade of prostaglandin biosynthesis (4), inhibits prostaglandin biosynthesis by 95% very early in perfusion (5). During the perfusions, however, there was a progressive increase in basal and bradykinin-stimulated prostaglandin biosynthesis after a 60- to 90-min delay which suggested that there was new cyclooxygenase being synthesized and further experiments confirmed that this was dependent on de novo synthesis of new enzyme (5). Inhibition * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. by aspirin does not affect the phosphatide acyl hydrolase responsible for the release of endogenous arachidonate. Thus, the above experiments did not exclude alterations in phosphatide acyl hydrolase activity. We therefore measured the release of endogenous arachidonic acid from peptide-stimulated perfused rabbit kidneys after 72-h ureter obstruction with the assumption that release of arachidonic acid is an indirect measure of phosphatide acyl hydrolase activity. MATERIALS AND METHODS

Serum stimulation of phospholipase A2 and prostaglandin release in 3T3 cells is associated with platelet-derived growth-promoting activity.

Sera from mouse, rat, and calf sources stimulate cellular phospholipase A2 activity (PLase; phosphatide 2-acylhydrolase, EC 3.1.1.4) and prostaglandin synthesis in 3T3 Swiss mouse fibroblasts, releasing up to 33% of biosynthetically incorporated [3H]arachidonic acid as hydrolysis products in 1 hr. The PLase stimulated by mouse serum exhibits specificity for arachidonic acid residues on phospholipids. It is stimulated 2.5-fold by 1.8 mM Ca2+ in the presence of 5 microM divalent cation ionophore A23187, consistent with a Ca2+-dependent enzyme possessing a cytoplasmic Ca2+ binding site. The percentage maximal PLase- and growth-stimulating activities of the three sera exhibit similar concentration dependencies, with the homologous (mouse) serum exhibiting the highest specific activities. Confluent 3T3 cells deplete PLase-stimulating activity from medium containing calf serum at a rate similar to the depletion of cell growth-stimulus activity. The PLase-stimulating activity in rat and mouse sera is derived from the leukocyte fraction of blood, presumably from platelets. In rat leukocyte lysates the PLase-stimulating activity exhibits properties similar to those reported for platelet-derived cell growth factors from rat and human sources--i.e., stability to exposure to 100 degrees C for 2 min or to pH 2 or pH 11, and cationic properties. Purified preparations of human platelet-derived growth factor also exhibit 3T3 PLase-stimulating activity.

Modulation of purified phospholipase A 2 activity from human platelets by calcium and indomethacin

A membrane bound phospholipase A 2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from human platelets has been purified 3500-fold, and partially characterized. Phospholipase A 2 activity was assayed using [1- 14C] oleate-labeled Escherichia coli or sonicated dispersions of synthetic phospholipids. The 2-acyl specificity of the phospholipase activity was confirmed using phosphatidylethanolamine labeled in the C-1 position as substrate. The purified enzyme was maximally active between pH 8.0 and 10.5, and had an absolute requirement for low concentrations of Ca 2+ Indomethacin, but not aspirin, inhibited phospholipase A 2 activity.

Presence of glycerophospholipid: cholesterol acyltransferase and phospholipase in culture supernatant of Aeromonas hydrophila.

Human erythrocyte membrane glycerophospholipids are deacylated by Aeromonas hydrophila 13-h culture supernatants, resulting in the production of cholesterol ester, free fatty acid, and water-soluble phosphates. This activity appears to be due to the actions of an acyltransferase (phosphatide:cholesterol acyltransferase, EC 2.3.1 group) and a phospholipase (phosphatide acyl-hydrolase). The enzyme activities are produced simultaneously in late exponential/early stationary phase, are precipitated together from the culture supernatant with 85% ammonium sulfate, and are eluted together near the void volume during gel filtration on Sepharose 6B. These results suggest that A. hydrophila produces a multienzyme complex with an unusual mode of action on membrane lipids. The complex is distinct from the hemolytic factor aerolysin, which is also produced by A. hydrophila.

Low concentrations of indomethacin inhibit phospholipase A2 of rabbit polymorphonuclear leukocytes.

Inhibition of prostaglandin synthesis by indomethacin, a drug with anti-inflammatory properties, has been attributed to its action on fatty acid cyclooxygenase. However, prostaglandin synthesis would also be inhibited if precursor fatty acids were not supplied. We find that indomethacin inhibits phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) of rabbit polymorphonuclear leukocytes in dose-dependent fashion. Inhibition is immediate and readily detected at 1 micrometer. The extent of inhibition is the same over a 10-fold range of substrate concentration and over a 500-fold range of enzyme purification. Inhibition is of the noncompetitive type, with an apparent Ki of 12 micrometer. Four other phospholipases A2--from venoms of Russell viper, Crotalus adamanteus, and bee, and from pig pancreas--are unaffected by 50 micrometer indomethacin, which inhibits leukocyte phospholipase A2 by 70%. This inhibition at low concentrations may well be important in the effects of the drug on protaglandin synthesis and inflammatory responses.

Cardiolipin: a stereospecifically spin-labeled analogue and its specific enzymic hydrolysis.

The spin-labeled cardiolipin 1-(3-sn-phosphatidyl)-3-[1-acyl-2-(16-doxylstearoyl)glycero(3)phosphol]-sn-glycerol has been prepared. The stereoselective synthesis makes use of the monolysocardiolipin 1-(3-sn-phosphatidyl)-3-[1-acyl-2-lyso-sn-glycero(3)phospho]-sn-glycerol, available from the stereospecific hydrolysis of cardiolipin by phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) of Trimeresurus flavoviridis. The results of treatment of the spin-labeled cardiolipin with the cardiolipin-specific phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) (Hemophilus parainfluenzae) of known specificity and with phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) of Bacillus cereus are consistent with the assigned structure. The spin-labeled cardiolipin is further characterized and the unique features of this diastereomer are discussed in the context of the unusual stereochemistry of the natural phospholipid.