DNA methylation and development.

(1) Isolated rat liver mitochondria were subjected to catalytic hydrogenation using a water-soluble Pd complex and molecular H2. This treatment resulted in a reduction of double bonds on phospholipid acyl chains as judged by gas chromatography of fatty acid methyl esters and HPLC of dinitrobenzoyldiacylglycerols. (2) After hydrogenation, mitochondria lost their ability to hydrolyze endogenous phospholipids in alkaline, Ca2+ containing medium, while phospholipase A2 retained full activity against exogenous substrates, regardless of whether those substrates were hydrogenated or not. (3) Inhibition by hydrogenation of endogenous phospholipid hydrolysis correlated with the loss of polyunsaturated fatty acyls, rather than with changes of the bulk membrane fluidity as measured by ESR and fluorescence studies. (4) These data suggest that the unsaturation of mitochondrial membrane lipids might be important for regulation of phospholipid breakdown by endogenous phospholipases. In particular, polyunsaturated molecular species seem to be involved in making phospholipids accessible to phospholipase A-mediated hydrolysis.     

Intramitochondrial phospholipase activity and the effects of Ca2+ plus N-ethylmaleimide on mitochondrial function.

Liver mitochondria treated with N-ethylmaleimide can accumulate Ca2+ but cannot retain it. Ca2+ loss following uptake occurs in parallel with a proton uptake and collapse of the membrane potential. Respiration is not activated during Ca2+ release and cannot be stimulated by uncoupler. After Ca2+ release and accompanying phenomena are nearly complete, the mitochondria undergo a large amplitude swelling. Nupercaine inhibits the premature release of Ca2+, proton uptake, decline in membrane potential, inhibition of uncoupler-stimulated respiration, and large amplitude swelling. Ruthenium red also prevents these effects. Neither Sr2+ or Mn2+ will substitute for Ca2+ to induce these effects in N-ethylmaleimide-treated mitochondria. The effects of N-ethylmaleimide plus Ca2+ on mitochondria are not accompanied by a significant alteration in the content or composition of phospholipids but are accompanied by small increases in the mitochondrial content of free fatty acids. Free fatty acids accumulate more rapidly in response to limited Ca2+ loading in the absence of N-ethylmaleimide than they do in its presence. In the absence of N-ethylmaleimide, polyunsaturated fatty acids and saturated plus monounsaturated fatty acids accumulate at nearly equal rates. In the presence of N-ethylmaleimide, polyunsaturated fatty acids accumulate more rapidly than saturated plus monounsaturated fatty acids. Any condition or agent tested which inhibited swelling and the other effects produced by Ca2+ plus N-ethylmaleimide also prevented the more rapid accumulation of polyunsaturated, compared to saturated plus monounsaturated, fatty acids. In the light of a positional analysis of phospholipid acyl moieties, these data suggest that 1-acyllysophospholipids accumulate in swelling mitochondria but not in response to noraml Ca2+ loading or when swelling is blocked by other agents. The free fatty acid accumulation, per se, is not responsible for swelling, but levels of exogenous palmitic acid as low as 1 nmol/mg of protein dramatically alter the dependence of swelling velocity on Ca2+ concentration, producing a shift from a sigmoidal- to a hyperbolic-like relationship. This same alteration is brought about by aging the mitochondrial preparation at 0 degrees C. Either pyruvate or DL-carnitine prevents the effect of exogenous palmitate and restores the Aa2+ swelling dependence of aged N-ethylmaleimide-treated mitochondria to that of fresh N-ethylmaleimide-treated mitochondria. Intramitochondrial acylcoenzyme A or acylcarnitine, or both, therefore, to be the modulator of Ca2+ sensitivity rather than free fatty acid. The findings are discussed in terms of the role of intramitochondrial phospholipase and other phospholipid metabolizing enzymes in the mechanisms of N-ethylmaleimide plus Ca2+ effects on mitochondria.     

Swelling of Phaseolus Mitochondria Induced by the Action of Phospholipase A

Phaseolus vulgaris mitochondria incubated in sucrose swell rapidly upon the addition of phospholipase A. Bovine serum albumin inhibits the swelling. The release of free fatty acids as a result of phospholipase A action on the mitochondria is detected only in the presence of bovine serum albumin, which promotes the hydrolysis of both mitochondrial phospholipids and purified lecithin. Either free fatty acid or lysolecithin is able to initiate an extensive mitochondrial swelling in sucrose. It is suggested that phospholipase A-induced swelling results from the release of lysophosphatides plus free fatty acids and their subsequent detergent action on the membranes rather than phospholipid loss per se.     

1-14C]oleate-labeled autoclaved yeast: a membranous substrate for measuring phospholipase A2 activity in vitro

Radiolabeled, autoclaved yeast were tested as a substrate for mammalian phospholipase A2 activity because the only other membranous substrate used for this purpose, autoclaved Escherichia coli, totally lacks a major mammalian phospholipid, phosphatidylcholine. Candida albicans were grown in the presence of [1-14C]oleate and then autoclaved. Sixty three percent of the incorporated label was in yeast phospholipid, and more than 95% of that was in the 2-acyl position. The distribution of label in the yeast phospholipids (phosphatidylcholine and -ethanolamine, -serine + -inositol, and phosphatidic acid corresponded closely to the chemical distribution of phosphorus in those phospholipids. Snake venom (Naja naja) and human synovial fluid phospholipase A2 hydrolyzed yeast phospholipid exclusively to release 14C-labeled fatty acid. When 50-60% of the yeast phospholipid was hydrolyzed, the radioactive fatty acids as determined by gas-liquid chromatographic analysis were predominantly oleate (45%) and linoleate (greater than 54%). Hydrolysis of yeast phospholipid by both enzymes was near-linear with protein and time under conditions of optimal pH (neutral-alkaline) and Ca2- (1-5 mM) previously reported for optimal hydrolysis of autoclaved E. coli phospholipid. N. naja phospholipase A2 showed less preference for phosphatidylethanolamine than -choline as liposomes or yeast phospholipid as compared to human synovial fluid phospholipase A2 which clearly preferred phosphatidylethanolamine to -choline as a liposome or yeast phospholipid. These results illustrate that radiolabeled phospholipids of autoclaved yeast, enriched in phosphatidylcholine, are readily hydrolyzed by snake venom and human nonpancreatic phospholipases A2 and may, therefore, be useful in the measurement of in vitro enzymatic activity.     

Phospholipases A1 and A2 of rat liver plasma membranes; mechanism of action.

While V/S plots of phospholipase A1 show a phase transition, kinetic behaviour of phospholipase A2 acting in the same concentration range is hyperbolic. However after phospholipase A2 has been solubilized from the plasma membranes by 1 M NaCl, the V/S curve shows a phase transition. Membrane-bound phospholipase A1 shows a narrow optimum pH at 8.5 -9, while phospholipase A2 activity presents only small variations between pH 7 and 9.5. Towards exogenous phospholipids at the optimum pH 8.5 of phospholipase A1, the specific activity of the latter is 3-fold higher than phospholipase A2 specific activity. On the contrary towards endogenous phospholipids, phospolipase A2 activity is higher than phospholipase A2 activity. Moreover labeled endogenous PE hydrolysis by phospholipase A2 is decreased by addition of non labeled exogenous PE into the incubation medium. All these data suggest that the active site of phospholipase A1 is turned to the outside and acts only on exogenous substrates: for phospholipase A2 it would be inside, and exogenous phospholipids could be hydrolyzed only after penetrating the membrane.     

Phospholipase activity in rat liver mitochondria studied by the use of endogenous substrates

The hydrolysis of endogenous phosphatidyl ethanolamine and lecithin in rat liver mitochondria has been studied by using mitochondria from rats injected with ethanolamine-1,2-(14)C or choline-1,2-(14)C. A phospholipase A-like enzyme has been demonstrated, which catalyzes the hydrolysis of one fatty acid ester linkage in phosphatidyl ethanolamine and lecithin. Phosphatidyl ethanolamine is hydrolyzed in preference to lecithin and the main reaction products are free fatty acids and lysophosphatidyl ethanolamine. The further breakdown of lysophospholipids appears to be limited in mitochondria, which indicates that lysophospholipase activity is mainly located extramitochondrially. The enzymic system is greatly stimulated by calcium ions, and also slightly by magnesium ions, while EDTA inhibits it almost completely. These findings are discussed in relation to previous observations on the effect of calcium and of EDTA on the functions of mitochondria. The possible function of the mitochondrial phospholipase for the formation of phospholipids with special fatty acids at the alpha- and -position is discussed.     

Changes in the phospholipid catabolism of mitochondria and microsomes during the development of rat liver.

The lipolytic activities of mitochondrial and microsomal fractions ('microsomes') isolated from foetal, suckling and adult rat liver were compared. The catabolism of endogenous phospholipids was followed by measuring the loss of phospholipids and the appearance of non-esterified fatty acids and lysophosphatides. The rate of mitochondrial phospholipid catabolism does not change significantly during development, but the rate of lipolysis of microsomal phospholipids increases 3-fold during development. Balance studies showed that, in mitochondria and microsomes of foetal, suckling and adult rat liver, fatty acid formation is greatly in excess of the fatty acids that can be accounted for by measuring phospholipid disappearance and lysophosphatide appearance. The hypothesis that this excess fatty acid formation resulted from the lipolysis of mitochondrial and microsomal triacylglycerols were tested and confirmed by preliminary experiments. Mitochondria and microsomes isolated from all developmental ages investigated had phospholipases with A1 and A2 activities. The degree of unsaturation of the fatty acids derived from the phospholipids of mitochondria did not vary significantly during development.     

Biosynthesis of mitochondrial phospholipids using endogenously generated diglycerides.

When isolated mitochondria or microsomes from rat liver were treated with phospholipase C, the incorporation of radioactive phospholipid precursors was markedly enhanced, presumably as a result of production of diglycerides by hydrolysis of endogenous phospholipids. Incorporation of CDP[14C]choline into lecithin in rat liver or BHK-21 mitochondria could be attributed to residual contamination from elements of the endoplasmic reticulum, with added diglycerides or with endogenous diglycerides produced by the phospholipase C treatment. A similar stimulation of [gamma32P]ATP incorporation into phospholipids was observed with exogenous or endogenous diglycerides, but the mitochondrial diglyceride kinase in either case was also related to the degree of microsomal contaminants. It was concluded that previous studies showing negligible capacity of mitochondria for lecithin biosynthesis de novo were not explainable on the basis of limited accessibility of added diglycerides, and that formation of phosphatidic acid by diglyceride kinase was not of significance in rat liver mitochondria.     

Lipids and lipolytic enzyme activities of rat heart mitochondria

The lipid composition and lipolytic enzyme activities in rat cardiac mitochondria were examined. Subsarcolemmal mitochondria were prepared by treatment of heart muscle with a Polytron tissue processor, while interfibrillar mitochondria were released by exposure of the remaining low-speed pellet to the protease, nagarse. These procedures are known to yield two functionally different populations of mitochondria. However, their phospholipid contents and compositions were identical, as were the positional distributions of the constituent fatty acids. Of the ethanolamine phospholipids, 20% were plasmalogens, and about 2% of the choline phospholipids consisted of this alkenylacyl species. Both subsarcolemmal and interfibrillar mitochondria contained a Ca²⁺-activated phospholipase A₂, as evidenced by the Ca²⁺-dependent release of unsaturated fatty acids and lysophosphatidylethanolamine from endogenous lipids. Ruthenium red prevented the activation of this enzyme by Ca²⁺, indicating that the activity is located in the matrix space or associated with the inner surface of the inner membrane. Both mitochondrial fractions produced free fatty acids and lysophosphatidylethanolamine in the absence of free Ca²⁺ apparently due to an outer membrane phospholipase A₁. The activity of this enzyme decreased with time, particularly in interfibrillar mitochondria, providing that Ca²⁺ was absent. Nagarse treatment of subsarcolemmal mitochondria resulted in a preparation with the same phospholipase A₁ properties as interfibrillar mitochondria. The possibility that differences in phospholipase A₁ properties account for some of the functional variations between the two mitochondrial types is discussed.     

Characterization of phospholipase activities in chromaffin granule ghosts isolated from the bovine adrenal medulla

Highly purified chromaffin granule membranes contain high levels (100 nmol/mg protein) of long-chain free fatty acids (Husebye, E.S. and Flatmark, T. (1984) J. Biol. Chem. 259, 15272-15276), as well as lysophosphatidylcholine (268 nmol/mg protein) and lysophosphatidylethanolamine (92 nmol/mg protein). The release of saturated and unsaturated long-chain fatty acids from endogenous phospholipids was 38 and 28 nmol/mg protein per h, respectively, at 37 degrees C and pH 7.5 (alkaline pH optimum). p-Bromophenacyl bromide inhibited the release of palmitate and oleate by 88 and 65%, respectively. The deacylation of membrane phospholipids was not significantly affected by micromolar free Ca2+. Based on experiments with pancreatic phospholipase A2, stearate and arachidonate were found to be suitable markers for deacylation at the sn-1 and sn-2 positions, respectively. Experiments with exogenously added labeled phosphatidylcholines confirmed that chromaffin granule ghosts contain a phospholipase A2 activity (alkaline pH optimum). The preparations also revealed a phospholipase A1 activity (acid pH optimum). Finally, the ghosts contain a lysophospholipase activity (alkaline pH optimum), that accounts for the major part of the deacylation of membrane phospholipids, notably the release of saturated fatty acids (stearate and palmitate). It is unlikely that the high content of lysophospholipids is an artifact of the procedure by which the granule ghosts are isolated.     

Pore formation and uncoupling initiate a Ca2+-independent degradation of mitochondrial phospholipids

Mitochondria contain a type IIA secretory phospholipase A(2) that has been thought to hydrolyze phospholipids following Ca(2+) accumulation and induction of the permeability transition. These enzymes normally require millimolar Ca(2+) for optimal activity; however, no dependence of the mitochondrial activity on Ca(2+) can be demonstrated upon equilibrating the matrix space with extramitochondrial Ca(2+) buffers. Ca(2+)-independent activity is seen following protonophore-mediated uncoupling, when uncoupling arises through alamethicin-mediated pore formation, or upon opening the permeability transition pore. Under the latter conditions, activity continues in the presence of excess EGTA but is somewhat enhanced by exogenous Ca(2+). The Ca(2+)-independent activity is best seen in media of high ionic strength and displays a broad pH optimum located between pH 8 and pH 8.5. It is strongly inhibited by bromoenol lactone but not by arachidonyl trifluoromethyl ketone, dithiothreitol, and other inhibitors of particular phospholipase A(2) classes. Immunoanalysis of mitochondria and mitochondrial subfractions shows that a membrane-bound protein is present that is recognized by antibody against an authentic iPLA(2) that was first found in P388D(1) cells. It is concluded that mitochondria contain a distinct Ca(2+)-independent phospholipase A(2) that is regulated by bioenergetic parameters. It is proposed that this enzyme, rather than the Ca(2+)-dependent type IIA phospholipase A(2), initiates the removal of poorly functioning mitochondria by processes involving autolysis.     

Relation of lipid structure of sarcotubular vesicles to Ca++ transport activity

The role of lipids of the sarcotubular membranes in their Ca(++) uptake and Mg-ATPase activities was investigated. Treatment of the membranes with phospholipase C inhibits both processes. Treatment with phospholipase A and phospholipase D, which results in massive hydrolysis of the sarcotubular phospholipids, does not inhibit either the Ca(++) uptake or the Mg-ATPase activities, nor does treatment with the polyene antibiotics affect these processes. Essential fatty acid deficiency alters sarcotubular membrane lipids; they contain much less stearic, linoleic, and arachidonic acids and much more oleic and eicosatrienoic acids than normally, but do not lose the ability to actively sequester Ca(++). It is concluded that neither nonpolar lipids nor the nonpolar regions of polar lipids are involved in Ca(++) sequestering and Mg-ATPase activities of the sarcotubular membranes. Of the polar components, the phosphoryl moiety of the phospholipids is required for both activities. However, the phosphoryl group appears to be required for the maintenance of the membranous structure necessary for Ca(++) sequestration rather than serving specifically in the active transport process. That treatment with phospholipase D, which results in the conversion of much of the sarcotubular phospholipid from a dipolar to an anionic structure, does not affect Ca(++) uptake activity is a most remarkable finding.     

Inactivation of mitochondrial 2-oxoglutarate dehydrogenase complex as a result of phospholipid degradation induced by freeze-thawing

The inactivation of 2-oxoglutarate dehydrogenase complex by freeze-thawing was examined along with alterations of membrane phospholipids, in order to elucidate the mechanism of freezing injury in mitochondria.The dehydrogenase complex activity in slowly frozen and thawed mitochondria decreased to 70% as compared to intact mitochondria and further decreased during incubation. This inactivation during incubation was temperature dependent, i.e., at temperatures up to 25°C there was a slight decrease, while at higher temperatures there was a marked decrease in the dehydrogenase complex activity. Simultaneously, there was a significant accumulation of free fatty acids, generated from mitochondrial phospholipids, which inhibited 2-oxoglutarate dehydrogenase and subsequently enzyme complex activity. Oxoglutarate dehydrogenase activity in mitochondria was markedly inhibited by exogenous phospholipase A, and this inhibition was partially prevented with bovine serum albumin. Furthermore, when intrinsic phospholipase A was either inhibited or stimulated, there was a respective decrease or increase in the enzyme complex inactivation.The activity of the purified enzyme complex decreased slightly after slow freezing, but remained constant even when incubated at temperatures up to 32°C. However, the activity of this enzyme complex was markedly reduced when incubated either in the presence of venom phospholipase A or with exogenous fatty acid.The relationship between inactivation of the 2-oxoglutarate dehydrogenase complex, phospholipase A activation and production of free fatty acids in frozen and thawed mitochondria is discussed.     

Inactivation of mitochondrial 2-oxoglutarate dehydrogenase complex as a result of phospholipid degradation induced by freeze-thawing.

The inactivation of 2-oxoglutarate dehydrogenase complex by freeze-thawing was examined along with alterations of membrane phospholipids, in order to elucidate the mechanism of freezing injury in mitochondria. The dehydrogenase complex activity in slowly frozen and thawed mitochondria decreased to 70% as compared to intact mitochondria and further decreased during incubation. This inactivation during incubation was temperature dependent, i.e., at temperatures up to 25 degrees C there was a slight decrease, while at higher temperatures there was a marked decrease in the dehydrogenase complex activity. Simultaneously, there was a significant accumulation of free fatty acids, generated from mitochondrial phospholipids, which inhibited 2-oxoglutarate dehydrogenase and subsequently enzyme complex activity. Oxoglutarate dehydrogenase activity in mitochondria was markedly inhibited by exogenous phospholipase A, and this inhibition was partially prevented with bovine serum albumin. Furthermore, when intrinsic phospholipase A was either inhibited or stimulated, there was a respective decrease or increase in the enzyme complex inactivation. The activity of the purified enzyme complex decreased slightly after slow freezing, but remained constant even when incubated at temperatures up to 32 degrees C. However, the activity of this enzyme complex was markedly reduced when incubated either in the presence of venom phospholipase A or with exogenous fatty acid. The relationship between inactivation of the 2-oxoglutarate dehydrogenase complex, phospholipase A activation and production of free fatty acids in frozen and thawed mitochondria is discussed.     

Regulatory aspects of mitochondrial phospholipase A2 from rat liver: effects of proteins, phospholipids and calcium ions

This paper deals with the search for specific inhibitors or activators of the mitochondrial phospholipase A2. Convincing evidence for the existence of proteins in the mitochondrial or cytosolic fraction that function as specific regulators of this enzyme was not obtained. The enzymatic activity appeared to be inhibited at low substrate concentrations by lipocortin isolated from human monocytes. However, at higher substrate concentrations, the inhibition disappeared, suggesting either that lipocortin sequestered the phospholipid substrate or that the putative inactive complex of enzyme and lipocortin dissociated in the presence of excess phospholipids. The hydrolysis of the neutral phospholipid phosphatidylethanolamine was stimulated by the presence of cardiolipin and phosphatidylglycerol. It is unlikely that this is caused merely by the negative charge of these phospholipids, since other negatively charged phospholipids did not show this effect. Using a phospholipid extract from mitochondria as substrate, the enzymatic activity as a function of the Ca2+ concentration was determined. Only one enzyme activity plateau was observed. The calculated KCa2+ value of 0.05 mM suggests that the mitochondrial phospholipase A2 could be regulated strictly by the modulation of the free Ca2+ concentration in vivo. The two activity plateaus observed previously upon variation of the Ca2+ concentration using phosphatidylethanolamine as substrate could be explained by a Ca2+-induced transition of the phospholipid structure.     

Effects of phospholipase A2 inhibitors on ruthenium red-induced Ca2+ release from mitochondria

The pharmacologic agents verapamil, nifedipine, diltiazem, prenylamine, N-oleoylethanolamine, R 24571, trifluoperazine, dibucaine, and quinacrine are examined as potential inhibitors of rat liver mitochondrial phospholipase A2 acting on endogenous phospholipid. Their potency as inhibitors of the enzyme is compared to their activities as inhibitors of phospholipase A2-dependent swelling and ruthenium red-induced Ca2+ release in intact mitochondria. For verapamil, diltiazem, trifluoperazine, dibucaine, and quinacrine, there is complete agreement between the relative potencies as inhibitors of phospholipase A2 and the two other processes. Nifedipine and prenylamine, which are weak inhibitors of phospholipase A2, produce a permeable inner membrane, provided that the mitochondrial have accumulated Ca2+. R 24571, which strongly inhibits the enzyme, disrupts mitochondria by a Ca2+-independent mechanism. N-Oleoylethanolamine, which is an effective inhibitor of swelling, does not inhibit phospholipase A2 or ruthenium red-induced Ca2+ release. The results support a proposed scheme wherein ruthenium red-induced Ca2+ release is viewed as reverse activity of the Ca2+-uptake uniporter occurring subsequent to decline in the proton motive force. The latter effect is proposed to arise from a specific phospholipase A2-dependent increase in inner-membrane H+ conductance of mitochondrial subpopulations. It is further shown that mitochondrial membranes display cyclic oscillations in free fatty acid content which are not dependent on the presence of Ca2+ or on the capacity to generate acylcoenzyme A.     

Fluidity and osmotic sensitivity changes of phospholipase A2-treated liposomes

Membrane fluidity and osmotic sensitivity were examined in DPPC liposomes treated with phospholipase A2 (PL.A2) in the presence of Ca2+ or Mg2+. The amount of liposome phospholipid hydrolyzed differed with the two ions. Embedded DPH, a rod-like fluorescent probe, was employed in the determination of membrane fluidity. Membrane fluidity decreased according to the degree of phospholipid hydrolization in liposomes by PL.A2. The reciprocal value of absorption at 450 nm was measured as the index of osmotic sensitivity of liposomes. Intact sonicated liposomes showed osmotic insensitivity. PL.A2-treated liposomes in which about 40% of total phospholipid was hydrolyzed showed osmotic sensitivity. No change in the membrane fluidity was obtained when PL.A2-treated liposomes were exposed to hypertonic or hypotonic solution. These results suggested that the motion of the acyl-chain of phospholipids and free fatty acids was resisted in PL.A2-treated liposomes. The resistance may be due to a phase separation between phospholipids and free fatty acids. The pore for water permeation might be induced in the border between phase-separated domains in PL.A2-treated liposomes.     

Action of Phospholipase A2 and Phospholipase C on Bacillus subtilis Protoplasts

Protoplasts prepared from Bacillus subtilis by lysozyme digestion lysed in the presence of pure pancreatic phospholipase A(2). The phospholipids cardiolipin, phosphatidylethanolamine, phosphatidylglycerol and lysylphosphatidylglycerol, which are present in the membrane, are degraded by phospholipase A(2) only after removal of the cell wall, giving free fatty acids and lyso derivatives. The four phospholipids are hydrolyzed equally well at a given enzyme concentration. Differences in the phospholipid composition of the protoplasts were obtained by variations in the growth medium, time of harvesting, and preincubation time with lysozyme. The extent of hydrolysis appeared to depend on the initial phospholipid composition. A relative increase in acidic phospholipids in the membrane facilitated the action of phospholipase A(2), whereas the rate of hydrolysis was diminished when protoplasts were tested which contained a relatively high amount of positively charged phospholipid. Pure phospholipase C from B. cereus preferentially hydrolyzed phosphatidyl-ethanolamine in the B. subtilis membrane. More than 80% of this phospholipid was converted into diglyceride, whereas only 30% of the cardiolipin was hydrolyzed. Such a loss of phospholipids, however, was not followed by lysis of the protoplasts. Liposomes were prepared from the lipid extracts of B. subtilis and incubated with both phospholipases. The hydrolysis pattern of the phospholipids in these model membrane systems was identical to the hydrolysis pattern of the phospholipids in the protoplast membrane. Phospholipase A(2) hydrolyzed all the phospholipids in the liposomes equally well, whereas phospholipase C preferentially degraded phosphatidylethanolamine.     

Hydrolysis of Endogenous Phospholipids by Rat Brain Microsomes

Phosphatidylcholine of rat brain microsomes was labeled in vivo by intracerebral injection of either [3H]oleic acid or [methyl-3H]choline chloride. These labeled microsomes served both as the enzyme source as well as a source of endogenously labeled substrate. Phospholipase D (PLD) activity was detected with these particles only in the presence of exogenous oleate, its activator. Ca2+ and the ionophore A 23187 inhibit PLD activity of oleate-labeled microsomes. In oleate-labeled particles, besides phosphatidic acid the product of PLD action radioactivity was also detected in diglyceride as a result of resident phosphatidate phosphohydrolase, which hydrolyzed the phosphatidic acid. The phosphatidate phosphohydrolase could not be completely inhibited by KF and propranolol. The release of endogenous fatty acids from labeled phospholipid by a mellitin-stimulated phospholipase A2 also present in these particulates produced minimal stimulation of endogenous PLD. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are hydrolyzed by 50% in the presence of mellitin and 90% of the radioactivity was found in the lyso-compounds. Mellitin and oleate together reduced the radioactivity found in lyso-PC and increased that in lyso-PE.     

Yeast mitochondria (Saccharomyces cerevisiae) contain Ca(2+)-independent phospholipase A1 and A2 activities: effect of respiratory state

We demonstrate that both phospholipase A1 and phospholipase A2 are associated with isolated yeast mitochondria (Saccharomyces cerevisiae). Activity assays indicate that, unlike most other mitochondrial phospholipases A, the yeast enzymes are Ca(2+)-independent with acidic (pH 4-5) as well as alkaline (pH 8-9) pH optima. Data obtained with mitochondria isolated from either fermenting or respiring cells, and initial observations with a petite strain, strongly suggest that a phospholipase A2 with an acidic pH optimum functions in the in vivo adaptation and maintenance of mitochondrial membranes required for respiration.