Hydrolysis of lipid mixtures by rat hepatic lipase

The hydrolysis of phospholipid mixtures by purified rat hepatic lipase, also known as hepatic triglyceride lipase, was studied in a Triton X-100/lipid mixed micellar system. Column chromatography of the mixed micelles showed elution of Triton X-100 and binary lipid mixtures of phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine as a single peak. This indicated that the mixed micelles were homogenous and contained all components in the designated molar ratios. The molar ratio of Triton X-100 to lipid was kept constant at 4 to 1. Labeling one lipid with 3H and the other lipid with 14C enabled us to determine the hydrolysis of both components of these binary lipid mixed micelles. We found that the hydrolysis of phosphatidylcholine was activated by the inclusion of small amounts of phosphatidic acid (2.5-fold), phosphatidylethanolamine (1.5-fold) or phosphatidylserine (1.4-fold). The maximal activation of phosphatidylcholine hydrolysis was observed when 5 mol% of phosphatidylethanolamine, 7.5 mol% phosphatidic acid or 5 mol% phosphatidylserine was added to Triton X-100 mixed micelles. The hydrolysis of phosphatidic acid was activated 30%, and that of phosphatidylserine was inhibited 30% when the molar proportion of phosphatidylcholine was less than 50 mol%. The hydrolysis of phosphatidylethanolamine was slightly activated when the mol% of phosphatidylcholine was below 5. The hydrolysis of phosphatidylserine was inhibited by phosphatidylethanolamine when the mol% of the latter was 50 or less whereas phosphatidylethanolamine hydrolysis was not affected by phosphatidylserine. Under the conditions used sphingomyelin and cholesterol did not have a significant effect on the hydrolysis of the phospholipids studied. In agreement with our previous study (Kucera et al. (1988) J. Biol. Chem. 263, 1920-1928) these studies show that the phospholipid polar head group is an important factor which influences the action of hepatic lipase and that the interfacial properties of the substrate play a role in the expression of the activity of this enzyme. The molar ratios of phosphatidic acid, phosphatidylethanolamine and phosphatidylserine which activated phosphatidylcholine hydrolysis correspond closely to the molar ratios of these lipids found in the surface lipid film of lipoproteins e.g., high density lipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hydrolysis of thioester analogs by rat liver phospholipase A1

The hydrolysis of thioester containing phospholipids by rat liver plasmalemma phospholipase A1 was measured in a continuous spectrophotometric assay. In this assay thioester substrates were employed which, upon hydrolysis, liberated a free thiol which was reacted with 4,4'-dithiopyridine to yield the product 4-thiopyridone that absorbs at 324 nm. Thioester substrates, prepared by chemical synthesis, were used in phospholipid and Triton X-100 micelles for kinetic analysis carried out according to the method of Hendrickson and Dennis (Hendrickson, H.S., and Dennis, E.A. (1984) J. Biol. Chem. 259, 5734-5739). Vmax, Ks, and Km values obtained for various isomers and racemic mixtures of the synthetic thioester analogs are compared with corresponding oxyester substrates. Unnatural sn-1 isomers competitively inhibited the hydrolysis of natural sn-3 isomers of phosphatidylethanolamine and phosphatidic acid. Furthermore, the sn-1 isomer of phosphatidic acid was hydrolyzed by phospholipase A1, but with lower catalytic efficiency than the sn-3 isomer. The presence of a thioester at the sn-1 position did not change the Vmax significantly, as compared to the oxyester phospholipids. When two thioesters were present on the phospholipid molecule, the Vmax was decreased significantly. A convenient synthesis of 1-monothioester analogs of phospholipids is reported. The results presented show the usefulness of the spectrophotometric assay for measuring phospholipase A1 activity as well as the influence of racemic mixtures and thioesters on the hydrolytic rate.     

Kinetic analysis of the dual phospholipid model for phospholipase A2 action

A kinetic scheme is proposed for the action of cobra venom phospholipase A2 on mixed micelles of phospholipid and the nonionic detergent Triton X-100, based on the "dual phospholipid model." (formula; see text) The water-soluble enzyme binds initially to a phospholipid molecule in the micelle interface. This is followed by binding to additional phospholipid in the interface and then catalytic hydrolysis. A kinetic equation was derived for this process and tested under three experimental conditions: (i) the mole fraction of substrate held constant and the bulk substrate concentration varied; (ii) the bulk substrate concentration held constant and the Triton X-100 concentration varied (surface concentration of substrate varied); and (iii) the Triton X-100 concentration held constant and the bulk substrate concentration varied. The substrates used were chiral dithiol ester analogs of phosphatidylcholine (thio-PC) and phosphatidylethanolamine (thio-PE), and the reactions were followed by reaction of the liberated thiol with a colorimetric thiol reagent. The initial binding (Ks = k1/k-1) was apparently similar for thio-PC and thio-PE (between 0.1 and 0.2 mM) as were the apparent Michaelis constants (Km = (k-2 + k3)/k2) (about 0.1 mol fraction). The Vmax values for thio-PC and thio-PE were 440 and 89 mumol min-1 mg-1, respectively. The preference of cobra venom phospholipase A2 for PC over PE in Triton X-100 mixed micelles appears to be an effect on k3 (catalytic rate) rather than an effect on the apparent binding of phospholipid in either step of the reaction.     

Phospholipid and guanine nucleotides sensitive properties of the smooth muscle adenylate cyclase catalytic unit

The adenylate cyclase catalytic unit was partially purified from uterine smooth muscle by chromatography on columns of SM-2 Bio-Beads and Sepharose 6B. Stimulation of catalysis by forskolin was much greater in the presence of Mn2+ than in the presence of Mg2+. Neither NaF nor guanine nucleotide stimulated catalysis in the presence of Mg2+ or Mn2+. These properties indicated the catalytic unit was not sensitive to regulation by the GS regulatory protein. Guanine nucleotide inhibited catalysis, however, and was a competitive inhibitor of the ATP substrate (Ki approximately 50 microM). Since inhibition affected Km but not Vmax, the catalytic unit also seemed insensitive to regulation by the Gi regulatory protein, which does not act like a competitive inhibitor in other enzyme systems. The catalytic unit was also phospholipid sensitive. Only phosphatidic acid (Pho-A) had a direct effect on catalysis and was a potent inhibitor. Its effects were antagonized by the concomitant addition of phosphatidylcholine (Pho-C) but not by phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol. Acyl chain composition had a marked effect on Pho-C binding when this was determined by antagonism of Pho-A-dependent inhibition. These properties suggest the catalytic unit has both polar head group and acyl chain requirements for phospholipid binding.     

Specificity reversal in phospholipase A2 hydrolysis of lipid mixtures

The activity and specificity of phospholipase A₂ from cobra venom ($\text{Naja naja naja}$) toward binary mixtures of phosphatidylcholine and phosphatidylethanolamine in mixed micelles with the nonionic surfactant Triton X-100 were examined. In mixtures containing 5–50 mol % phosphatidylcholine, the rate for phosphatidylethanolamine hydrolysis was enhanced greatly over that for phosphatidylcholine. This is in marked contrast to previous studies with individual phospholipid species in mixed micelles where phosphatidylcholine was found to be the preferred substrate and phosphatidylethanolamine was found to be a very poor substrate. Possible explanations for this specificity reversal are considered.     

Solubilization, purification, and characterization of a membrane-bound phospholipase A2 from the P388D1 macrophage-like cell line

The release of free arachidonic acid from membrane phospholipids is believed to be the rate-controlling step in the production of the prostaglandins, leukotrienes, and related metabolites in inflammatory cells such as the macrophage. We have previously identified several different phospholipases in the macrophage-like cell line P388D1 potentially capable of controlling arachidonic acid release. Among them, a membrane-bound, alkaline pH optimum, Ca2+-dependent phospholipase A2 is of particular interest because of the likelihood that the regulatory enzyme has these properties. This phospholipase A2 has now been solubilized from the membrane fraction with octyl glucoside and partially purified. The first two steps in this purification are butanol extractions that yield a lyophilized, stable preparation of phospholipase A2 lacking other phospholipase activities. This phospholipase A2 shows considerably more activity when assayed in the presence of glycerol, regardless of whether the substrate, dipalmitoylphosphatidylcholine, is in the form of sonicated vesicles or mixed micelles with the nonionic surfactant Triton X-100. Glycerol (70%) increases both the Vmax and the Km with both substrate forms, giving a Vmax of about 15 nmol min-1 mg-1 and an apparent Km of about 60 microM for vesicles and a Vmax of about 100 nmol min-1 mg-1 and an apparent Km of about 1 mM for mixed micelles. Vmax/Km is slightly greater for vesicles than for mixed micelles. The lyophilized preparation of the enzyme is routinely purified about 60-fold and is suitable for evaluating phospholipase A2 inhibitors such as manoalide analogues. Subsequent steps in the purification are acetonitrile extraction followed by high performance liquid chromatography on an Aquapore BU-300 column and a Superose 12 column. This yields a 2500-fold purification of the membrane-bound phospholipase A2 with a 25% recovery and a specific activity of about 800 nmol min-1 mg-1 toward 100 microM dipalmitoylphosphatidylcholine in mixed micelles. When this material was subjected to analysis on a Superose 12 sizing column, the molecular mass of the active fraction was approximately 18,000 daltons.     

Kinetic analysis of the Ca2+-dependent, membrane-bound, macrophage phospholipase A2 and the effects of arachidonic acid

The kinetics of the Ca2+-dependent, alkaline pH optimum, membrane-bound phospholipase A2 from the P388D1 macrophage-like cell line were studied using various phosphatidylcholine (PC) and phosphatidylethanolamine (PE) substrates. This enzyme exhibits "surface dilution kinetics" toward PC in Triton X-100 mixed micelles, and the "dual phospholipid model" was found to adequately describe its kinetic behavior. With substrate in the form of sonicated vesicles, the dual phospholipid model should give rise to Michaelis-Menten type kinetics. However, the hydrolysis of dipalmitoyl-PC, 1-palmitoyl-2-oleoyl-PC, and 1-stearoyl-2-arachidonoyl-PC vesicles exhibited two distinct activities. Below 10 microM, the data appeared to follow Michaelis-Menten behavior, while at higher concentrations, the data could best be fit to a Hill equation with a Hill coefficient of 2. These PCs had Vmax values for the low substrate concentration range of 0.2-0.6 nmol min-1 mg-1 and Km values of 1-2 microM. At the high substrate concentration range, the Vmax values were between 5 and 7 nmol min-1 mg-1. PC containing unsaturated fatty acids had an apparent Km, determined from the Hill equation, of about 15 microM, while the apparent Km of dipalmitoyl-PC was 0.6 microM. When 70% glycerol was included in the assays, a single Michaelis-Menten curve was obtained for both dipalmitoyl-PC and 1-stearoyl,2-arachidonoyl-PC. Possible explanations for these kinetic results include reconstitution of the membrane-bound phospholipase A2 in the phospholipid vesicle or the enzyme has tow distinct phospholipid binding function. The kinetics for both dipalmitoyl-PC and dipalmitoyl-PE hydrolysis in vesicles was very similar, indicating that the enzyme does not greatly prefer one of these head groups over the other. The enzyme also showed no preference for arachidonoyl containing phospholipid. Enzymatic activity toward PC containing saturated fatty acids was linear to about 15% hydrolysis while the hydrolysis of PC containing unsaturated fatty acids was linear to only about 5%. This loss of linearity was due to inhibition by released unsaturated fatty acids. Arachidonic acid was found to be a competitive inhibitor of dipalmitoyl PC hydrolysis with a K1 of 5 microM. This tight binding suggests a possible in vivo regulatory role for arachidonic acid. Three compounds of the arachidonic acid cascade, prostaglandin F2 alpha, 6-keto-prostaglandin F1 alpha, and thromboxane B2, showed no inhibition of enzymatic activity.     

High cooperativity, specificity, and multiplicity in the protein kinase C-lipid interaction

The number of phosphatidylserine molecules involved in activating protein kinase C was determined in a mixed micelle system where one monomer of protein kinase C binds to one detergent:lipid micelle of fixed composition. Unusually high cooperativity, specificity, and multiplicity in the protein kinase C-phospholipid interaction are demonstrated by examining the lipid dependence of enzymatic activity. The rates of autophosphorylation and substrate (histone) phosphorylation are specifically regulated by the phosphatidylserine content of the micelles. Hill coefficients of 8-11 were calculated for phosphatidylserine-dependent stimulation of enzyme activity, with a maximum occurring in micelles containing greater than or equal to 12 phosphatidylserine molecules. The high specificity that exists is illustrated by the fact that phosphatidylethanolamine and phosphatidylglycerol, but not phosphatidylcholine or phosphatidic acid, can replace only some of the phosphatidylserine molecules. We propose that Ca2+ and acidic phospholipids cause the protein to undergo a conformation change revealing multiple phosphatidylserine binding sites and resulting in the highly cooperative and specific interaction of protein kinase C with phosphatidylserine. Consistent with this, the proteolytic sensitivity of protein kinase C increases approximately 10-fold in the presence of phosphatidylserine and Ca2+ compared to Ca2+ alone. The high degree of cooperativity and specificity may provide a sensitive method for the physiological regulation of protein kinase C by phospholipid.     

Phospholipase A1 acting on phosphatidic acid in porcine platelet membranes

1-[14C]Palmitoyl-2-[3H]arachidonoyl-sn-glycerol 3-phosphate was hydrolyzed to form [14C]palmitic acid and 2-[3H]arachidonoyl-glycerophosphate by porcine platelet membranes. This phospholipase A1 activity was relatively specific for phosphatidic acid; the addition of several other phospholipids in equimolar amounts did not have a significant effect on the hydrolysis of radiolabeled phosphatidic acid, and the specific activity for phosphatidic acid hydrolysis was 20-fold higher than that of the hydrolysis of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol under the conditions used. This phospholipase A1 acting on phosphatidic acid has properties different from those reported for other phospholipases and lipases present in platelets.     

Effect of surface composition on triolein hydrolysis in phospholipid vesicles and microemulsions by a purified acid lipase

Sonicated dispersions of egg yolk phosphatidylcholine and triolein as vesicles and microemulsions have been used as substrates for the assay of a purified acid lipase. Previous studies have also shown that triolein localized in the surface phase of emulsions is the preferred substrate. In this study, we examined enzyme activity following several surface modifications using both vesicles and microemulsions. When the acidic phospholipids phosphatidylserine and phosphatidic acid were incorporated into both vesicles and microemulsions at up to 10 mol % of the total phospholipid, a dose-dependent reduction in the apparent Km was observed. Using the vesicles as substrate, a dose-dependent decrease in Vmax was also observed. Agarose gel electrophoresis was used to verify suspected changes in net particle charge. Analogous inclusion of phosphatidylethanolamine, sphingomyelin, or cholesterol did not affect kinetic parameters. Addition of oleic acid to sonication mixtures produced vesicles with a decreased apparent Km and Vmax, but triolein hydrolysis in microemulsions was not significantly altered. Triolein-containing vesicles prepared by using dimyristoyl- or dipalmitoylphosphatidylcholine were hydrolyzed maximally at the gel liquid-crystalline transition temperatures of the appropriate phospholipid. Differential scanning calorimetry was used to verify the temperatures of transition in these vesicles. The results indicate that acid lipase activity is influenced by the charge or physical state of the surface phase of model substrates and suggest that degradation of core components of naturally occurring substrates such as lipoprotein may be influenced by chemical changes on the surface of these particles.     

Interfacial reaction dynamics and acyl-enzyme mechanism for lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters

The fatty acyl (lipid) p-nitrophenyl esters p-nitrophenyl caprylate, p-nitrophenyl laurate and p-nitrophenyl palmitate that are incorporated at a few mol % into mixed micelles with Triton X-100 are substrates for bovine milk lipoprotein lipase. When the concentration of components of the mixed micelles is approximately equal to or greater than the critical micelle concentration, time courses for lipoprotein lipase-catalyzed hydrolysis of the esters are described by the integrated form of the Michaelis-Menten equation. Least square fitting to the integrated equation therefore allows calculation of the interfacial kinetic parameters Km and Vmax from single runs. The computational methodology used to determine the interfacial kinetic parameters is described in this paper and is used to determine the intrinsic substrate fatty acyl specificity of lipoprotein lipase catalysis, which is reflected in the magnitude of kcat/Km and kcat. The results for interfacial lipoprotein lipase catalysis, along with previously determined kinetic parameters for the water-soluble esters p-nitrophenyl acetate and p-nitrophenyl butyrate, indicate that lipoprotein lipase has highest specificity for the substrates that have fatty acyl chains of intermediate length (i.e. p-nitrophenyl butyrate and p-nitrophenyl caprylate). The fatty acid products do not cause product inhibition during lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles. The effects of the nucleophiles hydroxylamine, hydrazine, and ethylenediamine on Km and Vmax for lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl laurate are consistent with trapping of a lauryl-lipoprotein lipase intermediate. This mechanism is confirmed by analysis of the product lauryl hydroxamate when hydroxylamine is the nucleophile. Hence, lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles occurs via an interfacial acyl-lipoprotein lipase mechanism that is rate-limited by hydrolysis of the acyl-enzyme intermediate.     

Phospholipase C-delta3 binds with high specificity to phosphatidylinositol 4,5-bisphosphate and phosphatidic acid in bilayer membranes.

In order to acquire an understanding of phospholipase C-delta3 (PLC-delta3) action on substrate localized in lipid membrane we have studied the binding of human recombinant PLC-delta3 to large, unilamellar phospholipid vesicles (LUVs). PLC-delta3 bound weakly to vesicles composed of phosphatidylcholine (PtdCho) or PtdCho plus phosphatidylethanolamine (PtdEtn) or phosphatidylinositol (PtdIns). The enzyme bound strongly to LUVs composed of PtdEtn + PtdCho and phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The binding affinity (molar partition coefficient) of PLC-delta3 to PtdEtn + PtdCho + PtdInsP2 vesicles was 7.7 x 105 m-1. High binding of PLC-delta3 was also observed for LUVs composed of phosphatidic acid (PA). Binding of PLC-delta3 to phosphatidylserine (PtdSer) vesicles was less efficient. Calculated molar partition coefficient for binding of PLC-delta3 to PA and PtdSer vesicles was 1.6 x 104 m-1 and 9.4 x 102 m-1, respectively. Presence of PA in the LUVs containing PtdInsP2 considerably enhanced the binding of PLC-delta3 to the phospholipid membrane. Binding of PLC-delta3 to phospholipid vesicles was not dependent on Ca2+ presence. In the liposome assay PA caused a concentration-dependent increase in activity of PLC-delta3. The stimulatory effect of PA on PLC-delta3 was calcium-dependent. At Ca2+ concentrations lower than 1 microm, no effect of PA on the activity of PLC-delta3 was observed. PA enhanced PLC-delta3 activity by increasing the Vmax and lowering Km for PtdInsP2. As the mol fraction of PA increased from 0-40 mol% the enzyme Vmax increased 2.3-fold and Km decreased threefold. Based on the results presented, we assume that PA supports binding of PLC-delta3 to lipid membranes by interaction with the PH domain of the enzyme. The stimulatory effect of PA depends on calcium-dependent interaction with the C2 domain of PLC-delta3. We propose that binding of PLC-delta3 to PA may serve as a mechanism for dynamic membrane association and modulation of PLC-delta3 activity.     

Membrane lipids have multiple effects on interfacial catalysis by a phosphatidic acid-preferring phospholipase A1 from bovine testis

We previously purified a cytosolic phospholipase A1 that could catalyze the preferential hydrolysis of phosphatidic acid in mixed-micelle assays. Here we studied the enzyme's interactions with unilamellar lipid membranes and examined effects of the lipids on enzyme binding, stability, and catalysis. A major finding was that membrane lipids could influence the stability, activity, and specificity of the enzyme under conditions where enzyme binding to the membranes was likely to be saturated. Thus, the enzyme was unstable at 37 degrees C in the absence of membranes but bound to membranes that contained anionic phosphoglycerides and could be stabilized by these membranes in the presence of albumin. The overall activity of the bound enzyme toward membrane phosphoglycerides, assayed in the presence of albumin, increased when phosphatidylethanolamine was substituted for phosphatidylcholine. Furthermore, the enzyme's catalytic preference for phosphatidic acid increased when cholesterol and diacylglycerol were included in the membranes, sn-1-stearoyl-2-arachidonoylphosphatidylethanolamine was substituted for sn-1-palmitoyl-2-oleoylphosphatidylethanolamine, and the concentration of phosphatidic acid was increased from 0 to 10 mol % of the total membrane phosphoglycerides. Finally, changes in the relative contents of phosphatidylcholine and phosphatidylserine in the membranes influenced the enzyme's catalytic preference for different molecular species of phosphatidic acid. These results provide the first available information about the enzyme's ability to interact with membranes and identify conditions that yield high enzyme activity toward membrane-associated phosphatidic acid.     

Analysis of the kinetics of phospholipid activation of cobra venom phospholipase A2

A kinetic analysis of the "dual phospholipid model" for cobra venom phospholipase A2 (Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5734-5739) was applied to the activation of phospholipase A2-catalyzed hydrolysis of a thiol ester analog of phosphatidylethanolamine (thio - PE) in Triton X - 100/phospholipid mixed micelles by various phosphorylcholine-containing activators. Activation of thio-PE hydrolysis by didecanoylphosphatidylcholine (PC) was found to be a function of the surface concentration of activator rather than bulk concentration. Its presence did not affect the initial binding of enzyme to phospholipid in the micelle surface as determined kinetically. After initial binding of enzyme to the surface, the activation appears to be due to enzyme-lipid binding in the surface. Activation does not appear to affect the affinity of the enzyme for phospholipid substrate, but rather affects the catalytic efficiency of the enzyme as characterized by the value of Vmax. The monomeric phospholipid dibutyryl-PC, when used as an activator at 57 mM (bulk concentration), also showed effects of surface dilution with Triton X-100, which would not be expected unless the lipid is incorporated into the micelles to some extent at these high concentrations. A thiol ester analog of phosphatidylcholine, thio-PC, was less effective than didecanoyl-PC as an activator, but appeared to be more effective than decylphosphorylcholine. A conformational change of the enzyme upon binding of the activator, after enzyme is bound to substrate at the interface, is discussed as a possible mechanism for this activation.     

Structural characterization of the polar lipids of Clostridium novyi NT. Further evidence for a novel anaerobic biosynthetic pathway to plasmalogens

A study of the polar lipids of Clostridium novyi NT has revealed the presence of phosphatidylethanolamine (PE) and cardiolipin as major phospholipids with smaller amounts of phosphatidylglycerol (PG), lysyl-PG and alanyl-PG. Other minor phospholipids included phosphatidic acid, CDP-diacylglycerol, phosphatidylserine (PS) and phosphatidylthreonine (PT). PE, PG and amino acyl PG were present in both the diacyl and alk-1'-enyl acyl (plasmalogen) forms and cardiolipin plasmalogens were found to contain one or two alk-1'-enyl chains. In contrast, the precursor lipids phosphatidic acid, CDP-diacylglycerol and PS were present almost exclusively as diacyl phospholipids. These findings are consistent with the hypothesis that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid formation in Clostridium species. This novel pathway contrasts with the route in animals in which a saturated ether bond is formed at an early stage of plasmalogen biosynthesis and the alk-1-enyl bond is formed by an aerobic mechanism.     

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.     

Epidermal growth factor-induced hydrolysis of phosphatidylcholine by phospholipase D and phospholipase C in human dermal fibroblasts

The enzymatic pathways for formation of 1,2-diradylglyceride in response to epidermal growth factor in human dermal fibroblasts have been investigated. 1,2-Diradylglyceride mass was elevated 2-fold within one minute of addition of EGF. Maximal accumulation (4-fold) occurred at 5 minutes. Since both diacyl and ether-linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether-linked diglycerides following stimulation of fibroblasts with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2-diradylglyceride was diacyl, the remaining 20% being ether-linked. Addition of EGF caused accumulation of 1,2-diacylglyceride without alteration in the level of ether-linked diglyceride. Thus, the observed induction of 1,2-diradylglyceride by EGF was due exclusively to increased formation of 1,2-diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2-fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF-induced phospholipase D activity, cells were labelled with exogenous [3H]1-0-hexadecyl, 2-acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in 15 seconds after addition of EGF and was maximal (3-fold) at 30 seconds. Consistent with the presence of EGF-induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D-catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2-diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2-diglyceride. Thus, the induction of 1,2-diacylglycerol by EGF may occur primarily via phospholipase C-catalyzed hydrolysis of phosphatidylcholine.     

Regulatory aspects of mitochondrial phospholipase A2: correlation of hydrolysis rates with substrate configuration as evidenced by 31P-NMR

The influence of variation of the phospholipid composition in model membranes composed of phosphatidylcholine and phosphatidylethanolamine on the hydrolysis of these phospholipids by rat liver mitochondrial phospholipase A2 was investigated. With the pure phospholipids, phosphatidylethanolamine was hydrolyzed over 30-times faster than phosphatidylcholine. Upon increasing the mole percentage of phosphatidylethanolamine in mixtures, a gradual, though non-linear, increase in the initial rate of hydrolysis of this phospholipid was observed. By contrast, phosphatidylcholine hydrolysis remained constant up to about 50 mol% phosphatidylethanolamine, whereafter a sudden fall-off of activity was observed. This drop in the hydrolysis rate coincided with a transition of the phospholipid structure from bilayer to an as yet unidentified organization characterized by an isotropic signal in the 31P-NMR spectra recorded in the presence of Ca2+. The occurrence of this phase was clearly dependent on Ca2+, since mixtures with identical composition in the absence of Ca2+ remained largely in bilayer configuration. That the structure adopted by phospholipids is of importance for their susceptibility to attack by this intracellular phospholipase A2 became evident also in studies with the single phospholipids in the absence or presence of Triton X-100 above the critical micellar concentration. While phosphatidylcholine hydrolysis was inhibited in mixed micelles as compared to its bilayer organization, the hydrolysis of phosphatidylethanolamine in mixed micelles was 3-fold that in the hexagonal HII phase.     

Effect of unsaturated fatty acids and Ca2+ on phosphatidylinositol synthesis and breakdown

CDP-diglyceride : inositol transferase was inhibited by unsaturated fatty acids. The inhibitory activity decreased in the following order: arachidonic acid greater than linolenic acid greater than linoleic acid greater than oleic acid greater than or equal to palmitoleic acid. Saturated fatty acids such as myristic acid, palmitic acid, and stearic acid had no effect. Calcium ion also inhibited the activity of CDP-diglyceride : inositol transferase. In rat hepatocytes, arachidonic acid inhibited 32P incorporation into phosphatidylinositol and phosphatidic acid without any significant effect on 32P incorporation into phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Ca2+ ionophore A23187 also inhibited 32P incorporation into phosphatidylinositol. However, 32P incorporation into phosphatidic acid was stimulated with Ca2+ ionophore A23187. Phosphatidylinositol-specific phospholipase C was activated by unsaturated fatty acids. Polyunsaturated fatty acids such as arachidonic acid and linolenic acid had a stronger effect than di- and monounsaturated fatty acids. Saturated fatty acids had no effect on the phospholipase C activity. The phospholipase C required Ca2+ for activity. Arachidonic acid and Ca2+ had synergistic effects. These results suggest the reciprocal regulation of phosphatidylinositol synthesis and breakdown by unsaturated fatty acids and Ca2+.     

Partial purification and characterization of phosphatidic acid-specific phospholipase A(1) in porcine platelet membranes

We have shown previously that the phospholipase A (PLA) activity specific for phosphatidic acid (PA) in porcine platelet membranes is of the A(1) type (PA-PLA(1)) [J. Biol. Chem. 259 (1984) 5083]. In the present study, the PA-PLA(1) was solubilized in Triton X-100 from membranes pre-treated with 1 M NaCl, and purified 280-fold from platelet homogenates by sequential chromatography on blue-Toyopearl, red-Toyopearl, DEAE-Toyopearl, green-agarose, brown-agarose, polylysine-agarose, palmitoyl-CoA-agarose and blue-5PW columns. In the presence of 0.1% Triton X-100 in the assay mixture, the partially purified enzyme hydrolyzed the acyl group from the sn-1 position of PA independently of Ca(2+) and was highly specific for PA; phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) were poor substrates. The enzyme exhibited lysophospholipase activity for l-acyl-lysoPA at 7% of the activity for PA hydrolysis but no lipase activity was observed for triacylglycerol (TG) and diacylglycerol (DG). At 0.025% Triton X-100, the enzyme exhibited the highest activity, and PA was the best substrate, but PE was also hydrolyzed substantially. The partially purified PA-PLA(1) in porcine platelet membranes was shown to be different from previously purified and cloned phospholipases and lipases by comparing the sensitivities to a reducing agent, a serine-esterase inhibitor, a PLA(2) inhibitor, a Ca(2+)-independent phospholipase A(2) inhibitor, and a DG lipase inhibitor.