Hydrolysis of 2-acyl-sn-glycero-3-phosphocholines in guinea pig heart mitochondria

Although both 2-acyl-sn-glycero-3-phosphocholine and 1-acyl-sn-glycero-3-phosphocholine may be produced from phosphatidylcholine hydrolysis, studies on the former have lagged behind that of the latter. In this study a lysophospholipase A2 that hydrolyses 2-acyl-sn-glycero-3-phosphocholine has been characterized in guinea pig heart mitochondria. The lysophospholipase A2 activity was not dependent on Ca2+ and was inhibited differentially by saturated and unsaturated fatty acids. This lysophospholipase A2 activity was able to discriminate among different molecular species of 2-acyl-sn-glycero-3-phosphocholines when they were presented individually or in pairs. The order of decreasing rates of hydrolysis of different molecular species of 2-lysophosphatidylcholines, when the substrates were presented singly, was 18:2 greater than 20:4 greater than 18:1 greater than 16:0. A differential inhibition of the rate of hydrolysis of the individual substrates was observed when the substrates were presented in pairs. The degree of inhibition was dependent on the molar ratio of the mixed substrates. The characteristics of the enzyme suggest that involvement in the selective release of fatty acids from mitochondrial phosphatidylcholine would depend on a high selectivity of phospholipase A1 for different molecular species of phosphatidylcholine. A lysophospholipase A1 activity was also characterized in the mitochondria with a distinct acyl specificity from the lysophospholipase A2. Other characteristics of the two lysophospholipases suggest that the two reactions are not catalysed by the same enzyme.     

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.     

Substrate specificity and positional preference of a lipoprotein lipase

The substrate specificity and positional preference of a membrane-bound lipoprotein lipase with and without reconstitution in liposomes were studied. The enzyme showed the same preference for the acyl groups in the 1- and 3-positions of triglycerides, while its activity toward the 2-position was one-third of that toward the 1- or 3-positions. The substrate specificity of the enzyme toward fatty acids of different chain length and degree of unsaturation was in the order of C:14>C:16>C:18:1≥C:18. The enzyme with and without reconstitution showed the same positional preference and substrate specificity.     

Substrate specificity of lysophospholipase D which produces bioactive lysophosphatidic acids in rat plasma

Previously we reported that lysophospholipase D in rat plasma hydrolyzes endogenous unsaturated lysophosphatidylcholines (LPCs) preferentially to saturated LPCs to lysophosphatidic acids with growth factor-like and hormone-like activities. In this study, we examined the possibility that association of LPCs with different proteins in rat plasma has an effect on the preference of lysophospholipase D for unsaturated LPCs. Large portions of various LPCs were found to be recovered in the lipoprotein-poor bottom fraction. Furthermore, the percentages of LPCs associated with albumin isolated from rat plasma were shown not to be consistent with their percentage conversions to lysophosphatidic acids by lysophospholipase D on incubation of rat plasma at 37 degrees C. These results indicate that distinct distributions of LPCs in the plasma protein fractions are not critical factors for the substrate specificity of lysophospholipase D. Experiments with Nagase analbuminemic rats suggested that albumin-LPC complexes are not necessarily required for the hydrolysis by lysophospholipase D; lipoprotein-associate LPCs appeared to be good substrates for the phospholipase. We found that both saturated and unsaturated LPCs are present mainly as 1-acyl isomers in rat plasma. This result indicates that the preference of lysophospholipase D for unsaturated LPCs is not attributable to a difference in position of the acyl group attached to the glycerol backbone of LPC. In addition, lysophospholipase D was also found to attack choline phospholipids with a long chain group and a short chain alkyl group, although their percentage hydrolyses were low. Taken altogether, these results suggest that lysophospholipase D shows higher affinities for free forms of unsaturated acyl type LPCs equilibrated with albumin-bound and lipoprotein-associated forms, than for free forms of saturated acyl type LPCs and analogs of platelet-activating factor.     

Effects of substrate fatty acids on products of lecithin hydrolysis and acyl-CoA-independent transacylation with cholesterol by aortic enzyme preparations

The acyl composition of substrates and products of enzymatic hydrolysis and transacylation of lecithin with cholesterol in the arterial wall was investigated. Saturated acyl residues predominated in lysolecithin and unsaturated ones in acids released by hydrolysis of egg lecithin. In the reaction system with cholesterol, saturated acyls predominated in both lysolecithin and acids released whereas unsaturated ones were more abundant in newly formed acylcholesterols. Mainly unsaturated acyls were present in the hydrolysis products from soybean lecithin in the reaction systems with and without cholesterol. For acylcholesterols formed in the presence of either lecithin, the percent values are in the numerical order of C18:2 greater than C18:1 greater than C16:0 greater than or equal to C18:0. It It is concluded that acyl preferences and interactions in the enzyme-catalyzed reactions studied may contribute to the different accumulation and removal of the compounds involved from the artery.     

On the Status of Lysolecithin in Rat Cerebral Cortex During Ischemia

Abstract: Lysolecithin (lysoglycerophosphocholine, LPC) was isolated from rat cerebral cortex and quantitatively analyzed at various times after postdecapitative ischemic treatment. In addition, different procedures for extraction and analysis of the LPC in brain were evaluated. Results indicated that LPC can be quantitatively extracted into the organic phase using the conventional extraction procedure with chloroform-methanol (2:1, vol/ vol). However, care should be taken to avoid using strong acids, which can hydrolyze the alkenylether side chain of the plasmalogens, resulting in the release of 2-acyl-phospholipids. Quantitative GLC analysis using myris-toyl-LPC as internal standard revealed a level of 1.8 nmol LPC/mg protein in brain with acyl groups comprised mainly of 16:0, 18:0, and 18:1. The acyl group profile reflects that the LPC are derived mainly from phospho-lipase A₂ action. An increase of 46% in the LPC level was observed at 1 min after ischemic treatment, but this was followed by a steady decline. Ischemia induced an increase in the LPC species that are enriched in 18:0 and 18:1 fatty acids. The transient appearance of LPC during ischemia further suggests that this phospholipid is undergoing active turnover, possibly hydrolysis by the lysophospholipase. This mechanism of action may account, at least in part, for the increase in both saturated and unsaturated fatty acids during the early phase of the ischemic treatment.     

Partial purification of a lipolytic enzyme from Escherichia coli.

An enzyme with phospholipase Al activity was purified some 500-fold from Escherichia coli cell homogenates. Lipase, phospholipase A2, and lysophospholipase copurified with phospholipase A1 and the four activities displayed similar susceptibility to heat treatment. The phospholipase A and lipase activities were recovered in a single band when partially purified preparations were subjected to SDS gel electrophoresis. Phospholipase, lysophospholipase, and lipase all required Ca2+ for activity. Phosphatidylcholine, phosphatidylethanolamine, and their lyso analogues were all hydrolysed at equivalent rates and these were substantially greater than the rate of methylpalmitate or tripalmitoylglycerol hydrolyses under similar incubation conditions. Evidence for a direct but slow hydrolysis of the ester at position 2 of phosphoglyceride was obtained; however, release of fatty acid from this position is mostly indirect involving acyl migration to position 1 and subsequent release of the translocated fatty acid. Escherichia coli, therefore, appears to possess a lipolytic enzyme of broad substrate specificity acting mainly at position 1 but also at position 2 of phosphoglycerides and on triacylglycerols and methyl fatty-acid esters.     

Role of sphingomyelin and ceramide in the regulation of the activity and fatty acid specificity of group V secretory phospholipase A2

We previously showed that group V secretory phospholipase A(2) (sPLA(2)V) is inhibited by sphingomyelin (SM), but activated by ceramide. Here, we investigated the effect of sphingolipid structure on the activity and acyl specificity of sPLA(2)V. Degradation of HDL SM to ceramide, but not to ceramide phosphate, stimulated the activity by 6-fold, with the release of all unsaturated fatty acids being affected equally. Ceramide-enrichment of HDL similarly stimulated the release of unsaturated fatty acids. Incorporation of SM into phosphatidylcholine (PC) liposomes preferentially inhibited the hydrolysis of 16:0-20:4 PC. Conversely, SMase C treatment or ceramide incorporation resulted in preferential stimulation of hydrolysis of 16:0-20:4 PC. The presence of a long chain acyl group in ceramide was essential for the activation, and long chain diacylglycerols were also effective. However, ceramide phosphate was inhibitory. These studies show that SM and ceramide in the membranes and lipoproteins not only regulate the activity of phospholipases, but also the release of arachidonate, the precursor of eicosanoids.     

Isolation and characterization of three lysophospholipases from the murine macrophage cell line WEHI 265.1.

Anion exchange chromatography of WEHI 265.1 cell homogenates resolved the lysophospholipase activity into three peaks, when assayed using lysophosphatidylcholine as a substrate. Peaks 1 and 2 were purified by sequential hydrophobic interaction and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peaks 1 and 2 indicated homogeneous proteins with apparent masses of 28 and 27 kDa, respectively. Peak 3 lysophospholipases was partially purified by hydrophobic, hydroxyapatite and gel filtration chromatography. Peak 3 lysophospholipase also had calcium-dependent phospholipase A2 activity, which further co-purified with the lysophospholipase activity. The three lysophospholipases were characterized with respect to substrate specificity, additional enzymatic activities and the effects of lipids, metal ions and other compounds on enzymatic activity. Peaks 1, 2 and 3 hydrolyzed lysophosphatidylcholine most readily, but lysophosphatidylethanolamine also served as substrate for each enzyme. Furthermore, all three enzymes hydrolyzed platelet activating factor and acetylated lysophosphatidylcholine. Each lysophospholipase was inhibited by free fatty acids and by palmitoyl carnitine, although the relative sensitivities to these agents differed among the enzymes. The lysophospholipase activities of peaks 1 and 2, but not peak 3, were inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate and N-ethylmaleimide. Although they had similar masses, the amino acid compositions of peaks 1 and 2 differed, indicating that these are distinct proteins rather than posttranslational modifications of the same gene product.     

Kinetic properties of a high molecular mass arachidonoyl-hydrolyzing phospholipase A2 that exhibits lysophospholipase activity.

The first step in the production of eicosanoids and platelet-activating factor is the hydrolysis of arachidonic acid from membrane phospholipid by phospholipase A2. We previously purified from the macrophage cell line RAW 264.7 an intracellular phospholipase A2 that preferentially hydrolyzes sn-2-arachidonic acid. The enzyme exhibits a molecular mass of 100 kDa and an isoelectric point of 5.6. When assayed for other activities, the phospholipase A2 was found to exhibit lysophospholipase activity against palmitoyllysoglycerophosphocholine, and both activities copurified to a single band on silver-stained sodium dodecyl sulfate-polyacrylamide gels. An antibody against the macrophage enzyme was found to quantitatively immunoprecipitate both phospholipase A2 and lysophospholipase activities from a crude cytosolic fraction. When the immunoprecipitated material was analyzed on immunoblots, a single band at 100 kDa was evident, further suggesting that a single protein possessed both enzyme activities. When assayed as a function of palmitoyllysoglycerophosphocholine concentration and plotted as a double-reciprocal plot, two different slopes were apparent, corresponding to concentrations above and below the critical micellar concentration (7 microM) of the substrate. Above the critical micellar concentration, lysophospholipase exhibited an apparent Km of 25 microM and a Vmax of 1.5 mumol/min/mg. Calcium was not required for lysophospholipase activity, in contrast to phospholipase A2 activity. The enzyme, when assayed as either a phospholipase A2 or lysophospholipase, exhibited nonlinear kinetics beyond 1-2 min despite low substrate conversion. Readdition to more substrate after the activity plateaued did not result in further enzyme activity, ruling out substrate depletion. Readdition of enzyme, however, resulted in another burst of enzyme activity. The results are not consistent with product inhibition, but suggest that the enzyme may be subject to inactivation during catalysis.     

Acid lipase and carboxylesterases in EBV-transformed lymphoid cell line from Wolman's disease: influence of fatty acid structure of substrate

Lymphoid cell lines established by Epstein-Barr virus transformation of blood B lymphocytes from a patient with Wolman's disease exhibited the acid lipase deficiency characteristic for this disease. Comparison of hydrolysis by normal and Wolman's cells of 4-methylumbelliferyl-acyl esters with variable chain length demonstrates that: (1) the best substrates for acid lipase were characterized by an acyl chain length of 12-18 carbon atoms; (2) the acid residual activity in Wolman's cells showed a slightly different substrate specificity and this is probably due to an acid carboxylesterase different from the lysosomal acid lipase, and (3) the 'nonspecific' carboxylesterases (at pH 6.0 and 8.0) not inhibited by taurocholate showed a characteristic substrate specificity for short-chain fatty acids. In the used assay conditions (optimal for acid lipase), methylumbelliferyl-palmitate, -elaidate and -lignocerate are the most accurate synthetic substrates for the diagnostic of Wolman's disease.     

Purification and characterization of lysophospholipase L2 of Escherichia coli K-12

Lysophospholipase L2, which is bound to the inner membrane of Escherichia coli K-12, was produced in a large amount in cells bearing its cloned structural gene. Starting from these cells, the lysophospholipase L2 was purified approximately 700-fold to near homogeneity by solubilization with KCl, ammonium sulfate fractionation, chromatofocusing in the presence of a zwitterionic detergent, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and heparin-Sepharose affinity column chromatography. The final preparation showed a single protein band with a molecular weight of 38,500 daltons in SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the NH2-terminal portion of the purified enzyme was determined. It was in complete agreement with that deduced from the nucleotide sequence of the structural gene, pldB [Kobayashi, T., Kudo, I., Karasawa, K., Mizushima, H., Inoue, K., & Nojima, S. (1985) J. Biochem. 98, 1017-1025.] The purified enzyme hydrolyzes 2-acyl glycerophosphoethanolamine (GPE) and 2-acyl glycerophosphocholine (GPC) more effectively than 1-acyl GPE and 1-acyl GPC, but does not attack diacylphospholipids. The enzyme also catalyzes the transfer of an acyl group from lysophospholipid to phosphatidylglycerol for formation of acyl phosphatidylglycerol. The acyl group was more effectively transferred from 2-acyl lysophospholipid than from the 1-acyl derivative. This enzyme was heat-labile and was inactivated at 55 degrees C within 5 min. The present paper shows clearly that lysophospholipase L2 is a different enzyme protein from lysophospholipase L1 which was formerly purified from the supernatant of the wild strain of E. coli K-12 homogenates [Doi, O. & Nojima, S. (1975) J. Biol. Chem. 250, 5208-5214].     

Asymmetric short-chain phosphatidylcholines: defining chain binding constraints in phospholipases

Several short-chain asymmetric lecithins with a total of 14 carbons in the acyl chains (ranging from 1-lauroyl-2-acetylphosphatidylcholine to 1-hexanoyl-2-octanoylphosphatidylcholine) have been synthesized and characterized. The specific activities of phospholipase A2 from cobra venom, phospholipase A2 from porcine pancreas, and phospholipase C from Bacillus cereus toward these lecithins as micelles have been determined. The results of these kinetic studies allow the definition of hydrophobic binding requirements in the active sites of these water-soluble phospholipases. For phospholipase C, with the exception of monomyristoylphosphatidylcholine, each of the asymmetric short-chain lecithins exhibits high activity, comparable to the 14-carbon symmetric short-chain species, diheptanoylphosphatidylcholine. Therefore, for phospholipase C, in addition to the acyl linkages, only a certain degree of hydrophobicity in the fatty acyl chains is requisite for substrate binding and appreciable hydrolysis; there is no chain specificity. The activity of phospholipase A2 from cobra venom toward the same asymmetric lecithins is quite different. As the sn-2 chain lengthens, activity is increased to a maximum for diheptanoyl-PC. Further increase in the number of carbons in the sn-2 chain has no effect on hydrolysis rates. For this enzyme, seven carbons in the sn-2 chain are necessary for optimal activity. In contrast, porcine pancreatic phospholipase A2 activity shows very little dependence on sn-2 chain length.     

Interaction of phosphate analogues with glyceraldehyde-3-phosphate dehydrogenase.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate. A variety of phosphonates have been shown to substitute for phosphate in this reaction [Gardner, J. H., & Byers, L. D., (1977) J. Biol. Chem. 252, 5925--5927]. The dependence of the logarithm of the equilibrium constant for the reaction on the pKa2 value of the phosphonate is characterized by a Br?nsted coefficient, betaeq, of approximately 1. This represents the sensitivity of the transfer of the phosphoglyceroyl group between the active-site sulfhydryl residue (in the acyl-enzyme intermediate) and the acyl acceptor on the basicity of the acyl acceptor. Molybdate (MoO42-) can also serve as an acyl acceptor in the glyceraldehyde-3-phosphate dehydrogenase catalyzed reaction. The second-order rate constant for the reaction with molybdate is only approximately 12 times lower than the reaction with phosphate even though the pKa2 of molybdate is 3.1 units lower than the pKa2 of phosphate. The immediate product of the molybdate reaction is the acyl molybdate, 1-molybdo-3-phosphoglycerate. The acyl molybdate, like the acyl arsenate (the immediate product of the reaction when arsenate is the acyl acceptor), is kinetically unstable. At pH 7.3 (25 degrees C), the half-life for hydrolysis of the acyl molybdate, or the acyl arsenate, is less than 2.5 s. Thus, hydrolysis of 1-molybdo- and 1-arseno-3-phosphoglycerate is at least 2000 times faster than hydrolysis of 1,3-diphosphoglycerate under the same conditions. Glyceraldehyde-3-phosphate dehydrogenase has a fairly broad specificity for acyl acceptors. Most tetrahedral oxy anions tested are substrates for the enzyme (except SO4(2-) and SeO4(2-)). Tetrahedral monoanions such as ReO4- and GeO(OH)3- are not substrates but do bind to the enzyme. These results suggest the requirement of at least one anionic site on the acyl acceptor required for binding and another anionic group on the acyl receptor required for nucleophilic attack on the acyl enzyme.     

Kinetic characterization of Escherichia coli outer membrane phospholipase A using mixed detergent-lipid micelles

The substrate specificity of Escherichia coli outer membrane phospholipase A was analyzed in mixed micelles of lipid with deoxycholate or Triton X-100. Diglycerides, monoglycerides, and Tweens 40 and 85 in Triton X-100 are hydrolyzed at rates comparable to those of phospholipids and lysophospholipids. p-Nitrophenyl esters of fatty acids with different chain lengths and triglycerides are not hydrolyzed. The minimal substrate characteristics consist of a long acyl chain esterified to a more or less hydrophilic headgroup as is the case for the substrate monopalmitoylglycol. Binding occurs via the hydrocarbon chain of the substrate; diacyl compounds are bound three to five times better than monoacyl compounds. When acting on lecithins, phospholipase A1 activity is six times higher than phospholipase A2 activity or 1-acyl lysophospholipase activity. Activity on the 2-acyl lyso compound is about two times less than that on the 1-acyl lysophospholipid. The enzyme therefore has a clear preference for the primary ester bond of phospholipids. In contrast to phospholipase A1 activity, phospholipase A2 activity is stereospecific. Only the L isomer of a lecithin analogue in which the primary acyl chain was replaced by an alkyl ether group is hydrolyzed. The D isomer of this analogue is a competitive inhibitor, bound with the same affinity as the L isomer. On these ether analogues the enzyme shows the same preference for the primary acyl chain as with the natural diester phospholipids. Despite its broad specificity, the enzyme will initially act as a phospholipase A1 in the E. coli envelope where it is embedded in phospholipids.     

Influence of the degree of unsaturation of the acyl side chain upon the interaction of analogues of 1-arachidonoylglycerol with monoacylglycerol lipase and fatty acid amide hydrolase

Little is known as to the structural requirements of the acyl side chain for interaction of acylglycerols with monoacylglycerol lipase (MAGL), the enzyme chiefly responsible for the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain. In the present study, a series of twelve analogues of 1-AG (the more stable regioisomer of 2-AG) were investigated with respect to their ability to inhibit the metabolism of 2-oleoylglycerol by cytosolic and membrane-bound MAGL. In addition, the ability of the compounds to inhibit the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) was investigated. For cytosolic MAGL, compounds with 20 carbon atoms in the acyl chain and 2-5 unsaturated bonds inhibited the hydrolysis of 2-oleoylglycerol with similar potencies (IC50 values in the range 5.1-8.2 microM), whereas the two compounds with a single unsaturated bond were less potent (IC50 values 19 and 21 microM). The fully saturated analogue 1-monoarachidin did not inhibit the enzyme, whereas the lower side chain analogues 1-monopalmitin and 1-monomyristin inhibited the enzyme with IC50 values of 12 and 32 microM, respectively. The 22-carbon chain analogue of 1-AG was also potent (IC50 value 4.5 microM). Introduction of an alpha-methyl group for the C20:4, C20:3, and C22:4 compounds did not affect potency in a consistent manner. For the FAAH and the membrane-bound MAGL, there was no obvious relationship between the degree of unsaturation of the acyl side chain and the ability to inhibit the enzymes. It is concluded that increasing the number of unsaturated bonds on the acyl side chain of 1-AG from 1 to 5 has little effect on the affinity of acylglycerols for cytosolic MAGL.     

Stereospecific synthesis and enzyme studies of CDP-diacylglycerols

The fatty acid specificity of two enzymes that metabolize CDPdiacylglycerol, CDPdiacylglycerol hydrolase (EC 3.6.1.26) and CDPdiacylglycerol: inositol phosphatidyltransferase (EC 2.7.8.11), has been examined in guinea pig brain. Mixed CDPdiacylglycerols were stereospecifically synthesized by the following sequence: (i) hydrolysis of a homodiacyl lecithin to 1-acyl lysoPC by action of snake venom phospholipase A2, (ii) reacylation with the anhydride of the desired second fatty acid and dimethylaminopyridine, (iii) hydrolysis of the resultant heterodiacyl lecithin to phosphatidate with cabbage phospholipase D, and (iv) reaction of phosphatidate with CMPmorpholidate to give CDPdiacylglycerol. CDPdiacylglycerol: inositol phosphatidyltransferase showed the following rates of conversion of 40-microM suspensions of CDPdiacylglycerol in 0.15% Triton X-100 to phosphatidylinositol relative to the 1-stearoyl-2-oleoyl derivative (100%): dipalmitoyl, 70%; distearoyl, 38%; diarachidonoyl, 9%; 1-arachidonoyl-2-stearoyl, 6%; 1-stearoyl-2-arachidonoyl, 4%. These results indicate that the composition of isolated phosphatidylinositol and related lipids is not explained by the fatty acid specificity of the biosynthetic enzymes and supports the intervention of a deacylation-reacylation sequence. The rates of hydrolysis of the synthetic CDPdiacylglycerols at 76 microM, in 0.3% Triton X-100, by the CDPdiacylglycerol hydrolase relative to the 1-stearoyl-2-oleoyl derivative (100%) were: dipalmitoyl, 70%; distearoyl, 32%; 1-arachidonoyl-2-stearoyl, 30%; 1-stearoyl-2-arachidonoyl, 28%; diarachidonoyl, 22%. Inhibition of this enzyme by AMP was shown to be non-competitive, with a Ki of 40 microM. The lysosomal localization of the mammalian hydrolase was confirmed.     

A mutant of Arabidopsis deficient in c(18:3) and c(16:3) leaf lipids

Leaf tissue of a mutant of Arabidopsis thaliana contains reduced levels of both 16:3 and 18:3 fatty acids and has correspondingly increased levels of the 16:2 and 18:2 precursors due to a single recessive nuclear mutation. The kinetics of in vivo labeling of lipids with [¹⁴C]acetate and quantitative analysis of the fatty acid compositions of individual lipids suggests that reduced activity of a glycerolipid n-3 desaturase is responsible for the altered lipid composition of the mutant. The effects of the mutation are most pronounced when plants are grown at temperatures above 26°C but are relatively minor below 18°C, suggesting a temperature-sensitive enzyme. Since the desaturation of both 16- and 18-carbon fatty acids is altered, it appears that the affected enzyme lacks specificity with respect to acyl group chain length and that it is located in the chloroplast where 16:3-monogalactosyldiglyceride is synthesized. Because the degree of unsaturation of all the major glycerolipids was similarly affected by the mutation, it is inferred that either the affected desaturase does not exhibit head group specificity or there is substantial transfer of trienoic acyl groups between different lipid classes. Both chloroplast and extrachloroplast lipids are equally affected by the mutation. Thus, either the desaturase is located both outside and inside the chloroplast, or 18:3 formed inside the chloroplast is reexported to other cellular sites.     

Inhibition of lysophospholipase by cholesterol in rabbit aorta

Lysophospholipase activity was measured in rabbit aorta using 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine as a substrate. The enzyme did not require Ca2+ for its activation and the maximal activation was attained in the presence of EGTA. Cholesterol dose-dependently inhibited the lysophospholipase activity in the soluble fraction and IC50 value was approximately 15 microM. Lineweaver-Burk plot revealed that cholesterol competitively inhibited lysophospholipase and Km values in the presence and absence of cholesterol (15.5 microM) were 12.3 and 2.8 microM, respectively. Vmax values were approximately 475 pmol/min.mg. The results suggest that cholesterol can interact with the enzyme per se, resulting in the inhibition of the lysophospholipase activity in rabbit aorta.     

Negative charge at amino acid 149 is the molecular determinant for substrate specificity of lecithin: cholesterol acyltransferase for phosphatidylcholine containing 20-carbon sn-2 fatty acyl chains

We previously described a point mutation in human LCAT (E to A at residue 149; hE149A) that demonstrated greater activity with phosphatidylcholine (PC) substrate containing 20:4 in the sn-2 position compared with the wild-type enzyme [hLCAT; Wang et al. (1997) J. Biol. Chem. 272, 280-286], resulting in a human enzyme with the substrate specificity similar to that of rat LCAT. The purpose of the present study was to explore the molecular basis for the role of amino acid 149 in determining fatty acyl substrate specificity. In the first experiment, the reverse mutation in rat LCAT (rA149E) converted substrate specificity of rat LCAT toward that of the human enzyme, demonstrating that the mutation was context independent and reversible. In the second experiment, we found that hE149A compared with hLCAT demonstrated higher activity with PC species containing 20-carbon, but not 18-carbon, sn-2 fatty acyl chains. The increased activity of hE149A was due to an increase in apparent V(max) but not to apparent K(m) or LCAT binding to the PC surface. Substitution of different amino acids in the 149 position of hLCAT showed that activation of the enzyme with sn-2 20:4 containing PC substrate was only observed when the negative charge at residue 149 was removed. We conclude that the negative charge at amino acid 149 of LCAT is a critical determinant for the specificity of the enzyme for PC containing 18- vs 20-carbon sn-2 fatty acyl chains.