Properties of canine myocardial phosphatidylethanolamine N-acyltransferase

Dog heart contains a membrane bound N-acyltransferase (transacylase) which transfers acyl groups from the sn-1 position of membrane phospholipids to the amino group of ethanolamine phospholipids in the presence of millimolar Ca2+ concentrations. Using crude membrane preparations, we found this N-acyltransferase activity to be heat sensitive and inhibited by sulfhydryl reagents. Pretreatment of a membrane fraction with trypsin reduced N-acyltransferase activity to 60% while pretreatment with trypsin and Triton X-100 together reduced it to 30% of the control value. At pH 8.0 both Sr2+ and Mn2+ could fully substitute for Ca2+ with respect to optimum ion concentration and molecular species of the product formed in dog heart membranes from endogenous substrates. Ba2+ was equally effective in achieving N-acylation of ethanolamine phospholipids while other divalent cations were less effective or ineffective. The reaction exhibited a pH optimum of 8.5 to 9.0 with both Ca2+ and Sr2+ while Mn2+ precipitated above pH 8.0 resulting in decreased N-acylation activity. Both phosphatidylcholine and 1-acyl lysophosphatidylcholine could serve as acyl donors. Triton X-100 at a concentration of 0.1% stimulated acyl transfer from exogenous phosphatidylcholine but inhibited acyl transfer from lysophosphatidylcholine.     

Phospholipid-enzyme interactions of chitin synthase of Coprinus cinereus

Abstract The effect of phospholipids on chitin synthase activity has been studied with digitonin-solubilized and partially purified preparations from Coprinus cinereus . When cholate was used as detergent, it inhibited enzyme activity, but this inhibition was reversed by increasing concentrations of phospholipids. Preincubation with cholate and phospholipid caused irreversible loss of activity. When sonicated with solubilized enzyme preparation, dimyristoyl phosphatidyl choline strongly stimulated activity, while dioleoyl phosphatidyl choline was inhibitory. The Arrhenius plot of the effect of temperature on enzyme activity contained breaks, characteristic of a membrane-bound enzyme. It is suggested that chitin synthase requires an annulus of phospholipids for activity.     

Yeast dolichyl-phosphomannose synthase: reconstitution of enzyme activity with phospholipids.

The activity of purified recombinant yeast dolichyl-phosphomannose synthase (EC 2.4.1.83) was assessed following reconstitution of the enzyme with phospholipids. The yeast synthase, similar to the mammalian enzyme, was active when reconstituted with phosphatidylethanolamine dispersions but had little (less than 5%) activity in stable phosphatidylcholine bilayers. The enzyme was activated by adding increasing amounts of diacylglycerol to phospholipid bilayers, suggesting that activity of the yeast enzyme was dependent on lipid phase properties rather than specific phospholipids. The synthase could also be reconstituted as an active enzyme in bilayers prepared with a commercial crude lipid preparation containing 40% phosphatidylcholine, but at a rate 10% of that occurring in phosphatidylethanolamine. Vesicles composed of the 40% phosphatidylcholine lipid mixture, dolichyl phosphate, and enzyme were leaky in the presence of divalent cations, and dolichyl-phosphomannose synthase did not appear to catalyze the translocation of dolichyl phosphomannose across membranes at a catalytically significant rate under the assay conditions employed.     

Identification of a novel lysophospholipid acyltransferase in Saccharomyces cerevisiae

The incorporation of unsaturated acyl chains into phospholipids during de novo synthesis is primarily mediated by the 1-acyl-sn-glycerol-3-phosphate acyltransferase reaction. In Saccharomyces cerevisiae, Slc1 has been shown to mediate this reaction, but distinct activity remains after its removal from the genome. To identify the enzyme that mediates the remaining activity, we performed synthetic genetic array analysis using a slc1Delta strain. One of the genes identified by the screen, LPT1, was found to encode for an acyltransferase that uses a variety of lysophospholipid species, including 1-acyl-sn-glycerol-3-phosphate. Deletion of LPT1 had a minimal effect on 1-acyl-sn-glycerol-3-phosphate acyltransferase activity, but overexpression increased activity 7-fold. Deletion of LPT1 abrogated the esterification of other lysophospholipids, and overexpression increased lysophosphatidylcholine acyltransferase activity 7-fold. The majority of this activity co-purified with microsomes. To test the putative role for this enzyme in selectively incorporating unsaturated acyl chains into phospholipids in vitro, substrate concentration series experiments were performed with the four acyl-CoA species commonly found in yeast. Although the saturated palmitoyl-CoA and stearoyl-CoA showed a lower apparent Km, the monounsaturated palmitoleoyl-CoA and oleoyl-CoA showed a higher apparent Vmax. Arachidonyl-CoA, although not abundant in yeast, also had a high apparent Vmax. Pulse-labeling of lpt1Delta strains showed a 30% reduction in [3H]oleate incorporation into phosphatidylcholine only. Therefore, Lpt1p, a member of the membrane-bound o-acyltransferase gene family, seems to work in conjunction with Slc1 to mediate the incorporation of unsaturated acyl chains into the sn-2 position of phospholipids.     

Acyl transferase activities in dog lung microsomes

Mammalian lung has a high concentration of dipalmitoyl phosphatidylcholine and other phospholipids in which both fatty acid ester chains are saturated, as opposed to the usual asymmetric phospholipid (one saturated fatty acid and one unsaturated fatty acid). The acyl transferase system in dog lung microsomes was studied by determining the reactivities of various acyl CoA derivatives with 1-lyso-2-acyl- and 1-acyl-2-lyso-phosphatidylcholine. The 16:0 derivative had equal reactivity for both the 1- and 2-lyso positions. The 18:0 derivative also exhibited marked reactivity toward both positions, although the specific activity of the enzyme when palmitoyl CoA was used was approximately twice that compared to when stearoyl CoA was used. The 16:1 derivative showed approximately the same reactivity toward the 1-lyso position as did 16:0 but both 16:1 and 18:1 were more active with the 2-lyso position. These results suggest that acyl transferases may be important in the lung to insure that sufficient amounts of dipalmitoyl phosphatidylcholine will always be present for use in pulmonary surfactant biosynthesis. It is also conceivable that the acyl transferase system described acts on 1- and 2-lyso-palmitoyl phosphatidylcholine (produced by phospholipase hydrolysis of dipalmitoyl phosphatidylcholine) in order to produce phosphatidylcholine species needed for cellular purposes other than surfactant function.     

Effect of phospholipids on UDP-glucose: ceramide glucosyltransferase from Golgi membranes

1. The removal of phospholipids completely abolished the activity of the enzyme UDP-glucose:ceramide glucosyltransferase from Golgi membranes. 2. Modulation of enzyme activity by phospholipids was undertaken on the solubilized form of the enzyme. 3. Well-defined fatty acyl chains and polar head groups were necessary for maximal stimulation by phospholipids. 4. A specific requirement for phosphatidylcholine is suggested by preliminary experiments of reconstitution of enzyme activity with phosphatidylcholine vesicles.     

Turnover of phospholipid fatty acyl chains in cultured neuroblastoma cells: involvement of deacylation-reacylation and de novo synthesis in plasma membranes

Cultured neuroblastoma cells (NIE-115) rapidly incorporated the essential fatty acid, linoleic acid (18:2 (n = 6), into membrane phospholipids. Fatty acid label appeared rapidly (2-10 min) in plasma membrane phospholipids without evidence of an initial lag. Specific activity (nmol fatty acid/mumol phospholipid) was 1.5-2-fold higher in microsomes than in plasma membrane. In these membrane fractions phosphatidylcholine had at least 2-fold higher specific activity than other phospholipids. With 32P as radioactive precursor, the specific activity of phosphatidylinositol was 2-fold higher compared to other phospholipids in both plasma membrane and microsomes. Thus a differential turnover of fatty acyl and head group moieties of both phospholipids was suggested. This was confirmed in dual-label (3H fatty acid and 32P), pulse-chase studies that showed a relatively rapid loss of fatty acyl chains compared to the head group of phosphatidylcholine; the opposite occurred with phosphatidylinositol. A high loss of fatty acyl chain relative to phosphorus indicated involvement of deacylation-reacylation in fatty acyl chain turnover. The patterns of label loss in pulse-chase experiments at 37 and 10 degrees C indicated some independent synthesis and modification of plasma membrane phospholipids at the plasma membrane. Lysophosphatidylcholine acyltransferase and choline phosphotransferase activities were demonstrated in isolated plasma membrane in vitro. Thus, studies with intact cells and with isolated membrane fractions suggested that neuroblastoma plasma membranes possess enzyme activities capable of altering phospholipid fatty acyl chain composition by deacylation-reacylation and de novo synthesis at the plasma membrane itself.     

The purification and characterization of a phospholipase A in hamster heart cytosol for the hydrolysis of phosphatidylcholine

Phospholipases A1 and A2 catalyze the hydrolysis of acyl groups of phospholipids at C-1 and C-2, respectively. These phospholipases are important in phospholipid catabolism and the remodeling of the acyl groups of phospholipids. Phospholipase A from hamster heart cytosol was purified by a combination of ion-exchange and gel filtration chromatography. The purity of the enzyme was assessed by nondenaturing polyacrylamide gel electrophoresis, two-dimension polyacrylamide gel electrophoresis, and immunological studies. The purified enzyme exhibited both phospholipase A1 and A2 activities toward phosphatidylcholine and had the ability to hydrolyze the acyl groups of phosphatidylethanolamine. However, the enzyme was not active toward lysophosphatidylcholine, diacylglycerol, or triacylglycerol. By Sepharose 6B chromatography, the molecular weight of the purified enzyme was estimated to be 140,000. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was composed of identical Mr 14,000 subunits. At least six subunits in the native enzyme could be cross-linked by dimethyl suberimidate. Both phospholipase A1 and A2 activities showed similar pH profiles, exhibited no absolute requirements for divalent metallic cations, but displayed a high degree of specificity for the acyl groups of phosphatidylcholine at both C-1 and C-2. The Km of phospholipases A1 and A2 for 1-palmitoyl-2-arachidon-ylglycerophosphocholine was found to be identical (0.5 mM).     

Gut chitin synthase and sterols from larvae of Diaprepes abbreviatus (Coleoptera: Curculionidae)

Gut chitin synthase was characterized and the sterols and ecdysteroids in the sugarcane rootstalk borer weevil, Diaprepes abbreviatus, were identified. An in vitro cell-free chitin synthase assay was developed using larval gut tissues from D. abbreviatus. Subcellular fractionation experiments showed that the majority of chitin synthase activity was located in 10,000g pellets. The gut chitin synthase requires Mg²⁺ to be fully active: 7–8-fold increases in activity were obtained with 10 mM Mg²⁺ present in reaction mixture. Calcium also stimulated activity (4–5-fold with 10 mM Ca²⁺), while Cu⁺² completely inhibited at 1 mM. Other monovalent and divalent cations had little or no effect on activity. The pH and temperature optima were 7 and 25°C, respectively. Gut chitin synthesis was activated ca. 50% by trypsin treatments. GlcNAc stimulated chitin synthase activity, but Glc, GlcN and glycerin did not. Polyoxin D, UDP, and ADP inhibited the chitin synthase reaction with I₅₀'s of 75 μM, 2.3 mM, and 3.6 mM, respectively. Nikkomycin Z was a potent inhibitor of chitin synthase (91% inhibition at 10 μM). Tunicamycin and diflubenzuron had no effect on the enzyme. The apparent Km and V_max for the gut chitin synthase were, respectively, 122.5 ± 7.4 μM and 426 ± 19.7 pmol/h/mg protein utilizing UDP-GlcNAc as the substrate. Sterol analyses indicated that cholesterol was the major dietary and larval sterol. HPLC/RIA data indicated that 20-hydroxyecdysone was the major molting hormone.     

Substrate specificity of a CoA-dependent stearoyl transacylase from bovine testis membranes.

We identified a CoA-dependent stearoyl transacylase activity in bovine testis membranes, then examined the enzyme's specificity in mixed micelle systems containing the neutral detergent Triton X-100. The enzyme transferred stearoyl groups from a variety of phospholipids to sn-2-arachidonoyl lysophosphatidic acid (lysoPA), but showed very little palmitoyl transacylase activity. Its ability to transfer stearoyl groups was both donor- and acceptor-dependent. For example, it used weakly acidic phospholipids, such as sn-1-stearoyl-2-acyl species of phosphatidylinositol (PI), as donors, but did not use phosphatidylinositol-4,5-bisphosphate or sn-1-stearoyl-2-arachidonoyl phosphatidylcholine. Moreover, it used sn-2-acyl species of lysoPA and sn-2-arachidonoyl lysoPI as acceptors but did not use sn-2-arachidonoyl species of lysophosphatidylserine, lysophosphatidylethanolamine, or lysophosphatidylcholine. When taken together, our results raise the possibility that sn-1-stearoyl-2-acyl species of PI may be the primary acyl donors in the transacylase reaction in vivo, while sn-2-acyl species of lysoPA may be the primary acyl acceptors. Available evidence suggests that the PA that is formed may subsequently be converted into PI, but the metabolic fate of the other reaction product, sn-2-acyl lysoPI, remains to be determined.     

Phospholipid remodeling in human neutrophils. Parallel activation of a deacylation/reacylation cycle and platelet-activating factor synthesis

A23187 stimulated two enzymatic activities of human neutrophils (polymorphonuclear leukocytes), phospholipase A2 and fatty acyl-CoA acyltransferase, which resulted in a stimulated deacylation/reacylation cycle. The incorporation of fatty acids, other than arachidonic or eicosapentaenoic acid, into diacyl and alkylacyl species of choline phosphoglycerides was stimulated by 10-fold by A23187. These fatty acids were exclusively incorporated into the sn-2 position, and [3H]glycerol labeling showed there was no stimulation of de novo synthesis. A23187 also stimulated fatty acid incorporation into other phospholipids, but de novo synthesis accounted for a portion of this uptake. Inhibitors of protein kinase C prevented the stimulated recycling of phosphatidylcholine, and the simultaneous induction of platelet-activating factor synthesis, by inhibiting phospholipase A2 activation. They inhibited [3H]arachidonate release from prelabeled polymorphonuclear leukocytes, but had no effect on in vitro fatty acyl-CoA acyltransferase or acetyl-CoA acetyltransferase activity. Extracts from A23187-treated cells contained a fatty acyl-CoA acyltransferase, which did not utilize arachidonoyl-CoA, that was 2.3-fold more active than that of control extracts. Phosphatase treatment decreased this stimulated activity by 66%. Thus, A23187 stimulated a phospholipase A2 activity that generated both 1-alkyl and 1-acyl lysophosphatidylcholines. A stimulated acetyltransferase used a portion of the alkyl species for platelet-activating factor synthesis, while the acyl species and residual alkyl species were rapidly reacylated to phosphatidylcholine by a stimulated acyl-transferase. Arachidonate, an eicosanoid precursor, was spared by this process.     

Remodeling of rat hepatocyte phospholipids by selective acyl turnover

Acyl turnover of rat hepatocyte phospholipids and triacylglycerols was assessed by incubating the cells in media containing 40% H2(18)O and measuring the time-dependent incorporation of 18O into ester carbonyls by gas chromatography-mass spectrometry of hydrogenated methyl esters. Incorporation of 18O into 22-carbon acyl groups was low in phosphatidylcholine, phosphatidylinositol, and phosphatidylserine, whereas in phosphatidylethanolamine, it was about the same as in the other acyl groups. Incorporation of 18O into individual molecular species of phosphatidylcholine and phosphatidylethanolamine was determined after phospholipase C hydrolysis, derivatization to dinitrobenzoates, and separation by high-performance liquid chromatography. In most molecular species, acyl groups at the sn-1 and sn-2 positions became 18O-labeled at drastically different rates, indicating remodeling through deacylation-reacylation. Molecular species expected to arise de novo from acylation of glycerophosphate exhibited similar rates of 18O incorporation at the sn-1 and sn-2 positions. The data suggest that hepatocyte phospholipids are continually synthesized, remodeled by deacylation-reacylation at specific turnover rates up to 10-15%/h, and degraded. This acyl turnover probably does not involve the majority of intracellular unesterified fatty acids whose 18O incorporation was found to be very low. In contrast, the oxygens of extracellular unesterified fatty acids were readily exchanged with the media. This exchange was enzyme-catalyzed, possibly by lipases released into the media from damaged cells. Incorporation of 18O into exogenously added fatty acids was also rapid and resulted in enhanced uptake of 18O-labeled fatty acids into cellular lipids, primarily triacylglycerols and phosphatidylcholine, without drastic change of the intracellular free fatty acid pool.     

The isolation of plasma membrane and characterisation of the plasma membrane ATPase from the yeast Candida albicans

Plasma membrane ghosts were isolated from Candida albicans ATCC 10261 yeast cells following stabilisation of spheroplasts with concanavalin A, osmotic lysis and Percoll density gradient centrifugation. Removal of extrinsic proteins with NaCl and methyl alpha-mannoside gave increased ATPase and chitin synthase specific activities in the resultant plasma membrane fraction. Sonication of this fraction yielded unilamellar plasma membrane vesicles which exhibited ATPase and chitin synthase specific activities of 4.5-fold and 3.0-fold, respectively, over those of the plasma membrane ghosts. ATPase activity in the membrane ghosts was optimal at pH 6.4, showed high substrate specificity (for Mg X ATP) and was inhibited 80% by sodium vanadate but less than 4% by oligomycin and azide. The effects of a range of other inhibitors were also characterised. Temperature effects of ATPase activity were marked, with a maximum at 35 degrees C. Breaks in the Arrhenius plot, at 12.2 degrees C and 28.9 degrees C, coincided with endothermic heat flow peaks detected by differential scanning calorimetry. ATPase was solubilised from the plasma membranes with Zwittergent in the presence of glycerol and phenylmethylsulphonyl fluoride and partially purified by glycerol density gradient centrifugation. The solubilised enzyme hydrolysed Mg X ATP at Vmax = 20 mumol X min-1 X mg-1 in the presence of phospholipids, with optimal activity at pH 6.0--6.5.     

Phospholipids modulate the substrate specificity of soluble UDP-glucose:steroid glucosyltransferase from eggplant leaves.

UDP-glucose-dependent glucosylation of solasodine and diosgenin by a soluble, partially purified enzyme fraction from eggplant leaves is affected in a markedly different way by some phospholipids. While glucosylation of diosgenin and some closely related spirostanols, e.g. tigogenin or yamogenin, is strongly inhibited by relatively low concentrations of several phospholipids, the glucosylation of solasodine is unaffected or even slightly stimulated. These effects depend both on the structure of the polar head group and the nature of the acyl chains present in the phospholipid. The most potent inhibitors of diosgenin glucosylation are choline-containing lipids: phosphatidylcholine (PC) and sphingomyelin (SM) but the removal of phosphocholine moiety from these phospholipids by treatment with phospholipase C results in an almost complete recovery of the diosgenin glucoside formation by the enzyme. Significant inhibition of diosgenin glucoside synthesis and stimulation of solasodine glucosylation was found only with PC molecular species containing fatty acids with chain length of 12-18 carbon atoms. PC with shorter or longer acyl chains had little effect on glucosylation of either diosgenin or solasodine. Our results indicate that interaction between the investigated glucosyltransferase and lipids are quite specific and suggest that modulation of the enzyme activity by the nature of the lipid environment may be of importance for regulation of in vivo synthesis of steroidal saponins and glycoalkaloids in eggplant.     

Hydrolysis of phosphatidylcholine during LDL oxidation is mediated by platelet-activating factor acetylhydrolase

Degradation of phosphatidylcholine to lysophosphatidylcholine occurs during oxidative modification of low density lipoproteins (LDL). In this study, we have shown that this phospholipid hydrolysis is brought about by an LDL-associated phospholipase A2 that can hydrolyze oxidized but not intact LDL phosphatidylcholine. The chemical nature of the oxidized phospholipids that can act as substrates for this enzyme was not fully characterized, but we hypothesized that the specificity of the enzyme for oxidized LDL phosphatidylcholine might be explained by fragmentation of polyunsaturated sn-2 fatty acyl groups in LDL phosphatidylcholine during oxidation. To facilitate characterization of this enzyme, we therefore selected a fluorescent phosphatidylcholine substrate that had a short-chain, polar residue in the sn-2 position: 1-palmitoyl 2-(6-[7-nitrobenzoxadiazolyl]amino) caproyl phosphatidylcholine, (C6NBD PC). This substrate was efficiently hydrolyzed by LDL, but the dodecanoyl analogue of C6NBD PC, which differed only in that a 12-carbon rather than a 6-carbon acyl derivative was present in the sn-2 position, was not hydrolyzed. The phospholipase activity was heat-stable, calcium-independent, and was inhibited by the serine esterase inhibitors phenylmethylsulfonyl-fluoride and diisopropylfluorophosphate, but was resistant to p-bromophenacylbromide and dithiobisnitrobenzoic acid. The phospholipid hydrolysis could not be attributed to the action of lecithin:cholesterol acyltransferase or lipoprotein lipase. Nearly all of the activity in EDTA-anticoagulated normal plasma was physically associated with apoB-containing lipoproteins, but this apoprotein was not essential as enzyme activity was present in plasma from abetalipoproteinemic patients. These properties are very similar to those recently reported for human plasma platelet-activating factor (PAF) acetylhydrolase. In the present study, we found that acylhydrolase activity against C6NBD PC, PAF, and oxidized phosphatidylcholine copurfied through gel filtration and ion-exchange chromatography. Substrate competition was demonstrated between C6NBD PC, PAF, and oxidized 2-arachidonyl phosphatidylcholine, suggesting that a single enzyme was active against all three substrates. The enzyme had an apparent molecular weight of 40,000-45,000 by high pressure gel exclusion chromatography. Inhibition of this activity with disopropyfluorophosphate prior to oxidative modification of LDL prevented phospholipid hydrolysis but did not affect the production of thiobarbituric acid reactive compounds or the change in electrophoretic mobility. In addition, this inhibition of phospholipase did not prevent the rapid degradati     

Plant holo-(acyl carrier protein) synthase.

1. An improved method was developed for the assay of plant holo-(acyl carrier protein) synthase activity, using Escherichia coli acyl-(acyl carrier protein) synthetase as a coupling enzyme. 2. Holo-(acyl carrier protein) synthase was partially purified from spinach (Spinacia oleracea) leaves by a combination of (NH4)2SO4 fractionation and anion-exchange and gel-permeation chromatography. 3. The partially purified enzyme had a pH optimum of 8.2 and Km values of 2 microM, 72 microM and 3 mM for apo-(acyl carrier protein), CoA and Mg2+ respectively. Synthase activity was inhibited in vitro by the reaction product 3',5'-ADP. 4. Results from the fractionation of spinach leaf and developing castor-oil-seed (Ricinus communis) endosperm cells were consistent with a cytosolic localization of holo-(acyl carrier protein) synthase activity in plant cells.     

Diacylglycerol kinase from pig brain. Purification and phospholipid dependencies

Diacylglycerol kinase (EC 2.7.1.-) was purified 1,650-fold from pig brain cytosol. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the kinase was estimated to be 78,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar value (76,000) was obtained by Sephadex G-150 gel filtration. The activity of the purified enzyme was markedly enhanced by either deoxycholate or phospholipids. The extent of activation by phospholipids was in the order of phosphatidylcholine greater than lysophosphatidylcholine greater than phosphatidylethanolamine approximately equal to phosphatidylserine greater than sphingomyelin. Other phospholipids and unsaturated fatty acids were ineffective. Phosphatidylcholines from egg yolk and pig brain, and dioleoyl phosphatidylcholine were similarly effective. Saturated phosphatidylcholines with acyl chain lengths shorter than palmitate also gave a considerable activation. The activity with phosphatidylcholine was from 1.5- to 2.5-fold higher than that measured with deoxycholate. A very small amount of phosphatidylinositol or phosphatidylglycerol potently inhibited the phosphatidylcholine-dependent (but not deoxycholate-dependent) kinase activity. The inhibition by phosphatidylinositol was varied according to its molar ratio to phosphatidylcholine. As little as about 2.5 mol per cent of phosphatidylinositol resulted in 50% inhibition of the phosphatidylcholine-dependent kinase activity. The deoxycholate- and phosphatidylcholine-dependent kinase activities showed almost the same Km values for the substrates. In both cases, the apparent Km values for ATP and diacylglycerol were 300 microM and about 60 microM, respectively. The kinase required Mg2+ for its activity. When compared to deoxycholate, phosphatidylcholine was more effective at higher Mg2+ concentrations. The deoxycholate-dependent activity showed a broad pH optimum at around 8.0, whereas the phosphatidylcholine-dependent activity formed a clear peak at pH 7.4.     

Acyl specificity of hamster heart CDP-choline 1,2-diacylglycerol phosphocholine transferase in phosphatidylcholine biosynthesis

The acyl specificity of 1,2-diacylglycerol: CDP-choline phosphocholine transferase (EC 2.7.8.2) for the formation of phosphatidylcholine with the appropriate acyl groups in hamster heart was investigated. Enzyme activity was determined in the microsomal fraction with 1,2-diacylglycerols of known acyl content. Maximum enzyme activity was obtained with diacylglycerol containing a monoenoic acyl group at the C-2 position of the glycerol moiety, regardless of the acyl group at the C-1 position. The specificity of the enzymes was also investigated by perfusing the isolated hamster heart with labelled glycerol. Comparison of the molecular species of the labelled diacylglycerols and phosphatidylcholine subsequent to perfusion revealed that the specificity of phosphocholine transferase was not limited to the monoenoic species of diacylglycerol. The difference in specificity observed between the in vitro assay and the perfusion study may partly be attributed to the presence of detergent in the enzyme assay mixture (to facilitate solubility of diacylglycerol). It is concluded that in the hamster heart, phosphocholine transferase has only limited ability to select the appropriate acyl groups for phosphatidylcholine biosynthesis. It appears that the majority of the newly formed phosphatidylcholine in the heart via the CDP-choline pathway is subsequently resynthesized by deacylation-reacylation process.     

Activation of D-beta-hydroxybutyrate apodehydrogenase using molecular species of mixed fatty acyl phospholipids

D-beta-Hydroxybutyrate apodehydrogenase is a lipid-requiring enzyme with a specific requirement of lecithin for enzymatic function. The purified enzyme which is devoid of lipid can be reactivated with lecithin or mixtures of natural phospholipid-containing lecithin. However, it is mitochondrial phospholipid which activates the enzyme optimally and with kinetic parameters similar to that of the native membrane-bound enzyme. Mitochondrial phospholipid consists of three classes of phospholipid (lecithin:phosphatidylethanolamine:diphosphatidylglycerol in a ratio of approximately 2:2:1 by phosphorus); each class consists of a multiplicity of different molecular species due to diversity in the fatty acyl substituents. In this study, we have synthesized defined molecular species of mixed fatty acyl phospholipids to evaluate whether multiplicity of phospholipid molecular species are essential for optimal reactivation. We find that: 1) ternary mixtures of single molecular species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylpropan-1,3-diol in the liquid crystalline state mimic the optimal reactivation of the enzyme obtained with mitochondrial phospholipids; 2) although some negatively charged phospholipid appears necessary for optimizing the efficiency of activation, diphosphatidylglycerol can be replaced by phosphatidylpropan-1,3-diol, another negatively charged phospholipid; and 3) biphasic Arrhenius plots can be correlated with the liquid crystalline and gel states of the phospholipid.     

Methyl branching in short-chain lecithins: are both chains important for effective phospholipase A2 activity?

Several seven-carbon fatty acyl lecithins with varied acyl chain branching have been synthesized and characterized as potential phospholipase A2 substrates. Micellar bis(4,4-dimethylpentanoyl) phosphatidylcholine, bis(5-methylhexanoyl)phosphatidylcholine, bis(3-methylhexanoyl)phosphatidylcholine, and bis(2-methylhexanoyl)phosphatidylcholine are poor substrates for phospholipase A2 (Naja naja naja). These branched lecithins also inhibit the hydrolysis of diheptanoylphosphatidylcholine by the enzyme with Ki values comparable to or smaller than the apparent Km of the linear compound. The terminally branched lecithins are excellent substrates for another surface-active hydrolytic enzyme, phospholipase C from Bacillus cereus. When only one acyl chain bears a methyl group, the hybrid lecithins 1-heptanoyl-2-(2-methylhexanoyl)phosphatidylcholine and 1-(3-methylhexanoyl)-2-heptanoylphosphatidylcholine are substrates comparable to diheptanoylphosphatidylcholine. Analysis of micellar structure and dynamics by 1H and 13C NMR spectroscopy, quasi-elastic light scattering, and comparison of critical micellar concentrations indicates little significant difference in the conformation and dynamics of these seven-carbon fatty acyl lecithin micelles, even when the methyl groups are adjacent to the carbonyls. Phospholipase A2 UV difference spectra induced by phospholipid binding imply different enzyme conformations or aggregation states caused by linear-chain and asymmetric-chain lipids compared to bis(methylhexanoyl)phosphatidylcholines. The differences in hydrolytic activity of phospholipase A2 against the branched-chain micellar lecithins can then be attributed to an enzyme-lipid interaction at the active site. The species with both fatty acyl chains branched bind to phospholipase A2 but are not turned over rapidly. Since poor enzymatic activity only occurs for lecithins with both chains methylated, the interaction of both chains with the enzyme must be important for catalytic efficiency.