The effect of lipid environment and retinoids on the ATPase activity of ABCR, the photoreceptor ABC transporter responsible for Stargardt macular dystrophy

ABCR is a photoreceptor-specific ATP-binding cassette transporter that has been linked to various retinal diseases, including Stargardt macular dystrophy, and implicated in retinal transport across rod outer segment (ROS) membranes. We have examined the ATPase and GTPase activity of detergent-solubilized and reconstituted ABCR. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized ABCR had ATPase and GTPase activity (K(m) approximately 75 micrometer V(max) approximately 200 nmol/min/mg) that was stimulated 1.5-2-fold by all-trans-retinal and dependent on phospholipid and dithiothreitol. The K(m) for ATP decreased to approximately 25 micrometer after reconstitution, whereas the V(max) was strongly dependent on the lipid used for reconstitution. ABCR reconstituted in ROS phospholipid had a V(max) for basal and retinal activated ATPase activity that was 4-6 times higher than for ABCR in soybean or brain phospholipid. This enhanced activity was mainly due to the high phosphatidylethanolamine (PE) content of ROS membranes. PE was also required for retinoid-stimulated ATPase activity. ATPase activity of ABCR was stimulated by the addition of N-retinylidene-PE but not the reduced derivative, retinyl-PE. ABCR expressed in COS-1 cells also exhibited retinal-stimulated ATPase activity similar to that of the native protein. These results support the view that ABCR is an active retinoid transporter, the nucleotidase activity of which is strongly influenced by its lipid environment.     

Abnormal phospholipid composition impairs HDL biogenesis and maturation in mice lacking Abca1

Recent studies have demonstrated that the ATP-binding cassette transporter A1 (ABCA1) facilitates the efflux of phospholipids and cholesterol to apoprotein acceptors, leading to the synthesis of HDL. The purpose of this study was to determine the changes in the lipoprotein fractions in Abca1-deficient mice and study the mechanisms responsible for the low levels of HDL when ABCA1 is absent. Plasma phospholipid concentration was decreased by more than 75%, mostly due to a reduction of phosphatidylcholine (PC) in HDL. Abca1(-/-) HDL represents less than 2% of wild-type levels and is smaller and enriched in phospholipids (11.2-fold more than HDL from controls). Compared to wild-type littermates, Abca1(-/-) HDL had a 4-fold increase in PC, whereas lysophosphatidylcholine (LPC) (125-fold), sphingomyelin (SPH) (49-fold), and phosphatidylethanolamine (PE) (18-fold) showed even higher increases. As a consequence, the ratios of LPC/PC, SPH/PC, PE/PC, and phosphatidylinositol + phosphatidylserine (PI+PS)/PC were all much higher in HDL from Abca1(-/-), compared to wild-type HDL. Plasma phospholipid transfer protein (PLTP) and lecithin cholesterol acyltransferase (LCAT) activities were decreased by more than 80%, suggesting that the maturation of HDL is affected. To test this hypothesis, plasma from Abca1(-/-) mice was incubated with CHO cells that are known to express high levels of ABCA1 with the intent of restoring the flux of phospholipid and cholesterol onto apoAI. Compared to native plasma, no change in maturation of HDL was observed. In contrast, a 220% increase in the formation of mature HDL was observed when ABCA1 function and LCAT activities were restored. Taken together, these observations suggest that ABCA1 is necessary for the adequate lipidation of apoAI, which enables the interaction with LCAT and subsequent maturation.     

Regulation of the activity and fatty acid specificity of lecithin-cholesterol acyltransferase by sphingomyelin and its metabolites, ceramide and ceramide phosphate

Sphingomyelin (SM), the second most abundant phospholipid in plasma lipoproteins, was previously shown to be a physiological inhibitor of the lecithin-cholesterol acyltransferase (LCAT) reaction. In this study, we investigated the effects of its metabolites, ceramide and ceramide phosphate, on the activity and fatty acid specificity of LCAT in vitro. Treatment of SM-containing substrate with SMase C, which hydrolyzes SM to ceramide, abolished the inhibitory effect of SM, whereas treatment with SMase D, which hydrolyzes it to ceramide phosphate, increased the level of inhibition. Although incorporation of ceramide into the substrate in the absence of SM activated the LCAT reaction only modestly, its co-incorporation with SM neutralized the inhibitory effect of SM. Ceramide phosphate, on the other hand, inhibited the LCAT reaction more strongly than SM. The effects of the sphingolipids on the phospholipase A and cholesterol esterification reactions of the enzyme were similar, indicating that they regulate the binding of phosphatidylcholine (PC) to the active site, rather than the esterification step. Incorporation of ceramide into the substrate stimulated the synthesis of unsaturated cholesteryl esters at the expense of saturated esters. However, these effects on fatty acid specificity disappeared when the PC substrates were incorporated into an inert diether PC matrix, suggesting that ceramide increases the availability of polyunsaturated PCs to the enzyme by altering the macromolecular structure of the substrate particle. Since the plasma ceramide levels are increased during inflammation, these results indicate that the activity and fatty acid specificity of LCAT may be altered during the inflammatory response.     

Study of phospholipase A2 specificity in substrate-containing structures having different supramolecular organization

The specificity of snake venom phospholipase A2(PLA2) towards a number of phospholipid (PL) substrates, e. g., phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) organized in Triton X-100 mixed micelles, liposomes and proteoliposomes was studied. PC was shown to be more rapidly hydrolyzed in micelles. For other PLs, the rate of hydrolysis decreased in the following sequence: PC greater than PI greater than PE greater than PG. The incorporation into micelles of a non-hydrolyzable by PLA2 sphinogomyelin which, similar to PC, has a choline group, resulted in an increase of PLA2 specificity towards PL that are known to be devoid of this group: PE greater than PI greater than PG greater than PC. Quite a different picture was observed in bilayer liposomal structures: PI congruent to PE greater than PC greater than PG. The incorporation of cytochrome P-450 into liposomes caused the acceleration of PE and PG hydrolysis. The course of the PLA2-catalyzed hydrolysis in model membrane structures seems to be governed primarily by the supramolecular organization and localization of the substrate in the bilayer, but not by its chemical structure.     

Effects of phosphatidylcholine/phosphatidylethanolamine composition in cholesteryl ester-micellar substrates on neutral cholesterol esterase activity

The effect of phospholipid composition in cholesteryl ester (CE)-micellar substrates on neutral cholesterol esterase (N-CEase) activity was examined. N-CEase preparation was incubated with micelles composed of cholesteryl-[1-14C]-oleate, sodium taurocholate, and phosphatidylcholine (PC)/phosphatidylethanolamine (PE) at varying ratios (%PE:0 = PC only, 17, 33, 50, 66, 83). The activity increased dependently with the increase in PE content; the activity with the micelles containing the highest ratio of PE was 2.5-fold compared with the micelles consisting of PC only. Vmax with the micelles of 83, 66, and 50% PE was 3.1-, 2.7-, and 1.9-fold, respectively, compared with the micelles of PC only. Each micellar preparation was chromatographed through a Superose 6 column by the FPLC system. In 66 and 83% PE-containing micelles, PC, PE, CE, and part of sodium taurocholate eluted completely together in a single peak, whereas in micelles with 33 and 50% PE they eluted loosely together. The micelles with PC only or 17% PE formed PC-micelles without including CE and PE. It is concluded that PE plays a critical role in the formation of CE micelles with PC, and in the interaction with N-CEase. The CE-micelles with 66-83% PE serve as substrates for sensitive and reproducible N-CEase assay.     

Effects of age-dependent or liposome-induced alterations in the phospholipid composition on sphingomyelin biosynthesis in rat liver microsomal and plasma membranes.

1. The effects of age-dependent or liposome-induced alterations in the phospholipid composition of rat liver plasma and microsomal membranes on the phosphatidylethanolamine:ceramide-phosphoethanolamine (PE:Cer-PEt) and phosphatidylcholine:ceramide-phosphocholine (PC:Cer-PCh) transferase activities were studied. 2. In all cases under study the PE:Cer-PEt transferase activity was found to be several times higher than that of PC:Cer-PCh transferase in both plasma and microsomal rat liver membranes. 3. The presence of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) in plasma membranes was observed to enhance the PE:Cer-PEt transferase activity, while phosphatidylserine (PS) inhibited it.     

Head group precursors modify phospholipid synthesis in Schistosoma mansoni

The two predominant phospholipids in schistosomula of Schistosoma mansoni are phosphatidylcholine (PC) and phosphatidylethanolamine (PE) which are found in a molar ratio of 0.52 (PE/PC). The incorporation of four fatty acids (arachidonic, myristic, oleic, and palmitic) and glycerol into phospholipids of schistosomula was measured. In two different media (one containing ethanolamine, the other without), all four fatty acids were predominantly incorporated into PC with a PE/PC ratio of approximately 0.1 in a 90-min label. After a 24-h chase, PC remained the predominant labeled phospholipid but the fatty acid-labeled PE/PC ratio increased slightly, the specific activity of labeled neutral lipids decreased, and the specific activity of labeled PE increased. Glycerol was incorporated with a ratio of 0.55 in the presence of ethanolamine but only 0.19 in its absence. Schistosomula also incorporate fatty acids into phosphatidylmonomethylethanolamine (PMME) and phosphatidyldimethylethanolamine (PDME) at rates intermediate to that into PE and PC in the presence of the respective head group precursor; this incorporation was inhibited by choline. Relative to PC, oleic acid is incorporated into PE, PMME, and PDME at rates higher than for palmitic acid. These results suggest that schistosomula possess acyltransferase(s) with head group specificity and that acyl chains are transferred from neutral lipids to phospholipids over time.     

Peroxidation and phospholipase A2 hydrolytic susceptibility of liposomes consisting of mixed species of phosphatidylcholine and phosphatidylethanolamine.

The relationship between lipid peroxidation and phospholipase A2 (PLA2) hydrolytic activity was studied using unilamellar vesicles (liposomes) as model membranes. Hydrolytic specificity was examined using vesicles prepared with pure bovine heart phosphatidylcholine (PC), bovine heart phosphatidylethanolamine (PE), or mixtures of these phospholipids, using two preparative procedures, i.e., sonication or extrusion. Lipid peroxidation was induced by incubating vesicles with cumene hydroperoxide and hematin at 37 degrees C. Determinations of the extent of peroxidation by means of diene conjugate content derived from second derivative spectra or by polarographic measurement of oxygen consumption rates provided a basis for comparing the extent of peroxidation of each phospholipid species to their subsequent hydrolysis by PLA2 (from Crotalus adamanteus). The extent of hydrolysis was determined through the release of arachidonic acid from either PC or PE. The PE distribution among the outer vs. inner leaflet of the membrane bilayer was nearly equal in sonicated vesicles, whereas most of the phospholipid was incorporated into the inner leaflet in extruded vesicles. The proportion of PE found in the inner leaflet progressively increased as the ratio of PE to PC increased in both sonicated and extruded vesicle preparations. Lipid peroxidation had no effect on PE distribution under the conditions examined. There was a clear preference for PC peroxidation for all vesicle compositions tested and PC was preferentially hydrolyzed by PLA2. This effect is proposed to result from a perturbation of membrane structure following peroxidation with assimilation of PC into PLA2-susceptible domains whereas PE peroxidation and hydrolysis is less affected in mixed PC/PE vesicles. Lipid peroxidation imposes an additional hydrolytic susceptibility over the effects exerted through the mixing of these phospholipids which is based on structural changes rather than formation of specific substrates for PLA2.     

Effect of a fatty acid moiety of phospholipid and ceramide on purified GalT-3 (UDP-Gal:GM2 beta 1-3 galactosyltransferase) activity from embryonic chicken brain

Galactosyltransferase, GalT-3 (UDP-Gal:GM2 beta 1-3 galactosyltransferase) has been characterized and solubilized from 19-day-old embryonic chicken brain, and purified to over 2000-fold using mixed-modal chromatography on a omega-aminohexyl Sepharose column and affinity chromatography on a UDP-hexanolamine Sepharose column. The activity of purified GalT-3 was modulated by phospholipids in vitro with stimulation observed specifically with dipalmitoyl phosphatidylethanolamine (PE). All natural phospholipids tested (PE, PC and PI) inhibited GalT-3 activity. Enzyme activity was affected by the structure of the phospholipid vesicle. It was stabilized by the hexagonal (dipalmitoyl PE) structure and inhibited by the bilayer (dielaidoyl PE) structure. The long-chain fatty acid moiety of the glycosphingolipid substrate, GM2, was found to be necessary for optimum enzyme activity. In the absence of fatty acid, the modified substrates, lyso-GM2 and acetyl-GM2, had a 10-fold increased Km and a 4-8 fold decreased Vmax compared to the normal substrate. We postulate that GalT-3 belongs to a group of glycosyltransferases having recognition for both the carbohydrate as well as the hydrophobic domains (HY-CARS) of their substrates and that the fatty acid moiety of either the substrate (GM2) or a heterotropic effector (phospholipid) plays an important role in regulating the activity of this enzyme.     

Partial purification and characterization of phospholipid N-methyltransferases from murine thymocyte microsomes

Significant amounts of phospholipid N-methyltransferase activity in murine thymocytes were found to be distributed on the plasma membrane. The enzyme activity had an optimum pH of 9. The presence of divalent cations, Mg2+ (10 mM) or Ca2+ (1 mM), and EGTA separately in the assay had only a small effect on the enzyme activity. However, addition of both 10 mM Mg2+ and 1 mM Ca2+ increased the enzyme activity. The presence of two enzymes for each conversion of phosphatidylethanolamine (PE) to phosphatidylmonomethylethanolamine (PME) and PME to phosphatidylcholine (PC) was suggested by the result of the determination of the incorporated radioactivity into PME, phosphatidyldimethylethanolamine (PDE) and PC; the apparent Km values for S-adenosyl-L-methionine were 20 and 400-500 microM for the conversion of PE to PME and for the conversion of PME to PC they were 5 microM and 40 microM. S-Adenosyl-L-homocysteine (AdoHcy), a known inhibitor of enzymatic methylation, competitively inhibited [14C]methyl incorporation into total lipid. The apparent Ki value for AdoHcy was 44.7 microM. Two phospholipid N-methyltransferases were partially purified by extraction with sodium deoxycholate, gel filtration on Sephadex G-75, and affinity column chromatography on AdoHcy-Sepharose. One enzyme, mainly catalyzing the formation of PME, was purified approximately 1548-fold and the other catalyzing the formation of PDE and PC, was purified approximately 629- to 703-fold. However, the former still contained a little activity for PDE and PC formation and the latter contained a little activity for PME formation. In these partially purified phospholipid N-methyltransferase preparations, little contaminating protein O-carboxylmethyltransferase activity was observed; however, significant PC-phospholipase A2 activity was detected. This result may suggest that phospholipid N-methyltransferases associate with phospholipase A2 in the thymocyte plasma membrane.     

Properties of phosphatidylethanolamine-containing phospholipid-apolipoprotein complexes modified by lecithin-cholesterol acyltransferase

The effect of the inclusion of phosphatidylethanolamine (PE), a phospholipid with unusual packing properties, on the substrate properties of protein-lipid complexes toward lecithin-cholesterol acyltransferase (LCAT) has been studied. Recombinant particles of apolipoprotein A-I with dimyristoylphosphatidylcholine (DMPC), dilauroylphosphatidylethanolamine (DLPE) and cholesterol were prepared at a molar ratio of 1:140:14 (A-I/DMPC/cholesterol) or 1:70:70:14 (A-I/DMPC/DLPE/cholesterol); the efficiency of cholesterol incorporation into complexes containing phosphatidylethanolamine was found to be very pH-dependent, with enhanced cholesterol incorporation at elevated pH values. By incubating the complexes with either purified human LCAT or the d greater than 1.21 g/ml fraction of rat serum as a source of LCAT activity, it was found that a high degree of cholesterol esterification could be achieved with either complex; however, the DLPE-containing complex possessed a much smaller Stokes' diameter than the DMPC-only particle despite compositional similarities between these complexes. With respect to particle diameter the DLPE-containing particles behaved more like complexes prepared with egg yolk lecithin than did complexes prepared with DMPC alone. When human LDL was added to the incubations to provide a source of additional cholesterol, the products were markedly different. Concomitant with an increased cholesteryl ester core was an increase in the protein stoichiometry in both types of particles, from 2 to 3 or 4 apo A-I per particle. The proportion of DLPE to DMPC in the products was reduced from 1:1 to 0.3:1, reflecting a preferential hydrolysis of PE by LCAT, and the Stokes' diameters of the DMPC-only and the DLPE-containing complexes were closely similar. We conclude that the presence of elevated proportions of certain phospholipid species may significantly alter both the physical properties of the particles and their substrate properties with regard to reactions with enzymes of lipid metabolism.     

Arachidonic acid turnover in response to lipopolysaccharide and opsonized zymosan in human monocyte-derived macrophages.

Macrophages are an important source of the lipid mediators, arachidonic acid metabolites and platelet-activating factor (PAF), produced during inflammation. Studies were undertaken to identify the phospholipid substrates that can serve as a source of arachidonic acid in human monocyte-derived macrophages exposed to the inflammatory stimuli bacterial lipopolysaccharide (LPS) and opsonized zymosan (OpZ). Since PAF is derived from 1-alkyl-2-acyl-glycerophosphocholine, it was of interest to determine if this phospholipid precursor could also serve as a source of arachidonic acid. The day-5 macrophages incorporated 38% of the available [3H]arachidonic acid into lipid by 4 h, 54% of which was in phospholipid [phosphatidylcholine (PC) greater than phosphatidylethanolamine (PE) greater than phosphatidylinositol (PI)]. The proportion of label incorporated into ether-linked PC and PE increased with time. After prelabelling with [3H]arachidonic acid, the effect of stimuli on the redistribution of label within phospholipids was followed. Without stimulus there was a loss of label from PC, PI and phosphatidic acid by 3 h, but an increase of label in PE. The [3H]arachidonic acid that was lost from PC in the absence of stimulus was derived solely from the 1-acyl-linked species of PC, whereas an increase in label occurred in the 1-alkyl-linked species of PC. By contrast, LPS stimulation resulted in a preferential, dose-dependent loss of label from PC and PI, which was maximal between 1 and 3 h after adding the LPS. In addition, LPS induced a 35% decrease in the molar quantity of PI in the macrophages but had no effect on the quantity of PC, PE or phosphatidylserine. Stimulation with OpZ also resulted in a loss of label, mainly from PC and PI. Of the total label lost from PC in response to LPS or OpZ, approx. 50% was derived from the 1-alkyl-linked species. The results suggest that phospholipase C- and phospholipase A2-mediated mechanisms for arachidonic acid release are activated in human macrophages exposed to the inflammatory stimuli LPS and OpZ. In addition, 1-alkyl-linked PC can serve as a source of arachidonic acid and as a precursor for PAF production in the stimulated macrophages.     

Phospholipid-transfer proteins in human liver and primary liver carcinoma

Investigations have been carried out on phospholipid-transfer activity of the cytosol and the phospholipid composition of subcellular membranes from human liver and primary liver carcinoma. In both human liver and primary liver carcinoma cytosolic fractions, the transfer activity for phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin has been observed for the first time. The transfer rate of PC and PE in normal human liver was almost equal, whereas sphingomyelin-transfer activity was much slower. In carcinoma cells, the transfer activity for PE and PC was significantly enhanced, while sphingomyelin transfer remained unchanged. Comparative investigations with HepG2 cultured cells have revealed a high PE-transfer activity in this cell line. Parallel with the phospholipid-transfer activity modifications in neoplasic cells, changes in the phospholipid composition of microsomes and mitochondria have been observed. The content of PC and PE in hepatocarcinoma cells was decreased in microsomes, while in the mitochondria it was increased. The possible role of the phospholipid-transfer proteins in the maintenance of membrane composition and structure is discussed.     

Incorporation of radioiodinated fatty acids into cardiac phospholipids of normoxic canine myocardium

The aim of this study was to assess the phospholipid distribution of radioiodinated 17-iodoheptadecanoic acid (IHDA), 15-(p-iodophenyl)pentadecanoic acid (p-IPPA) and 15-(p-iodophenyl)-3,3-dimethylpentadecanoic acid (DMIPPA) under normoxic conditions and to compare these data with the fatty acid composition of the phospholipid classes. After simultaneous i.v. injection of the radioiodinated fatty acids (1-123-IHDA; 1-131-p-IPPA; 1-125 DMIPPA) in open-chest dogs seven myocardial biopsies were taken over 40 min (n = 26). After lipid extraction of the biopsies the organic phase was analyzed for both neutral and polar lipids by two different TLC systems. The following polar lipid fractions were analyzed: lysophopshatidylcholine (LPC), sphingomyelin (SPH), phosphatidy1choline (PC; lecithin), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG; cardiolipin) and neutral lipids. Fractions were counted in a gamma well counter and corrected for cross-over and recovery. Results of the polar phospholipids analysis showed that IHDA has the highest incorporation into the phospholipids. The IHDA was mainly incorporated into PI (45.6%) followed by PC (30.9%), PE (14.0%) and PS (5.6%). The p-IPPA was predominantly incorporated incorporated into PC (37.2%), followed by PS (20.1%) and PE (13.7%). In contrast to IHDA, incorporation of p-IPPA into PI was small (6.4%). The DMIPPA analogue was incorporated into phopsholipids to only a very small degree, compared to IHDA and p-IPPA. PS (27.4%) was the only considerable phospholipid fraction into which DMIPPA was incorporated.The results clearly demonstrated that these radioiodinated fatty acid analogues have entirely different patterns of phospholipid incorporation. Major resemblances have been found between the incorporation into phospholipids of IHDA and the phospholipid distribution of the natural counterpart: stearic acid. The p—IPPA phospholipid incorporation only partly resembles the phospholipid distribution of palmitic acid. DMIPPA is because of its modified structure, incorporated into phospholipids to a low extent, mainly into PS. (Mol Cell Biochem116: 79–87, 1992)     

An assessment of phospholipid methylation in sarcolemma and sarcoplasmic reticulum of the aging myocardium

The stepwise N-methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) (phospholipid methylation) was assessed in cardiac sarcolemma and sarcoplasmic reticulum of aging rats. This phenomenon was depressed in aging hearts relative to young ones. A decrease in activity of catalytic sites appears to be involved in the depressed phospholipid methylation of aging myocardium.     

Cerebral Phospholipid Content and Na+, K+-ATPase Activity During Ischemia and Postischemic Reperfusion in the Mongolian Gerbil

Using bilateral carotid artery occlusion in adult gerbils we examined the effects of ischemia and ischemia/reperfusion on cerebral phospholipid content and Na+,K+-ATPase (EC 3.6.1.3) activity. In contrast to the large changes in phospholipid content and membrane-bound enzyme activity that have been observed in liver and heart tissues, we observed relatively small changes in the cerebral content of total phospholipid, phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) following ischemic intervals of up to 240 min. Following 15 min of ischemia the cerebral content of sphingomyelin (SM) was decreased to less than 50% of control values but returned to near-normal levels with longer ischemic periods. Significant decreases in the cerebral content of phosphatidylinositol (PI) and phosphatidic acid (PA) were observed following shorter intervals of ischemia (15-45 min). Na+,K+-ATPase activity of cerebral homogenates prepared from the brains of gerbils subjected to 30-240 min of ischemia was decreased but significantly different from control activity only after 30 min of ischemia (-29%, p less than or equal to 0.05). With the exception of PS, reperfusion for 60 min following 60 min of ischemia resulted in marked increases in cerebral phospholipid content with PC, SM, PI, and PA levels exceeding and PE levels equal to preischemic values. Longer periods of reperfusion (180 min) resulted in decreases in cerebral phospholipid content toward (PC, SM, PI, and PA) or below (PE) preischemic levels. In contrast, the cerebral content of PS significantly decreased during reperfusion (-51% at 60 min, p less than or equal to 0.05) and remained below preischemic values even after 180 min of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane properties induced by anionic phospholipids and phosphatidylethanolamine are critical for the membrane binding and catalytic activity of human cytochrome P450 3A4

Human cytochrome P450 (CYP) 3A4, a membrane anchoring protein, is the major CYP enzyme present in both liver and small intestine. The enzyme plays a major role in the metabolism of many drugs and procarcinogens. The roles of individual phospholipids and membrane properties in the catalytic activity, membrane binding, and insertion into the membrane of CYP3A4 are poorly understood. Here we report that the catalytic activity of testosterone 6beta-hydroxylation, membrane binding, and membrane insertion of CYP3A4 increase as a function of anionic phospholipid concentration in the order phosphatidic acid (PA) > phosphatidylserine (PS) in a binary system of phosphatidylcholine (PC)/anionic phospholipid and as a function of phosphatidylethanolamine (PE) content in ternary systems of PC/PE/PA or PC/PE/PS having a fixed concentration of anionic phospholipids. These results suggest that PA and PE might help the binding of CYP3A4 to the membrane and the interaction with NPR. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4 in all tested phospholipids vesicles with various compositions. Phospholipid-dependent changes of the CYP3A4 conformation were also revealed by altered Trp fluorescence and CD spectra. We also found that PE induced the formation of anionic phospholipid-enriched domains in ternary systems using extrinsic fluorescent probes incorporated into lipid bilayers. Taken together, it can be suggested that the chemical and physical properties of membranes induced by anionic phospholipids and PE are critical for the membrane binding and catalytic activity of CYP3A4.     

The role of cholesterol in the activity of reconstituted Ca-ATPase vesicles containing unsaturated phosphatidylethanolamine

The effect of cholesterol on the activity of sarcoplasmic reticulum Ca-ATPase, reconstituted in proteoliposomes containing soybean phosphatidylethanolamine (PE), egg phosphatidylcholine (PC), and cholesterol, was examined. The protein incorporation efficiency increased with PE content but appeared to be independent of cholesterol content. At low cholesterol, PE stimulated calcium uptake. The coupling efficiency of the proteoliposomes increased with an increase in cholesterol content at each PC/(PC + PE) ratio and was more pronounced for those proteoliposomes containing high PE. Dynamic fluorescence measurements of the incorporated lipophilic probe, diphenyl-1,3,5-hexatriene, revealed a decrease in the motion and an increase in the order of the phospholipid fatty acyl chains in proteoliposomes with high cholesterol content. A complementary observation was made using electron spin resonance of the spin label, 2,2-dimethyl-5-dodecyl-5-methyloxazolidine N-oxide. Freeze-fracture electron microscopy studies on proteoliposomes containing 0.20 molar ratio of PC/(PC + PE) and cholesterol revealed predominantly vesicular structures with occasional bilayer defects at high cholesterol content. It is postulated that the cholesterol-induced enhancement of the Ca-transport function of the Ca-ATPase is related to the hydration-related bilayer-destabilizing characteristic of the cholesterol molecule as revealed by 31P NMR.     

Cardiolipin synthases of Escherichia coli have phospholipid class specific phospholipase D activity dependent on endogenous and foreign phospholipids

E. coli has three Cls-isoenzymes for cardiolipin (CL) synthesis but the differences between these three enzymes remain unresolved. All three Cls enzymes contain the phospholipase D (PLD) characteristic HKD motive and synthesize CL using PLD activity. Here, using LC-MS we show the effect of overexpressing or deletion of the three individual Cls enzymes on the lipidome, which included changes in lipid class distribution and CL species profiles. We demonstrate, for the first time, that overexpression of only ClsB resulted in the appreciable synthesis of a variety of phosphatidylalcohols, thereby establishing a ‘classic’ PLD activity for this enzyme: phospholipid headgroup exchange. Endogenous E. coli lipids and primary alcohols were substrates for this trans-phosphatidylation reaction. Furthermore, we show that endogenous levels of ClsA mediated a similar trans-phosphatidylation reaction to form phosphatidylalcohols, however this reaction was dependent on the presence of the foreign phospholipid class phosphatidylcholine (PC). This allows us to clarify the different specificities of the cardiolipin synthases.     

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.