Rapid transbilayer movement of fluorescent phospholipid analogues in the plasma membrane of endocytosis-deficient yeast cells does not require the Drs2 protein.

Evidence is presented that endocytosis-deficient Saccharomyces cerevisiae end4 yeast cells rapidly internalize the fluorescent phospholipid analogues 1-palmitoyl-2-{6-[7-nitro-2,1, 3-benzoxadiazol-4-yl(NBD)amino] caproyl}phosphatidylcholine (P-C6-NBD-PtdCho) and P-C6-NBD-phosphatidylserine (P-C6-NBD-PtdSer). Both analogues redistributed between the exoplasmic and cytoplasmic leaflet with a half-time of < 15 min at 0 degrees C. The plateau of internalized analogues was about 70%. Transbilayer movement is probably protein-mediated, as the flip-flop of both analogues was very slow in liposomes composed of plasma-membrane lipids. Rapid analogue internalization was not abolished on depletion of intracellular ATP by about 90%. For P-C6-NBD-PtdCho only was a moderate decrease in the plateau of internalized analogues of about 20% observed, while that of P-C6-NBD-PtdSer was not affected. The Drs2 protein plays only a minor role, if any, in the rapid transbilayer movement of analogues in S. cerevisiae end4 cells. In S. cerevisiae end4 Deltadrs2 cells harbouring both an end4 allele and a drs2 null allele, about 60% and 50% of P-C6-NBD-PtdCho and P-C6-NBD-PtdSer, respectively, became internalized within 15 min at 0 degrees C. The preferential orientation of P-C6-NBD-PtdSer to the cytoplasmic leaflet is in qualitative agreement with the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet, as assessed by binding of annexin V. Virtually no binding of annexin V to spheroplasts of the parent wild-type strain or the mutant strains was observed. Likewise, no difference in the exposure of endogenous aminophospholipids to the exoplasmic leaflet between these strains was found by labelling with trinitrobenzenesulfonic acid. Thus, lipid asymmetry, at least of aminophospholipids, was preserved in S. cerevisiae end4 cells independently of the presence of the Drs2 protein.     

Lipid domains in the exoplasmic and cytoplasmic leaflet of the human erythrocyte membrane: a spin label approach

The existence of different lipid domains in the monolayers of the human erythrocyte membrane was investigated at 4 °C by employing spin-labelled phospholipid analogues. Spectra of analogues located exclusively either in the exoplasmic or in the cytoplasmic leaflet of erythrocyte membranes were recorded. Spectra were simulated by variation of order parameter describing the average amplitude of motion of the long molecular axis of the nitrogen 2pπ orbital of the spin label and of the respective correlation times. For both leaflets at least three components were required to fit the experimental spectra, differing mainly in the order parameter. While the parameters of each component are not very different between both membrane halves, the relative contribution of each component to the spectrum is different between the exoplasmic and cytoplasmic leaflet. The order parameter of the most fluid component, presumably resembling the lipid bulk phase, is smaller in the cytoplasmic leaflet in comparison to the exoplasmic one. The lateral coexistence of different lipid domains in the human red blood cell membrane is concluded. The molecular nature of those domains is discussed. Received: 6 November 1998 / Revised version: 25 January 1999 / Accepted: 29 January 1999     

Dynamics of lipid chain attached fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) in negatively charged membranes determined by NMR spectroscopy

We have determined the average location and dynamic reorientation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) attached to a C12 sn-2 chain of a phosphatidylserine (PS) analogue (C12-NBD-PS) in zwitterionic phosphatidylcholine (PC) and negatively charged phosphatidylserine (PS) host membranes. (1)H magic angle spinning nuclear Overhauser enhancement spectroscopy indicates a highly dynamic reorientation of the aromatic molecule in the membrane. The average location of NBD is characterized by a broad distribution function along the membrane director with a maximum indicating the location of the probe in the lipid/water interface of the lipid membrane. This behavior can be explained by a backfolding of the sn-2 chain towards the aqueous phase. Small differences in the distribution profiles of the NBD group along the membrane normal between PC and PS host membranes were found: in a PC host membrane, the NBD distribution has its maximum in the glycerol region; in a PS host membrane, NBD resides mostly in the upper chain region. These differences may be accounted for by packing differences in the PC versus PS host membranes. As seen by (2)H NMR order parameters, PS bilayers show a much higher packing density compared to PC membranes. Consequently, backfolding of the sn-2 chain with the NBD group attached causes a larger decrease of molecular order of the sn-1 chain in PS than in PC membranes. The broad distributions obtained for lipid chain attached NBD molecules reflect the motional freedom and molecular disorder in the liquid-crystalline lipid membrane.     

Separation of intact plasmalogens and all other phospholipids by a single run of high-performance liquid chromatography

Plasmalogens are a unique subclass of glycerophospholipids characterized by the presence of a vinyl ether bond at the sn-1 position of the glycerol backbone, and they are found in high concentration in cellular membranes of many mammalian tissues. However, separation of plasmalogens as intact phospholipids has not been reported. This article describes a high-performance liquid chromatographic method that can separate intact ethanolamine plasmalogens (pl-PEs) and choline plasmalogens (pl-PCs) as well as all other phospholipid classes usually found in mammalian tissues by a single chromatographic run. The separation was obtained using an HPLC diol column and a gradient of a hexane/isopropanol/water system containing 1% acetic acid and 0.08% triethylamine. The HPLC method allowed a clear separation of plasmalogens from their diacyl analogues. The HPLC method, as applied to the study of peroxidation in human erythrocytes by a hydroperoxide, demonstrated that pl-PEs were targeted twice as much as their diacyl analogues.     

The role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes.

To elucidate the role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes, we examined the interaction of erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion of human erythrocytes was monitored by light microscopy as well as spectrophotometrically by the octadecylrhodamine dequenching assay. Phospholipid translocation and distribution between the inner and the outer leaflet of intact red blood cells were determined with spin-labeled phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Significant fusion of lipid-asymmetric red blood cells where PS and PE are predominantly oriented to the inner leaflet was only observed at Ca2+ concentrations greater than or equal to 10 mM (in the presence of 10 mM phosphate buffer) while fusion of lipid-symmetric erythrocyte membranes was established at greater than or equal to 1.5 mM Ca2+. The Ca2+ threshold of fusion of lipid-asymmetric red blood cells was significantly reduced (i) after exposure of PS to the outer layer but not after redistribution of PE alone, and (ii) upon incorporation of spin-labeled PS into the outer leaflet of red blood cells. Spin-labeled PE or PC did not affect fusion, suggesting that the serine headgroup is an important factor in calcium-phosphate-induced fusion.     

Headgroup-specific exposure of phospholipids in ABCA1-expressing cells

ABCA1 has been established to be required for the efflux of cholesterol and phospholipids to apolipoproteins such as apoA-I. At present, it is unclear whether ABCA1-mediated lipid exposure is specific with regard to lipid headgroups and whether it requires calcium activation and the presence of a lipid acceptor. In the present work, we found exofacial exposure of endogenous phosphatidylserine in the absence of apoA-I to be enhanced in ABCA1-GFP expressing MDCKII and HeLa cells compared with control cells. By using C6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-labeled phospholipid analogues, we observed elevated redistribution of phosphatidylserine and phosphatidylethanolamine but not of phosphatidylcholine analogues from the cytoplasmic to the exoplasmic leaflet of the plasma membrane of ABCA1-GFP expressing cells. Whereas glyburide affected neither the level of exofacial endogenous PS nor the outward movement of the amino phospholipid analogues, the latter was sensitive to intracellular Ca2+ in ABCA1-GFP expressing cells, further enhancing outward analogue redistribution with respect to control cells. Both receptor-mediated endocytosis and fluidphase endocytosis were reduced in MDCKII cells expressing ABCA1-GFP. Glyburide raised the level of receptor-mediated endocytosis in the ABCA1-GFP expressing cell to the level of control cells in the absence of glyburide. In control cells, however, fluid-phase endocytosis but not receptor-mediated endocytosis was significantly reduced upon glyburide treatment.     

Type IV P-type ATPases Distinguish Mono- versus Diacyl Phosphatidylserine Using a Cytofacial Exit Gate in the Membrane Domain

Type IV P-type ATPases (P4-ATPases) use the energy from ATP to “flip” phospholipid across a lipid bilayer, facilitating membrane trafficking events and maintaining the characteristic plasma membrane phospholipid asymmetry. Preferred translocation substrates for the budding yeast P4-ATPases Dnf1 and Dnf2 include lysophosphatidylcholine, lysophosphatidylethanolamine, derivatives of phosphatidylcholine and phosphatidylethanolamine containing a 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) group on the sn-2 C6 position, and were presumed to include phosphatidylcholine and phosphatidylethanolamine species with two intact acyl chains. We previously identified several mutations in Dnf1 transmembrane (TM) segments 1 through 4 that greatly enhance recognition and transport of NBD phosphatidylserine (NBD-PS). Here we show that most of these Dnf1 mutants cannot flip diacylated PS to the cytosolic leaflet to establish PS asymmetry. However, mutation of a highly conserved asparagine (Asn-550) in TM3 allowed Dnf1 to restore plasma membrane PS asymmetry in a strain deficient for the P4-ATPase Drs2, the primary PS flippase. Moreover, Dnf1 N550 mutants could replace the Drs2 requirement for growth at low temperature. A screen for additional Dnf1 mutants capable of replacing Drs2 function identified substitutions of TM1 and 2 residues, within a region called the exit gate, that permit recognition of dually acylated PS. These TM1, 2, and 3 residues coordinate with the “proline + 4” residue within TM4 to determine substrate preference at the exit gate. Moreover, residues from Atp8a1, a mammalian ortholog of Drs2, in these positions allow PS recognition by Dnf1. These studies indicate that Dnf1 poorly recognizes diacylated phospholipid and define key substitutions enabling recognition of endogenous PS.     

Phosphatidylserine exposure in living cells

Abstract

P4-ATPases, a subfamily of P-type ATPases, translocate cell membrane phospholipids from the exoplasmic/luminal leaflet to the cytoplasmic leaflet to generate and maintain membrane lipid asymmetry. Exposure of phosphatidylserine (PS) in the exoplasmic leaflet is well known to transduce critical signals for apoptotic cell clearance and platelet coagulation. PS exposure is also involved in many other biological processes, including myoblast and osteoclast fusion, and the immune response. Moreover, mounting evidence suggest that PS exposure is critical for neuronal regeneration and degeneration. In apoptotic cells, PS exposure is induced by irreversible activation of scramblases and inactivation of P4-ATPases. However, how PS is reversibly exposed and restored in viable cells during other biological processes remains poorly understood. In the present review, we discuss the physiological significance of reversible PS exposure in living cells, and the putative roles of flippases, floppases, and scramblases.     

Exposure of endogenous phosphatidylserine at the outer surface of stimulated platelets is reversed by restoration of aminophospholipid translocase activity

Phosphatidylserine (PS) in the plasma membrane of nonactivated human platelets is almost entirely located on the cytoplasmic side. Stimulation of platelets with the Ca2+ ionophore A23187 or combined action of collagen plus thrombin results in a rapid loss of the asymmetric distribution of PS. Also, treatment with the sulfhydryl-reactive compounds diamide and pyridyldithioethylamine (PDA) causes exposure of PS at the platelet outer surface. PS exposure is sensitively measured as the catalytic potential of platelets to enhance the rate of thrombin formation by the enzyme complex factor Xa-factor Va, since this reaction is essentially dependent on the presence of a PS-containing lipid surface. In this paper we demonstrate that endogenous PS, previously exposed at the outer surface during cell activation or sulfhydryl oxidation, can be translocated back to the cytoplasmic leaflet of the membrane by addition of dithiothreitol (DTT) but not by nonpermeable reducing agents like reduced glutathione. Treatment of platelets with trypsin or chymotrypsin, prior to addition of DTT, inhibits the inward transport of exposed PS. Moreover, severe depletion of metabolic ATP, as obtained by platelet stimulation with A23187 in the presence of metabolic inhibitors, though not inhibiting PS exposure at the outer surface, blocks the translocation of endogenous PS to the internal leaflet of the plasma membrane. These results strongly indicate the involvement of a membrane protein in the inward transport of endogenous PS. Recently, an aminophospholipid-specific translocase in the platelet membrane was postulated on the basis of the inward transport of exogenously added PS (analogues) [Sune, A., Bette-Bobillo, P., Bienvenue, A., Fellmann, P., & Devaux, P.F. (1987) Biochemistry 26, 2972-2978].(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP and GSH dependence of MRP1-mediated outward translocation of phospholipid analogs in the human erythrocyte membrane

The active outward translocation of phospholipid analogues from the inner to the outer membrane leaflet of human erythrocytes by the multi-drug resistance protein MRP1 (ABCC1) depends on intracellular reduced glutathione (GSH). Entrapment of ATP and increasing amounts of GSH inside resealed ghosts prepared from erythrocytes resulted in an up to six-fold increase of the translocation rate. Entrapped oxidized glutathione (GSSG) acted inhibitory but produced stimulation after addition of the disulphide-reducing reagent dithioerythritol. Modification of GSH by esterification of the C-terminal carboxylate of Gly, removal of the N-terminal Glu or substitution of the SH group by an anionic S-dicarboxyethyl or sulphonate group abolished stimulation. The effect of S-alkylation of GSH depended on the length of the alkyl group. S-methyl GSH was somewhat more effective than GSH, but maximal stimulation was similar. S-butyl GSH acted poorly stimulatory while S-hexyl GSH was essentially ineffective. Analyses of the kinetic data of translocation revealed K(m) values for GSH and methyl-GSH of respectively 7.4 +/- 2.4 and 4.9 +/- 1.1 mmol l(-1). At high GSH levels and defined constant ATP levels using an ATP-regenerating system, the Km for ATP of the outward translocation was 0.16 +/- 0.02 mmol l(-1). In the same system lacking GSH, the Km for ATP of the inward translocation by the aminophospholipid flippase was 0.53 +/- 0.23 mmol l(-1).     

Effect of oxidative stress on membrane phospholipid and protein organization in human erythrocytes

Membrane phospholipid and protein organization was studied in intact human erythrocytes exposed to phenylhydrazine, an oxidative agent inducer. The evaluation of the membrane phospholipid and protein organization was carried out in terms of asymmetric distribution across the membrane bilayer for the phospholipids, and in terms of accessibility of cleavable sites present on the outer membrane surface for the proteins. Treatment of phenylhydrazine-exposed erythrocytes either with bee venom phospholipase A2 or with trinitrobenzenesulfonic acid indicated that phosphatidylserine (PS), which is the only phospholipid not formally present on the outer leaflet of the membrane, was translocated to the outer surface of the cell membrane. The extent of this phenomenon was directly proportional to the concentration of the oxidant having a peak value at 0.1 mM. Phosphatidylcholine and phosphatidylethanolamine conserved their original distribution across the erythrocyte membrane throughout the study. The oxidant, at a dose which did not induce any modification of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis cytoskeleton membrane protein pattern, did not provoke any alteration of the membrane protein surface architecture, although the translocation of PS to the membrane outer leaflet in intact erythrocytes was present.     

A molecular switch in SecA protein couples ATP hydrolysis to protein translocation

SecA, the dimeric ATPase subunit of bacterial protein translocase, catalyses translocation during ATP-driven membrane cycling at SecYEG. We now show that the SecA protomer comprises two structural modules: the ATPase N-domain, containing the nucleotide binding sites NBD1 and NBD2, and the regulatory C-domain. The C-domain binds to the N-domain in each protomer and to the C-domain of another protomer to form SecA dimers. NBD1 is sufficient for single rounds of SecA ATP hydrolysis. Multiple ATP turnovers at NBD1 require both the NBD2 site acting in cis and a conserved C-domain sequence operating in trans. This intramolecular regulator of ATP hydrolysis (IRA) mediates N-/C-domain binding and acts as a molecular switch: it suppresses ATP hydrolysis in cytoplasmic SecA while it releases hydrolysis in SecY-bound SecA during translocation. We propose that the IRA switch couples ATP binding and hydrolysis to SecA membrane insertion/deinsertion and substrate translocation by controlling nucleotide-regulated relative motions between the N-domain and the C-domain. The IRA switch is a novel essential component of the protein translocation catalytic pathway.     

ATP and GSH dependence of MRP1-mediated outward translocation of phospholipid analogs in the human erythrocyte membrane

The active outward translocation of phospholipid analogues from the inner to the outer membrane leaflet of human erythrocytes by the multi-drug resistance protein MRP1 (ABCC1) depends on intracellular reduced glutathione (GSH). Entrapment of ATP and increasing amounts of GSH inside resealed ghosts prepared from erythrocytes resulted in an up to six-fold increase of the translocation rate. Entrapped oxidized glutathione (GSSG) acted inhibitory but produced stimulation after addition of the disulphide-reducing reagent dithioerythritol. Modification of GSH by esterification of the C-terminal carboxylate of Gly, removal of the N-terminal Glu or substitution of the SH group by an anionic S-dicarboxyethyl or sulphonate group abolished stimulation. The effect of S-alkylation of GSH depended on the length of the alkyl group. S-methyl GSH was somewhat more effective than GSH, but maximal stimulation was similar. S-butyl GSH acted poorly stimulatory while S-hexyl GSH was essentially ineffective. Analyses of the kinetic data of translocation revealed K_m values for GSH and methyl-GSH of respectively 7.4±2.4 and 4.9±1.1 mmol l^?1. At high GSH levels and defined constant ATP levels using an ATP-regenerating system, the K_m for ATP of the outward translocation was 0.16±0.02 mmol l^?1. In the same system lacking GSH, the K_m for ATP of the inward translocation by the aminophospholipid flippase was 0.53±0.23 mmol l^?1.     

Loss of P4 ATPases Drs2p and Dnf3p disrupts aminophospholipid transport and asymmetry in yeast post-Golgi secretory vesicles

Eukaryotic plasma membranes generally display asymmetric lipid distributions with the aminophospholipids concentrated in the cytosolic leaflet. This arrangement is maintained by aminophospholipid translocases (APLTs) that use ATP hydrolysis to flip phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external to the cytosolic leaflet. The identity of APLTs has not been established, but prime candidates are members of the P4 subfamily of P-type ATPases. Removal of P4 ATPases Dnf1p and Dnf2p from budding yeast abolishes inward translocation of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS, PE, and phosphatidylcholine (PC) across the plasma membrane and causes cell surface exposure of endogenous PE. Here, we show that yeast post-Golgi secretory vesicles (SVs) contain a translocase activity that flips NBD-PS, NBD-PE, and NBD-PC to the cytosolic leaflet. This activity is independent of Dnf1p and Dnf2p but requires two other P4 ATPases, Drs2p and Dnf3p, that reside primarily in the trans-Golgi network. Moreover, SVs have an asymmetric PE arrangement that is lost upon removal of Drs2p and Dnf3p. Our results indicate that aminophospholipid asymmetry is created when membrane flows through the Golgi and that P4-ATPases are essential for this process.     

ATP9B, a P4-ATPase (a putative aminophospholipid translocase), localizes to the trans-Golgi network in a CDC50 protein-independent manner

Type IV P-type ATPases (P4-ATPases) are putative phospholipid flippases that translocate phospholipids from the exoplasmic (lumenal) to the cytoplasmic leaflet of lipid bilayers and are believed to function in complex with CDC50 proteins. In Saccharomyces cerevisiae, five P4-ATPases are localized to specific cellular compartments and are required for vesicle-mediated protein transport from these compartments, suggesting a role for phospholipid translocation in vesicular transport. The human genome encodes 14 P4-ATPases and three CDC50 proteins. However, the subcellular localization of human P4-ATPases and their interactions with CDC50 proteins are poorly understood. Here, we show that class 5 (ATP10A, ATP10B, and ATP10D) and class 6 (ATP11A, ATP11B, and ATP11C) P4-ATPases require CDC50 proteins, primarily CDC50A, for their exit from the endoplasmic reticulum (ER) and final subcellular localization. In contrast, class 2 P4-ATPases (ATP9A and ATP9B) are able to exit the ER in the absence of exogenous CDC50 expression: ATP9B, but not ATP11B, was able to exit the ER despite depletion of CDC50 proteins by RNAi. Although ATP9A and ATP9B show a high overall sequence similarity, ATP9A localizes to endosomes and the trans-Golgi network (TGN), whereas ATP9B localizes exclusively to the TGN. A chimeric ATP9 protein in which the N-terminal cytoplasmic region of ATP9A was replaced with the corresponding region of ATP9B was localized exclusively to the Golgi. These results indicate that ATP9B is able to exit the ER and localize to the TGN independently of CDC50 proteins and that this protein contains a Golgi localization signal in its N-terminal cytoplasmic region.     

Aminophospholipid translocase of human erythrocytes: phospholipid substrate specificity and effect of cholesterol

The outside-inside translocation rate and transmembrane equilibrium distribution, at 37 degrees C, of 16 different amphiphilic spin-labeled phospholipids have been determined in human erythrocytes. The transmembrane distribution was assessed by bovine serum albumin extraction of the spin-labels present in the outer monolayer. Within 15 min, more than 90% of the phosphatidylserine analogue was found in the inner monolayer; the equilibrium distribution of phosphatidylethanolamine spin-label was approximately 85-90% inside, with a half-time for translocation of approximately 50 min. In contrast, phosphatidylcholine reached a distribution corresponding to approximately 30% of the labels inside with a half-time of approximately 8 h, and only traces of sphingomyelin were found in the inner monolayer after 16 h. Thus, the spin-label analogues distributed themselves like endogenous phospholipids in red cells with a spontaneous segregation between the amino lipids and the choline-containing phospholipids. Progressive methylation of the amine group of phosphatidylethanolamine resulted in a stepwise decrease of the specific transport; modification of the beta-carbon of the serine also decreased the efficiency of the rapid translocation without abolishing it. Phosphatidyl-propanolamine was not transported. Substitution of the glyceride group by a ceramide abolished the rapid outside-inside translocation even with a molecule bearing a serine head group. Also it was found that esterification of the sn-2 position of the glycerol component was necessary for a rapid translocation since lysophosphatidylserine was only slowly transported from outside to inside.(ABSTRACT TRUNCATED AT 250 WORDS)     

Natural phosphatidylcholine is actively translocated across the plasma membrane to the surface of mammalian cells

The cell surface of eukaryotic cells is enriched in choline phospholipids, whereas the aminophospholipids are concentrated at the cytosolic side of the plasma membrane by the activity of one or more P-type ATPases. Lipid translocation has been investigated mostly by using short chain lipid analogs because assays for endogenous lipids are inherently complicated. In the present paper, we optimized two independent assays for the translocation of natural phosphatidylcholine (PC) to the cell surface based on the hydrolysis of outer leaflet phosphoglycerolipids by exogenous phospholipase A2 and the exchange of outer leaflet PC by a transfer protein. We report that PC reached the cell surface in the absence of vesicular traffic by a pathway that involved translocation across the plasma membrane. In erythrocytes, PC that was labeled at the inside of the plasma membrane was translocated to the cell surface with a half-time of 30 min. This translocation was probably mediated by an ATPase, because it required ATP and was vanadate-sensitive. The inhibition of PC translocation by glibenclamide, an inhibitor of various ATP binding cassette transporters, and its reduction in erythrocytes from both Abcb1a/1b and Abcb4 knockout mice, suggest the involvement of ATP binding cassette transporters in natural PC cell surface translocation. The relative importance of the outward translocation of PC as compared with the well characterized fast inward translocation of phosphatidylserine for the overall asymmetric phospholipid organization in plasma membranes remains to be established.     

Synthetic branched-chain analogues of distearoylphosphatidylcholine: structure-activity relationship in inhibiting and activating protein kinase C

A series of distearoylphosphatidylcholine (DSPC) analogues having various branched alkyl chains were synthesized and tested for their abilities to regulate protein kinase C (PKC). The greatest improvement (about 3-fold) in the PKC inhibitory activity over that seen for the parental lipid (i.e., DSPC) was accomplished by substitution of 8-methylstearate at sn-2 and 16-methylstearate at both sn-1 and sn-2 positions of glycerol; substitutions at both sn-1 and sn-2 with 8-methylstearate, on the other hand, caused a decrease (about 4-fold) in its inhibitory activity. Introduction of butyl, phenyl, or keto functions to various positions in the fatty alkyl chain substituted at both sn-1- and sn-2 positions imparted upon the DSPC analogues an ability to potently stimulate PKC to an extent comparable to those attainable by diacylglycerol or phorbol ester; the analogues having substitution only at the sn-2 position, in comparison, had no or reduced stimulatory activity. The butyl, phenyl, and keto analogues of DSPC, as with DSPC itself and its methyl analogues, inhibited PKC at high concentrations. Kinetic analysis indicated that the methyl DSPC analogues inhibited the enzyme competitively with respect to phosphatidylserine (PS; a phospholipid cofactor) and Ca2+. The butyl analogues activated the enzyme without affecting its affinity for PS or Ca2+, indicating a mechanism different from that seen for diacylglycerol or phorbol ester. The inhibitory activity of the methyl DSPC analogues and the stimulatory activity of the butyl DSPC analogues were reduced when PKC was activated by phorbol ester. Both classes of the analogues were unable to compete for the binding of [3H]phorbol dibutyrate to PKC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate specificity and partial characterization of the PAF-acylhydrolase in human serum that rapidly inactivates platelet-activating factor

The structure of the potent inflammatory mediator, platelet-activating factor, is 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC, PAF-acether). Human sera contain an acid labile factor (ALF) that is a Ca+2-independent 2-acylhydrolase-specific for AGEPC and AGEPC-like molecules. The enzyme functions by catalytically removing the sn-2 acetyl moiety from AGEPC, producing the biologically inactive sn-2 hydroxy form or 2-lyso-GEPC. Incubation of ALF with sn-2 acyl PAF analogs indicated that the enzyme hydrolyzes the sn-2 fatty acid only if the chain length is five carbons or less, the sn-1 position fatty acid length is greater than 10 carbon units, and at least one methyl group is present on the terminal amine of the choline group. The enzyme was active with either an ether or ester linkage at the sn-1 position. ALF is inactivated by heating to 65 degrees C for 30 min. It is pronase and trypsin sensitive but resistant to papain and papain with dithiothreitol. Further characteristics of human ALF indicated a broad pH range of activity with an optimum of pH 6.2 and an isoelectric point of 6.2 to 6.7. The specificity and Ca+2 independence of human ALF sets it apart from phospholipase A2. It is proposed that human ALF be called human serum PAF-acylhydrolase to distinguish it from other hydrolases currently known to exist.     

Phosphatidylserine synthase 2: high efficiency for synthesizing phosphatidylserine containing docosahexaenoic acid

Phosphatidylserine (PS), the major anionic phospholipid in eukaryotic cell membranes, is synthesized by the integral membrane enzymes PS synthase 1 (PSS1) and 2 (PSS2). PSS2 is highly expressed in specific tissues, such as brain and testis, where docosahexaenoic acid (DHA, 22:6n-3) is also highly enriched. The purpose of this work was to characterize the hydrocarbon-chain preference of PSS2 to gain insight on the specialized role of PSS2 in PS accumulation in the DHA-abundant tissues. Flag-tagged PSS2 was expressed in HEK cells and immunopurified in a functionally active form. Purified PSS2 utilized both PE plasmalogen and diacyl PE as substrates. Nevertheless, the latter was six times better utilized, indicating the importance of an ester linkage at the sn-1 position. Although no sn-1 fatty acyl preference was noted, PSS2 exhibited significant preference toward DHA compared with 18:1 or 20:4 at the sn-2 position. Preferential production of DHA-containing PS (DHA-PS) was consistently observed with PSS2 purified from a variety of cell lines as well as with microsomes from mutant cells in which PS synthesis relies primarily on PSS2. These findings suggest that PSS2 may play a key role in PS accumulation in brain and testis through high activity toward DHA-containing substrates that are abundant in these tissues.