Bilayer membrane destabilization induced by dolichylphosphate

Small vesicles containing the fluorescent probe calcein were used to investigate the effect of dolichyl phosphate (Dol-P) on phospholipid bilayer stability. In the absence of Dol-P, phospholipid vesicles retained the fluorescent probe upon the addition of divalent cations. Small vesicles containing Dol-P, however, exhibited calcein leakage when incubated in the presence of divalent cations. This effect was observed in liposomes composed of a mixture of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and Dol-P, but not in PC/Dol-P liposomes. The rate of calcein leakage was proportional to divalent cation concentration and to temperature, but was independent of vesicle concentration. These results demonstrate that Dol-P has significant effects on the stability of PE containing phospholipid bilayers. Vesicle leakage was also promoted by the addition of rat liver Dol-P-mannose synthase (EC 2.4.1.83) to intact PE/PC/Dol-P vesicles. Enzyme induced leakage from phospholipid vesicles required the presence of both unsaturated PE and Dol-P. The phospholipid composition of leaky vesicles could be correlated with the lipid matrix required for maximal transferase activity of the rat liver synthase. The destabilizing effects of Dol-P on phospholipid bilayers may therefore be involved in the translocation of activated sugars across biological membranes.     

Phosphatidylinositol synthase from Saccharomyces cerevisiae. Reconstitution, characterization, and regulation of activity

Purified membrane-associated phosphatidylinositol synthase (CDP diacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) from Saccharomyces cerevisiae was reconstituted into unilamellar phospholipid vesicles. Reconstitution of the enzyme was performed by removing detergent from an octylglucoside/phospholipid/Triton X-100/enzyme mixed micelle mixture by Sephadex G-50 superfine column chromatography. The average diameter of the vesicles was 40 nm and chymotrypsin treatment of intact vesicles indicated that over 90% of the reconstituted enzyme had its active site facing outward. The enzymological properties and reaction mechanism of reconstituted phosphatidylinositol synthase were determined in the absence of detergent. The reconstituted enzyme was used as a model system to study the regulation of activity. Phosphatidylinositol synthase was constitutive in wild type cells grown in the presence of water-soluble phospholipid precursors as determined by enzyme activity and immunoblotting. Reconstituted enzyme was not effected by water-soluble phospholipid precursors or nucleotides. Maximum activity was found when the enzyme was reconstituted into phosphatidylcholine: phosphatidylethanolamine: phosphatidylinositol: phosphatidylserine vesicles. Phosphatidylserine stimulated reconstituted activity, suggesting that the local phospholipid environment may regulate phosphatidylinositol synthase activity.     

Utilization of diacylglycerol in phospholipid bilayers by pig brain diacylglycerol kinase and Rhizopus arrhizus lipase

Diacylglycerol kinase purified from pig brain cytosol could use sonication-dispersed diacylglycerol in the presence of its activator, phosphatidylcholine vesicles. However, the kinase failed to significantly use diacylglycerol cosonicated with phosphatidylcholine. Similarly, the kinase could not use diacylglycerol generated in microsomes by the back reaction of diacylglycerol choline phosphotransferase, though phospholipase C treatment of microsomes yielded effective substrate for the kinase. In order to elucidate the mechanism of these discrepant findings, we studied the activity of the purified kinase and Rhizopus arrhizus lipase utilizing dioleoylglycerol incorporated into various phospholipid vesicles. The inaccessibility of diacylglycerol contained in phospholipid vesicles was observed similarly for the two different enzymes. We considered that the apparent enzymic latency of diacylglycerol could be best accounted for by an extremely limited solubility of diacylglycerol in the outer leaflet of phospholipid bilayers. The experimental bases for this interpretation are: 1) diacylglycerol cosonicated with dihexanoyl phosphatidylcholine was exceptionally effective as substrate for the kinase; 2) the enzyme activities with cosonicated and separately sonicated lipids became similar when bile salts were present; 3) both enzymes could use diacylglycerol generated on phosphatidylcholine vesicles by a limited phospholipase C hydrolysis; and 4) phosphatidylcholine diacylglycerol vesicles at widely different molar ratios (from 1:0.014 to 1:0.2) were similarly ineffective as substrate for both enzymes.     

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.     

Regulation of plasma membrane Ca(2+)-ATPase activity by acetylated tubulin: influence of the lipid environment

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca(2+)-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300μg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50μg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300μg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca(2+) transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50μg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.     

Involvement of nonlamellar-prone lipids in the stability increase of human cytochrome P450 1A2 in reconstituted membranes

The effect of nonlamellar-prone lipids, diacylglycerol (DG) and phosphatidylethanolamine (PE), on the stability of human cytochrome P450 1A2 (CYP1A2) was examined. When 100% phosphatidylcholine (PC) in standard vesicles was gradually replaced with either DG or PE, the stability of CYP1A2 increased; the incubation time-dependent destruction of spectrally detectable P450, decrease of catalytic activity, reduction of intrinsic fluorescence, and increased sensitivity to trypsin digestion were significantly alleviated. The ternary system of PC/PE/DG increased the stability of CYP1A2 more, even at lower concentrations of each nonlamellar-prone lipid, than that of the binary lipid mixture (PC/nonlamellar lipid). By incorporating the nonlamellar-prone lipids, the CYP1A2-induced increase of the surface pressure of the lipid monolayer was much higher compared to that for 100% PC. Increased surface pressure indicates a deep insertion of the protein into lipid monolayers. Nonlamellar lipids also increased the transition temperature of CYP1A2 in thermal unfolding and reduced the incubation time-dependent detachment of membrane-bound CYP1A2 from vesicles. Taken together, these results suggest that nonlamellar lipids per se and/or the phase properties of the membrane containing these lipids are important in the enhanced stability of CYP1A2 and the concomitant maintenance of catalytic activity of the protein.     

Modulation of dolichyl-phosphomannose synthase activity by changes in the lipid environment of the enzyme

Rat liver dolichyl-phosphomannose synthase (GDP mannose-dolicholphosphate mannosyltransferase; EC 2.4.1.83) was previously shown to catalyze optimal rates of mannosyl transfer to dolichyl-P when the polyprenol acceptor was incorporated into a phosphatidylethanolamine (PE) matrix that has a tendency to adopt a nonbilayer (hexagonal HII) phase [Jensen, J. W., & Schutzbach, J. S. (1985) Eur. J. Biochem. 153, 41-48]. The present investigations now further define the properties of the lipid environment that are essential for mannosyltransferase activity. Monogalactosyl diglyceride (MGDG), a glycoglycerolipid that prefers a nonbilayer-phase organization in isolation, was shown to provide a suitable lipid matrix for synthase activity. By comparison, the enzyme was not activated by digalactosyl diglyceride (DGDG), which forms stable bilayer structures upon hydration. Enzyme activity in MGDG/DGDG mixtures decreased as the proportion of DGDG in the dispersion was increased. Although bilayer-forming phospholipids supported low rates of mannosyl transfer, enzyme activity was stimulated by the addition of MGDG to either phosphatidylcholine (PC) or PE/PC (1:1) membranes. The incorporation of agents known to destabilize bilayer structures including dolichols, ubiquinone, dodecane, and cholesterol into PE/PC (1:1) membranes also increased the rate of mannosyl transfer. Enzyme activity in PC membranes was stimulated by the presence of gramicidin and also by greatly increased concentrations of the substrate, dolichyl-P. The results demonstrate that the enzyme does not have a requirement for PE and suggest that the physical state of the lipid matrix is an important determinant for reconstitution of the synthase and polyprenol phosphate substrate in a productive complex. The formation of an enzyme/lipid complex was demonstrated by sucrose density gradient centrifugation and could be correlated with the lipid requirements for enzyme activity.     

Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting ordered-disordered and gel-fluid domains

The binding and early stages of activity of a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa on giant unilamellar vesicles (GUV) have been monitored using fluorescence confocal microscopy. Both the lipids and the enzyme were labeled with specific fluorescent markers. GUV consisted of a mixture of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and cholesterol in equimolar ratios, to which 5-10 mol% of the enzyme end-product ceramide and/or diacylglycerol were occasionally added. Morphological examination of the GUV in the presence of enzyme reveals that, although the enzyme diffuses rapidly throughout the observation chamber, detectable enzyme binding appears to be a slow, random process, with new bound-enzyme-containing vesicles appearing for several minutes. Enzyme binding to the vesicles appears to be a cooperative process. After the initial cluster of bound enzyme is detected, further binding and catalytic activity follow rapidly. After the activity has started, the enzyme is not released by repeated washing, suggesting a "scooting" mechanism for the hydrolytic activity. The enzyme preferentially binds the more disordered domains, and, in most cases, the catalytic activity causes the disordering of the other domains. Simultaneously, peanut- or figure-eight-shaped vesicles containing two separate lipid domains become spherical. At a further stage of lipid hydrolysis, lipid aggregates are formed and vesicles disintegrate.     

Regulation of yeast phosphatidylserine synthase and phosphatidylinositol synthase activities by phospholipids in Triton X-100/phospholipid mixed micelles

The regulation of purified yeast membrane-associated phosphatidylserine synthase (CDP-diacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) and phosphatidylinositol synthase (CDP-diacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) activities by phospholipids was examined using Triton X-100/phospholipid mixed micelles. Phosphatidate, phosphatidylcholine, and phosphatidylinositol stimulated phosphatidylserine synthase activity, whereas cardiolipin and the neutral lipid diacylglycerol inhibited enzyme activity. Phosphatidate was a potent activator of phosphatidylserine synthase activity with an apparent activation constant (0.033 mol %) 88-fold lower than the apparent Km (2.9 mol %) for the surface concentration of CDP-diacylglycerol. Phosphatidate caused an increase in the apparent Vmax and a decrease in the apparent Km for the enzyme with respect to the surface concentration of CDP-diacylglycerol. Phosphatidylcholine and phosphatidylinositol caused an increase in the apparent Vmax for phosphatidylserine synthase with respect to CDP-diacylglycerol with apparent activation constants of 3.4 and 3.2 mol %, respectively. Cardiolipin and diacylglycerol were competitive inhibitors of phosphatidylserine synthase activity with respect to CDP-diacylglycerol. The apparent Ki value for cardiolipin (0.7 mol %) was 4-fold lower than the apparent Km for CDP-diacylglycerol, whereas the apparent Ki for diacylglycerol (7 mol %) was 2.4-fold higher than the apparent Km for CDP-diacylglycerol. Phosphatidylethanolamine and phosphatidylglycerol did not affect phosphatidylserine synthase activity. Phosphatidylinositol synthase activity was not significantly effected by lipids. The role of lipid activators and inhibitors on phosphatidylserine synthase activity is discussed in relation to overall lipid metabolism.     

In vitro alterations of L-asparaginase activity of Tetrahymena pyriformis by lipids

A membrane-bound L-asparaginase (EC 3.5.1.1) of Tetrahymena pyriformis was purified to homogeneity. The purified enzyme is a lipoprotein, since it is inactivated by phospholipase C and its activity is restored by the addition of naturally occuring lipids, such as phosphatidylcholine, triolein and oleyl acetate. The relative effectiveness of a variety of phospholipids, free saturated and unsaturated fatty acids, or neutral lipids, such as esters of fatty, acids and glycerides, with respect to the activation of purified L-asparaginase is compared. Enzyme activity is reconstituted in the presence of lipids and evidence for the formation of an enzyme-phospholipid complex is presented. The data of this report suggest that L-asparaginase may have a requirement for lipids that reconstitute a physiological hydrophobic environment, similar to the one existing in vivo.Abbreviations DPPC Dipalmitoylphosphatidylcholine - DPPE Dipalmitoylphosphatidylethanolamine - DMPC Dimyristoylphosphatidylcholine - PS Phosphatidylserine - PI Phosphatidylinositol - IPC Lysophosphatidylcholine - PC Phosphatidylcholine - PE Phosphatidylethanolamine     

Effects of physical states of phospholipids on the incorporation and cytochalasin B binding activity of human erythrocyte membrane proteins in reconstituted vesicles

Proteoliposomes were reconstituted from a Triton extract of human erythrocyte membrane proteins and a mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of varying ratios. With mixtures of egg PC and soybean PE, the protein/lipid ratio of the reconstituted vesicles was maximal at 25% PC and 75% PE, the composition which is known to have a maximum bilayer disruption (highest occurrence of lipidic particles seen by freeze-fracture electron microscopy). With mixtures of 1-palmitoyl-2-oleoyl-PC and dilinoleoyl-PE, which gave vesicles with few isolated lipidic particles at room temperature, the effect was less pronounced. The specific activity of the cytochalasin B (CB) binding protein in the reconstituted vesicles, on the other hand, was increased monotonically up to severalfold as the PC content was increased in the egg PC/soybean PE mixture. A similar increase was observed when soybean PE was partially substituted by dimyristoyl-PC, cholesterol, or transphosphatidylated PE from egg PC. These findings indicate that preexisting defects in the lipid bilayer promote protein incorporation into the bilayer during reconstitution whereas reduction of the bilayer fluidity facilitates the CB binding activity in the reconstituted vesicles.     

Diacylglycerol generated in the phospholipid vesicles by phospholipase C is effectively utilized by diacylglycerol lipase in rat liver cytosol

Diacylglycerol was generated in phosphatidylcholine vesicles by incubation with Clostridium welchii phospholipase C. Newly formed diacylglycerol was rapidly converted to monoacylglycerol and glycerol when rat liver cytosol fraction was present in the incubation mixture, suggesting the presence of di- and monoacylglycerol lipase activities in this subcellular fraction. On the other hand, 3H-labeled diacylglycerol co-emulsified with non-radioactive phosphatidylcholine was found to be a poor substrate for the diacylglycerol lipase. These results indicate that enzymatic generation of diacylglycerol provide a substrate having a suitable physical state for the expression of diacylglycerol lipase activity. It was also found that the rate of diacylglycerol hydrolysis was dependent upon the rate of diacylglycerol generation, but not upon the absolute concentration in the incubation mixture. When the rate of diacylglycerol hydrolysis was plotted against the rate of diacylglycerol generation, a saturation curve was obtained and the double-reciprocal plot gave a straight line. It is not known why a relationship similar to Michaelis-Menten type kinetics was obtained between the rate of diacylglycerol hydrolysis and diacylglycerol generation instead of diacylglycerol concentration, but it may be best explained by the following assumptions: (1) diacylglycerol molecules are generated at the surface of the lipid vesicles where they are readily accessible to diacylglycerol lipase; (2) soon after the generation, diacylglycerol molecules migrate into inside the vesicles where they are inaccessible to the enzyme; (3) the effective concentration of diacylglycerol, i.e., the concentration of diacylglycerol located in the surface layer of the vesicles is proportional to the rate of diacylglycerol generation.     

End-products diacylglycerol and ceramide modulate membrane fusion induced by a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

A phospholipase C/sphingomyelinase from Pseudomonas aeruginosa has been assayed on vesicles containing phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and cholesterol at equimolar ratios. The enzyme activity modifies the bilayer chemical composition giving rise to diacylglycerol (DAG) and ceramide (Cer). Assays of enzyme activity, enzyme-induced aggregation and fusion have been performed. Ultrastructural evidence of vesicle fusion at various stages of the process is presented, based on cryo-EM observations. The two enzyme lipidic end-products, DAG and Cer, have opposite effects on the bilayer physical properties; the former abolishes lateral phase separation, while the latter generates a new gel phase [Sot et al., FEBS Lett. 582, 3230-3236 (2008)]. Addition of either DAG, or Cer, or both to the liposome mixture causes an increase in enzyme binding to the bilayers and a decrease in lag time of hydrolysis. These two lipids also have different effects on the enzyme activity, DAG enhancing enzyme-induced vesicle aggregation and fusion, Cer inhibiting the hydrolytic activity. These effects are explained in terms of the different physical properties of the two lipids. DAG increases bilayers fluidity and decreases lateral separation of lipids, thus increasing enzyme activity and substrate accessibility to the enzyme. Cer has the opposite effect mainly because of its tendency to sequester sphingomyelin, an enzyme substrate, into rigid domains, presumably less accessible to the enzyme.     

Sodium channel activity of lobster nerve membrane treated with purified phospholipase A2

Sodium channel activity was determined by measuring the veratridine-tetrodotoxin-sensitive sodium influx in reconstituted vesicles prepared from lobster nerve membrane and soybean lipids. The sodium channel activity was abolished by treatment of membranes, prior to reconstitution, with purified phospholipase A2. When the treatment with phospholipase A2 was carried out in the presence of bovine serum albumin the channel activity was fully preserved. Electron microscopy revealed that the normal vesicular appearance of the membranes was changed to an amorphous mass by treatment of the membranes with enzyme alone. A population of preserved vesicular structures was observed when bovine serum albumin was present during the enzyme treatment. Analysis of the membrane components indicate that there is no relationship between phospholipid composition and sodium channel activity.     

Cholesterol displacement by ceramide in sphingomyelin-containing liquid-ordered domains, and generation of gel regions in giant lipidic vesicles

Fluorescence confocal microscopy and differential scanning calorimetry are used in combination to study the phase behaviour of bilayers composed of PC:PE:SM:Chol equimolecular mixtures, in the presence or absence of 10mol% egg ceramide. In the absence of ceramide, separate liquid-ordered and liquid-disordered domains are observed in giant unilamellar vesicles. In the presence of ceramide, gel-like domains appear within the liquid-ordered regions. The melting properties of these gel-like domains resemble those of SM:ceramide binary mixtures, suggesting Chol displacement by ceramide from SM:Chol-rich liquid-ordered regions. Thus three kinds of domains coexist within a single vesicle in the presence of ceramide: gel, liquid-ordered, and liquid-disordered. In contrast, when 10mol% egg diacylglycerol is added instead of ceramide, homogeneous vesicles, consisting only of liquid-disordered bilayers, are observed.     

Phospholipase C and diacylglycerol lipase activities associated with plasma membranes of chromaffin cells isolated from bovine adrenal medulla

The plasma membranes of bovine adrenal chromaffin cells were isolated and the activities of enzymes involved in arachidonic acid liberation were investigated. Only a minute activity of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) could be detected using externally added phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as substrate. When membranes were treated with exogenous phospholipase C (orthophosphoric acid diester phosphohydrolase, EC 3.1.4.1) there was a liberation of free fatty acids from the sn-2 position of PC. The enzyme responsible for this effect could be demonstrated to be a diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) localized in the plasma membrane. Using phosphatidylinositol (PI) as a substrate, it was found that an endogenous phospholipase C exists which co-purifies with the membrane preparation. The produced diacylglycerol is subsequently hydrolyzed by diacylglycerol lipase liberating arachidonic acid. The two enzymes, phospholipase C and diacylglycerol lipase were characterized. Phospholipase C was found to be calcium dependent and PI specific, showing an activity of 60 pmol/micrograms protein per h (1.2 mM Ca2+), whereas the diacylglycerol lipase was calcium independent hydrolyzing diacylglycerol at a rate of 7.2 pmol/micrograms protein per h. The lipase but not the phospholipase C was inhibited 50% by 1.7 mM para-bromophenacylbromide.     

Phosphatidylcholine metabolism in endothelial cells: evidence for phospholipase A and a novel Ca2+-independent phospholipase C

The metabolism of phosphatidylcholine (PC) was investigated in sonicated suspensions of bovine pulmonary artery endothelial cells and in subcellular fractions using two PC substrates: 1-oleoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho[14C]choline. When these substrates were incubated with the whole cell sonicate at pH 7.5, all of the metabolized 3H label was recovered in [3H]oleic acid (95%) and [3H]diacylglycerol (5%). All of the 14C label was identified in [14C]lysoPC (92%) and [14C]phosphocholine (8%). These data indicated that PC was metabolized via phospholipase(s) A and phospholipase C. Substantial diacylglycerol lipase activity was identified in the cell sonicate. Production of similar proportions of diacylglycerol and phosphocholine and the low relative activity of phospholipase C compared to phospholipase A indicated that the phospholipase C-diacylglycerol lipase pathway contributed little to fatty acid release from the sn-2 position of PC. Neither phospholipase A nor phospholipase C required Ca2+. The pH profiles and subcellular fractionation experiments indicated the presence of multiple forms of phospholipase A, but phospholipase C activity displayed a single pH optimum at 7.5 and was located exclusively in the particulate fraction. The two enzyme activities demonstrated differential sensitivities to inhibition by p-bromophenacylbromide, phenylmethanesulfonyl fluoride and quinacrine. Each of these agents inhibited phospholipase A, whereas phospholipase C was inhibited only by p-bromophenacylbromide. The unique characteristics observed for phospholipase C activity towards PC indicated the existence of a novel enzyme that may play an important role in lipid metabolism in endothelial cells.     

Phosphatidylethanolamine and phosphatidylcholine are sources of diacylglycerol in ras-transformed NIH 3T3 fibroblasts

Ras-transformation of cells is accompanied by an increase of the level of diacylglycerol (DAG), which participates in the signal transduction pathways. DAG could be generated from phospholipids either by activation of phospholipase C or by a more complex pathway involving phospholipase D and phosphatidate phosphohydrolase. To clarify which phospholipids produce DAG and which pathways are involved, we examined the DAG generating enzyme activities, using phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) as substrates. The study showed that the breakdown of PC and more markedly of PE by phospholipases C and D was stimulated in membranes from ras-transformed cells. Phosphatidate phosphohydrolase activity was also elevated in oncogene-expressing cells. The increase in glycerol uptake was most pronounced in cells given PE, followed by PC. The fatty acid analysis revealed apparent similarities between the acyl chains of PE and DAG only in the transformed cells. These findings suggest that PE is a source of DAG in ras-fibroblasts but does not rule out the role of PC in DAG production, due to the activation of the PC-specific phospholipases C and D.     

Effect of phospholipids on thyroid oligosaccharyltransferase activity and orientation. Evaluation of structural determinants for stimulation of N-glycosylation

Oligosaccharyltransferase solubilized by Nonidet P-40 was found to have a highly specific lipid requirement which is consistent with the lability of the enzyme when removed from its membrane association. Enzyme activity as measured by the N-glycosylation of a hexapeptide acceptor was greatly stimulated and stabilized by phosphatidylcholine (PC) while other naturally occurring phosphoglycerides had minimal effect. The quaternary ammonium group of PC was observed to be involved in the interaction with the enzyme as modification of the choline moiety by removal of methyl groups resulted in a progressive loss of the stimulatory effect (choline greater than N,N-dimethylethanolamine greater than N-monomethylethanolamine greater than ethanolamine) which was reflected primarily in the Vmax rather than the Km values. Evaluation of a number of PC and choline derivatives indicated that the nonpolar domain of the lipid also played an important specifying role. Two hydrophobic chains attached to the phosphoglycerol backbone were found to be essential, and furthermore the length and degree of unsaturation of the fatty acid substituents as well as their position of attachment on the glycerol moiety greatly affected the extent of activation. Since the L-isomer of PC brought about a 3-fold greater stimulation than the D-isomer the interaction of the enzyme with the phospholipid appears to be stereoselective. Upon chromatography of the PC-stabilized enzyme on concanavalin A-agarose almost complete retention occurred at 0.4% Nonidet P-40, while no binding took place at a detergent concentration of 0.075%; this suggested that upon dilution in the presence of PC, the oligosaccharyltransferase was reconstituted into vesicles in an asymmetric fashion with its N-linked carbohydrate located internally. Enzymatic assay of these vesicles demonstrated that the active site of the enzyme was also oriented toward the interior. These studies indicate that the activity as well as the membrane insertion of the oligosaccharyltransferase are to a large measure influenced by its interaction with PC.     

Phosphatidylcholine/sphingomyelin metabolism crosstalk inside the nucleus

It is known that phospholipids represent a minor component of chromatin. It has been highlighted recently that these lipids are metabolized directly inside the nucleus, thanks to the presence of enzymes related to their metabolism, such as neutral sphingomyelinase, sphingomyelin synthase, reverse sphingomyelin synthase and phosphatidylcholine-specific phospholipase C. The chromatin enzymatic activities change during cell proliferation, differentiation and/or apoptosis, independently from the enzyme activities present in nuclear membrane, microsomes or cell membranes. This present study aimed to investigate crosstalk in lipid metabolism in nuclear membrane and chromatin isolated from rat liver in vitro and in vivo. The effect of neutral sphingomyelinase activity on phosphatidylcholine-specific phospholipase C and sphingomyelin synthase, which enrich the intranuclear diacylglycerol pool, and the effect of phosphatidylcholine-specific phospholipase C activity on neutral sphingomyelinase and reverse sphingomyelin synthase, which enrich the intranuclear ceramide pool, was investigated. The results show that in chromatin, there exists a phosphatidylcholine/sphingomyelin metabolism crosstalk which regulates the intranuclear ceramide/diacylglycerol pool. The enzyme activities were inhibited by D609, which demonstrated the specificity of this crosstalk. Chromatin lipid metabolism is activated in vivo during cell proliferation, indicating that it could play a role in cell function. The possible mechanism of crosstalk is discussed here, with consideration to recent advances in the field.