Effect of phosphatidylinositol replacement by diacylglycerol on various physical properties of artificial membranes with respect to the role of phosphatidylinositol response

In an attempt to gain insight into the physiological role of phosphatidylinositol turnover enhanced by extracellular stimuli, the physical properties of artificial membranes (egg yolk phosphatidylcholine/bovine brain phosphatidylserine) containing phosphatidylinositol or diacylglycerol were studied by ESR using spin probes and freeze-fracture electron microscopy. Diacylglycerol lost both the ability to form lipid bilayer structures and its susceptibility to calcium ions. Yeast phosphatidylinositol included in dipalmitoylphosphatidylcholine liposomes lowered the phase transition temperature of dipalmitoylphosphatidylcholine and expanded the temperature range of phase transition. However, diacylglycerol at the same concentration did not undergo the effects caused by phosphatidylinositol but the phase transition temperature was slightly raised. Phase separation of phosphatidylserine induced by calcium ions was enhanced when the phosphatidylinositol was replaced by diacylglycerol in phosphatidylcholine/ phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) mixtures. The mobility of phosphatidylcholine spin probe was decreased in phosphatidylcholine/ phosphatidylserine/diacylglycerol (3:5:2, by molar ratio) liposomes compared with phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) liposomes. An additional component from protonated stearic acid spin probes was observed in phosphatidylcholine/phosphatidylinositol (8:2, by molar ratio) liposomes at 40°C, whereas the component was not seen in phosphatidylcholine/diacylglycerol (8:2, by molar ratio) liposomes. This may indicate the alteration of surface charge induced by the replacement of phosphatidylinositol by diacylglycerol. Indeed, in the presence of 1 mM Ca²⁺, the additional component was removed by an electrostatic interaction between Ca²⁺ and phosphatidylinositol molecules in phosphatidylcholine/phosphatidylinositol liposomes at 40°C. These results support the hypothesis that the enhanced turnover of phosphatidylinositol may play a triggering role for various cellular responses to exogenous stimuli by altering membrane physical states.     

Physical properties of phosphatidylcholine-phosphatidylinositol liposomes in relation to a calcium effect

Physical properties of binary mixtures of dipalmitoylphosphatidylcholine and yeast phosphatidylinositol were studied by ESR analysis using TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) and lipid spin probes, freeze-fracture electronmicroscopy and particle microelectrophoresis, and they were compared with those of phosphatidylcholine/bovine brain phosphatidylserine mixtures. The phase diagram of the binary mixtures of dipalmitoylphosphatidylcholine and phosphatidylinositol was obtained from the thermal features of TEMPO spectral parameter in the lipid mixtures. The phase diagram provided evidence that these two phospholipids in various combinations were miscible in the crystalline state. The addition of 10 mM Ca2+ slightly shifted the phase diagram upward. TEMPO titration of the binary mixture of dipalmitoylphosphatidylcholine and bovine brain phosphatidylserine revealed that 10 mM Ca2+ caused the complete phase separation of this lipid mixture. Studies of phase separations using phosphatidylcholine spin probe manifested that 10 mM Ca2+ induced almost complete phase separation in egg yolk phosphatidylcholine/bovine brain phosphatidylserine mixtures but only slight phase separation in egg yolk phosphatidylcholine/yeast phosphatidylinositol mixtures. However, some phase changes around the fluidus and the solidus curves were visualized by the freeze-fracture electronmicroscopy. The molecular motion of lipid spin probe was decreased by the addition of Ca2+ in the liposomes containing phosphatidylinositol. The temperature dependence of electrophoretic mobility was also examined in the absence and presence of 1 mM Ca2+. Liposomes of dipalmitoylphosphatidylcholine-phosphatidylinositol (90 : 10, mol/mol) exhibited a clear transition in the thermal features of electrophoretic mobilities. Raising the phosphatidylinositol content up to 25 mol% rendered the transition broad and unclear. The addition of 1 mM Ca2+ decreased the electrophoretic mobility but did not change its general profile of the thermal dependence. These results suggest that the addition of calcium ions induced a small phase change in the binary mixture of phosphatidylcholine and phosphatidylinositol while Ca2+ causes a remarkable phase separation in phosphatidylcholine/phosphatidylserine mixture. The physical role of phosphatidylinositol is discussed related to the formation of diacylglycerol.     

Phospholipid dependence of calcium ion effects on electrophoretic mobilities of liposomes

Electrophoretic light scattering (ELS) and depolarization of fluorescence have been used to determine the effect of membrane fluidity on the binding of Ca2+ to liposomes. ELS was used to measure the electrophoretic mobilities of the liposomes. Fluorescence depolarization was used to determine membrane fluidity. Zero to 30 mol% phosphatidylserine (PS) was incorporated into liposomes containing, as bulk phospholipids, one of the following: dimyristoyl-phosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (PC), or hydrogenated egg phosphatidylcholine (H egg PC). The binding of Ca2+ to the liposomes appears to be influenced by membrane fluidity. Liposomes containing bulk phospholipids whose phase transition temperature is higher than the experimental temperature exhibit enhanced binding of CA2+.     

Reconstitution of the skeletal sarcoplasmic reticulum Ca2(+)-pump: influence of negatively charged phospholipids.

The Ca2(+)-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in the presence of phosphatidyl choline using the freeze-thaw sonication technique. The effect of incorporation of negatively charged phospholipids, phosphatidylserine and phosphatidylinositol phosphate, into the phosphatidylcholine proteoliposomes was investigated. Various ratios of phosphatidylserine or phosphatidylinositol phosphate to phosphatidylcholine were used, while the total amount of phospholipid in the reconstituted vesicles was kept constant. Enrichment of phosphatidylcholine proteoliposomes by phosphatidylserine or phosphatidylinositol phosphate was associated with activation of Ca2(+)-uptake and Ca2(+)-ATPase activities. The highest activation was obtained at a 50:50 molar ratio of phosphatidylserine:phosphatidylcholine and at a 10:90 molar ratio of phosphatidylinositol phosphate:phosphatidylcholine. The initial rates of Ca2(+)-uptake obtained at 1 microM Ca2+ were 2.6 +/- 0.1 mumol/min per mg of phosphatidylserine:phosphatidylcholine proteoliposomes and 1.5 +/- 0.1 mumol/min per mg of phosphatidylinositol phosphate:phosphatidylcholine proteoliposomes, compared to 0.9 +/- 0.05 mumol/min per mg of phosphatidylcholine proteoliposomes. These findings suggest that negatively charged phospholipids may be involved in the activation of the reconstituted skeletal muscle sarcoplasmic reticulum Ca2(+)-pump.     

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.     

Interaction of bovine brain phospholipid exchange protein with liposomes of different lipid composition

The major phospholipid exchange protein from bovine brain catalyzes the transfer of phosphatidylinositol and phosphatidylcholine between rat liver microsomes and sonicated liposomes. The effect of liposomal lipid composition on the transfer of these phospholipids has been investigated. Standard liposomes contained phosphatidylcholine-phosphatidic acid (98:2, mol%); in general, phosphatidylcholine was substituted by various positively charged, negatively charged, or zwitterionic lipids. The transfer of phosphatidylinositol was essentially unaffected by the incorporation into liposomes of phosphatidic acid, phosphatidylserine, or phosphatidylglycerol (5–20 mol%) but strongly depressed by the incorporation of stearylamine (10–40 mol%). Marked stimulation (2–4-fold) of transfer activity was observed into liposomes containing phosphatidylethanolamine (2–40 mol%). The inclusion of sphingomyelin in the acceptor liposomes gave mixed results: stimulation at low levels (2–10 mol%) and inhibition at higher levels (up to 40 mol%). Cholesterol slightly diminished transfer activity at a liposome cholesterol/phospholipid molar ratio of 0.81. Similar effects were noted for the transfer to phosphatidylcholine from microsomes to these various liposomes. Compared to standard liposomes, the magnitude of $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ tended to increase for liposomes which depressed phospholipid transfer and to decrease for those which stimulated; little change was observed in the values of $\text{V}$. Single phospholipid liposomes of phosphatidylinositol were inhibitory when added to standard liposomes.     

Regulation of plasma membrane Ca2+ ATPases by lipids of the phosphatidylinositol cycle

When the erythrocyte plasma membrane Ca2+ pump is reconstituted into phosphatidylcholine liposomes, the inclusion of small amounts of phosphatidic acid or phosphatidylinositol 4,5-bisphosphate stimulates the enzyme's activity. Other lipids of the phosphatidylinositol cycle (diacylglycerol, phosphatidylinositol) have little effect. The stimulatory effect of phosphatidylinositol 4,5-bisphosphate is greater than that of calmodulin; this lipid also stimulates the plasma membrane Ca2+ ATPase from rat brain.     

Involvement of the membrane fluidity of lactosylceramide-targeted liposomes in their intrahepatic uptake

Incorporation of N-lignoceroyldihydrolactocerebroside (lactosylceramide) enhanced liver uptake of small unilamellar liposomes consisting of dipalmitoylphosphatidylcholine, cholesterol and dicetyl phosphate (molar ratio, 4:5:1). The increase in liver uptake was mostly accounted for by an enhanced uptake into the parenchymal cells. The enhancing effects of lactosylceramide on uptake of the liposomes into liver in vivo and into isolated parenchymal cells in vitro were greater with dipalmitoylphosphatidylcholine-based liposomes than with dimyristoylphosphatidylcholine-based ones. In contrast, addition of lactosylceramide had no significant effect on egg phosphatidylcholine vesicle uptake. The stimulated uptake of lactosylceramide liposomes by parenchymal cells was counteracted by added asialofetuin. These observations suggest that transfer of the targeted liposomes via a galactose-specific receptor into parenchymal cells may be controlled by the membrane fluidity of the liposomes.     

Ca2+-translocation activities of phosphatidylinositol, diacylglycerol and phosphatidic acid inferred by quin-2 in artificial membrane systems

Ca2+-translocating activities of phosphatidylinositol, diacylglycerol and phosphatidic acid were investigated in phosphatidylcholine liposomes. Using a fluorescent indicator of Ca2+ concentration, quin-2, release of encapsulated Ca2+ from egg yolk phosphatidylcholine liposomes containing 2 mol% of one of these lipids was measured at 37 degrees C. The rate of Ca2+ translocation across the liposomal membrane mediated by phosphatidic acid was about 3-fold larger than those mediated by phosphatidylinositol and diacylglycerol. The result implies that phosphatidic acid has Ca2+-ionophore activity in the agonist dependent metabolism of inositol phospholipids. The ionophoretic activity depended on the degree of unsaturation of the fatty acyl chains. The Ca2+ translocation rate was smallest in dipalmitoylphosphatidic acid, and it increased in the order of dioleoyl-, dilinoleoyl- and dilinolenoyl-phosphatidic acid. Ca2+ mobilization of a stimulated cell is discussed in the light of Ca2+-ionophore activity of phosphatidic acid converted from inositol phospholipids.     

Binding of CTP: phosphocholine cytidylyltransferase to large unilamellar vesicles

We have studied the binding of CTP: phosphocholine cytidylyltransferase from HeLa cell cytosol to large unilamellar vesicles of egg phosphatidylcholine (PC) or HeLa cell phospholipids that contain various amounts of oleic acid. A fatty acid/phospholipid molar ratio exceeding 10% was required for CTP: phosphocholine cytidylyltransferase binding to liposomes. At a fatty acid/phospholipid molar ratio of 1; 85% of the cytosolic CTP: phosphocholine cytidylyltransferase was bound. The enzyme also bound to liposomes with at least 20 mol% palmitic acid, monoolein, diolein or oleoylacetylglycerol. Oleoyl-CoA did not promote enzyme binding to liposomes. Binding to oleate-PC vesicles was blocked by Triton X-100 but not by 1 M KCl, and was reversed by incubation of the vesicles with bovine serum albumin. Cytidylyltransferase bound to egg PC vesicles that contained 33 mol% oleic acid equally well at 4 degrees C and 37 degrees C. The enzyme also bound to dimyristoyl- and dipalmitoylphosphatidylcholine vesicles containing oleic acid at temperatures below the phase transition for these liposomes. Binding of the cytidylyltransferase to egg PC vesicles containing oleic acid, monoolein, oleoylacetylglycerol or diolein resulted in enzyme activation, as did binding to dipalmitoylPC-oleic acid vesicles. However, binding to egg PC-palmitic acid vesicles did not fully activate the transferase. Various mechanisms for cytidylyltransferase interaction with membranes are discussed.     

Lipid composition of miniature pig platelets

1. Analyses of platelet lipid composition were carried out on material pooled from male and female miniature pigs. 2. The cholesterol/phospholipid molar ratio was 0.6. 3. Phosphatidylcholine represents the major class of phospholipids (47%) and phosphatidylinositol the minor (2%). 4. The main fatty acids of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and sphingomyelin were: palmitic, stearic, oleic, linoleic and arachidonic acids. 5. The ratios of saturated to unsaturated fatty acids were: sphingomyelin, 1.7; phosphatidylcholine, 1.2; phosphatidylserine, 0.9; phosphatidylethanolamine and phosphatidylinositol, 0.6. 6. Our results suggests that human and miniature pig platelet lipids bear several characteristics in common. This fact would allow miniature pig to be used as a new experimental model.     

Diacylglycerol causes major structural transitions in phospholipid bilayer membranes

We demonstrate for the first time that major structural changes are imposed on various phospholipid bilayers by diacylglycerol, a product of phosphatidylinositol metabolism. By 5 mole percent in phosphatidylethanolamine a lamellar to hexagonal transition starts that is complete at 10 mole percent. At 30 mole percent it causes the same transition in phosphatidylcholine and forms a cubic phase at 80 mole percent. Diacylglycerol disorders the phosphatidylserine lamellar phase. We view the formation of the non-lamellar phases as diagnostic of the destabilizations that diacylglycerol can cause in membranes. We suggest how DAG may act both in its specific activation of membrane enzymes and in inducing membrane fusion.     

Purification and Characterization of Phospholipase C Preferentially Hydrolysing Phosphatidylcholine in Tetrahymena Membranes

A phospholipase C (PLC) activity that preferentially hydrolyses phosphatidylcholine to diacylglycerol and phosphorylcholine was found to be present in Tetrahymena pyriformis, strain W and most of its activity was recovered in the membrane fraction. This enzyme was extracted with 1% Triton X-100 from the membrane fraction and purified to apparent homogeneity by sequential chromatographies on Fast Q-Sepharose, hydroxyapatite HCA-100S, Mono Q and Superose 12 gel filtration columns. The purified enzyme had specific activity of 2083 nmol of diacylglycerol released/mg of protein/min for dipalmitoylphosphatidylcholine hydrolysis. Its apparent molecular mass was 128 kDa as determined by SDS-polyacrylamide gel electrophoresis and was 127 kDa by gel filtration chromatography, indicating that the enzyme is present in a monomeric form. The enzyme exhibited an optimum pH 7.0 and the apparent K_m value was determined to be 166 μM for dipalmitoylphosphatidylcholine. A marked increase was observed in phosphatidylcholine hydrolytic activity in the presence of 0.05% (1.2 mM) deoxycholate. Ca²⁺ but not Mg²⁺ enhanced the activity at a concentration of 2 mM. This purified phospholipase C exhibited a preferential hydrolytic activity for phosphatidylcholine but much less activity was observed for phosphatidylinositol (～ 9%) and phosphatidylethanolamine (～ 2%).     

Brain cortex phosphatidylserine inhibits phosphatidylinositol turnover in rat anterior pituitary glands

The in vitro effect of bovine brain cortex phosphatidylserine on 32Pi incorporation into phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine of rat anterior pituitary glands was studied. Phosphatidylserine (0.1 to 66.6 microM) decreased the incorporation of 32Pi into phosphatidylinositol, but not phosphatidylcholine or phosphatidylethanolamine, in a concentration-related manner. The inhibitory effect of phosphatidylinositol was similar to that of dopamine in the same experimental conditions. The combined effects of submaximal concentrations of dopamine and phosphatidylserine elicited an apparently additive inhibitory effect on phosphatidylinositol synthesis. The inhibitory effect of phosphatidylserine was completely reversed by haloperidol and sulpiride and only partially by pimozide, antidopaminergic agents which per se do not affect phosphatidylinositol synthesis. The stimulatory effect of TRH to increase 32Pi incorporation into phosphatidylinositol was decreased by phosphatidylserine. These observations suggest that the decrease in prolactin release in the presence of phosphatidylserine may be evoked through a dopaminergic mechanism.     

The cancer chemopreventive agent resveratrol is incorporated into model membranes and inhibits protein kinase C alpha activity

Resveratrol is a phytoalexin found in grapes and other foods that cancer chemopreventive and other biological activities have been attributed recently. We report that resveratrol is able to incorporate itself into model membranes in a location that is inaccessible to the fluorescence quencher, acrylamide. Differential scanning calorimetry revealed that resveratrol considerably affected the gel to liquid-crystalline phase transition of multilamellar vesicles made of phosphatidylcholine/phosphatidylserine and increased the temperature at which the fluid lamellar to H(II) inverted hexagonal transition took place in multilamellar vesicles made of 1,2-dielaidoyl-sn-phosphatidylethanolamine. Such a transition totally disappeared at 2.5 mM of resveratrol (resveratrol/lipid molar ratio of 2:1). This effect on 1, 2-dielaidoyl-sn-phosphatidylethanolamine polymorphism was confirmed through (31)P-NMR, which showed that an isotropic peak appeared at high temperature instead of the H(II)-characteristic peak of 42 mM of resveratrol (resveratrol/lipid molar ratio of 1.5:1). Finally, resveratrol inhibited PKCalpha when activated by phosphatidylcholine/phosphatidylserine vesicles with an IC(50) of 30 microM, whereas when the enzyme was activated by Triton X-100 micelles the IC(50) was 300 microM. These results indicate that the inhibition of PKCalpha by resveratrol can be mediated, at least partially, by membrane effects exerted near the lipid-water interface.     

Synthesis, liposomal formulation and thermal effects on phospholipid bilayers of leuprolide.

A novel liposomal formulation was developed for the encapsulation of the oligopeptide leuprolide (GlpHisTrpSerTyr-D-LeuLeuArgProNHEt), a potent analogue of gonadotropin releasing hormone used in the treatment of advanced prostate cancer, endometriosis and precocious puberty. Leuprolide was synthesized using solid phase methodology on a {3-[(ethyl-Fmoc-amino)-methyl]-1-indol-1-yl}-acetyl AM resin and Fmoc/tBu chemistry. The new liposomal formulation, called 'liposomes in liposomes' is composed of egg phosphatidylcholine:dipalmitoylphosphatidylglycerol in a molar ratio of 98.91:1.09 (internal liposomes) and egg phosphatidylcholine:dipalmitoylphosphatidylglycerol:cholesterol in a molar ratio of 68.71:0.76:30.53 (external liposomes). It offers high encapsulation efficiency (73.8% for leuprolide); it can provide new delivery characteristics and it may have possible advantages in future applications regarding the encapsulation and delivery of bioactive peptides to target tissues. Furthermore, the physicochemical characteristics (size distribution and zeta-potential) of the liposomal formulations and the thermal effects on leuprolide in model lipidic bilayers composed of dipalmitoylphosphatidylcholine were studied using differential scanning calorimetry. Finally, the dynamic effects of leuprolide in an egg phosphatidylcholine/cholesterol system were examined using solid state 13C MAS NMR spectroscopy.     

Membrane properties modulate the activity of a phosphatidylinositol transfer protein from the yeast, Saccharomyces cerevisiae

A phospholipid transfer protein from yeast (Daum, G. and Paltauf, F. (1984) Biochim. Biophys. Acta 794, 385-391) was 2800-fold enriched by an improved procedure. The specificity of this transfer protein and the influence of membrane properties of acceptor vesicles (lipid composition, charge, fluidity) on the transfer activity were determined in vitro using pyrene-labeled phospholipids. The yeast transfer protein forms a complex with phosphatidylinositol or phosphatidylcholine, respectively, and transfers these two phospholipids between biological and/or artificial membranes. The transfer rate for phosphatidylinositol is 19-fold higher than for phosphatidylcholine as determined with 1:8 mixtures of phosphatidylinositol and phosphatidylcholine in donor and acceptor membrane vesicles. If acceptor membranes consist only of non-transferable phospholipids, e.g., phosphatidylethanolamine, a moderate but significant net transfer of phosphatidylcholine occurs. Phosphatidylcholine transfer is inhibited to a variable extent by negatively charged phospholipids and by fatty acids. Differences in the accessibility of the charged groups of lipids to the transfer protein might account for the different inhibitory effects, which occur in the order phosphatidylserine which is greater than phosphatidylglycerol which is greater than phosphatidylinositol which is greater than cardiolipin which is greater than phosphatidic acid which is greater than fatty acids. Although mitochondrial membranes contain high amounts of negatively charged phospholipids, they serve effectively as acceptor membranes, whereas transfer to vesicles prepared from total mitochondrial lipids is essentially zero. Ergosterol reduces the transfer rate, probably by decreasing membrane fluidity. This notion is supported by data obtained with dipalmitoyl phosphatidylcholine as acceptor vesicle component; in this case the transfer rate is significantly reduced below the phase transition temperature of the phospholipid.     

Effect of N-methylation of phosphatidylethanolamine on the fluidity of phospholipid bilayers

The effect of N-methylphosphatidylethanolamine on phase transition and the fluidity of the liposomes made of dipalmitoylphosphatidylcholine or phosphatidylethanolamine was studied by the steady-state fluorescence polarization method and differential scanning calorimetry. N-methylation of phosphatidylethanolamine caused a decrease of fluidity of liposomes made of dipalmitoylphosphatidylcholine, but had little effect on dipalmitoylphosphatidylethanolamine. The liposomes prepared with both phosphatidylcholine and N-methylphosphatidylethanolamine and also phosphatidylethanolamine and N-methylphosphatidylethanolamine could be composed of solid solution and exhibited symmetric phase diagram.     

The protein-mediated net transfer of phosphatidylinositol in model systems

The phospholipid monolayer technique has been used to study the transfer activity of the phospholipid exchange protein from beef brain. In measuring the transfer between a monolayer consisting of equimolar amounts of phosphatidylcholine and phosphatidylinositol and liposomes consisting of 98 mol% phosphatidylcholine and 2 mol% phosphatidylinositol, the beef brain protein demonstrates an 8-fold higher transfer activity for phosphatidylinositol than for phosphatidylcholine. Under similar conditions the phosphatidylcholine exchange protein from beef liver showed a great preference for phosphatidylcholine. Phosphatidylcholine liposomes devoid of phosphatidylinositol still functioned as receptors of phosphatidylinositol when the beef brain exchange protein was present. This indicates that this protein can catalyse a net transfer of phosphatidylinopsitol. Binding of both phosphatidylinositol and phosphatidylcholine to the beef brain protein was shown.     

Diacylglycerol kinase from pig brain. Purification and phospholipid dependencies

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