Modulation of bovine milk galactosyltransferase activity by lipids

The effect of lipids singly and in combination on the ability of galactosyltransferase to transfer galactose to N-acetyl-D-glucosamine-forming lactosamine and to glucose forming lactose has been studied. Lecithins, as egg phosphatidylcholine (PC), or saturated as dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine stimulated the activity of the enzyme to form lactosamine to different extents. Egg PC produced the greatest stimulation of all the lecithins tested. Egg phosphatidic acid (PA) inhibited the activity of the enzyme at very low concentrations of lipid. In mixed vesicles of gel phase or liquid crystalline phase lecithins and egg PA, the acidic lipid was able to overcome the stimulation produced by the lecithins. The dominant effect of the head group was demonstrated by the effects of gel phase dimyristoylphosphatidic acid (DMPA). In mixtures with PC, DMPA also was able to inhibit the enzyme for lactosamine synthesis but higher concentrations of the gel phase DMPA were required for inhibition compared to the liquid crystalline PA. Although the head group appeared to dominate the inhibition, the nature of the acyl chains of the lipid played a secondary role at least. Other acid lipids, phosphatidylserine (PS) and phosphatidylinositol (PI) were much less effective than PA. PS alone inhibited the activity of the enzyme. However, in mixed lipids (PS and egg PC), PS was unable to reverse the stimulation produced by PC while PC was able to reverse the inhibition produced by PS. PI alone had no effect on the enzyme activity. In mixtures with egg PC, the stimulating effect of PC was dominant. In the lactose synthetase reaction, the effect of lipids was similar to that of the lactosamine synthetase, i.e. PC stimulated and PA inhibited activity and in mixtures of PC and PA, the inhibitory effect of PA was dominant.     

The protein-mediated transfer of phosphatidylcholine between membranes. The effect of membrane lipid composition and ionic composition of the medium.

The phosphatidylcholine exchange protein from bovine liver catalyzes the transfer of phosphatidylcholine between rat liver mitochondria and sonicated liposomes. The effect of changes in the liposomal lipid composition and ionic composition of the medium on the transfer have been determined. In addition, it has been determined how these changes affected the electrophoretic mobility i.e. the surface charge of the membrane particles involved. Transfer was inhibited by the incorporation of negatively charged phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol into the phosphatidylcholine-containing vesicles; zwitterionic phosphatidyl-ethanolamine had much less of an inhibitory effect while positively charged stearylamine stimulated. The cation Mg2+ and, to a lesser extent, K+ overcame the inhibitory effect exerted by phosphatidic acid, in that concentration range where these ions neutralized the negative surface charge most effectively. Under conditions where Mg2+ and K+ affected the membrane surface charge relatively little inhibition was observed. In measuring the protein-mediated transfer between a monolayer and vesicles consisting of only phosphatidylcholine, cations inhibited the transfer in the order La3+ greater than Mg2+ larger than or equal to Ca2+ greater than K+ = Na+. Inhibition was not related to the ionic strength, and very likely reflects an interference of these cations with an electrostatic interaction between the exchange protein and the polar head group of phosphatidylcholine.     

Hydrolysis of lipid mixtures by rat hepatic lipase

The hydrolysis of phospholipid mixtures by purified rat hepatic lipase, also known as hepatic triglyceride lipase, was studied in a Triton X-100/lipid mixed micellar system. Column chromatography of the mixed micelles showed elution of Triton X-100 and binary lipid mixtures of phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine as a single peak. This indicated that the mixed micelles were homogenous and contained all components in the designated molar ratios. The molar ratio of Triton X-100 to lipid was kept constant at 4 to 1. Labeling one lipid with 3H and the other lipid with 14C enabled us to determine the hydrolysis of both components of these binary lipid mixed micelles. We found that the hydrolysis of phosphatidylcholine was activated by the inclusion of small amounts of phosphatidic acid (2.5-fold), phosphatidylethanolamine (1.5-fold) or phosphatidylserine (1.4-fold). The maximal activation of phosphatidylcholine hydrolysis was observed when 5 mol% of phosphatidylethanolamine, 7.5 mol% phosphatidic acid or 5 mol% phosphatidylserine was added to Triton X-100 mixed micelles. The hydrolysis of phosphatidic acid was activated 30%, and that of phosphatidylserine was inhibited 30% when the molar proportion of phosphatidylcholine was less than 50 mol%. The hydrolysis of phosphatidylethanolamine was slightly activated when the mol% of phosphatidylcholine was below 5. The hydrolysis of phosphatidylserine was inhibited by phosphatidylethanolamine when the mol% of the latter was 50 or less whereas phosphatidylethanolamine hydrolysis was not affected by phosphatidylserine. Under the conditions used sphingomyelin and cholesterol did not have a significant effect on the hydrolysis of the phospholipids studied. In agreement with our previous study (Kucera et al. (1988) J. Biol. Chem. 263, 1920-1928) these studies show that the phospholipid polar head group is an important factor which influences the action of hepatic lipase and that the interfacial properties of the substrate play a role in the expression of the activity of this enzyme. The molar ratios of phosphatidic acid, phosphatidylethanolamine and phosphatidylserine which activated phosphatidylcholine hydrolysis correspond closely to the molar ratios of these lipids found in the surface lipid film of lipoproteins e.g., high density lipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of phospholipids on the activity of sialosyl lactosylceramide (GM3): N-acetylgalactosaminyl transferase from chick embryo brain

Summary A preparation of UDP-Ga1NAc:sialosyl lactosylceramide N-acetylgalactosaminyl transferase (E.C. 2.4.1.92) obtained from 14-day chick embryo brain was delipidated partially by treatment with cold acetone in the presence of varying amounts of sodium dodecyl sulphate (SDS). The lipid content and the enzyme activity of the preparation decreased as the concentration of SDS increased. At 0.3% SDS the lipid content was about 30% and the enzyme activity about 15% of the original. The activity could be restored up to 60% of the original by added phospholipids, provided the removal of endogenous lipids did not exceed 70%. Phospholipids with diferent composition showed different abilities to restore the enzyme activity. Among phosphatidylcholines the decreasing order of effectivity was dilauroyl dimiristoyl dipalmitoyl distearoyl-choline. Dimiristoyl phosphatidylcholine, dimiristoyl phosphatidylglycerol and dipalmitoyl phosphatidylglycerol activated the enzyme more effectively than dimiristoyl phosphatidyl ethanolamine, dimiristoyl phosphatidic acid or brain phosphatidylserine. No correlation was found between the activating ability and the charge in the polar head group of the lipid added. Addition of dilauroyl phosphatidylcholine to the delipidated preparation increased about 5-fold the Vmax without affecting the apparent Km for both the donor nucleotide and acceptor glycolipid. The data suggest that the lipid composition of the enzyme environment constitutes a potential level of regulation of the activity of this key enzyme of ganglioside biosynthesis.     

Phosphatidylinositol transfer protein from bovine brain: Substrate specificity and membrane binding properties

A recently developed fluorimetric transfer assay (Somerharju, P., Brockerhoff, H. and Wirtz, K.W.A. (1981) Biochim. Biophys. Acta 649, 521–528) has been applied to study the substrate specificity and membrane binding of the phosphatidylinositol-transfer protein from bovine brain. The substrate specificity was investigated by measuring the rate of transfer, either directly or indirectly, for a series of phosphatidylinositol analogues which included phosphatidic acid, phosphatidylglycerol as well as three lipids obtained from yeast phosphatidylinositol by partial periodate oxidation and subsequent borohydride reduction. Phosphatidylglycerol and the oxidation products of phosphatidylinositol were transferred at about one tenth of the rate observed for phosphatidylinositol while phosphatidic acid was not transferred. It is concluded that an intact inositol moiety favours the formation of the putative transfer protein-phosphatidylinositol complex. In addition to phosphatidylinositol, the transfer protein also transfers phosphatidylcholine. In order to obtain information on the possible occurrence of two sites of interaction, vesicles consisting of either pure 1-acyl-2-parinaroylphosphatidylinositol or 1-acyl-2-parinaroylphosphatidylcholine were titrated with the protein. Binding of labeled phospholipid to the protein was represented by an increase of lipid fluorescence and found to be much more efficient for phosphatidylinositol than for phosphatidylcholine. This is interpreted to indicate that the protein contains an endogenous phosphatidylinositol molecule which can be easily replaced by exogenous phosphatidylinositol but not by phosphatidylcholine, a lipid with a lower affinity for this protein. Thus the binding sites for the two phospholipids are mutually exclusive, i.e. phosphatidylinositol and phosphatidylcholine cannot be bound to the protein simultaneously. Finally, the effect of acidic phospholipids on the transfer protein activity was studied either by varying the content of phosphatidic acid in the acceptor vesicles or by adding vesicles of pure acidic phospholipids to the normal assay system. The latter vesicles consisted of either phosphatidic acid, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or cardiolipin. In both instances the transfer protein activity was inhibited, obviously through the enhanced association of the protein with the negatively charged vesicles. These findings strongly suggest that relatively nonspecific ionic forces rather than specific protein-phospholipid headgroup interactions contribute to the association of the phosphatidylinositol-transfer protein with membranes.     

Phosphatidylinositol transfer protein from bovine brain. Substrate specificity and membrane binding properties

A recently developed fluorimetric transfer assay (Somerharju, P., Brockerhoff, H. and Wirtz, K.W.A. (1981) Biochim. Biophys. Acta 649, 521-528) has been applied to study the substrate specificity and membrane binding of the phosphatidylinositol-transfer protein from bovine brain. The substrate specificity was investigated by measuring the rate of transfer, either directly or indirectly, for a series of phosphatidylinositol analogues which included phosphatidic acid, phosphatidylglycerol as well as three lipids obtained from yeast phosphatidylinositol by partial periodate oxidation and subsequent borohydride reduction. Phosphatidylglycerol and the oxidation products of phosphatidylinositol were transferred at about one tenth of the rate observed for phosphatidylinositol while phosphatidic acid was not transferred. It is concluded that an intact inositol moiety favours the formation of the putative transfer protein-phosphatidylinositol complex. In addition to phosphatidylinositol, the transfer protein also transfers phosphatidylcholine. In order to obtain information on the possible occurrence of two sites of interaction, vesicles consisting of either pure 1-acyl-2-parinaroylphosphatidylinositol or 1-acyl-2-parinaroylphosphatidylcholine were titrated with the protein. Binding of labeled phospholipid to the protein was represented by an increase of lipid fluorescence and found to be much more efficient for phosphatidylinositol than for phosphatidylcholine. This is interpreted to indicate that the protein contains an endogenous phosphatidylinositol molecule which can be easily replaced by exogenous phosphatidylinositol but not by phosphatidylcholine, a lipid with a lower affinity for this protein. Thus the binding sites for the two phospholipids are mutually exclusive, i.e. phosphatidylinositol and phosphatidylcholine cannot be bound to the protein simultaneously. Finally, the effect of acidic phospholipids on the transfer protein activity was studied either by varying the content of phosphatidic acid in the acceptor vesicles or by adding vesicles of pure acidic phospholipids to the normal assay system. The latter vesicles consisted of either phosphatidic acid, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or cardiolipin. In both instances the transfer protein activity was inhibited, obviously through the enhanced association of the protein with the negatively charged vesicles. These findings strongly suggest that relatively nonspecific ionic forces rather than specific protein-phospholipid headgroup interactions contribute to the association of the phosphatidylinositol-transfer protein with membranes.     

Modulation of the in vitro activity of lysosomal phospholipase A1 by membrane lipids

Lysosomal phospholipases play a critical role for degradation of cellular membranes after their lysosomal segregation. We investigated the regulation of lysosomal phospholipase A1 by cholesterol, phosphatidylethanolamine, and negatively-charged lipids in correlation with changes of biophysical properties of the membranes induced by these lipids. Lysosomal phospholipase A1 activity was determined towards phosphatidylcholine included in liposomes of variable composition using a whole-soluble lysosomal fraction of rat liver as enzymatic source. Phospholipase A1 activity was then related to membrane fluidity, lipid phase organization and membrane potential as determined by fluorescence depolarization of DPH, 31P NMR and capillary electrophoresis. Phospholipase A1 activity was markedly enhanced when the amount of negatively-charged lipids included in the vesicles was increased from 10 to around 30% of total phospholipids and the intensity of this effect depended on the nature of the acidic lipids used (ganglioside GM1相似文献   

Effect of basic protein from human central nervous system myelin on lipid bilayer structure

Summary The effect of myelin basic protein from normal human central nervous system on lipid organization has been investigated by studying model membranes containing the protein by differential scanning calorimetry or electron spin resonance spectroscopy. Basic protein was found to decrease the phase transition temperature of dipalmitoyl phosphatidyl-glycerol, phosphatidic acid, and phosphatidylserine. The protein had a greater effect on the freezing temperature, measured from the cooling scan, than on the melting temperature, measured from the heating scan. These results are consistent with partial penetration of parts of the protein into the hydrocarbon region of the bilayer in the liquid crystalline state and partial freezing out when the lipid has been cooled below its phase transition temperature.The effect of the protein on fatty acid chain packing was investigated by using a series of fatty acid spin labels with the nitroxide group located at different positions along the chain. If the protein has not yet penetrated, it increases the order throughout the bilayer in the gel phase, probably by decreasing the repulsion between the lipid polar head groups. Above the phase transition temperature, when parts of it are able to penetrate, it decreases the motion of the lipid fatty acid chains greatly near the polar head group region, but has little or no effect near the interior of the bilayer. Upon cooling again the protein still decreases the motion near the polar head group region but increases it greatly in the interior. Thus, the protein penetrates partway into the bilayer, distorts the packing of the lipid fatty acid chains, and prevents recrystallization, thus decreasing the phase transition temperature.The magnitude of the effect varied with the lipid and was greatest for phosphatidic acid and phosphatidylglycerol. It could be reversed upon cooling for phosphatidylglycerol but not phosphatidic acid. The protein was only observed to decrease the phase transition temperature of phosphatidylserine upon cooling. It had only a small effect on phosphatidylethanolamine and no effect on phosphatidylcholine. Thus, the protein may penetrate to a different extent into different lipids even if it binds to the polar head group region by electrostatic interactions.     

Stimulation and inhibition of the activity of rat liver cytosolic phosphatidate phosphohydrolase by various phospholipids.

The influence of phospholipids on the activity of the soluble phosphatidate phosphohydrolase from rat liver was studied. Phosphatidylethanolamine stimulated the enzyme activity whereas phosphatidylglycerol, phosphatidylserine, and phosphatidylinositol were inhibitory. At a phospholipid concentration of 0.7 mg/ml, phosphatidylglycerol inhibited phosphatidate phosphohydrolase activity by 75%, while the enzyme activity was stimulated twofold in the presence of phosphatidylethanolamine. Both lysophosphatidylglycerol and lysophosphatidylethanolamine inhibited phosphatidate phosphohydrolase activity as did octylglucoside, sodium cholate, and Tween 20. The finding that phospholipids influence hepatic phosphatidate phosphohydrolase activity indicates that changes in the lipid environment may modulate the enzyme activity.     

Segregation of phosphatidic acid-rich domains in reconstituted acetylcholine receptor membranes

Purified Acetylcholine Receptor (AcChR) from Torpedo has been reconstituted at low (approximately 1:3500) and high (approximately 1:560) protein to phospholipid molar ratios into vesicles containing egg phosphatidylcholine, cholesterol, and different dimyristoyl phospholipids (dimyristoyl phosphatidylcholine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid) as probes to explore the effects of the protein on phospholipid organization by differential scanning calorimetry, infrared, and fluorescence spectroscopy. All the experimental results indicate that the presence of the AcChR protein, even at the lower protein to phospholipid molar ratio, directs lateral phase separation of the monoanionic phosphoryl form of the phosphatidic acid probe, causing the formation of specific phosphatidic acid-rich lipid domains that become segregated from the bulk lipids and whose extent (phosphatidic acid sequestered into the domain, out of the total population in the vesicle) is protein-dependent. Furthermore, fluorescence energy transfer using the protein tryptophan residues as energy donors and the fluorescence probes trans-parinaric acid or diphenylhexatriene as acceptors, establishes that the AcChR is included in the domain. Other dimyristoyl phospholipid probes (phosphatidylcholine, phosphatidylserine, phosphatidylglycerol) under identical conditions could not mimic the protein-induced domain formation observed with the phosphatidic acid probe and result in ideal mixing of all lipid components in the reconstituted vesicles. Likewise, in the absence of protein, all the phospholipid probes, including phosphatidic acid, exhibit ideal mixing behavior. Since phosphatidic acid and cholesterol have been implicated in functional modulation of the reconstituted AcChR, it is suggested that such a specific modulatory role could be mediated by domain segregation of the relevant lipid classes.     

The modulating effects of lipids on purified rat liver Golgi galactosyltransferase

Galactosyltransferase was purified from rat liver Golgi membranes. The Triton X-100, used to solubilize the enzyme was removed immediately prior to the lipid interaction studies. In lipid vesicles, prepared from a variety of phosphatidylcholines (PCs), including egg PC, DOPC, DMPC, DPPC and DSPC, the ability of the lipids to stimulate the enzyme decreased in the order egg PC greater than DOPC greater than DMPC greater than DPPC greater than DSPC, i.e. the lower the transition temperature (Tc) the greater the stimulation of the enzyme. A second, neutral lipid, phosphatidylethanolamine was used to permit a comparison of the effect of a different head group of the same net charge at neutral pH. The PEs included, egg PE, soy PE, Pl-PE, PE(PC) and DPPE in order of increasing Tc. The effect of the PEs was opposite to that of the PCs, i.e. the higher the Tc, the greater the stimulation of the enzyme. In fact egg PE and soy PE which have the lowest Tc values were inhibitory. Thus the modulation of the Golgi membrane galactosyltransferase by these lipids was different from that reported earlier for the bovine milk galactosyltransferase. The effects of two acidic lipids, egg phosphatidic acid (PA) and egg phosphatidylglycerol (PG) were studied also. Both totally inhibited the enzyme even at low concentrations of lipid, however, the PA was more effective than PG. In mixtures of neutral lipid (PC) and acidic lipid (PA or PG), the effect of the acidic lipid dominated. Even in the presence of excess PC, total inhibition of the enzyme was observed. It was concluded that the enzyme bound the acidic lipid preferentially to itself. The choice of the lipids allowed us to make several direct comparisons concerning the effect of the nature of the lipid head group on the activity of the enzyme. For example PE(PC), egg PA and egg PG would have fatty acid chains identical to egg PC since these three lipids are all prepared by modification of egg PC. As well, DPPE differs from DPPC only by nature of the head group. These comparisons indicated that not only the net charge but also chemical nature of the head group were important in the lipid modulation of Golgi galactosyltransferase.     

The stimulation and binding of CTP: phosphorylcholine cytidylyltransferase by phosphatidylcholine-oleic acid vesicles

The activity of the low molecular weight form of cytidylyltransferase from fetal lung cytosol and adult liver cytosol was stimulated more by phosphatidylcholine-oleic acid (1:1 molar ratio) vesicles than by phosphatidylglycerol vesicles. Phosphatidylcholine alone did not stimulate the activity, while oleic acid alone produced only slight stimulation. Vesicles prepared from phosphatidylinositol, phosphatidylglycerol-cholesterol (2:1) and phosphatidylglycerol-phosphatidylcholine (1:1) all stimulated the activity to the same extent. Phosphatidylcholine-oleic acid vesicles (molar ratio 2:1) produced less stimulation than 1:1 vesicles. Phosphatidylcholine-palmitic acid vesicles (2:1) were about 50% as active as the corresponding phosphatidylcholine-oleic acid vesicles. All vesicles were in the size range of small unilamellar vesicles as judged by Sephacryl S-1000 chromatography. Stimulation also occurred when phosphatidylcholine vesicles and oleic acid were added separately to the assay. The stimulation by phospholipid vesicles was correlated with the ability of the vesicles to bind cytidylyltransferase, determined by sucrose density centrifugation of the enzyme-vesicles mixtures. We conclude that the stimulation of soluble cytidylyltransferase occurs through binding of the enzyme to anionic membrane surfaces. Suitable anionic membranes can be prepared either from anionic phospholipids, or by the addition of anionic lipids (unesterified fatty acids or phosphatidylglycerol) to phosphatidylcholine.     

The stimulation and binding of CTP : Phosphorylcholine cytidylyltransferase by phosphatidylcholine-oleic acid vesicles

The activity of the low molecular weight form of cytidylyltransferase from fetal lung cytosol and adult liver cytosol was stimulated more by phosphatidylcholine-oleic acid (1:1 molar ratio) vesicles than by phosphatidylglycerol vesicles. Phosphatidylcholine alone did not stimulate the activity, while oleic acid alone produced only slight stimulation. Vesicles prepared from phosphatidylinositol, phosphatidylglycerol-cholesterol (2:1) and phosphatidylglycerol-phosphatidylcholine (1:1) all stimulated the activity to the same extent. Phosphatidylcholine-oleic acid vesicles (molar ratio 2:1) produced less stimulation than 1:1 vesicles. Phosphatidylcholine-palmitic acid vesicles (2:1) were about 50% as active as the corresponding phosphatidylcholine-oleic acid vesicles. All vesicles were in the size range of small unilamellar vesicles as judged by Sephacryl S-1000 chromatography. Stimulation also occurred when phosphatidylcholine vesicles and oleic acid were added separately to the assay. The stimulation by phospholipid vesicles was correlated with the ability of the vesicles to bind cytidylyltransferase, determined by sucrose density centrifugation of the enzyme-vesicles mixtures. We conclude that the stimulation of soluble cytidylyltransferase occurs through binding of the enzyme to anionic membrane surfaces. Suitable anionic membranes can be prepared either from anionic phospholipids, or by the addition of anionic lipids (unesterified fatty acids or phosphatidylglycerol) to phosphatidylcholine.     

Inhibition of chymase activity by phosphoglycerides

The activity of chymase was markedly inhibited by phosphoglycerides such as phosphatidic acid, phosphatidylserine, and phosphatidylinositol, but was not affected by acylglycerides, phosphoglyceroserine, serine, inositol, or glycerol. These results suggest that both the nonpolar hydrophobic hydrocarbon tails and the polar hydrophilic head are essential for the inhibitory effects of phosphoglycerides. Binding of a primary amine to an anionic polar head of phosphatidic acid, such as in phosphatidylserine and phosphatidylethanolamine, slightly decreased the inhibitory effect of phosphatidic acid and, conversely, binding of a strong cation to the head, such as in phosphatidylcholine, resulted in its activation of chymase. Phosphatidic acid containing an unsaturated fatty acid, such as dioleoyl phosphatidic acid, caused the same extent of inhibition as natural phosphatidic acid from bovine brain, but was 20 times more inhibitory than phosphatidic acid containing a saturated fatty acid, such as distearoyl phosphatidic acid. The inhibition by phosphatidylserine was noncompetitive and pseudoirreversible, and the K_i value was 0.54 μm. The inhibition of chymase by phosphatidylserine was pH dependent, being strong at pH 8.5 to 9.5 but weak below pH 7.5. Phosphatidylserine specifically inhibited chymase and elastase; it did not inhibit the other chymotrypsin-type serine endopeptidases tested, trypsin, papain, collagenase, carboxypeptidase A, or cathepsin D.     

Identification and characterization of the nuclear isoform of Drosophila melanogaster CTP:phosphocholine cytidylyltransferase

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the conversion of phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline for the eventual synthesis of phosphatidylcholine (PC). The enzyme is regulated by reversible association with cellular membranes, with the rate of catalysis increasing following membrane association. Two isoforms of CCT appear to be present in higher eukaryotes, including Drosophila melanogaster, which contains the tandem genes Cct1 and Cct2. Before this study, the CCT1 isoform had not been characterized and the cellular location of each enzyme was unknown. In this investigation, the cDNA encoding the CCT1 isoform from D. melanogaster has been cloned and the recombinant enzyme purified and characterized to determine catalytic properties and the effect of lipid vesicles on activity. CCT1 exhibited a V max of 23904 nmol of CDP-choline min (-1) mg (-1) and apparent K m values for phosphocholine and CTP of 2.29 and 1.21 mM, respectively, in the presence of 20 muM PC/oleate vesicles. Cytidylyltransferases require a divalent cation for catalysis, and the cation preference of CCT1 was found to be as follows: Mg (2+) > Mn (2+) = Co (2+) > Ca (2+) = Ni (2+) > Zn (2+). The activity of the enzyme is stimulated by a variety of lipids, including phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, diphosphatidylglycerol, and the fatty acid oleate. Phosphatidylethanolamine and phosphatidic acid, however, did not have a significant effect on CCT1 activity. The cellular location of both CCT1 and CCT2 isoforms was elucidated by expressing green fluorescent fusion proteins in cultured D. melanogaster Schneider 2 cells. CCT1 was identified as the nuclear isoform, while CCT2 is cytoplasmic.     

Synthesis of saturated phosphatidylcholine and phosphatidylglycerol by freshly isolated rat alveolar type II cells

Saturated phosphatidylcholine and phosphatidylglycerol are important components of pulmonary surface active material, but the relative contributions of different pathways for the synthesis of these two classes of phospholipids by alveolar type II cells are not established. We purified freshly isolated rat type II cells by centrifugal elutriation and incubated them with [1-14C]palmitate as the sole exogenous fatty acid in one series of experiments or with [9,10-3H]palmitate, mixed fatty acids (16:0, 18:1 and 18:2), and [U-14C]glucose in another series of experiments. Type II cells readily incorporated [1-14C]palmitate into saturated phosphatidic acid (55-59% of total phosphatidic acid), saturated diacylglycerol (82-87% of total diacylglycerol), saturated phosphatidylcholine (69-76% of total phosphatidylcholine), and saturated phosphatidylglycerol (55-59% of total phosphatidylglycerol). Saturated phosphatidic acid, diacylglycerol and phosphatidylglycerol were nearly equally labeled in the sn-1 and sn-2 positions, whereas saturated phosphatidylcholine was preferentially labeled in the sn-2 position. With [9,10-3H]palmitate and [U-14C]glucose, the labeling patterns of phosphatidic acid, diacylglycerol and phosphatidylglycerol were similar to each other but different from that of phosphatidylcholine. The glucose label was found predominantly in the unsaturated phosphatidylcholines at early times (3-10 min) and in the saturated phosphatidylcholines at later times (30-90 min). Similarly, the 3H/14C ratio was very high in saturated phosphatidylcholine and always above that in saturated diacylglycerol. We conclude that freshly isolated type II cells synthesize saturated phosphatidic acid, diacylglycerol, phosphatidylcholine and phosphatidylglycerol and that under our in vitro conditions the deacylation-reacylation pathway is important for the synthesis of saturated phosphatidylcholine but is less important for the synthesis of saturated phosphatidylglycerol. By the assumptions stated in the text during the pulse chase experiment de novo synthesis of saturated phosphatidylcholine from saturated diacylglycerol accounted for 25% of the total synthesis of saturated phosphatidylcholine.     

Transfer properties of the bovine brain phospholipid transfer protein. Effect of charged phospholipids and of phosphatidylcholine fatty acid composition

The monolayer technique has been used to study the transfer of [¹⁴C]phosphatidylinositol from the monolayer to phosphatidylcholine vesicles. An equivalent transfer rate was found for egg phosphatidylcholine, dioleoylphosphatidylcholine, dielaidoylphosphatidylcholine and dipalmitoylphosphatidylcholine. A reduced transfer rate was found for a shorter-chain derivative, dimyristoylphosphatidylcholine, and for species with two polyunsaturated fatty acid chains such as dilinoleoylphosphatidylcholine, diheptadecadienoylphosphatidylcholine, dilinolenoylphosphatidylcholine and diether and dialkyl derivatives. No activity was found for 1,3-dipalmitoylphosphatidylcholine. The presence of up to 5 mol% phosphatidylinositol in egg phosphatidylcholine vesicles had no effect on the transfer rate. Introduction of more than 5 mol% phosphatidylinositol or phosphatidic acid into the phosphatidylcholine vesicles gradually decreased the rate of phosphatidylinositol transfer from the monolayer. 20 mol% acidic phospholipid was nearly completely inhibitory. Transfer experiments between separate monolayers of phosphatidylcholine and phosphatidylinositol showed that the protein-bound phosphatidylcholine is readily exchanged for phosphatidylinositol, but the protein-bound phosphatidylinositol exchange for phosphatidylcholine occurs at a 20-times lower rate. The release of phosphatidylinositol is dependent on the lipid composition and the concentration of charged lipid in the acceptor membrane, but also on the ratio between donor and acceptor membranes. The main transfer protein from bovine brain which transfer phosphatidylinositol and phosphatidylcholine transfers also phosphatidylglycerol, but not phosphatidylserine or phosphatidic acid. The absence of significant changes in the surface pressure indicate that the phosphatidylinositol and phosphatidylcholine transfer is not accompanied by net mass transfer.     

Phospholipids differently modulate the activity of cytosolic protein-tyrosine kinase from porcine spleen

Effect of membrane phospholipids on the activity of cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) has been studied. Using poly(Glu Na, Tyr)4:1 as a substrate, phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine had stimulatory effects on that phosphorylation activity, however phosphatidic acid had inhibitory and phosphatidylinositol had no effects. Similar results were obtained using[Val5]angiotensin II as a substrate. On the other hand using basic protein (H2B histone and myelin basic protein) as substrates, phosphatidic acid stimulated the activity of CPTK-40, while phosphatidylinositol inhibited the activity. Phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine caused different effect on the activity of CPTK-40 depending on the substrate employed. However using acidic protein (tubulin and casein) as substrates, the activity of CPTK-40 was neither stimulated nor inhibited by any phospholipids. These results suggest that phospholipids may modulate the activity of CPTK-40.     

Head group and chain length dependence of phospholipid self-assembly studied by spin-label electron spin resonance

The critical micelle concentrations (cmc's) of a variety of spin-labeled phospholipids, 1-acyl-2-[4-(4,4-dimethyloxazolidine-N-oxyl)valeryl]-sn-glycero-3-pho sph o derivatives, have been determined by electron spin resonance (ESR) spectroscopy. The narrow, three-line ESR spectra of the rapidly tumbling monomers are clearly distinguished from the spin-spin broadened spectra of the micellar aggregates, allowing a direct determination of the concentrations of the two species. The influence of both the hydrocarbon chain length and the polar head group on the energetics of self-assembly has been studied. For phosphatidylcholine, 1n [cmc] decreases linearly with the length of the sn-1 chain. The gradient of this linear dependence corresponds to a free energy of transfer of the monomer from the aqueous phase to the micelle of delta Gtr = -1.1RT per CH2 group. The cmc's of the 1-lauroyl derivatives of both phosphatidylcholine and phosphatidylglycerol have relatively shallow, biphasic temperature dependences with a minimum at approximately 20 degrees C. Both of these properties are characteristic of the hydrophobic effect, with the free energy of transfer being slightly less than that for the solubility of n-hydrocarbons in water, corresponding to the reduced configurational entropy of the lipid chains in the micellar state. The cmc's of the 1-lauroyl derivatives of the phospholipids in 0.15 M NaCl, for their various charge states, are as follows: phosphatidic acid(2-), 0.77 mM; phosphatidic acid(1-), 0.13 mM; phosphatidylserine(1-), 0.24 mM; phosphatidylglycerol(1-), 0.17 mM; phosphatidylcholine, 0.10 mM; phosphatidylethanolamine, 0.05 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vanadate-sensitive phosphatidate phosphohydrolase activity in a purified rabbit kidney Na,K-ATPase preparation.

Reconstitution of purified rabbit kidney Na,K-ATPase in phosphatidylcholine/phosphatidic acid liposomes resulted in the absence of ATP in a time-, temperature- and protein-dependent formation of inorganic phosphate. This formation of inorganic phosphate could be attributed to a phosphatidate phosphohydrolase activity present in the Na,K-ATPase preparation. A close interaction of the enzyme with the substrate phosphatidic acid was important, since no or little Pi production was observed under any of the following conditions: without reconstitution, after reconstitution in the absence of phosphatidic acid, with low concentrations of detergent or at low lipid/protein ratios. The hydrolysis of phosphatidic acid was not influenced by the Na,K-ATPase inhibitor ouabain but was completely inhibited by the P-type ATPase inhibitor vanadate. Besides Pi diacylglycerol was also formed, confirming that a phosphatidate hydrolase activity was involved. Since the phosphatidate phosphohydrolase activity was rather heat- and N-ethylmaleimide-insensitive, we conclude that the phosphatidic acid hydrolysis was not due to Na,K-ATPase itself but to a membrane-bound phosphatidate phosphohydrolase, present as an impurity in the purified rabbit kidney Na,K-ATPase preparations.