Microsomal CTP:choline phosphate cytidylyltransferase: kinetic mechanism of fatty acid stimulation.

Fatty acids are known to cause an increase in the incorporation of radioactive choline into phosphatidylcholine. A coincident increase in membrane cytidylyltransferase activity is well documented. The purpose of the present studies was to determine the direct effects of oleic acid on the kinetic properties of membrane cytidylyltransferase. An examination of the reaction characteristics of membrane cytidylyltransferase revealed that membranes from adult rat lung contained high CTPase activity. This activity prevented the determination of reaction velocities at low CTP concentrations. The CTPase activity was blocked by the addition of ADP or ATP to the reaction. The addition of 6.0 mM ADP to the assay mixture enabled us to determine the effect of oleate on the CTP Km. Oleate (122 microM) caused a significant decrease in CTP Km for microsomal cytidylyltransferase (0.99 mM to 0.33 mM) and H-Form cytidylyltransferase (1.04 mM to 0.27 mM). Oleate did not decrease the CTP Km for L-Form cytidylyltransferase. Oleate had no effect on the choline phosphate Km in microsomal, H-Form or L-Form cytidylyltransferase. Oleate also increased the Vmax for cytidylyltransferase. The increase was dependent upon the concentration of oleate with a maximal increase of 50-60% at 100-130 microM oleate. We conclude that oleate has a direct stimulatory effect on cytidylyltransferase when it is in the active form (membrane bound or H-Form lipoprotein complex). We suggest that the kinetic effects operate synergistically with other regulatory mechanisms such as translocation or conversion of inactive to active species. The direct effect of oleate on the cytidylyltransferase may be an important regulatory mechanism when CTP concentrations are limiting.     

Regulation of CTP:phosphocholine cytidylyltransferase by lipids. 1. Negative surface charge dependence for activation.

The activity of phosphocholine cytidylyltransferase (CT), the regulatory enzyme in phosphatidylcholine synthesis, is dependent on lipids. The enzyme, obtained from rat liver cytosol, was purified in the presence of Triton X-100 [Weinhold et al. (1986) J. Biol. Chem. 261, 5104]. The ability of lipids to activate CT when added as Triton mixed micelles was limited to anionic lipids. The relative effectiveness of the lipids tested suggested a dependence on the negative surface charge density of the micelles. The mole percent lipid in the Triton mixed micelle required for activation decreased as the net charge of the lipid varied from 0 to -2. Evidence for the physical association of CT with micelles and vesicles containing phosphatidylglycerol was obtained by gel filtration. The activation by micelles containing PG was influenced by the ionic strength of the medium, with a higher surface charge density required for activation at higher ionic strength. The micelle surface potential required for full activation of CT was calculated to be -43 mV. A specificity toward the structure of the polar group of the acidic lipids was not apparent. CT was activated by neutral lipids such as diacylglycerol or oleyl alcohol when included in an egg PC membrane, but the activities were reduced by dilution with as little as 10 mol % Triton. Thus Triton mixed micelles are not suitable for studying the activation of CT by these neutral lipid activators. We conclude that one way that lipid composition can control CT-membrane binding and activity is by changing the surface potential of the membrane. Other distinct mechanisms involved in the activation by neutral lipids are discussed.     

Metabolism of monoacylglycerol and diacylglycerol by enzyme preparations from spinach leaves

Acetone powders prepared from a 20,000g participate preparation from spinach leaf catalyzed several reactions involving monoacylglycerol and diacylglycerol. When these substrates were presented as Triton X-100-mixed micelles, diacylglycerol gave rise to free fatty acids, monoacylglycerol, triacylglycerols, and steryl esters, and in the presence of ethanol, small amounts of ethyl esters of fatty acid. Monoacylglycerol gave rise to free fatty acids and diacylglycerol, and in the presence of ethanol, large amounts of ethyl esters of fatty acid. In the presence of bovine serum albumin, the conversion of monoacylglycerol to free fatty acid was retarded. In the presence of bovine serum albumin, steryl ester was an important product from diacylglycerol. The system containing Triton X-100-mixed micelles and bovine serum albumin permitted analysis of reaction products which showed diacylglycerol to be an acyl donor in steryl ester biosynthesis. All reactions observed in the mixed micelle system were transacylation reactions involving various acceptors: dipalmitoylglycerol → monopalmitoylglycerol + palmitate; monopalmitoylglycerol → glycerol + palmitate; dipalmitoylglycerol + sterol → monopalmitoylglycerol + steryl palmitate; monopalmitoylglycerol + ethanol → ethyl palmitate + glycerol; monopalmitoylglycerol → dipalmitoylglycerol (+glycerol); dipalmitoylglycerol → tripalmitoylglycerol (+monopalmitoylglycerol).     

Modulation of CTP:phosphocholine cytidylyltransferase translocation by oleic acid and the antitumoral alkylphospholipid in HL-60 cells.

Short time effect of oleate and 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphocholine (AMGPC) on choline incorporation into phosphatidylcholines were studied in HL-60 cells. The non lytic concentration of 50 microM oleate induced a three-fold increase in [3H]choline incorporation into phosphatidylcholine. This stimulation was accompanied by a translocation of the CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) from cytosol to membranes. By contrast, the ether-lipid AMGPC inhibited [3H]choline incorporation into phosphatidylcholine by 60% at 10 microM. AMGPC had no effect on choline kinase or choline phosphotransferase activities. When AMGPC was added separately to an homogenate, a particulate or a cytosolic fraction, cytidylyltransferase inhibition was observed only in the homogenate. However on particulates recovered from homogenates treated with increasing concentrations of AMGPC, membranous cytidylyltransferase activity decreased dose-dependently. Thus AMGPC had no effect on cytidylyltransferase activity itself but inhibited its translocation from cytosol to membrane. At variance with the well-established positive effect on cytidylyltransferase translocation induced by fatty acids, this is the first demonstration that AMGPC can inhibit cytidylyltransferase translocation in cell-free system.     

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.     

Membrane transport in relation to net uptake of glucose in the perfused rat hindlimb. Stimulatory effect of insulin, hypoxia and contractile activity.

The assay for the fucose-binding protein described by Lehrman & Hill [(1983) Methods Enzymol. 98, 309-319] was adapted for the measurement of the asialoglycoprotein receptor in rat liver. The amount of ligand bound to the plasma-membrane-associated or affinity-purified receptor was acutely sensitive to the concentrations of Triton X-100 and NaCl in the assay: 0.02% (v/v) Triton X-100 increased ligand binding to the two preparations by 100% and 40% respectively. Higher concentrations of detergent progressively decreased binding, and in 0.32% Triton X-100 it was about 30% of the value obtained in detergent-free buffer. The addition of increasing concentrations of NaCl to the assay progressively inhibited ligand binding to the membrane-associated receptor, whereas there was a 60% increase in binding to the pure receptor in the presence of 0.1-0.2 M-NaCl. These effects could not be identified in the original assay procedure described by Hudgin, Pricer, Ashwell, Stockert & Morell [(1974) J. Biol. Chem. 249, 5536-5543]. Using optimal assay conditions the binding of 125I-beta-D-galactosyl-bovine serum albumin to both the membrane-associated and purified receptor was inhibited by 50% by 1 nM-beta-D-galactosyl-bovine serum albumin and -asialoorosomucoid and by approx. 100 microM-beta-L-fucosyl-bovine serum albumin, whereas beta-D-galactose, lactose and beta-L-fucose had no effect on ligand binding up to concentrations of 1 mM, 500 microM and 5 mM respectively. KD values of 0.94 and 1.25 nM and Bmax. values of 40 and 1660 pmol of D-galactosyl-bovine serum albumin bound/mg of receptor were obtained for the membrane-bound and purified receptor respectively. Hill-plot analysis of the same data gave slopes of 0.96 and 1.01. Scatchard analysis of saturation-binding studies with other subcellular fractions indicated that the receptor was distributed in the proportions 72:23:2.5:2.5 between total microsomal fractions, plasma membrane, Golgi and canalicular membrane respectively. The receptor was about 1% of the total protein in each compartment and was estimated to be about 0.3% of the total liver protein.     

Phosphoinositide reorganization in human erythrocyte membrane upon cholesterol depletion.

The effect of cholesterol depletion on the activity of phosphatidylinositol/phosphatidylinositol 4-phosphate and diacylglycerol kinases and polyphosphoinositide phosphodiesterase has been studied in isolated membranes of human normal and cholesterol-depleted erythrocytes. Polyphosphoinositide synthesis (phosphatidylinositol/phosphatidylinositol 4-phosphate kinase activities) were found to depend on the permeability and sidedness characteristics of the membrane vesicles, which could limit the accessibility of ATP for the enzymes. When measured under proper conditions, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate synthesis were decreased in cholesterol-depleted membranes as compared with control membranes. The same level of synthesis could be obtained in both membranes by the addition of phosphatidylinositol (and Triton X-100) or of phosphatidylinositol 4-phosphate. Phosphatidic acid synthesis (diacylglycerol kinase activity) was also decreased in cholesterol-depleted membranes as compared with control membranes when measured in the presence of Ca2+. Addition of diolein (and Triton X-100) caused a large increase in phosphatidic acid synthesis which reached approximately the same level in both membranes. This showed that the apparent inhibition of polyphosphoinositide and phosphatidic acid synthesis was not due to a loss or to an inactivation of the kinases. Ca2+-activated polyphosphoinositide phosphodiesterase promoted the hydrolysis of 65-70% of the polyphosphoinositides in control and of only 45-55% in cholesterol-depleted membranes without changing the Ca2+ concentration for half-maximum hydrolysis (1 microM). Upon addition of sodium oleate, the extent of polyphosphoinositide hydrolysis became identical in both membranes, indicating again that there was no loss nor inactivation of the polyphosphoinositide phosphodiesterase in the cholesterol-depleted membranes. Since the concentration of the polyphosphoinositides was not changed by cholesterol depletion [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200], the reduction in both their synthesis and degradation observed here could be attributed to a reorganization of the phosphoinositides in membrane domains where they were not accessible to the kinases and phosphodiesterase. The reduction in phosphatidic acid synthesis was likely caused by a reduction in the total amount of the substrate diacylglycerol in cholesterol-depleted membranes as already shown [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200].     

CTP:phosphocholine cytidylyltransferase in rat lung: relationship between cytosolic and membrane forms

The purpose of these studies was to determine the properties of the membrane-bound cytidylyltransferase in adult lung and to assess the relationship between the microsomal enzyme and the two forms of cytidylyltransferase in cytosol. Microsomes, isolated by glycerol density centrifugation, contained significantly less cytidylyltransferase than microsomes isolated by differential centrifugation (11.6 +/- 3.2 vs. 30 +/- 11 nmol/min per g lung). The released activity was recovered as H-form cytidylyltransferase. Cytidylyltransferase activity was not removed from microsomes by washing of the microsomal pellet with homogenizing buffer. Triton X 100 extracted all of the cytidylyltransferase from microsomes. The extracted activity was similar to H-form. Chlorpromazine dissociated microsomal enzyme to L-form. Chlorpromazine has been shown previously to dissociate H-form to L-form. These results suggested that microsomal cytidylyltransferase existed in a form similar if not identical to cytosolic H-form. In vitro translocation experiments demonstrated that the L-form of cytidylyltransferase was the species which binds to microsomal membranes. Triton X 100 extraction of microsomes from translocations experiments removed the bound enzyme activity. Glycerol density fractionation indicated that the activity in the Triton extract was H-form cytidylyltransferase. We concluded that the active lipoprotein form of cytidylyltransferase (H-form) is the membrane-associated form of cytidylyltransferase in adult lung; that it is formed after the L-form binds to microsomal membranes and that cytosolic H-form is released from the membrane.     

Synthesis of polyphosphoinositides in vertebrate photoreceptor membranes

Rod outer segments isolated from bovine retinas incorporated 32P into phospholipids after incubation with [gamma-32P]ATP in a Mg2+-containing medium. Only phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidate were labelled. The incorporation of label into lipids was detected as early as 20 s after the start of incubation and the products were stable for at least 10 min. The reactions were time, protein and ATP-concentration dependent. Entire rod outer segments showed higher diacylglycerol kinase and lower phosphatidylinositol and phosphatidylinositol 4-phosphate kinase activities than the disc membranes obtained from them. Exogenously added phosphatidylinositol (up to 1 mM) in the presence of Triton X-100 increased phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate labelling in rod outer segments (8- and 6-fold, respectively). Triton X-100 at a concentration of 0.4% stimulated phosphorylation of endogenous phosphoinositides. Diacylglycerol kinase activity was largely suppressed by the detergent, but this effect was partially reversed by addition of phosphatidylinositol. It is suggested that the rod outer segments contain phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase bound to disc membranes, as well as an active diacylglycerol kinase occurring either as a soluble or a peripherally bound protein in disc membranes.     

Activation of protein kinase C by oleic acid. Determination and analysis of inhibition by detergent micelles and physiologic membranes: requirement for free oleate.

Sodium oleate is able to activate soluble protein kinase C (Murakami, K., Chan, S. Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429) but is unable to activate membrane-bound enzyme (El Touny, S., Khan, W., and Hannun, Y. (1990) J. Biol. Chem. 265, 16437-16443). Because physiologic interactions of fatty acids with protein kinase C occur in the presence of membranes, the following studies were conducted to evaluate the effects of surfaces (detergent micelles or platelet membranes) on the activation of protein kinase C by oleate. At concentrations at or above the critical micellar concentration (CMC) of Triton X-100, oleate was present primarily in Triton X-100/oleate-mixed micelles, as determined by gel permeation chromatography and equilibrium dialysis binding studies. At concentrations slightly below the CMC for Triton X-100, the presence of oleate caused the formation of a limited number of mixed micelles. Studies of the dose-dependent activation of purified platelet protein kinase C by sodium oleate in the presence of different concentrations of Triton X-100 indicated that only unbound oleate was able to activate protein kinase C. Platelet protein kinase C was resolved into two major isoenzymes (types II (beta) and III (alpha)) which displayed nearly identical interaction with oleate. Activation of protein kinase C by oleate in a physiologic setting employing platelet substrates and endogenous platelet protein kinase C was investigated. Oleate potently activated protein kinase C in the cytosolic compartment. In platelet homogenates as well as in a reconstituted platelet cytosol and membrane system, the dose dependence of protein kinase C on oleate showed a significant shift to the right. Approximately 30% of oleate was associated with platelet cytosol and 70% was associated with platelet membranes. Partitioning of oleate into the two platelet compartments showed little change with pH, temperature, or duration of incubation. When corrected for free oleate concentration, activation of protein kinase C by oleate showed identical dose dependence in cytosol and homogenate. Arachidonate, a potential physiologic activator of protein kinase C, showed similar behavior as oleate although only 30% of arachidonate partitioned into platelet membranes with the majority of arachidonate (70%) remaining in the cytosolic fraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

The inhibitory effects of polyoxyethylene detergents on human erythrocyte acetylcholinesterase and Ca2+ + Mg2+ ATPase

The activities of acetylcholinesterase and Ca2+ + Mg2+ ATPase were measured following treatment of human erythrocyte membranes with nonsolubilizing and solubilizing concentrations of Triton X-100. A concentration of 0.1% (v/v) Triton X-100 caused a significant inhibition of both enzymes. The inhibition appears to be caused by perturbations in the membrane induced by Triton X-100 incorporation. No acetylcholinesterase activity and little Ca2+ + Mg2+ ATPase activity were detected in the supernatant at 0.05% Triton X-100 although this same detergent concentration induced changes in the turbidity of the membrane suspension. Also, no inhibition of soluble acetylcholinesterase was observed over the entire detergent concentration range. The inhibition of these enzymes at 0.1% Triton X-100 was present over an eightfold range of membrane protein in the assay indicating an independence of the protein/detergent ratio. The losses in activities of these two enzymes could be prevented by either including phosphatidylserine in the Triton X-100 suspension or using Brij 96 which has the same polyoxyethylene polar head group but an oleyl hydrophobic tail instead of the p-tert-octylphenol group of Triton X-100. The results are discussed in regard to the differential recovery of enzyme activities over the entire detergent concentration range.     

Carnitine palmitoyltransferase. Activation by palmitoyl-CoA and inactivation by malonyl-CoA

Extraction of rat liver mitochondria twice with 0.5% Triton X-100 in a salt-free medium leaves less than 10% of the carnitine palmitoyltransferase membrane bound. The remaining membrane-bound enzyme is inhibited virtually completely by 10 microM malonyl-CoA. Preincubation of the extracted membranes with palmitoyl-CoA and salts (KCI) for several minutes activates the enzyme and makes it increasingly insensitive to malonyl-CoA. Addition of malonyl-CoA to the preincubation reverses this desensitization. In albumin-containing media salts also decrease the binding of palmitoyl-CoA to albumin and stimulate carnitine palmitoyltransferase by increasing substrate availability in free solution. The reverse reaction shows accelerated desensitization by palmitoylcarnitine and resensitization by malonyl-CoA.     

Proteolipid formation in Mycoplasma capricolum. Influence of cholesterol on unsaturated fatty acid acylation of membrane proteins

Mycoplasma capricolum, a procaryotic sterol and fatty acid auxotroph was grown on media supplemented with [3H]palmitate or [3H]oleate. The isolated bacterial membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the more than 50 membrane polypeptides revealed by Coomassie blue staining, approximately 25 were labeled with [3H]palmitate and only about 6 were labeled with [3H]oleate. Exhaustive delipidation of the membranes with chloroform:methanol did not alter the labeling pattern. Treatment of delipidated membranes by mild alkaline hydrolysis released up to 71% of the [3H]palmitate and 93% of the [3H]oleate. The data suggest that numerous membrane proteins of M. capricolum are covalently modified by acylation with saturated and unsaturated fatty acids. Cerulenin, a specific inhibitor of fatty acid synthesis had no effect on the labeling of mycoplasma membrane proteins by either [3H]palmitate or [3H]oleate. A small amount of membrane-associated cholesterol previously shown to stimulate sequentially the synthesis of unsaturated phospholipid, RNA, and protein (Dahl, J. S., and Dahl, C. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 692-696) specifically enhances the acylation of certain proteolipids by oleate but not by palmitate.     

Chemical cross-linking reveals a dimeric structure for CTP:phosphocholine cytidylyltransferase

CTP:phosphacholine cytidylyltransferase (EC 2.7.7.15) was purified from rat liver according to the method of Weinhold et al. (Weinhold, P. A., Rounsifer, M. E., and Feldman, D. A. (1986) J. Biol. Chem. 261, 5104-5110). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without beta-mercaptoethanol revealed a single major band of 42,000 daltons. This band corresponds to the 45-kDa catalytic subunit isolated by Feldman and Weinhold (Feldman, D. A., and Weinhold, P. A. (1987) J. Biol. Chem. 262, 9075-9081). A minor component of 84,000 daltons was intensified in nonreducing gels when the sulfhydryl reducing agent, dithiothreitol, was removed from the enzyme preparation by dialysis. Reduction with dithiothreitol and electrophoresis in the second dimension showed that this 84-kDa protein was derived from the 42-kDa protein. This result suggested that the 42 kDa protein can be converted to an 84-kDa protein by disulfide bond formation. Reaction with the thiol-cleavable cross-linking reagents, dithiobis(succimidyl propionate) or dimethyl-3,3'-dithiobispropionimidate, converted the 42-kDa cytidylyltransferase subunit into a diffuse band approximately twice its molecular mass. Disulfide reduction and electrophoresis in the second dimension showed that this band was derived exclusively from the 42-kDa subunit. This cross-linking pattern was observed when cytidylyltransferase was bound to a Triton X-100 micelle or when bound to a membrane vesicle containing phosphatidylcholine, oleic acid, and Triton X-100. Reaction of the fully reduced enzyme with glutaraldehyde also generated a cross-linked dimer. All three cross-linking reagents inactivated the enzyme. Reduction of the disulfide cross-linkers with dithiothreitol partially reactivated the transferase. When Triton was removed from the enzyme preparation by DEAE-Sepharose chromatography, reaction of the detergent-depleted enzyme with glutaraldehyde generated a band corresponding to a hexamer and higher molecular weight aggregates. The dimeric form was regenerated by addition of either Triton X-100 or phosphatidylcholine-oleic acid vesicles. We conclude that the purified, native cytidylyltransferase, when bound to a detergent micelle or membrane vesicle, is a dimer composed of two noncovalently linked 42-kDa subunits. In the absence of a membrane or micelle, the dimers self-aggregate in a reversible manner.     

Phosphatidylcholine synthesis in castor bean endosperm: the localization and control of CTP: choline-phosphate cytidylyltransferase activity

The reaction catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) has been postulated to be a control reaction in the synthesis of phosphatidylcholine (PtdCho) in many animal tissues and some plants. In 3-day-old castor bean (Ricinus communis L. var. Hale) endosperm the majority of cytidylyltransferase activity resided in a 12,000gav 10-min pellet. Following density-gradient fractionation, 60 to 70% of the enzyme activity was associated with the endoplasmic reticulum (ER) fraction, with the remainder in the particulate fraction being in an unidentified membrane band (band A), less than occurred in the soluble fractions. The properties and kinetics of the forward and reverse reactions are described. About 40% of the total ER activity could be solubilized by treatment of the fraction with 0.32 M KCl, which resulted in a threefold increase in the specific activity of the enzyme. The Michaelis constants of the solubilized enzyme were similar to those of the ER activity. The activity of the solubilized enzyme was stimulated 35% by addition of phosphatidylglycerol or phosphatidylinositol to the assay. Addition of a number of other phospholipids to the incubation medium caused only a small change in activity (+/- 10%) but the enzyme could be stimulated up to 60% by the addition of 0.01-1 mM sodium oleate. A combination of 0.25 mM PtdCho with oleate in the assay resulted in additional stimulation at all concentrations of oleate. Oleate had no effect on the ER activity. These results are discussed in relation to the regulation of cytidylyltransferase activity in plants.     

The incorporation of [14C]acetate into the constituent fatty acids of monogalactosyldiglyceride by isolated spinach chloroplasts

The incorporation into diglycerides of the acyl products synthesized from acetate by spinach chloroplasts was greatly stimulated by the addition of glycerol 3-phosphate. When UDP-galactose was added also, monogalactosyldiglycerides became the major products. Palmitate biosynthesis was stimulated about twofold by these additions, while oleate biosynthesis decreased slightly, so that oleate:palmitate ratios were in the range 0.6 to 0.8 rather than about 1.6 when glycerol 3-phosphate and UDP-galactose were not added. On the other hand, Triton X-100 greatly stimulated both oleate and palmitate biosynthesis to give oleate:palmitate ratios of about 2.0. The proportions of oleate and palmitate in the newly synthesized diglycerides, or in monogalactosyldiglycerides when exogenous UDP-galactose was added, did not always reflect the proportions of these two fatty acids synthesized from acetate. When oleate:palmitate ratios were ?1, equal amounts were incorporated into diglycerides or into monogalactosyldiglycerides. When oleate:palmitate ratios were <1, incorporation of palmitate into diglycerides and monogalactosyldiglycerides exceeded that of oleate. 1-Oleoyl, 2-palmitoyl glycerol compounds were the principal products under all conditions but 1,2-dipalmitoyl compounds were also quantitatively important when glycerol 3-phosphate alone, or glycerol 3-phosphate together with UDP-galactose, was added. The distribution of label in the constituent glycerol and fatty acid moieties when monogalactosyldiglycerides were synthesized from diglycerides is consistent with galactosylation occurring without modification or exchange of fatty acids. The distribution of 16- and 18-carbon acyl residues between the 1 and 2 stereospecific positions of newly synthesized monogalactosyldiglyceride was typical of the endogenous polyene monogalactosyldiglycerides. However when palmitate synthesis was in excess of oleate synthesis some palmitate was esterified in position 1, whereas in the endogenous monogalactosyldiglycerides hexadecatrienoate is confined to position 2.     

Diacylglycerol signals the translocation of CTP:choline-phosphate cytidylyltransferase in HeLa cells treated with 12-O-tetradecanoylphorbol-13-acetate.

The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated phosphatidylcholine biosynthesis in HeLa cells was investigated. TPA caused a 3-fold increase in particulate CTP:phosphocholine cytidylyltransferase activity in HeLa cells which correlated with decreased cytidylyltransferase activity in the cytosol. The increase in membrane-associated cytidylyltransferase was confirmed by immunoblotting. Immunoprecipitation studies suggested that TPA had no effect on the phosphorylation state of cytidylyltransferase. Enhanced binding of cytidylyltransferase to diacylglycerol-enriched membranes has previously been shown. Diacylglycerol levels in TPA-treated HeLa cells increased approximately 2-fold (2.29 to 4.02 nmol/mg of protein) after 1 h of TPA treatment. A time course experiment showed a temporal relationship in which production of diacylglycerol appeared to signal translocation of cytidylyltransferase to membranes followed by a stimulation of phosphatidylcholine biosynthesis. Diacylglycerol was further evaluated as a translocator of cytidylyltransferase by depleting HeLa cells of protein kinase C and incubating with dioctanoylglcerol. This treatment increased both membrane-associated cytidylyltransferase activity and the rate of phosphatidylcholine biosynthesis approximately 2-fold. A time course experiment with dioctanoylglycerol showed a strong positive correlation (r2 = 0.89) between the amount of particulate cytidylyltransferase activity and the rate of phosphatidylcholine biosynthesis. Therefore, TPA stimulates phosphatidylcholine biosynthesis by causing a translocation of cytidylyltransferase from the cytosol to membranes, which appears to be mediated by increased diacylglycerol.     

Separation and characterization of the acid lipase and neutral esterases from human liver.

Electrophoresis of human liver homogenates followed by reaction with 4-methylumbelliferyl palmitate reveals the presence of two major electrophoretic forms with esterase (lipase) activity toward this substrate. The two enzymes were isolated and partially purified based on their solubility differences and their relative affinities for the lectin column concanavalin A-Sepharose 4B. Lipase A was particulate with an acidic pH optimum (5.2) and could be solubilized with the non-ionic surfactant Triton X-100. Lipase B was soluble and had a more neutral pH optimum (6.3--6.6). Both forms bound to immobilized concanavalin A and could be specifically eluted. Buffers containing alpha-methylmannoside eluted lipase B, and buffers with alpha-methylmannoside and Triton X-100 eluted lipase A, giving a 22- and 257-fold purification, respectively, over whole-tissue homogenates. Cholesterol oleate, trioleoylglycerol, and 4-methylumbelliferyl palmitate were substrates for solubilized lipase A. Lipase B hydrolyzed 4-methylum-belliferyl palmitate but not trioleoylglycerol or cholesterol oleate. Lipase B was more thermolabile than lipase A, and it was selectively inhibited by diethyl-p-nitrophenyl phosphate at low concentrations. We conclude that lipase A and B are distinctly different enzymes and that they are probably not related polymorphic forms of one another.     

Regulation of the translocation of phosphatidate phosphohydrolase between the cytosol and the endoplasmic reticulum of rat liver. Effects of unsaturated fatty acids, spermine, nucleotides, albumin and chlorpromazine.

The translocation of phosphatidate phosphohydrolase between the cytosol and the microsomal membranes was investigated by using a cell-free system from rat liver. Linoleate, alpha-linolenate, arachidonate and eicosapentenoate promoted the translocation to membranes with a similar potency to that of oleate. The phosphohydrolase that associated with the membranes in the presence of [14C]oleate or 1mM-spermine coincided on Percoll gradients with the peak of rotenone-insensitive NADH-cytochrome c reductase, and in the former case with a peak of 14C. Microsomal membranes were enriched with the phosphohydrolase activity by incubation with [14C]oleate or spermine and then incubated with albumin. The phosphohydrolase activity was displaced from the membranes by albumin, and this paralleled the removal of [14C]oleate from the membranes when this acid was present. Chlorpromazine also displaced phosphatidate phosphohydrolase from the membranes, but it did not displace [14C]oleate. The effects of spermine in promoting the association of the phosphohydrolase with the membranes was inhibited by ATP, GTP, CTP, AMP and phosphate. ATP at the same concentration did not antagonize the translocating effect of oleate. From these results and previous work, it was concluded that the binding of long-chain fatty acids and their CoA esters to the endoplasmic reticulum acts as a signal for more phosphatidate phosphohydrolase to associate with these membranes and thereby to enhance the synthesis of glycerolipids, especially triacylglycerol. The translocation of the phosphohydrolase probably depends on the increased negative charge on the membranes, which could also be donated by the accumulation of phosphatidate. Chlorpromazine could oppose the translocation by donating a positive charge to the membranes.     

Diacylglycerol kinase from suspension cultured plant cells : purification and properties

Diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) from suspension-cultured Catharanthus roseus cells was extracted from a membrane fraction with 0.6% Triton X-100 and 150 millimolar NaCl and was purified about 900-fold by DEAE-cellulose, blue Sepharose, gel permeation, and phenyl-Sepharose chromatography. The enzyme is obviously membrane bound as activity in the cytosol could not be detected. In the presence of detergents such as Triton X-100 (3-[3-cholamidopropyl]dimethylamino)-1-propanesulfonate (Chaps), or deoxycholate, a molecular weight of about 250,000 was determined by gel filtration. In glycerol density gradients, the enzyme sedimented slightly more slowly than bovine serum albumin, indicating a molecular weight of less than 68,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis enzyme activity could be assigned to a protein of 51,000 daltons. As found previously for bacterial and animal diacylglycerol kinases, the purified enzyme was completely devoid of activity without the addition of phospholipids or deoxycholate. Cardiolipin was found to be most effective, whereas higher amounts of detergent were inhibitory. The enzyme needs divalent cations for activity, with Mg²⁺ ions being the most effective. Apparent K_m values for ATP and diacylglycerol were determined as 100 and 250 micromolar, respectively.