Solubilization and partial purification of cholinephosphotransferase in hamster tissues

CDP choline:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) is located on the cytoplasmic side of the endoplasmic reticulum, and catalyzes the final step in the synthesis of phosphatidylcholine via the CDP choline pathway. The enzyme was solubilized from hamster liver microsomes by 3% Triton QS-15, and partially purified by DEAE-Sepharose chromatography and Sepharose 6B chromatography. The microsomal and partially purified enzymes displayed similar pH profile, and both showed absolute requirement for Mg⁺⁺ or other divalent cations. The Km values of CDP choline were similar between microsomal and partially purified enzyme, whereas the Km value for diacylglycerol was substantially lowered when the enzyme was partially purified. Hamster heart cholinephosphotransferase was not solubilized by Triton QS-15.     

Factors enhancing diacylglycerol acyltransferase activity in microsomes from cell-suspension cultures of oilseed rape

Several factors, including an unidentified endogenous component, were found to stimulate microsomal diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) from a microspore-derived cell-suspension culture of oilseed rape (Brassica napus L. cv. Jet Neuf). At a concentration of 25mM, MgSO₄ and MgCl₂ stimulated microsomal DGAT 25- and 10-fold, respectively. AIP and CoA at concentrations of 2 and 1 mM stimulated the enzyme 2.4- and 12-fold, respectively, although the effects were lessened in the presence of higher Mg²⁺ concentrations. Although microsomal DGAT activity was increased only slightly by the addition of exogenous sn-1,2-diacylglycerol to the reaction mixture, it was increased substantially by the addition of exogenous phosphatidate. sn-Glycerol-3-phosphate and other phospholipids tested did not have this stimulatory effect. DGAT activity did not decrease when microsomes were incubated with ATP in the presence of the cytosolic fraction. This fraction, however, contained a small organic compound(s) that stimulated microsomal DGAT activity.     

Dietary ω3 fatty acids and cholesterol modify enterocyte microsomal membrane phospholipids,cholesterol content and phospholipid enzyme activities in diabetic rats

Diabetes-associated changes in intestinal uptake of nutrients are modified by isocaloric variations in the type of dietary lipids, and are associated with alterations in the phospholipid and fatty acyl content of the intestinal brush border membrane. The present study was designed to test the hypothesis that diet- and diabetes-associated changes in enterocyte microsomal membrane phospholipids are due to variations in the activity of two phospholipid metabolizing enzymes, 1,2-diacylglycerol: CDP choline cholinephosphotransferase (CPT) and phosphatidylethanolamine methyltransferase (PEMT). Adult female Wistar rats were fed one of four semisynthetic diets—beef tallow low in cholesterol (BT), beef tallow high in cholesterol (BTC), fish oil low in cholesterol (FO) or fish oil high in cholesterol. In half of the animals, diabetes mellitus was produced by injection of streptozotocin. Jejunal and ileal enterocyte microsomes (EMM) were isolated and analyzed for cholesterol and phospholipids, as well as for CPT and PEMT activities. In control animals, feeding FO reduced EMM total phospholipids including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol. Feeding FO resulted in a greater than 95% reduction in the activity of CPT. Diabetes was associated with increased jejunal EMM total phospholipids including sphingomyelin (SM) and PE, without associated changes in CPT or PEMT. Dietary cholesterol supplementation did not effect EMM total cholesterol or phospholipid composition in control rats fed BT or FO, but was associated with an increase in EMM cholesterol in diabetic rats fed BT or FO. A decrease in total phospho-lipids due to a decline in SM, PC and PE in diabetic rats fed FO was not associated with changes in the activities of CPT or PEMT in EMM. Thus (i) enterocyte microsomal membrane cholesterol and phospholipid contents are influenced by diabetes, dietary cholesterol and the type of fatty acid in the diet, and (ii) changes in phospholipid composition are not fully explained by alterations in the activities of CPT and PEMT.     

Phospholipid requirement of alkylglycerol monooxygenase from rat liver microsomes

The alkylglycerol monooxygenase catalyzing the cleavage of the ether bond in alkylglycerol resides in rat liver microsomes. The enzyme preparation was freed of phospholipids by sodium deoxycholate treatment followed by gel filtration in the presence of deoxycholate. The removal of phospholipids markedly decreased the alkylglycerol monooxygenase activity. The activity of the delipidated enzyme, however, could be completely restored by the addition of phospholipid vesicles without detergent. When individual phospholipids were added, anionic phospholipids such as phosphatidylglycerol and diphosphatidyl-glycerol were the most effective. These findings, along with our previous observation of a similar effect of liposomes on the purified enzyme, indicate that the amphipathic nature of the protein is responsible for the lipid dependence of enzymatic activity.     

Formation of diacyl and alkylacyl glycerophosphocholine in rabbit alveolar macrophages

The incorporation of various labeled precursors into alkenylacyl, alkylacyl and diacyl phospholipids in rabbit alveolar macrophages was studied. The incorporation rates of the individual precursors were shown to be quite different among the three subclasses of phospholipids. [³H] Glycerol, [¹⁴C]16∶0, [¹⁴C]18∶1, [¹⁴C]18∶2 and [³²P]-orthophosphate were preferentially incorporated into choline glycerophospholipids (CGP), especially into diacyl glycerophosphocholine (GPC), indicating that the de novo synthesis of diacyl GPC is extremely high. Considerable portions of the radioactiveties of [¹⁴C]16∶0, [¹⁴C]18∶1, [¹⁴C]18∶2 and [³²P] orthophosphate were also found in alkylacyl GPC, the incorporation being higher than or comparable to that in the case of diacyl glycerophosphoethanolamine (GPE). We then examined the activities of cholinephosphotransferase and ethanolaminephosphotransferase, and found that the activity of cholinephosphotransferase was remarkably high in macrophage microsomes compared with that in microsomes from several other tissues. This suggests that diradylglycerols were preferentially utilized by cholinephosphotransferase, which is consistent with the results obtained for intact cells. We confirmed that a considerably higher amount of diacyl GPC as well as alkylacyl GPC was formed through this enzyme reaction with macrophage microsomes than with brain microsomes. The high formation of alkylacyl GPC could be responsible, at least in part, for the accumulation of this unique ether phospholipid, a stored precursor form of plateletactivating factor in macrophages. Fatty chains are designated in terms of number of carbon atoms: number of double bonds, e.g., 18:1 for oleic acid.     

Membrane-bound phospholipid desaturases

This review covers studies on membrane-bound phospholipid desaturases in yeast and rat liver carried out in this laboratory. In yeast the desaturase system was shown to effect the direct desaturation of dioleoyl-lecithin to dilinoleoyl-lecithin. In rat liver the desaturase was capable of converting 2-eicosatrienoyl-lecithin to 2-arachidonoyl-lecithin. Both systems required reduced pyridine nucleotides, O₂ and cytochrome b₅. Eicosatrienoyl-lecithin desaturase along with eicosatrienoyl-CoA desaturase of rat liver microsomes was solubilized with detergents and purified 7–8-fold from the microsomal pellets. Both activities were reconstituted in the presence of deoxycholate on addition of the other components of the cytochrome b₅-electron transport chain (cytochrome b₅ and NADH-cytochrome b₅ reductase) to the solubilized desaturase; addition of lecithin further stimulated the activities. The demonstration of desaturation of eicosatrienoyl-lecithin by a solubilized and partially purified desaturase provides strong evidence for the direct desaturation of the lecithin substrate without prior conversion to the acyl-CoA thiolester.     

Phospholipid reactivation of plasmalogen metabolism

This report is concerned mainly with the properties of an enzyme from rat liver microsomes which hydrolyzes the alkenyl ether bond of 1-(1′-alk-1′-enyl)-glycero-3-phosphoryl-choline (alkenyl-GPC hydrolase). Destruction of the normal environment of the microsomes by treatment with phospholipases A or C caused inactivation of the alkenyl-GPC hydrolase, which was then partially reactivated by the addition of exogenous phospholipids. Both sphingomyelin and diacyl-GPC were efficient in restoring activity; diacyl-GPE was less effective; and monoacyl-GPC and monoacyl-GPE were ineffective. The presence of two long hydrocarbon chains in the lipid activator is apparently required for reactivation, suggesting that interaction of hydrophobic areas of the enzyme with the phospholipid is necessary for maximal activity. High concentrations of sucrose mimicked the effect of phospholipids, and because the sucrose and diacyl-GPC did not show an additive effect, they may reactivate the enzyme in a similar manner. Disrupting the enzyme's environment by freezing and thawing the preparation also resulted in a loss of enzymatic activity, which was restored by added exogenous phospholipids. The alkenyl-GPC hydrolase was inhibited by imidazole and some of its derivatives. Histidine and N-acetyl histidine did not inhibit the enzyme, presumably due to the presence of a negative charge on the carboxyl group rather than the steric bulk of that group, since histidine methyl ester did inhibit the enzyme. Kinetic evidence showed imidazole to be a competitive inhibitor. The enzymatic activity of imidazole-treated microsomes also increased following addition of exogenous phospholipids. Imidazole inhibition differed from the phospholipase A-inactivation in that it was partially reversed by KCl, but not by sucrose. Imidazole did not inhibit other microsomal enzymes tested, indicating that it is not a general inhibitor of membrane-associated enzymes.     

The effect of lidocaine onde novo phospholipid biosynthesis in the isolated hamster heart

Lidocaine is used clinically as an antiarrhythmic agent, but its effect on cardiac phospholipid metabolism has not been defined. In this study, hamster hearts were perfused with [1,3-³H]glycerol in the presence of 0.5 mg/mL lidocaine. The incorporation of radioactivities into lysophosphatidic acid, phosphatidic acid, phosphatidylethanolamine, cytidine diphosphate diacylglycerol, phosphatidylinositol, phosphatidylserine, diacylglycerol and triacylglycerol were enhanced by lidocaine treatment, whereas the labelling of phosphatidylcholine was reduced. Analyses of enzyme activities in the heart after perfusion with lidocaine revealed that the activities of phosphatidate phosphatase and acyl-coenzyme A (CoA):1,2-diacylglycerol acyltransferase were enhanced. The presence of lidocaine in the assay did not directly stimulate these enzymes. However, the activity of acyl-CoA:glycerol-3-phosphate acyltransferase was stimulated by lidocaine whereas the activity of cytidine diphosphocholine:1,2-diacylglycerol cholinephosphotransferase was inhibited by lidocaine. We conclude that lidocaine affects the regulation of phospholipid biosynthesis in the heart by both direct and indirect modulation of phospholipid biosynthetic enzymes.     

Phosphatidate phosphatases of mammals,yeast, and higher plants

Phosphatidate phosphatase (EC 3.1.3.4) catalyzes the hydrolysis of phosphatidate to yieldsn-1,2-diacylglycerol and inorganic phosphate. In mammalian systems, forms of phosphatidate phosphatase involved in glycerolipid synthesis and signal transduction have been identified. Forms of the enzyme involved in signal transduction have been purified and partially characterized. In yeast, phosphatidate phosphatases associated with the endoplasmic reticulum and mitochondria have also been purified and partially characterized. Information on phosphatidate phosphatases from mammals and yeast is useful in characterizing the enzyme from plant systems. This review examines progress on the characterization of phosphatidate phosphatases from mammals, yeast, and higher plants. The purification and characterization of the phosphatidate phosphatase involved in glycerolipid synthesis in developing oilseeds may lead to the identification of the encoding gene. Increasing our understanding of the enzymes of lipid synthesis in developing seeds will aid in the development of biotechnological strategies for seed oil modification.     

Properties of diacylglycerol acyltransferase from spinach leaves

Diacylglycerol-acyltransferase (EC 2.3.1.20) was partially purified and characterized from spinach leaves. The enzyme had a pH optimum of 8.0 and activity was stimulated 2-fold by the addition of 20 mM Mn²⁺ or Mg²⁺. Diolein and dipalmitin were examined assn-1,2-diacylglycerol substrates. Only diolein gave detectable triacylglycerol synthesis. In addition, the saturation kinetics of the enzyme with 16∶0-CoA, 18∶0-CoA and 18∶1-CoA were examined. The highest apparent, Km was observed with 18∶1-CoA, the lowest with 16∶0-CoA. Endogenous spinach leaf glycerolipids were extracted and analyzed. The leaves contained 70 nmol triacylglycerol (g fresh weight)⁻.     

Reversibility of cholinephosphotransferase in lung microsomes

The effect of cytidine 5′-monophosphate (CMP) on the incorporation of cytidine 5′-diphosphate (CDP) [methyl-¹⁴C]choline or [1-¹⁴C]dipalmitoylglycerol into phosphatidylcholine (PC) catalyzed by rabbit lung microsomal CDP choline:1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied. In the presence of 0.85 mM CMP and nonsaturating diacylglycerol concentration, the incorporation of CDP[¹⁴C]choline into PC was markedly stimulated, but the incorporation of [¹⁴C]dipalmitoylglycerol into PC was inhibited. This was due to the increase of endogenous diacylglycerol generated from microsomal PC by the cholinephosphotransferase reverse reaction. However, the newly synthesized PC was not readily hydrolyzed in the presence of CMP. The results of this study suggest that the endogenous membranous diacylglycerol is utilized more preferentially for PC synthesis than the exogenous diacylglycerol and that the newly synthesized PC could rapidly equilibrate with the endogenous membrane PC pool.     

Methylating activity of (Methyl-14C)-S-adenosylmethionine by microsomes of the insectCeratitis capitata

The methylating activity of (methyl-14C)-S-adenosylmethionine by microsomes from different stages of development of the insect Ceratitis capitata was studied in a series of in vitro experiments. Larval and pharate adult microsomal preparations were used in the in vitro conditions, and the utilization of the methyl group of the S-adenosylmethionine for the synthesis of phospholipids was evaluated. Incorporation of radioactivity in lipids by pharate adult microsomes was significantly higher than that by larval preparations. In both cases, phosphatidyl ethanolamine showed the highest levels of radioactivity incorporation. Free bases from total lipid hydrolysates were resolved by paper chromatography, and the labeling was investigated by radioactivity scanning of paper chromatograms. Larval and pharate adult preparations showed clearly the presence of dimethylethanolamine and choline. The presence in the incubation media of phosphatidyldimethylethanolamine and deoxycholate enhanced the radioactivity incorporation into choline, whereas, the first stages of methylation were inhibited. These findings confirmed previous results using insect homogenates.     

A continuous assay forO-alkylglycerol monooxygenase (E.C. 1.14.16.5)

The antitumor activity of alkyl lysophospholipids has raised some questions concerning the degradation ofO-alkyl bonds in naturally occurring ether lipids. In this report, we describe the first continuous assay forO-alkylglycerol monooxygenase (AGMO), the only enzyme known to cleave theO-alkyl bond in saturated ether lipids and ether phospholipids. AGMO activity was monitored at 340 nm by coupling the NADH redox reaction to the tetrahydropteridine cofactor of the rat liver microsomal enzyme. Turnover rates as low as 0.6 nmol/min could be measured. Using radiolabeled substrates, the products were identified with a TLC-Linear-Analyzer. The only interference with this assay can arise from other reducing agents, e.g. dithiothreitol. The assay was used to develop protocols for the solubilization of AGMO from membrane preparations in the presence of detergents.     

Glutathione and antioxidants protect microsomes against lipid peroxidation and enzyme inactivation

The study investigated the relationship between lipid peroxidation and enzyme inactivation in rat hepatic microsomes and whether prior inactivation of aldehyde dehydrogenase (ALDH) exacerbated inactivation of other enzymes. In microsomes incubated with 2.5 μM iron as ferric sulfate and 50 μM ascorbate, ALDH, glucose-6-phosphate (G6Pase) and cytochrome P450 (Cyt-P450) levels decreased rapidly and concurrently with increased levels of thiobarbituric acid-reactive substances. Microsomal glutathioneS-transferase and nicotinamide adenine dinucleotide phosphate-cytochromec reductase were little affected during 1 hr of incubation. Addition of reduced glutathione partially protected, andN,N′-diphenyl-p-phenylenediamine and butylated hydroxytoluene completely protected microsomes against inactivation of ALDH, G6Pase and Cyt-P450, as well as lipid peroxidation induced by iron and ascorbate. ALDH was more susceptible than G6Pase to inactivation by iron and ascorbate, and was thus an excellent marker for oxidative stress. Inhibition of ALDH by cyanamide injection of rats exacerbated the inactivation of G6Pase in microsomes incubated with 0.1 mM, but not 25 μM 4-hydroxynonenal (4-HN). 4-HN did not stimulate lipid peroxidation. Thus, 4-HN may play a minor role in microsomal enzyme inactivation. In contrast, lipid, peroxyl radicals play an important role in microsomal enzyme inactivation, as evidenced by the prevention of both lipid peroxidation and enzyme inactivation by chain-breaking antioxidants.     

Preparation and characterization of mouse intestinal phospholipase

Phospholipase has been prepared in a stable, partially purified form from the small intestine of mice infected with the tapewormHymenolepis nana. The enzyme(s) attacks diacylphosphatides with liberation of free fatty acids and a corresponding decrease in phospholipid phosphorus, without accumulation of lysophosphatides. The reactivity with various substrates is strongly influenced by their physical state, the presence of other lipids, proteins or detergents. The phospholipids of some biomembranes (mitochondria, microsomes, red cell ghosts) are readily hydrolyzed under customary reaction conditions. The ensuing biochemical, morphological and functional alterations have been documented. In contrast, the diacylphospholipids of the cell membrane (intact erythrocytes,Pseudomonas aerunginosa) are not accessible to phospholipase action unless some alteration of the integrity of the cell is induced by physical or chemical means (hemolysis, polymyxin). The enzyme is proposed as a tool for the investigation of biomembranes and as a model for the study of phospholipase activity.     

Characterization of phospholipase A2 from rabbit lung microsomes

A phospholipase A₂ activity associated with the microsomal fraction of rabbit lung homogenates was studied. The enzyme showed specificity for thesn ⁻² ester bond of phosphatidylcholine, had an alkaline pH optimum and required Ca²⁺ for activity. Other divalent cations were unable to support hydrolysis. In the absence of detergents, exogenous phosphatidylethanolamine was deacylated at a rate sevenfold higher than phosphatidylcholine. The activity toward both substrates could be enhanced by sodium deoxycholate or, more effectively, by sodium taurodeoxycholate. Phosphatidylethanolamine required higher detergent/phospholipid molar ratios than phosphatidylcholine. Under these conditions, the preference for the former substrate over the latter was nearly abolished. The zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and the nonionic detergent Triton X-100 were either ineffective (phosphatidylcholine) or inhibitory (phosphatidylethanolamine). Addition of KCl produced opposite effects on the activity depending on the bile salt used to disperse the substrate. The phospholipase A₂ activity was inhibited byp-bromophenacyl bromide but remained unaffected after treatment with diisopropylfluorophosphate or NaF. N-Ethylmaleimide, but not other thiol reagents, partially inhibited the activity. Presented in part at the 29th CFBS meeting, Guelph, Canada, June 1986, and at the symposium “25 Years Lipids and Biomembranes,” Utrecht, The Netherlands, June 1986.     

Dietary control of the chain elongation of palmityl-CoA in rat liver microsomes

The rate of chain elongation of palmityl-CoA to stearyl-CoA in rat liver microsomes was studied in connection with the nutritional status of the rats. The microsomal chain elongation activity, which had been decreased by starvation for 48 hr, was rapidly increased to a high level on refeeding. The apparent Km value for malonyl-CoA in both normal and refed rats was the same, 1.2×10⁻⁴ M. Both cycloheximide and actinomycin D prevented the induction of microsomal chain elongation activity which was associated with refeeding. In addition, the activity of acyl-CoA hydrolase and the rates of esterification of acyl-CoA into phospholipids and neutral lipids in microsomes were not changed by the dietary alteration. These results support the conclusion that changes of the activity of microsomal chain elongation of palmityl-CoA in various nutritional status result from a rapid synthesis of new enzyme(s).     

Simultaneous quantitation of ceramides and 1,2-diacylglycerol in tissues by latroscan thin-layer chromatography-flame-ionization detection

Ceramides and 1,2-diacylglycerol have been demonstrated in intracellular signaling pathways. A method of simultaneous mass determination of ceramides and 1,2-diacylglycerol in tissues was developed using the latroscan which combines thin layer chromatography and flame ionization detection (TLC/FID) techniques. Because of relatively low amounts of these components in tissues, the fraction of nonpolar lipids, which included ceramides and glycerides, was eluted with chloroform/acetone mixture (3∶1, vol/vol) through a silicic acid column to eliminate the polar phospholipids. Development of Chromarods was carried out using three solvent systems in a four-step development technique. The relationship of the peak area ratio to weight ratio compared with cholesteryl acetate added as an internal standard was linear. The amount of ceramides increased with incubation of rat heart homogenate and human erythrocyte membranes in the presence of sphingomyelinase (E.C. 3.1.4.12). The latroscan TLC/FID system provided a quick and reliable assessment of ceramides and 1,2-diacylglycerol.     

Zinc deficiency-induced changes in the composition of microsomal membranes and in the enzymatic regulation of glycerolipid synthesis

The effects of zinc deficiency and/or castration on the lipid composition of microsomal membranes of liver, small intestine and testes were studied in rats. The results showed that feeding a zinc-deficient diet to castrated rats decreased phospholipid content and consequently increased the cholesterol-to-phospholipid ratio in liver microsomes. An increase in cholesterol-to-phospholipid ratio occurred also in small intestine and testes microsomes from rats fed the zinc-deficient diet. It is postulated, therefore, that zinc deficiency alters the lipid composition and fluidity of microsomal membranes. Zinc deficiency also affected the activities of the enzymes involved in the formation of triglycerides and phospholipids. There was a large increase in total and specific activity of phosphatidate phosphatase and the changes in the total activity of choline phosphotransferase correlated well with the changes observed in serum or liver triglycerides and phospholipids. Stearoyl CoA desaturase, which is a control enzyme for hepatic lipogenesis, was also increased by more than 200% in zinc-deficient states, as was the diglyceride content of hepatic microsomes. These results indicate that the increased synthesis of triglycerides and phospholipids in zinc deficiency may be due to the increased availability of substrates as well as to increased activities of the enzymes involved in these processes.     

Quantitation of 1,2-diacylglycerol in rat heart by latroscan TLC/FID

1,2-Diacylglycerol, which has been recognized as one of the intracellular second messengers, was measured quantitatively in the lipid extract from rat hearts using the thin layer chromatography and flame ionization detection (TLC/FID) method. Cholesterol acetate was added to the tissue as an internal standard, and the crude lipids from the tissue were purified with silicic acid column to eliminate phospholipids. Development of Chromarods was carried out using two solvent systems and a three-step development technique. The relationship of the peak area ratio detected by flame ionization detector to weight ratio was linear compared with cholesteryl acetate. The 1,2-diacylglycerol content in the rat heart in the unstimulated condition was 72.5±15.3 ng/mg wet wt (mean±SD).