Enzyme and phospholipid asymmetry in liver microsomal membranes

The transverse distribution of enzyme proteins and phospholipids within microsomal membranes was studied by analyzing membrane composition after treatment with proteases and phospholipases. Upon trypsin treatment of closed microsomal vesicles, NADH- and NADPH-cytochrome c reductases as well as cytochrome b5 were solubilized or inactivated, while cytochrome P-450 was partially inactivated. When microsomes were exposed to a concentration of deoxycholate which makes them permeable to macromolecules but does not disrupt the membrane, the detergent alone was sufficient to release four enzymes: nucleoside diphosphatase, esterase, beta-glucuronidase, and a portion of the DT-diaphorase. Introduction of trypsin into the vesicle lumen inactivated glucose-6-phosphatase completely and cytochrome P-450 partially. The rest of this cytochrome, ATPase, AMPase, UDP-glucuronyltransferase, and the remaining 50% of DT-diaphorase activity were not affected by proteolysis from either side of the membrane. Phospholipase A treatment of intact microsomes in the presence of albumin hydrolyzed all of the phosphatidylethanolamine, phosphatidylserine, and 55% of the phosphatidylcholine. From this observation, it was concluded that these lipids are localized in the outer half of the bilayer of the microsomal membrane; Phosphatidylinositol, 45% of the phosphatidylcholine, and sphingomyelin are tentatively assigned to the inner half of this bilayer. It appears that the various enzyme proteins and phospholipids of the microsomal membrane display an asymmetric distribution in the transverse plane.     

Influence of cholesterol on sphingomyelin metabolism and hemileaflet fluidity of rat liver plasma membranes.

The objective of this study was to examine the effect of dietary Chol supplementation on SM metabolism in rat liver plasma membranes, as well as on membrane leaflet fluidity characteristics. The membrane Chol content increased significantly during the first 20 days of dietary feeding, but returned to the level of the control group when the diet was continued for another ten days. The initially more fluid outer leaflet of the membrane rigidified as a result of the diet, obliterating the natural asymmetry in the fluidity of the membrane bilayer. Changes in the neutral SMase activity were also observed. These changes were in strong negative correlation (r = -0.978) with the Chol/Pr ratio and are consistent with the in vitro inhibition of SMase activity reported earlier. In contrast, the SM synthesizing enzymes, PC:Cer-PCh and PE:Cer-PEt transferase, were stimulated in course of the dietary Chol feeding. The activity of PC:Cer-PCh transferase was more strongly affected. Our results support the concept that SM metabolism is regulated coordinately with that of Chol. The present work could contribute to the better understanding of the parallel accumulation of SM and Chol observed in a variety of pathological conditions such as atherosclerosis and Niemann-Pick disease.     

Lipid composition and turnover of rough and smooth microsomal membranes in rat liver

Subfractions of rat liver microsomes (rough, smooth I, and smooth II), isolated in a cation-containing sucrose gradient system, were analyzed. After removal of adsorbed and luminal protein, these subfractions had the same phospholipid/protein ratio, about 0.40. Both the classes and the relative amounts of phospholipids were similar in the three subfractions, but the relative amounts of neutral lipids (predominantly free cholesterol and triglycerides) were higher in smooth I and especially in smooth II than in rough microsomes. Various pieces of evidence indicate that the neutral lipids are tightly bound to the membranes. Glycerol-(3)H was incorporated into the phospholipids of the rough and smooth I microsomes significantly faster than into those of the smooth II membranes; (32)P incorporation followed a similar but less pronounced pattern. Acetate-(3)H was incorporated into the free cholesterol of smooth I microsomes only half as fast as into the other two subfractions. Injection of phenobarbital increased the cellular phospholipid and neutral lipid content in the rough and smooth I, but not in the smooth II microsomes. Consequently, the neutral lipid/phospholipid ratio of all three subfractions remained unchanged after phenobarbital treatment. It is concluded that the membranes of the rough and the two smooth microsomal subfractions from rat liver have a similar phospholipid composition, but are dissimilar in their neutral lipid content and in the incorporation rate of precursors into membrane lipids.     

Acidic phospholipid species inhibit adenylate cyclase activity in rat liver plasma membranes.

Incubation of rat liver plasma membranes with liposomes of dioleoyl phosphatidic acid (dioleoyl-PA) led to an inhibition of adenylate cyclase activity which was more pronounced when fluoride-stimulated activity was followed than when glucagon-stimulated activity was followed. If Mn2+ (5 mM) replaced low (5 mM) [Mg2+] in adenylate cyclase assays, or if high (20 mM) [Mg2+] were employed, then the perceived inhibitory effect of phosphatidic acid was markedly reduced when the fluoride-stimulated activity was followed but was enhanced for the glucagon-stimulated activity. The inhibition of adenylate cyclase activity observed correlated with the association of dioleoyl-PA with the plasma membranes. Adenylate cyclase activity in dioleoyl-PA-treated membranes, however, responded differently to changes in [Mg2+] than did the enzyme in native liver plasma membranes. Benzyl alcohol, which increases membrane fluidity, had similar stimulatory effects on the fluoride- and glucagon-stimulated adenylate cyclase activities in both native and dioleoyl-PA-treated membranes. Incubation of the plasma membranes with phosphatidylserine also led to similar inhibitory effects on adenylate cyclase and responses to Mg2+. Arrhenius plots of both glucagon- and fluoride-stimulated adenylate cyclase activity were different in dioleoyl-PA-treated plasma membranes, compared with native membranes, with a new 'break' occurring at around 16 degrees C, indicating that dioleoyl-PA had become incorporated into the bilayer. E.s.r. analysis of dioleoyl-PA-treated plasma membranes with a nitroxide-labelled fatty acid spin probe identified a new lipid phase separation occurring at around 16 degrees C with also a lipid phase separation occurring at around 28 degrees C as in native liver plasma membranes. It is suggested that acidic phospholipids inhibit adenylate cyclase by virtue of a direct headgroup specific interaction and that this perturbation may be centred at the level of regulation of this enzyme by the stimulatory guanine nucleotide regulatory protein NS.     

Alterations in microsomal and plasma membranes during liver regeneration.

Investigations have been carried out on the alterations of membrane lipids and some enzyme activities during liver regeneration. The results indicated that 32 h after partial hepatectomy the membrane phospholipids per mg protein were augmented. The cholesterol esters were also increased in both microsomal and plasma membranes. The specific radioactivity of the separate phospholipid fractions, estimated by incorporation of 14C-palmitate into the phospholipid molecules, was higher in membranes from partially hepatectomized rats, compared to sham-operated ones, indicating an enhanced phospholipid synthesis. The content and specific radioactivity of diacylglycerols and triacylglycerols was enhanced in both types of membranes from regenerating liver. Moreover, we observed a fluidization of these membranes, which is illustrated by the decrease of the structural order parameter (SDPH) of the lipid bilayer as well as by the elevation of the excimer to monomer fluorescent ratio (IE/IM). 1,6-Diphenyl-1,3,5-hexatriene and pyrene were used as fluorescent probes for determination of the membranes physical state. Palmitoyl-CoA and oleoyl-CoA synthetase, acyl-CoA: lysophosphocholine and acyl-CoA:lysophosphoethanolamine acyltransferase as well as phospholipase C activities were augmented in membranes from partially hepatectomized rats. The biological significance of these alterations in the process of liver regeneration is discussed.     

The role of sphingomyelin synthetase and sphingomyelinase in 1,2-dimethylhydrazine-induced lipid alterations of rat colonic plasma membranes

Recently, our laboratory, utilizing the 1,2-dimethylhydrazine model of colonic adenocarcinoma, demonstrated alterations in the 'dynamic component' of fluidity in brush-border membranes prepared from distal colonocytes of rats administered this agent for 5, 10 and 15 weeks, i.e., before the development of colon cancer. Furthermore, changes in the sphingomyelin content and sphingomyelin/phosphatidylcholine molar ratio of these membranes appeared, at least partially, to be responsible for these fluidity alterations. In an attempt to elucidate the mechanism(s) involved in these dimethylhydrazine-induced lipid changes, in the present studies the activities of sphingomyelin synthetase and magnesium-dependent neutral sphingomyelinase, enzymes involved in the synthesis and degradation of this phospholipid, respectively, were examined and compared in distal colonic brush-border membranes prepared from rats after 5, 10 or 15 weeks administration of dimethylhydrazine or diluent. The results of these studies demonstrate that alterations in both these enzymatic activities can be detected after administration of dimethylhydrazine and appear to, at least in part, be responsible for the changes in membrane sphingomyelin composition noted previously. These results as well as a discussion of their possible serve as the basis for the present report.     

Purification of phospholipid methyltransferase from rat liver microsomal fraction.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.     

Studies on plasma membranes XVII. On the chemical composition of plasma membranes prepared from rat and mouse liver and hepatomas

Summary Plasma membranes were isolated under hypotonic conditions from rat and mouse livers and five hepatomas, i.e. one rather anaplastic rat hepatoma (and its subline) and three well-differentiated mouse hepatomas. All these membranes contained some 25% protein soluble in 0.15m NaCl. Evidence is presented that this protein is mainly, if not exclusively of nonmembranous origin. Protein/phospholipid P (P=phosphorus) ratios did not differ significantly for the various plasma membrane species except the rat-hepatoma subline, which showed a markedly lower ratio and was thus identified. Hepatoma membranes contained more P of a nonphospholipid nature than did liver membranes and to this increase contributed in all instances an increased RNA content and in some cases also an increased DNA content. The presence of DNA in these plasma membranes is artefactual, but that of RNA is more complicated. Artefactually, Ca²⁺-associated RNA of low mol wt and soluble in 0.15m NaCl, and residual RNA (genuine?, in liver membranes less than 1% in respect of protein) have been demonstrated. The increase in hepatoma-membrane RNA is attributed to the ribosomal RNA of the few microsomal vesicles which are structurally connected with these plasma membranes. The sialic acid content and the percentage of neuraminidase-resistant sialic acid of hepatoma as compared with liver membranes was either similar or changed, depending on the hepatoma strain. Gelfiltration of trypsin-released peptides of liver plasma membranes showed hexosamine and hexose to be confined to the sialic acidcontaining fractions. In spite of quantitative differences among fractions, the relative contents of the three carbohydrates in the combined fractions were (about) similar to those in intact liver membranes. Similar experiments with the rat-hepatoma membranes showed a changed carbohydrate expression.     

Influence of phospholipid environment on the phosphatidylethanolamine: ceramide-phosphorylethanolamine transferase activity in rat liver plasma membranes.

1. Plasma membranes were treated with phospholipase A2, phospholipase C or phospholipase D. The phosphatidylethanolamine:ceramide-phosphorylethanolamine transferase was deactivated by phospholipase C treatment, whereas phospholipase A2 and phospholipase D did not affect the enzyme. 2. Incorporation of phosphatidylethanolamine and phosphatidylglycerol into partially delipidated plasma membranes resulted in significant stimulation of the transferase, whereas inclusion of sphingomyelin and phosphatidylserine suppressed the enzyme activity. Our results suggest that phosphatidylserine is a regulator of sphingomyelin level in membranes. 3. The activity of phosphatidylethanolamine:ceramide-phosphorylethanolamine transferase was not influenced by the fluidity of its lipid environment.     

Copper deficiency-induced changes in the fatty acid composition of mitochondrial and microsomal membranes of rat liver

The effects of copper deficiency on the fatty acid composition of mitochondrial and microsomal phospholipids in rat liver were studied. Copper deficiency was induced by a milk powder diet. To evaluate the effect of the milk diet on the fatty acid pattern of mitochondrial and microsomal phospholipids, one group of rats was fed Cusupplemented powdered milk. A decrease in the relative proportion of linoleic acid and an increase in the level of oleic and docosahexaenoic acids in membrane phospholipids were found in this group. However, no changes in the fatty acid pattern characteristic of essential fatty acid deficiency were observed. Dietary copper deficiency produced a significant decrease in the relative amounts of linoleic and arachidonic acids, as well as an increase in the docosahexaenoic acid content in both mitochondrial and microsomal membranes compared to the nondeficient controls. The disproportionate quantities of polyunsaturated fatty acids are discussed with a view to the disturbances of membrane function in copper deficiency.     

Recycling of glucosylceramide and sphingosine for the biosynthesis of gangliosides and sphingomyelin in rat liver.

It was previously shown that sphingomyelin and gangliosides can be biosynthesized starting from sphingosine or sphingosine-containing fragments which originated in the course of GM1 ganglioside catabolism. In the present paper we investigated which fragments were specifically re-used for sphingomyelin and ganglioside biosynthesis in rat liver. At 30 h after intravenous injection of GM1 labelled at the level of the fatty acid ([stearoyl-14C]GM1) or of the sphingosine ([Sph-3H]) moiety, it was observed that radioactive sphingomyelin was formed almost exclusively after the sphingosine-labelled-GM1 administration. This permitted the recognition of sphingosine as the metabolite re-used for sphingomyelin biosynthesis. Conversely, gangliosides more complex than GM1 were similarly radiolabelled after the two treatments, thus ruling out sphingosine re-utilization for ganglioside biosynthesis. For the identification of the lipid fragment re-used for ganglioside biosynthesis, we administered to rats neutral glycosphingolipids (galactosylceramide, glucosylceramide and lactosylceramide) each radiolabelled in the sphingosine moiety or in the terminal sugar residue. Thereafter we compared the formation of radiolabelled gangliosides in the liver with respect to the species administered and the label location. After galactosylceramide was injected, no radiolabelled gangliosides were formed. After the administration of differently labelled glucosylceramide, radiolabelled gangliosides were formed, regardless of the position of the label. After lactosylceramide administration, the ganglioside fraction became more radioactive when the long-chain-base-labelled precursors were used. These results suggest that glucosylceramide, derived from glycosphingolipid and ganglioside catabolism, is recycled for ganglioside biosynthesis.     

Effect of hydrocortisone on the phospholipid composition and activity of various enzymes of phospholipid metabolism in the nuclear membranes of the rat liver

It was shown that hydrocortisone injections markedly increase the total content of liver nuclear membrane phospholipids, the greatest increase being observed in the phosphatidyl choline level. It was found also that nuclear membranes contain phospholipid metabolism enzymes. The hormone-induced increase in the phospholipid content is accompanied by a marked decrease of the activity of phospholipase A2 (5.3 times against control), phospholipase C (9.3 times) and acyl-CoA: lysophosphatidylcholine transferase (2.5 times). The results obtained are suggestive of appreciable metabolic changes in nuclear membrane phospholipids caused by hydrocortisone which, in turn, may be due to hormonal activation of the genome.