Rate of microsomal protein metabolism in the mouse liver

NaH14CO3, a poorly reutilized biosynthesis precursor, was used to study the rate of whole microsomal protein degradation in mouse liver. The use of the precursors, however, does not prevent the reutilization of labeled amino acids on phenobarbital administration. To avoid reutilization, a new method has been developed. It was shown that phenobarbital injections have no effect on the degradation rate of the whole microsomal protein. The effect of amidopyrine, a monooxygenase microsomal system substrate, on the rate of whole microsomal protein degradation was examined. An experimental model was developed, in which the monooxygenase microsomal system substrate does not exhibit the properties of its inducer. Amidopyrine administration to mice simultaneously with phenobarbital induction has no effect on the degradation rate of the whole microsomal protein.     

Phosphatidylcholine mobility in liver microsomal membranes

Purified phosphatidylcholine exchange protein from bovine liver was used to exchange rat liver microsomal phosphatidylcholine for egg phosphatidylcholine. It was found that at 25 and 37°C rat liver microsomal phosphatidylcholine was completely and rapidly available for replacement by egg phosphatidylcholine. In contrast, phosphatidylcholine in vesicles prepared from total microsomal lipids could only be exchanged for about 60%. At 8 and 0°C complex exchange kinetics were observed for phosphatidylcholine in rat liver microsomes. The exchange process had neither effect on the permeability of the microsomal membrane to mannose 6-phosphate, nor on the permeability of the phosphatidylcholine vesicles to neodymium (III) cations.Purified phospholipase A₂ from Naja naja could hydrolyze some 55–60% of microsomal phosphatidylcholine at 0°C, but 70–80% at 37°C. Microsomal phosphatidylcholine, remaining after phospholipase treatment at 37°C, could be exchanged for egg phosphatidylcholine at 37°C, but at a slower rate than with intact microsomes. Microsomal phosphatidylcholine remaining after phospholipase treatment at 0 and 37°C had a lower content of arachidonic acid than the original phosphatidylcholine.These results are discussed with respect to the localization and transmembrane movement of phosphatidylcholine in liver microsomes.     

Phosphatidylcholine mobility in liver microsomal membranes

Analysis of the 35SO4-labelled macromolecules synthesized by cultures of normal )NIL8) and transformed (NIL8-HSV) hamster fibroblasts has revealed the following differences between the two cell lines: (1) The proportion of sulfate incorporated into cell-associated macromolecules is three times higher in normal than in transformed cells. In addition, normal fibroblasts incorporate more sulfate into extracellular, middle and low molecular weight species than do transformed cells. Transformed cells, however, incorporate more sulfate into extracellular, very high molecular weight species than do normal cells. (2) Normal fibroblasts, which synthesize much more extracellular dermatan sulfate than do transformed cells, produce a class of extracellular heterogeneous sulfated proteoglycans absent from transformed cultures. This macromolecular species consists largely of dermatan sulfate. The transformed cells instead release a lower molecular weight class of proteoglycans which consist of chondroitin sulfates A and C. (3) The large, external, transformation-sensitive glycoprotein is sulfated in NIL8 cultures. This macromolecular species is present on the surface membrane of normal cells, but absent from transformed cells. Sulfated large, external transformation-sensitive protein is also present in the conditioned medium from normal cultures. A similar species is present in the conditioned medium from transformed cultures, but has a slightly higher apparent molecular weight and differs in other properties from the large, external, transformation-sensitive protein of normal cells.     

Effect of t-butyl hydroperoxide on liver microsomal membranes and microsomal calcium sequestration

In vitro exposure of hepatocytes or liver microsomes to t-butyl hydroperoxide resulted in a marked decrease of liver microsomal calcium pump activity. Decreased calcium pump activity was dependent upon both concentration and time. Liver microsomes could be protected from this effect by glutathione or dithiothreitol. In addition to decreased calcium pump activity, exposure of liver microsomes to t-butyl hydroperoxide produced a concentration-dependent aggregation of microsomal membrane protein as determined by polyacrylamide gel electrophoresis. Inhibition of microsomal calcium pump activity was observed when intact hepatocytes were incubated, in vitro, with t-butyl hydroperoxide. However, aggregation of microsomal membrane protein was not observed when hepatocytes were incubated with t-butyl hydroperoxide. The effects produced by exposure of liver microsomes to this compound do not appear to be a complete model of actions of the compound on intact cells.     

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.     

Sidedness of ceramide-phosphoethanolamine synthesis on rat liver and brain microsomal membranes

Phosphatidylethanolamine:ceramide-ethanolamine-phosphotransferase catalyzes the synthesis of ceramide-phosphoethanolamine, a sphingomyelin analogue. Its localization was studied in rat liver and brain microsomes. After testing the integrity and the sidedness of microsomal vesicles, trypsin treatment of intact or deoxycholate-disrupted microsomes made it possible to conclude that both the transferase and the ceramide-phosphoethanolamine are located in the cisternal leaflet of the membrane bilayer. Using trinitrobenzenesulfonic acid as a probe, no trace of newly synthesized ceramide-phosphoethanolamine was detectable on the cytoplasmic side of the microsomes.     

D-galactosamine induced changes in rat liver plasma membranes lipid composition and some enzyme activities

The influence of D-galactosamine administration on rat liver plasma membranes lipid composition, fluidity and some enzyme activities was investigated. D-Galactosamine was found to induce an increase of the total phospholipids, the cholesterol level and membrane rigidity. In liver plasma membranes of D-galactosamine-treated rats the exogenous phospholipase A2 activity was enhanced about 2 fold, whereas the endogenous activity was slightly decreased. No alteration of the neutral sphingomyelinase activity was observed.     

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.     

Theophylline metabolism by rat liver microsomal monooxygenases

Theophylline metabolism has been studied in a reconstituted monooxygenase system with purified forms of cytochrome P-450: P-450a, P-450b, P-450d and P-450k as well as in liver microsomes of control and 3-methylcholanthrene-induced rats. Cytochrome P-450 isoforms, P-450a and P-450b, had no effect on theophylline metabolism, whereas forms P-450d and P-450k induced the synthesis of 1.3-dimethyluric acid (1.3-DMA) at the rates of 900 and 330 pmol/min/nmol of protein, respectively. The catalytic activity of these isoforms was fully inhibited by homologous monospecific antibodies. P-450c catalyzed the formation of a nonidentified metabolite. In microsomes of control animals antibodies specifically directed to cytochrome P-450k suppressed the rate of 1.3-DMA synthesis by 73%, whereas antibodies specifically raised against P-450c+d--by 11%. In microsomes of methylcholanthrene-induced animals the rate of 1.3-DMA synthesis was increased two-fold. This activity was inhibited by 61% by antibodies to cytochrome P-450k and by 18% by anti-P-450c+d antibodies.     

Association of diamine oxidase with microsomal membranes in rabbit liver

Diamine oxidase (diamine O₂ oxidoreductase, EC 1.4.3.6, DAO) of rabbit liver is localized in the microsomal fraction (1, 2). Microsomes, freed of adsorbed and intraluminal proteins by washing procedures, still retain all DAO activity. This indicates that DAO belongs to the microsomal membrane itself. While the release of DAO activity from washed microsomes occurs exclusively in the presence of high concentrations of deoxycholate, tight binding of this enzyme to the membrane structure is apparent.     

Free radical metabolism of mutagenic acridines and binding to microsomal membranes

Free radical metabolism of the acridine derivatives, quinacrine and 9-amino-acridine, has been studied using horseradish peroxidase-H₂O₂ (HRP-H₂O₂) and prostaglandin-arachidonic acid systems. In the presence of HRP-H₂O₂ quinacrine rapidly formed a free radical intermediate consisting of three lines which collapsed into a single line with a g-value of 2.0055. Under similar conditions no radical was detected with 9-aminoacridine. In contrast, incubation of either quinacrine or 9-aminoacridine with ram seminal vesicle microsomes and arachidonic acid gave a single line spectrum with g-values of 2.0055. Although no radical could be detected with rat hepatic microsomes, incubation of the acridines resulted in covalent binding to microsomal membranes which was NADPH-dependent. Free radical metabolism and covalent binding may play a significant role in the mutagenic properties of quinacrine and 9-aminoacridine.     

Activity of enzymes participating in xenobiotic metabolism and the condition of microsomal membranes of rat liver in vitamin K deficiency

Alimentary deficiency or vitamin K (vitamin K-poor diet) as well as the vitamin deficiency resulting from sinkumar administration are accompanied by a decreased activity of microsomal demethylases, hydroxylase, NADH- and nNADPH-reductases of dichlorophenolindophenol and neotrazolium. The activity of cytosolic enzymes (only glutathione-S-transferases, aryl- and allyl esterases) is diminished in a lesser degree. Vitamin K deficiency does not significantly interfere with the effect of the xenobiotic metabolism enzyme inducer (phenobarbital) or the cytochrome P-450 inhibitor (cobalt chloride). The changes in the enzyme activity result in a decrease of acetanilide biotransformation. A possible reason for the observed changes in the activity of microsomal enzymes is the weakening of hydrophobic and polar interactions in microsomal membranes. This hypothesis was confirmed by experiments with the use of membrane perturbants as well as by solubilization of membrane-bound enzymes.     

Cytoplasmic location of linoleoyl-CoA desaturase in microsomal membranes of rat liver

The intramembrane localization of linoleoyl-CoA desaturase in rat liver microsomes was examined by various methods, such as digestion by proteases, effect of detergents, and inhibition by the antibodies against purified terminal desaturase. Exposure of the desaturase on the surface of microsomal vesicles was suggested by the fact that the enzyme activity in the intact microsomes was susceptible to tryptic digestion, and considerably inhibited by anti-desaturase antibodies. When microsomes were previously treated with trypsin, the enzyme became more susceptible to the antibodies. Furthermore, it was demonstrated that the protein fragments cleaved from microsomal membranes by tryptic digestion formed a single precipitin line with the antibodies by the double-immunodiffusion test. These findings suggest the presence of linoleoyl-CoA desaturase on the cytoplasmic surface in the endoplasmic reticulum, since tryptic digestion liberates only the protein components situated on the surface area of membranes. In addition, desaturase activity in the intact microsomes was not stimulated by addition of the detergent, indicating the further outside location of the active site of the enzyme in microsomal vesicles. The pretreatment of microsomes with a low concentration (0.05%) of sodium deoxycholate, which destroys the permeability barrier for macromolecules without membrane disassembly, did not increase the susceptibility to tryptic digestion and the antibodies. These results show that linoleoyl-CoA desaturase is not present in a latent state in the membrane.