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

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.     

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.     

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.     

Effects of clofibrate on the metabolism of progesterone and oestradiol in rat liver microsomal fraction

1. The substrate conversion of [4-(14)C]progesterone and [4-(14)C]oestradiol during incubation with the liver microsomal fraction from both control and clofibrate-treated rats amounted to about 10-15 and 20-25% respectively. 2. The metabolites of progesterone formed by preparations from control rats were hydroxylated in the 16alpha-position (14%), the 6beta-position (12%) and the 2alpha-position (7%). Of the products formed from oestradiol 12% were recovered as a 16alpha-hydroxylated derivative whereas 5% had a 6beta- and 2% a 6alpha-hydroxyl group. 3. Clofibrate affected the microsomal metabolism of both progesterone and oestradiol. It induced 7alpha-hydroxylation of both compounds, metabolic conversions not found in control rats. The 6beta-hydroxylation of progesterone and the 6alpha-hydroxylation of oestradiol were enhanced by a factor of 2 and 3.5 respectively. The 2alpha-hydroxylation, and the 20alpha- and 20beta-hydroxy steroid reduction of progesterone were significantly decreased as were the 16alpha- and the 6beta-hydroxylation of oestradiol.     

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.     

束缚应激对D-氨基半乳糖联合脂多糖诱导小鼠肝损伤的保护作用

目的探讨束缚应激对D-氨基半乳糖联合脂多糖（D–galactosamine and lipopolysaccharide combination,D＋L）诱导的小鼠肝损伤的保护作用。方法正常BALB/c小鼠随机分为正常对照组（con）、应激对照组（str）、模型组（D＋L）、束缚应激组（D＋L＋str）。con组小鼠常规饲养;str组小鼠给予定时定量的束缚应激;D＋L组小鼠腹腔注射D-氨基半乳糖和脂多糖的混合溶液,1次/2天;D＋L＋str组小鼠腹腔注射等量D＋L混合液后,给予与str组相同的束缚应激。第8周,各组小鼠取血检测血清AST、ALT,肝脏固定后HE及Masson染色观察小鼠肝脏结构、细胞形态及纤维化程度。结果第8周D＋L＋str组与D＋L组小鼠相比,血清ALT和AST显著降低（P〈0.01）,AST/ALT显著增高（P〈0.01）;HE及Masson染色显示,D＋L组小鼠肝小叶结构紊乱,出现结节性增生及大量上皮细胞核浓缩、溶解、坏死,枯否氏细胞浸润,而D＋L＋str组未见明显病理变化;纤维化程度评分显示,D＋L＋str组与D＋L组小鼠相比,病理评分与纤维显色吸光度值均显著降低（P〈0.05）。结论束缚应激对D＋L诱导的小鼠肝损伤具有一定保护作用。     

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.     

Cyclic AMP metabolism by swine adipocyte microsomal and plasma membranes

Extracellular cyclic AMP is source of extracellular adenosine in brain and kidney. Whether this occurs in adipose tissue is unknown. The present study evaluated the capacity of swine adipocyte plasma membranes to metabolize cyclic AMP to AMP and adenosine, via phosphodiesterase (PDE) and 5'-nucleotidase (5'-NT), respectively. Plasma membranes (PM) and microsomal membranes (MM) were isolated from over-the-shoulder subcutaneous adipose tissue of 3 month-old male miniature swine. The purity of the membrane fractions was determined and PDE and 5'-NT activities in PM and MM fractions were corrected for cross-contamination. The maximal activity of MM-PDE was 7-fold greater than that of PM-PDE. MM-PDE was 100% inhibited by 5 microM cilostamide, while PM-PDE was unaffected by this PDE3B inhibitor. Inhibitors of PDE1, PDE2, PDE4 and PDE5 also failed to inhibit PM-PDE. However, 1 mM DPSPX inhibited PM-PDE activity by 72%. When PM were incubated with 0.8 microM cyclic AMP for 20 min, AMP accumulation was four times that of adenosine. These data demonstrate that cyclic AMP can be converted to AMP and adenosine by the PM-bound enzymes 5'-NT and PDE, and suggest that the PM-PDE responsible for extracellular cyclic AMP metabolism to AMP is distinct from the intracellular MM-PDE.     

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.     

Vanadate-dependent oxidation of pyridine nucleotides in rat liver microsomal membranes

An enzymatic Na₃VO₄-dependent system for the oxidation of reduced pyridine nucleotides in purified rat liver microsomes was characterized. The system has a pH optimum of 6.5, and appears to be specific for vanadate, since activity in the presence of a related transition metal, molybdate, was not detected. Vanadate-dependent oxidation occurred with a concomitant consumption of O₂ and, contrary to previous reports, preferred NADPH over NADH. At pH 6.5, the NADPH/NADH oxidase activity ratio was greater than 2:1. Sodium vanadate-dependent oxidation of NADH was inhibited by rotenone, antimycin A, NaN₃, and NaCN. Conversely, Na₃VO₄-dependent NADPH oxidation was slightly affected by rotenone, but was insensitive to antimycin A, NaN₃, NaCN, or quinacrine. Vanadate-dependent oxidation of either pyridine nucleotide was inhibited by the addition of either Superoxide dismutase or catalase, indicating that both superoxide and hydrogen peroxide may be intermediates in the process. Linear sucrose gradient purification of the microsomes showed that the vanadate-dependent system for NADPH oxidation resides primarily in the endoplasmic reticulum. These studies indicate the existence of separate and distinct enzymatic systems for vanadate-stimulated oxidation of NADPH and NADH in mammalian microsomal membranes, and argue against an exclusive role of endogenous Superoxide in the process.     

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.     

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.