Vanadate-dependent NAD(P)H oxidation by microsomal enzymes

Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.     

Oxidation of 7-dehydrocholesterol by a mouse liver microsomal system dependent on reduced pyridine nucleotides

Aerobic incubation of 7-dehydrocholesterol with mouse liver microsomes in the presence of a detergent, an iron salt, and NADH or NADPH resulted in the conversion of the sterol to more polar products. In the presence of Fe(3+) or low levels of Fe(2+) the reaction was dependent upon reduced pyridine nucleotide and a microsomal enzyme system. At high levels of Fe(2+) or in the presence of Fe(2+) or Fe(3+) and ascorbic acid, nonenzymatic oxidation of 7-dehydrocholesterol occurred in the absence of NADH or NADPH. Chromatograms of products resulting from the enzyme-dependent and enzyme-independent reactions were similar. The enzymatic reaction was inhibited by certain chelating agents, by antioxidants, and by menadione, phenazine methosulfate, and ferricyanide. Low concentrations of EDTA stimulated the reaction and high concentrations inhibited it. In the complete system sterol oxidation was correlated with the peroxidation of microsomal lipids, but peroxidation of microsomal lipids proceeded more rapidly when either the sterol, the detergent, or both were omitted. Ergosterol was resistant to oxidation under conditions that caused extensive loss of 7-dehydrocholesterol. Microsomes from tissues other than liver were relatively inactive.     

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.     

Hydroperoxide-induced loss of pyridine nucleotides and release of calcium from rat liver mitochondria

It has been previously reported (L?tscher, H. R., Winterhalter, K. H., Carafoli, E., and Richter, C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4340-4344) that in Ca2+-loaded mitochondria hydroperoxides induce a release of Ca2+ from mitochondria and an irreversible oxidation of mitochondrial pyridine nucleotides. Here we show that in the presence of Ca2+ oxidized mitochondrial pyridine nucleotides are hydrolyzed inside mitochondria and that nicotinamide is released from mitochondria. The extent of the hydrolysis of NAD(P)+ is dependent on the amount of both hydroperoxide and Ca2+. The hydrolysis is reversible in the presence of added nicotinamide. The release of Ca2+ from mitochondria is electroneutral, and is directly or indirectly dependent on oxidized mitochondrial pyridine nucleotides. By contrast, the uptake of Ca2+ most probably does not require the present of reduced pyridine nucleotides. Control experiments show that even under the most drastic conditions employed in this study (100 nmol of Ca2+ and 85 nmol of t-butylhydroperoxide/mg of protein) mitochondria retain a considerable degree of functional integrity.     

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.     

Mechanism of release of integral proteins from rat liver microsomal membranes

The release of three integral enzymatic activities (NADH- and NADPH-cytochrome c reductase and 5'-nucleotidase) and total protein from washed rat liver microsomal membranes, upon simple incubation at 37 degrees C in aqueous media, was investigated. Release does not depend on contaminating proteases and is enhanced by alkaline pH. Total protein and enzyme release is consistent with a loss of phospholipids which are not recovered in the soluble phase. Following incubation at pH 9.0 large amounts of free fatty acids were recovered in the soluble phase, accounting for a ratio of 1/1 (w/w) with released protein. This evidence, together with the data available about densities (1.07-1.08 g/ml) and molecular weights (1 700 000-700 000) of the released enzymes, suggests that they are solubilized from microsomal membranes in the form of mixed micelles mostly formed by free fatty acids and integral proteins, probably owing to the activity of endogenous phospholipases on membrane lipids. Release of total protein and enzymatic activities is decreased by Ca2+, whose possible role in the phenomenon is discussed.