Properties of Reconstituted Cholesterol 7α‐Hydroxylase System from Rat and Rabbit Liver Microsomes

Hansson, Ronnie; Wikvall, Kjell

doi:10.1111/j.1432-1033.1979.tb12838.x

Cited by 50 publications

(10 citation statements)

References 34 publications

(14 reference statements)

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“…Standards for IgG subclass determination were kindly donated by Pharmacia. Preparations and sources for steroid analogues have, been described previously [4]. …”

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Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

Bergman

Postlind

1990

Biochemical Journal

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The cytochrome P-450 enzyme which catalyses 25-hydroxylation of vitamin D3 (cytochrome P-45025) from pig kidney microsomes [Postlind & Wikvall (1988) Biochem. J. 253,[549][550][551][552] has been further purified. The specific content of cytochrome P-450 was 15.0 nmol -mg of protein-', and the protein showed a single spot with an apparent isoelectric point of 7.4 and an M, of 50500 upon two-dimensional isoelectric-focusing/SDS/PAGE. The 25-hydroxylase activity towards vitamin D3 was 124pmol min-' nmol of cytochrome P-450-1 and towards la-hydroxyvitamin D3 it was 1375 pmol min-' nmol-1. The preparation also catalysed the 25-hydroxylation of 5/J-cholestane-3a,7a-diol at a rate of 1000 pmol min-' nmol ofcytochrome P-450-1 and w -1 hydroxylation of lauric acid at a rate of 200 pmol min-I nmol of cytochrome P-450-1. A monoclonal antibody raised against the 25-hydroxylating cytochrome P-450, designated mAb 25E5, was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-45025 from kidney as well as from pig liver microsomes, and to immunoprecipitate the activity for 25-hydroxylation of vitamin D3 and 5/3-cholestane-3a,7a-diol when assayed in a reconstituted system. The hydroxylase activity towards lauric acid was not inhibited by the antibody. By SDS/PAGE and immunoblotting with mAb 25E5, cytochrome P-45025 was detected in both pig kidney and pig liver microsomes. These results indicate a similar or the same species of cytochrome P-450 in pig kidney and liver microsomes catalysing 25-hydroxylation of vitamin D3 and C27 steroids. The N-terminal amino acid sequence of the purified cytochrome P-45025 from pig kidney microsomes differed from those of hitherto isolated mammalian cytochromes P-450. INTRODUCTION25-Hydroxylation is involved in the bioactivation of vitamin D and in bile acid metabolism [1,2]. The 25-hydroxylation of vitamin D3is the first step in the bioactivation of vitamin D3 into its hormonal form, and microsomal 25-hydroxylation is involved in an alternative pathway for bile acid synthesis [2]. So far, no extrahepatic enzyme systems able to catalyse 25-hydroxylation of bile acid intermediates have been described.In a previous study [3], the isolation of a cytochrome P-450 (cytochrome P-45025) from pig kidney microsomes which is active in the 25-hydroxylation of vitamin D3 was described. In the present study the enzyme is further characterized with a monoclonal antibody and by determination of the N-terminal amino acid sequence. The properties of the enzyme are compared with those of liver microsomal 25-hydroxylase. EXPERIMENTAL MaterialsTSK DEAE-5PW and ampholines were from LKB, and Mono S HR 10/10 and Protein A-Sepharose were from Pharmacia. Freund's complete adjuvant was from Difco. Standards for IgG subclass determination were kindly donated by Pharmacia. Preparations and sources for steroid analogues have, been described previously [4]. Enzyme purificationCytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids from pig kidney microsomes was purified to...

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“…Standards for IgG subclass determination were kindly donated by Pharmacia. Preparations and sources for steroid analogues have, been described previously [4]. …”

mentioning

confidence: 99%

Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

Bergman

Postlind

1990

Biochemical Journal

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“…Sources and preparations of the various other labeled substrates have been described previously [2,11]. Octylamine-Sepharose 4B was prepared [12J and materials were obtained as described previously [2].…”

Section: Methodsmentioning

confidence: 99%

Cytochromes P‐450 LM_3b and LM₄ in Biosynthesis of Bile Acids

Boström¹,

Hansson²,

Jönsson³

et al. 1981

European Journal of Biochemistry

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Cytochromes P-450 LM3b and LM4 were prepared from untreated and cholestyramine-treated rabbits. The catalytic properties of these cytochrome P-450 fractions towards substrates in bile acid biosynthesis were studied in reconstituted systems containing NADPH -cytochrome P-450 reductase and phospholipid. Cytochrome P-450 LM3b showed no hydroxylase activity towards cholesterol and only low activity towards some other Cz7-steroids whereas it catalyzed efficient hydroxylation of testosterone and demethylation of ethylmorphine. Preparations of cytochrome P-450 LM4 catalyzed hydroxylation of cholesterol and other Cz7-steroids more efficiently than microsomes. Cytochrome hs had no stimulatory effect on the Cz,-steroid hydroxylase activities.

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“…Lipid peroxidation can become autocatalytic, but the chain reaction can be prevented by the action of vitamin E. Vitamin C has a sparing activity on vitamin E. The hypocholesterolemic effect of vitamin E in rats may be explained in that vitamin E enhances the activity of cholesterol 7 α-hydroxylase since vitamin E stimulates cytochrome P-450, a necessary component in the 7 α-hydroxylase system. 31 Vitamins C and E exert their plasma lipid lowering action in rats fed atherogenic diet only after four or five weeks of supplementation since three weeks or less had no significant changes on the plasma lipid concentrations. This may reflect a depletion of liver cholesterol stores during the four weeks of vitamin administration.…”

Section: Discussionmentioning

confidence: 99%

Effect of Vitamins C and E Intake on Plasma Lipid Concentrations in Rats

Khoja

Marzouki

1994

Annals of Saudi Medicine

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Changes in the plasma lipid levels were investigated among rats fed an atherosclerotic-promoting diet containing 0.5% cholesterol and rats fed the same diet with added vitamin C (ascorbic acid), vitamin E (a-tocopherol) and vitamins C + E from one to seven weeks. Total cholesterol (TC) and triglycerides (TG) were significantly increased in rats fed a hyperlipidemic diet from the third week to the seventh week, whereas high density lipoprotein cholesterol (HDL-C) was not affected. Rats supplemented with 5 mg vitamin C, 5 mg vitamin E or 5 mg vitamin C + 5 mg vitamin E per day for four to seven weeks showed significant decrease in the concentration of TC and TG. HDL-C was only affected at the seventh week with vitamin C alone, whereas it was significantly increased with vitamin E alone and vitamins C + E at five to seven weeks. However, supplementation of vitamins C, E or C + E for less than four weeks has no significant effect on plasma lipid concentrations. The antioxidant effect of vitamins C and E is probably a time-dependent process that significantly lowers plasma lipids between week four and week seven following administration of these vitamins. It is therefore suggested that the incidence of coronary heart disease (CHD) may be reduced in lowering plasma lipid levels by dietary supplementation of vitamins C or E. Ann Saudi Med 1994;14(5):371-374. SM Khoja, ZMH Marzouki, Effect of Vitamins C and E Intake on Plasma Lipid Concentrations in Rats. 1994; 14(5): 371-374Coronary heart disease (CHD) is one of the major causes of morbidity and mortality. A positive correlation has been demonstrated between raised serum lipids and the incidence of CHD and atherosclerosis in humans. Cholesterol is the lipid most frequently implicated in this relationship. The cholesterol-rich low density lipoprotein (LDL) has met all criteria of a primary risk factor for CHD. However, the levels of the various lipoproteins and their apolipoproteins have been found to be altered in patients with CHD. Recent research in the pathophysiology of atherosclerosis focused on modifications of lipoproteins increasing their atherogenic potential.1 Those cells that are responsible for the formation of atherosclerotic lesions, namely the macrophages, are normally not able to take up LDL in considerable amounts. Only after having modified LDL by acetylation, Goldstein and Brown succeeded in loading macrophages with LDL.2 This process leads to the formation of so-called lipid laden foam cells. The Σ-amino groups of the lysine residues on apolipoprotein B (Apo B) become blocked by acetylation and the net negative charge of the lipoprotein is strongly enhanced. Looking for other physiological mechanisms of LDL modification, Henriksen et al. 3 observed that macrophages, previously incubated with cultured endothelial cells, demonstrate enhanced degradation of LDL and storage of cholesterol esters. In following reports, it was shown that lipid peroxidation is involved in this modification of LDL by cultured cells. For reviews see references 4 to 6.Recent ...

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Properties of Reconstituted Cholesterol 7α‐Hydroxylase System from Rat and Rabbit Liver Microsomes

Cited by 50 publications

References 34 publications

Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

Cytochromes P‐450 LM_3b and LM₄ in Biosynthesis of Bile Acids

Effect of Vitamins C and E Intake on Plasma Lipid Concentrations in Rats

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Properties of Reconstituted Cholesterol 7α‐Hydroxylase System from Rat and Rabbit Liver Microsomes

Cited by 50 publications

References 34 publications

Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

Cytochromes P‐450 LM3b and LM4 in Biosynthesis of Bile Acids

Effect of Vitamins C and E Intake on Plasma Lipid Concentrations in Rats

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Cytochromes P‐450 LM_3b and LM₄ in Biosynthesis of Bile Acids