Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Highly selective chromatography of microsomal enzymes has been carried out on columns of immobilized cytochrome b5 that was obtained by detergent solubilization (d-b5) of the complete amphipathic molecule. Several partially purified isozymes of cytochrome P-450 are resolved on d-b5 columns, and one high-affinity isozyme has been readily purified to homogeneity. Chromatographic selectivity and correlation of elution order of isozymes of cytochrome P-450 with direct spectral measurements of affinity constants suggests affinity chromatography on d-b5 columns. Substantial one-step enrichments of NADH-cytochrome-b5 reductase and an unstable cytochrome b5-dependent oxidase of cholesterol synthesis, 4-methyl sterol oxidase, have been obtained on d-b5 columns which further supports this conclusion. Comparison of chromatographic behavior on columns of immobilized cytochrome b5 that was obtained by trypsin solubilization (t-b5) with d-b5 columns shows marked differences which must be attributed to the absence of the hydrophobic domain of the t-b5 molecule. NADH-cytochrome-b5 reductase and the high affinity isozyme of cytochrome P-450 purified by d-b5 affinity chromatography are poorly retained on t-b5 columns. A different cytochrome P-450 isozyme with lower affinity for cytochrome b5 is only retained on d-b5 columns. Cytochrome-P-450 reductase is not retained on either column. Because affinity chromatography is suggested on d-b5 columns, the procedure may be generally applicable for predicting protein-protein interactions of microsomal electron transport components that either donate electrons to, or receive electrons from, cytochrome b5. In addition, the procedure should have considerable utilitarian application for enzyme enrichment.
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PMID:Affinity chromatography of microsomal enzymes on immobilized detergent-solubilized cytochrome b5. 394 90

Electron transfer to rat liver microsomal cytochrome P-450 of 14 alpha-methyl group demethylation of 24,25-dihydrolanosterol (C30-sterol) has been studied with a new radio-high-performance liquid chromatography assay. The monooxygenase is dependent upon NADPH plus oxygen, insensitive to CN-, and sensitive to CO. Microsomal oxidation is also sensitive to trypsin digestion, and reactivation is dependent upon the addition of purified, detergent-solubilized cytochrome P-450 reductase. Electron transport of C-32 sterol demethylation can be fully supported by very low concentrations of NADPH (approximately 10 microM) only in the presence of saturating concentrations of NADH (approximately 200 microM) suggesting involvement of cytochrome b5-dependent electron transfer in addition to the NADPH-supported pathway. The cytochrome P-450 of 14 alpha-demethylation has been solubilized with detergents, resolved chromatographically from cytochrome P-450 reductase and cytochrome b5, and fully active C-32 demethylase reconstituted. Incubation of intact microsomes with NADH and very low concentrations of NADPH described above leads to interruption of demethylation without 14 alpha-methyl group elimination. Under these conditions, C-32 oxidation products of the C30-sterol substrate accumulate at the expense of formation of demethylated, C29-sterol products. This enzymic interruption of C-32 demethylation, accumulation of oxygenated C30-sterols, along with subsequent demethylation of the isolated C30-oxysterols under similar oxidative conditions supports the suggestion that 14 alpha-hydroxymethyl and aldehydic sterols are metabolic intermediates of sterol 14 alpha-demethylation. Only very modest inductions of the constitutive cytochrome P-450 isozyme of 14 alpha-methyl sterol oxidase can be obtained with just 2 out of 12 known, potent inducers of mammalian hepatic cytochrome P-450s. Alternatively, administration of complete adjuvant in mineral oil drastically reduces amounts of total microsomal cytochrome P-450 while activity of 14 alpha-methyl sterol oxidase is not affected dramatically. Thus, as much as 2.5-fold enhancement of C-32 oxidase specific activity is obtained when expressed per unit of cytochrome P-450.
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PMID:Cytochrome P-450-dependent oxidation of lanosterol in cholesterol biosynthesis. Microsomal electron transport and C-32 demethylation. 620 95

Methyl sterol oxidase of microsomal synthesis of cholesterol from lanosterol is a mixed-function oxidase that is dependent upon reduced pyridine nucleotide. The methyl sterol oxidase, as well as NADH-cytochrome c reductase, in intact rat liver microsomes are inhibited by anti-cytochrome b5 immunoglobulin, but NADPH-cytochrome c reductase is not affected. There is a decreased time lag prior to onset of reoxidation of steady state levels of reduced cytochrome b5 when 4-methyl sterol oxidase substrates are present. Trypsin treatment of microsomes destroys cytochrome b5 with loss of methyl sterol oxidase activity. Activity is restored by addition of purified cytochrome b5 to trypsin-treated microsomes. Initial attempts to solubilize and purify 4-methyl sterol oxidase have been only partially successful due to the extreme lability of the oxidase. However, DEAE-cellulose column chromatography of a detergent extract of microsomes yields a fraction that contains the oxidase, lipids, and NADH-cytochrome b5 reductase but is free of cytochrome b5. Oxidation of 4 alpha [30-3H] methyl-5 alpha-cholest-7-en-3 beta-ol by methyl sterol oxidase in this isolated fraction can be fully restored by the addition of purified liver microsomal cytochrome b5. These results strongly support the suggestion that membrane-bound cytochrome b5 of rat liver microsomes is an obligatory electron carrier from NADH to 4-methyl sterol oxidase.
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PMID:Total enzymic synthesis of cholesterol from lanosterol. Cytochrome b5-dependence of 4-methyl sterol oxidase. 722 57