Publications

• Investigation of the component reactions of oxidative sterol demethylation. Study of the aerobic and anaerobic processes.

  Miller W.L., Kalafer M.E., Gaylor J.L., Delwiche C.V.

  Biochemistry 6:2673-2678(1967) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Mixed function oxidases in sterol metabolism. Source of reducing equivalents.

  Brady D.R., Crowder R.D., Hayes W.J.

  Either NADH or NADPH can serve as a cofactor for oxidative demethylation of [30,31-14C]4,4-dimethyl-5 alpha-cholest-7-en-3 beta-ol and oxidative deformylation of 4-hydroxy[14C]methylene-5 alpha-cholest-7-en-3-one. This report suggests that the cofactors interact with these two oxidase systems diff ... >> More

  Either NADH or NADPH can serve as a cofactor for oxidative demethylation of [30,31-14C]4,4-dimethyl-5 alpha-cholest-7-en-3 beta-ol and oxidative deformylation of 4-hydroxy[14C]methylene-5 alpha-cholest-7-en-3-one. This report suggests that the cofactors interact with these two oxidase systems differently depending upon whether the reduced cofactor arises intra- or extramicrosomally. Marked differences in oxidative activity are observed depending on whether NADPH is generated in the microsomes or is added as an exogenous cofactor. Thus, the concentration of added NADPH required to yield maximal rates of sterol oxidation is 500 muM or greater. Nearly equivalent rates of sterol oxidation are obtained from NADPH generated in the microsomes where the NADPH concentration is no greater than 0.454 muM. Similar results are observed with NADH. In this case, NADH is generated in the microsomes from added NAD+ by microsomal reactions. The rate of sterol oxidation when NADH is generated from added NAD+ is nearly the same as that obtained from added NADH; although the concentration of NADH generated from NAD+ is 0.403 muM, the concentration of added NADH is 100 muM, and Km for added NADH is 1.7 muM. << Less

  J Biol Chem 255:10624-10629(1980) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Investigation of the component reactions of oxidative sterol demethylation. Evidence against participation of cytochrome P-450.

  Gaylor J.L., Mason H.S.

  J Biol Chem 243:4966-4972(1968) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Affinity chromatography of microsomal enzymes on immobilized detergent-solubilized cytochrome b5.

  Kawata S., Trzaskos J.M., Gaylor J.L.

  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 on ... >> More

  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. << Less

  J Biol Chem 261:3790-3799(1986) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Total enzymic synthesis of cholesterol from lanosterol. Cytochrome b5-dependence of 4-methyl sterol oxidase.

  Fukushima H., Grinstead G.F., Gaylor J.L.

  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 immunog ... >> More

  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. << Less

  J. Biol. Chem. 256:4822-4826(1981) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Biological demethylation of 4,4-dimethyl sterols, Evidence for enzymic epimerization of the 4beta-methyl group prior to its oxidative removal.

  Sharpless K.B., Snyder T.E., Spencer T.A., Maheshwari K.K., Nelson J.A.

  J Am Chem Soc 91:3394-3396(1969) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.