Affinity chromatography of microsomal enzymes on immobilized detergent-solubilized cytochrome b5.
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.
D002846:Chromatography, Affinity / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification; D003579:Cytochrome Reductases / Q000302:isolation & purification; D003573:Cytochrome b Group; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5; D003902:Detergents / Q000494:pharmacology; D004579:Electron Transport; D004800:Enzymes, Immobilized; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification; D008861:Microsomes / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000302:isolation & purification; D012995:Solubility; D014357:Trypsin / Q000494:pharmacology
D003573:Cytochrome b Group; D003902:Detergents; D004800:Enzymes, Immobilized; D007527:Isoenzymes; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C027532:methylsterol monooxygenase; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D014357:Trypsin