Discrimination between conformational states of mitochondrial cytochrome P-450scc by selective modification with pyridoxal 5-phosphate

• Published in: Biochemistry (Moscow) Vol. 63; no. 5; pp. 568 - 572
• Main Authors: Lapko, A G, Ruckpaul, K
• Format: Journal Article
• Language: English
• Published: United States 01.05.1998
• Subjects: Adrenal Cortex - enzymology; Animals; Cattle; Cholesterol Side-Chain Cleavage Enzyme - chemistry; Cholesterol Side-Chain Cleavage Enzyme - isolation & purification; Cholesterol Side-Chain Cleavage Enzyme - metabolism; In Vitro Techniques; Mitochondria - enzymology; Protein Conformation - drug effects; Pyridoxal Phosphate - pharmacology; Spectrophotometry
• ISSN: 0006-2979

Electrophoretically homogeneous cytochrome P-450scc preparation isolated by the standard method from adrenal cortex mitochondria comprises two protein forms differing in the accessibility of their amino groups to specific chemical modification with pyridoxal 5-phosphate. The protein form whose lysine amino groups are accessible to the modifier constitutes about 60-70% of the preparation. Being covalently bound to pyridoxal 5-phosphate, this protein form loses enzymatic activity and affinity for adrenodoxin. This protein form can be separated by affinity chromatography on adrenodoxin-Sepharose. The cytochrome P-450scc form whose amino groups are not accessible to the modifier is retained on the affinity matrix, and after elution from adrenodoxin-Sepharose has the absorption spectrum typical of the high-spin protein with a spectral homogeneity index A392/A278 = 1.0. The enzymatic activity of the hemoprotein form whose lysine amino groups are inaccessible to the modification is identical to that of the initial unmodified protein.