Side-chain cleavage P-450 from bovine adrenocortical mitochondria. Reconstitution of enzyme activity.
The subunit structure of the cytochrome P-450 from bovine adrenocortical mitochondria responsible for the conversion of cholesterol to pregnenolone (side-chain cleavage) has been studied. Isoelectric focusing in 6 M urea reveals two fractions of identical amino acid composition which differ in apparent isoelectric points and in phospholipid content: fraction SI shows 0.6-1.8 nmol phospholipid per 53 000 daltons and pI approx. 4.0; SII shows 6.6-8.9 nmol phospholipid per 53 000 daltons and pI approx. 7.0. SII can be made to behave on isoelectric focusing like SI by removal of phospholipid and SI like SII when the extracted phospholipid is added to the protein (SI). Enzymatic activity can be restored to SII by addition of heme and to SI by addition of heme together with the phospholipid extracted from P-450 from the fractions SI and SII. This phospholipid contains at least four classes of phospholipid of which two have been tentatively identified as phosphatidylcholine and phosphatidylethanolamine. A variety of phospholipids from commercial sources do not permit reconstitution of enzyme activity. Evidence is presented to show that minor contaminants seen on polyacrylamide SDS gels are not essential for enzyme activity nor do they appear greatly to influence enzymatic activity. The possible role of phospholipid in reconstituting cytochrome P-450 activity is considered.
D000302:Adrenal Cortex / Q000201:enzymology*; D000311:Adrenal Glands / Q000201:enzymology*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000302:isolation & purification / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D007525:Isoelectric Focusing; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D010088:Oxidoreductases / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis / Q000494:pharmacology
D000596:Amino Acids; D046911:Macromolecular Substances; D010743:Phospholipids; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D002786:Cholesterol Side-Chain Cleavage Enzyme