Protein kinase stimulation of a reconstituted cholesterol side chain cleavage enzyme system in the bovine corpus luteum.
A solubilized preparation of cytochrome P-450, obtained by treatment of mitochondria from bovine corpora lutea with phospholipase A, contained all of the necessary components for the cholesterol side chain cleavage activity. The solubilized cytochrome -450 preparation could be isolated essentially free of endogenous cholesterol side chain cleavage activity by various fractionation techniques. A cholesterol side chain cleavage enzyme system was reconstituted using the isolated cytochrome P-450 preparation and purified adrenodoxin and adrenodoxin reductase (components of the enzyme system purified from the adrenal cortex). Protein kinase was partially purified from the cytosol fraction of bovine corpora lutea. It was purified 43-fold and the activity was highly dependent on cyclic adenosine 3:5-monophosphate (cyclic AMP). When ATP and this partially purified cyclic AMP-dependent protein kinase were added to the reconstituted cholesterol side chain cleavage enzyme assay in which cytochrome P-450 was limiting, a stimulation (20 to 74%) of the conversion of cholesterol into pregnenolone was observed. This stimulation was statistically significant with p value less than 0.001. The stimulatory effect of the protein kinase appeared to be dependent on ATP and was not mimicked by bovine serum albumin, indicating that the effect was specific for protein kinase. Protein kinase caused a phosphorylation of the cytochrome P-450 preparation when large amounts of this preparation were used in the assay. It is concluded from these results that the direct activation of the cytochrome P-450 component of the cholesterol side chain cleavage by protein kinase may be one of the ways by which cyclic AMP mediates the effect of luteinizine.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000325:Adrenodoxin / Q000378:metabolism; D000818:Animals; D002250:Carbon Radioisotopes; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D003338:Corpus Luteum / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005260:Female; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011247:Pregnancy; D011284:Pregnenolone / Q000096:biosynthesis; D011494:Protein Kinases / Q000378:metabolism*; D014316:Tritium
D002250:Carbon Radioisotopes; D010761:Phosphorus Radioisotopes; D014316:Tritium; D000325:Adrenodoxin; D011284:Pregnenolone; D000255:Adenosine Triphosphate; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D000242:Cyclic AMP; D010088:Oxidoreductases; D005954:Glucosephosphate Dehydrogenase; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005287:Ferredoxin-NADP Reductase; D011494:Protein Kinases