Temperature dependence of cholesterol binding to cytochrome P-450scc of the rat adrenal. Effect of adrenocorticotropic hormone and cycloheximide.
A type I absorbance change is observed in suspensions of adrenal cortical mitochondria as the temperature is increased from 0-22 degrees. This "heat-generated" type I absorbance change is similar in magnitude to the pregnenolone-induced type II absorbance change of these mitochondria. Studies with inhibitors of cholesterol side chain cleavage indicate that the heat-generated type I absorbance change represents the specific interaction of cytochrome P-450scc with endogenous cholesterol in the mitochondria. This finding is confirmed by low temperature EPR spectroscopy on temperature-equilibrated, quick frozen adrenal mitochondrial samples. The EPR resonance at g = 8.2, which is that of the high spin cholesterol-bound cytochrome P-450scc, is absent in the samples incubated at 0 degrees and increases in magnitude with increasing temperature of incubation. Studies of the pH dependence of the heat-generated type I and pregnenolone-induced type II absorbance changes reveal that both are diminished by increasing pH over the range 6 to 8. Adrenocorticotropic hormone (ACTH) treatment of rats results in adrenal mitochondria which show a greatly increased heat-generated type I absorbance change. The latter correlates with an increased pregnenolone-induced type II absorbance change and increased EPR g = 8.2 signal. Prior treatment of animals with cycloheximide eliminated the ACTH-induced increase in the heat-generated type I absorbance change, the pregnenolone-induced type II absorbance change and the EPR g = 8.2 signal. We estimate that the hydrophobic bonding of cholesterol to cytochrome P-450scc occurs with a deltaH0' of approximately +15 kcal/mol and a deltaS0' of approximately +55 cal/mol deg. Our data support the concept of a labile protein which participates directly in this process.
D000311:Adrenal Glands / Q000378:metabolism*; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology*; D000818:Animals; D002784:Cholesterol / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D006863:Hydrogen-Ion Concentration; D007016:Hypophysectomy; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D051381:Rats; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013256:Steroids / Q000494:pharmacology; D013696:Temperature
D013256:Steroids; D000324:Adrenocorticotropic Hormone; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D003513:Cycloheximide