Temperature dependence of cholesterol binding to cytochrome P-450(scc) 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°. 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 450(scc) with endogenous cholesterol in the mitochondria. This findings 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 450(scc), is absent in the samples incubated at 0° 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 binding of cholesterol to cytochrome P 450(scc) occurs with a ΔH ⁰' of approximately +15 kcal/mol and a ΔS ⁰' of approximately +55 cal/mol deg. Our data support the concept of a labile protein which participates directly in this process.