Characterization of an Adrenal Activator for Cholesterol Side Chain Cleavage

Abstract

Abstract The enzyme system which converts cholesterol to pregnenolone by side chain cleavage is present in a soluble buffer extract of an acetone powder of adrenal mitochondria. An activation factor remains in the soluble buffer extract after heating at 100° for 2 min, whereas the known components of the P-450 enzyme system are inactivated. The addition of this soluble, heat-stable factor with the active enzyme will enhance pregnenolone formation 5- to 10-fold. The activator is precipitable with acetone, and saturated (NH4)2SO4, is not dialyzable, and is inactivated partially with trypsin. The factor is present in 105,000 x g supernatant but not the microsome fraction. Bovine serum albumin or rat serum does not stimulate. A heat-stable soluble liver cholesterol carrier protein (liver-SCP) has been shown to stimulate cholesterol side chain cleavage using the adrenal mitochondrial enzyme system. The binding properties of the activator with cholesterol on Sephadex G-25 are consistent with its behavior as a carrier protein for cholesterol. Pregnenolone and other adrenal steroids bind less than 1%. Evidence is presented for a cholesterol protein complex which may serve to transport cholesterol within the cell and to participate at the active site for the enzymatic cleavage of the cholesterol side chain.