Preparation of antiserum to rat cytochrome P-450 cholesterol side chain cleavage, and its use for ultrastructural localization of the immunoreactive enzyme by protein A-gold technique

Abstract

Rabbit antiserum to rat cytochrome P-450 cholesterol side chain cleavage (P-450scc) was produced without a previous biochemical purification of the enzyme. Instead, for immunization we used a single protein band of mol wt 53,000, which was isolated from sodium dodecyl sulfate polyacrylamide gel electrophoresis of rat steroidogenic mitochondrial membranes. The resulting antiserum cross-reacted in a protein-blotting test with affinity purified and biologically active bovine adrenocortical P-450scc enzyme. The antiserum to the rat P-450scc also substantially blocked the conversion of cholesterol to pregnenolone in sonicated steroidogenic mitochondria, suggesting a successful cross-reactivity with the native form of the enzyme, despite the fact that the immunizing antigen was sodium dodecyl sulfate-denatured protein. The antiserum was applied for ultrastructural immunocytochemical visualization of the P-450scc in thin sections of adrenal cortex and immature ovary. Immunoreactive enzyme was identified by the protein-A-gold technique which showed that the gold particles concentrated exclusively in the steroidogenic mitochondria of adrenal zona glomerulosa and fasciculata cells. In the immature ovary, the only zone which was heavily stained with colloidal gold was the population of the interstitial cells. Part of the theca cell population contained P-450scc before PMSG treatment. The granulosa cells were devoided of the enzyme in any follicles before the preovulatory stage. The high resolution of the pAg technique allowed to visualize the localization of the P-450scc antigen in the matrix side of the inner mitochondrial membranes. Moreover, a clear coupling could be demonstrated between the morphological and functional maturation of the steroidogenic mitochondrion in the ovary: from a few lamella cristae devoid of P-450scc in the unstimulated granulosa mitochondria, to numerous tubulovesicular inner membranes, heavily loaded with the enzyme, in the mitochondria of the interstitial cells.