Electron microscopic immunocytochemistry on the colocalization of ATP synthase and cytochrome P-450 of side chain cleavage enzymes in mitochondria of rat and bovine adrenal cortical cells.

To clarify whether ATP synthase and side chain cleavage enzymes (SCC) can coexist in a mitochondrion of the adrenal cortical cell, and whether the functional heterogeneity of mitochondria can occur in an adrenal cortical cell, the immunostainability of these two enzymes in mitochondria was compared by electron microscope using pre- and post-embedding methods. In the pre-embedding method, the inner membrane of most mitochondria in the adrenal cortical cell was positively stained for SCC, 11 beta-hydroxylase, and ATP synthase, but among these immunopositive mitochondria we often observed negatively immunoreacted ones in the same adrenal cortical cell. In the positively stained mitochondria, immunoreaction products were evenly localized along the inner mitochondrial membrane. We therefore think that these differences in the immunostainability are caused by technical artifacts, namely the inadequate penetration of the antibody. In the post-embedding immunocytochemistry, gold particles showing the presence of the enzymes were observed on all mitochondria. Two different antibodies, anti-ATP synthase antibody and anti-cytochrome P-450scc antibody, which were labelled with gold particles of varying diameter (5 nm, 10 nm each) could be observed on all the mitochondria of the bovine adrenal cortical cells. No heterogeneities as to the stainability for both ATP synthase and SCC were detected in mitochondria of the rat and bovine adrenal cortical cells. These results indicate that in the rat and bovine adrenal cortical cells, both ATP synthase and SCC are evenly and simultaneously present on the inner membrane of all mitochondria. Hence, mitochondria in the adrenal cortical cell seem to be homogeneous in their steroid and ATP synthesizing abilities.