Studies on P-450: V. On the Substrate-Induced Spectral Change of P-450 Solubilized from Bovine Adrenocortical Mitochondria<xref ref-type="fn" rid="fn1"><sup>*</sup></xref>

Abstract

Procedures for solubilization and purification of P-450 from bovine adrenocortical mitochondria were examined and an improved method for the preparation of partially purified P-450 was described. By measuring the “substrate-induced difference spectrum”, the substrate-binding specificity of the solubilized preparation was compared with those of P-450 in bovine adrenocortical mitochondria, bovine adrenocortical microsomes, and rat liver microsomes. The specificity of the solubilized preparation was mostly the same as that of P-450 in adrenocortical mitochondria. The specificity of mitochondrial P-450 and that of P-450 in adrenocortical microsomes were similar in qualitative aspects, but the relative intensity of spectral change and the affinity for substrate were frequently different. On the other hand, the binding specificity of hepatic microsomal P-450 was rather different qualitatively from those of adrenal P-450's. The effect of successive addition of two different substrates to the solubilized preparation was also examined. The intensity of spectral change induced by a type I substrate was inhibited non-competitivcly by the addition of another type I substrate, but was not affected by the addition of a type II substrate. The significance of these results was discussed. In addition to these observations, spectral evidence was presented indicating the change in spin state of P-450 by binding with a substrate.