Microsomal electron transfer system of bovine retinal pigment epithelium

Abstract

Pigment epithelium microsomes are capable of catalyzing oxidation of NADH and NADPH in the absence of l-ascorbate. Ascorbate increases the oxidase activity, and the ascorbate-stimulated fraction of the activity is cyanide insensitive. Reduction of epithelium microsomes by the pyridine nucleotides under anaerobic conditions reveals the presence of a hemoprotein, which has absorption maxima at 429, 528 and 559 mμ in the (reduced vs. oxidized) difference spectrum. 3,4-Dihydroxyphenylalanine, which stimulates the pyridine nucleotide oxidation, is also capable of reducing the hemoprotein. The reduced form is rapidly autoxidizable and combines with carbon monoxide. From the carbon monoxide spectrum the microsome-bound hemoprotein was identified with P-450, a CO-binding hemoprotein originally found in liver microsomes. The hemoprotein could be solubilized by trypsinolysis of microsomes, purified on Sephadex and identified with P-420, a solubilized form of P-450. The spectral characteristics and amino acid composition of the solubilized component agree well with those of P-420 that we previously obtained from heart microsomes. Cytochrome b5, if present, must be a very minor hemoprotein component in pigment epithelium microsomes.