Existence of multiple forms of cytochrome P-450 scc purified from bovine adrenocortical mitochondria

Abstract

Three fractions of cytochrome P-450 scc (denoted as fractions a, b, and c) were purified by a new procedure from bovine adrenocortical mitochondria. The amino-acid content analyses of these three fractions showed no difference. NH 2-terminal amino-acid sequences of cytochrome P-450 scc fractions, a and b agreed completely with the sequence deduced by nucleotide sequence of cDNA of cytochrome P-450 scc mRNA (Morohashi, K.; Fujii-Kuriyama, Y.; Okada, Y., Sogawa, K.; Hirose, T.; Inayama, S. and Omura, T. (1984) Proc. Natl. Acad. Sci. USA 81, 4647–4651), whereas t he sequence of fraction c showed a missing of isoleucine at the NH 2-terminal. COOH-terminal amino-acid sequences of fraction a, b and c were -Gln-Ala-COOH, identical with the deduced sequence from the cDNA. Measurements of the enzymatic activities of cholesterol side-chain cleavage reaction revealed no distinct difference among these three fractions. Although each of these fractions appeared as a single protein staining band upon SDS-polyacrylamide gel electrophoresis, these fractions showed heterogeneities upon two-dimensional electrophoresis and chromatofocusing. Fraction a contained the major form of cytochrome P-450 scc, and its isoelectric point was estimated to be pH 7.8 by isoelectric focusing under both native and denatured conditions, and this value was confirmed by chromatofocusing. Neither of the carbohydrate-specific stainings (such as periodic acid-Schiff staining and lectin-peroxidase stainings using concanavalin A, wheat-germ agglutinin, and soybean agglutinin) of purified cytochrome P-450 scc fractions after the electrophoretic resolution on SDS-polyacrylamide gel could show cytochrome P-450 scc fractions as glycoproteins, suggesting that the heterogeneities were not due to the glycosylation state.