A Form of Reduced Nicotinamide Adenine Dinucleotide-Cytochrome b5 Reductase Containing Both the Catalytic Site and an Additional Hydrophobic Membrane-binding Segment

Abstract

Cytochrome b5 reductase from calf liver has been purified by a nonhydrolytic procedure which involves Triton X-100 extraction of microsomes and DEAE-chromatography in the presence of Triton X-100 and deoxycholate. The reductase obtained in this manner has a monomeric molecular weight of 43,000 as compared to a molecular weight of 33,000 for reductase obtained by lysosomal extraction of microsomes. The additional mass present in the new preparation is due to an additional 99 amino acid residues, about 65% of which are hydrophobic and most of which occur in a single amino acid sequence. This hydrophobic amino acid sequence confers upon the reductase a marked tendency to polymerize in aqueous media and permits it to bind to microsomal membranes. The detergent-extracted reductase is catalytically similar to lysosomal-extracted reductase with ferricyanide as an electron acceptor, but its reaction with cytochrome b5 is slowed by polymerization. Chymotryptic cleavage of detergent-extracted reductase releases a core enzyme, which is chromatographically indistinguishable from lysosomal-extracted reductase, and polymers of hydrophobic peptides, the largest of which has a monomeric molecular weight of about 10,000. This is similar to our earlier studies with detergent-extracted cytochrome b5 and constitutes the second example of a membrane protein which has a distinctly amphipathic structure.