Purification and properties of NADH-cytochrome b5 reductase solubilized by lysosomes from rat liver microsomes.

Abstract

A new method for the purification of NADH-cytochrome b5 reductase [EC 1.6.2.2] from rat liver microsomes is presented. Solubilization of the reductase was achieved by incubating liver microsomes with lysosomes of the same tissue, and external addition of solubilizing agents was not needed. The method is suitable for a large scale preparation of the purified reductase starting from 1 kg or more of liver. The purification ratio of the reductase activity was 600 to 700. The overall yield from microsomes was about 40%. The properties of the purified enzyme were almost identical with those reported by previous workers for “snake venom-solubilized” enzyme. The purified enzyme preparations obtained by this method still contained two protein components with identical enzymatic activities and very similar physicochemical properties. These two components were immunologically indistinguishable. Examination of solubilization treatment suggested that these two NADH-cytochrome b5 reductase components had been derived from a single enzyme present in microsomes, and long incubation with lysosomes was responsible for the appearance of two components. NADH-ferricyanide reductase activity of liver microsomes could be mostly explained by NADH-cytochrome b5 reductase. The reduction of neotetrazolium by microsomes was, however, dependent on the interaction of the reductase with cytochrome b5 in microsomal membranes as in the case of microsomal NADH-cytochrome c reductase.