Purification and properties of the intact form of NADH-cytochrome b5 reductase from rabbit liver microsomes.
NADH-cytochrome b5 reductase [EC 1.6.2.2] has been solubilized with Triton X-100 and purified to homogeneity from rabbit liver microsomes. The purified enzyme is essentially free of the detergent and phospholipids and exists in aqueous media as an oligomeric aggregate of about 13 S. Its monomeric molecular weight is about 33,000 and 1 mole of FAD is associated with 1 mole of the monomeric unit. The enzyme catalyzes the reductions by NADH of ferricyanide and 2,6-dichlorophenol indophenol at an activity ratio of 1 : 0.09. Although the intact form of cytochrome b5 is a poorer electron acceptor than its hydrophilic fragment for the purified flavoprotein, electron transfer from the reductase to the intact cytochrome can be markedly stimulated by detergents or phospholipids, which also cause profound enhancement of the NADH-cytochrome c reductase activity reconstituted from the reducatse and cytochrome b5. Upon digestion with trypsin [EC 3.4.21.4], the ability of the reductase to form an active NADH-cytochrome c reductase system with the intact form of cytochrome b5 and Triton X-100 is rapidly lost. This loss of the reconstitution capability can be prevented by preincubation of the reductase with phosphatidylcholine liposomes. Trypsin digestion also results in the cleavage of the reductase molecule to a protein having a molecular weight of about 25,000 and a smaller fragment. The purified flavoprotein can bind to liver microsomes, liver mitochondria, sonicated human erythrocyte ghosts, and phosphatidylcholine liposomes. The reductase solubilized directly from liver microsomes by lysosomal digestion however, is devoid of membrane-binding capacity. It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety, which is removable by lysosomal digestion, is responsible for the tight binding of the reductase to microsomes and for its normal functioning in the membrane.
D000818:Animals; D003579:Cytochrome Reductases / Q000302:isolation & purification / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D005292:Ferricyanides / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D046911:Macromolecular Substances; D008566:Membranes / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010713:Phosphatidylcholines; D011092:Polyethylene Glycols / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D014357:Trypsin / Q000494:pharmacology
D005292:Ferricyanides; D008081:Liposomes; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D009243:NAD; D011092:Polyethylene Glycols; D003579:Cytochrome Reductases; D014357:Trypsin