Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase: location of the hydrophobic, membrane-binding region at the carboxyl-terminal end and the masked amino terminus.
Microsomal NADH-cytochrome b5 reductase is an amphiphilic protein consisting of a hydrophilic (catalytic) region and a hydrophobic (membrane-binding) segment. Digestion of the reductase purified from rabbit liver microsomes with carboxypeptidase Y (CPY), but not with aminopeptidases, resulted in the abolishment of the capacities of the reductase to bind to phosphatidylcholine liposomes and to reconstitute an active NADH-cytochrome c reductase system upon mixing with cytochrome b5. The NADH-ferricyanide reductase activity of the flavoprotein was, however, inactivated only slightly by the CPY digestion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid analyses indicated that the CPY treatment removed about 30 amino acid residues from the tcooh terminus of the reductase and that about 70% of the amino acids released were hydrophobic. It is concluded that the hydrophobic region of the reductase, responsible for both membrane binding and effective reconstitution of NADH-cytochrome c reductase activity, is located at the COOH-terminal portion of the molecule. No NH2-terminal residue could be detected in the intact and CPY-modified reductase preparations. The location of the hydrophobic, membrane-binding segment at the COOH-terminal end and the masked NH2 terminus have also been reported for cytochrome b5, another microsomal membrane protein.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002268:Carboxypeptidases; D002942:Circular Dichroism; D003579:Cytochrome Reductases / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008862:Microsomes, Liver / Q000201:enzymology; D009243:NAD; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits
D000596:Amino Acids; D008081:Liposomes; D009243:NAD; D003579:Cytochrome Reductases; D002268:Carboxypeptidases