Isolation and partial characterization of the NH2-terminal membrane-binding domain of NADH-cytochrome b5 reductase.
The amphipathic flavoprotein NADH-cytochrome b5 reductase from steer liver was converted into the membrane-binding and soluble catalytic domains by controlled subtilisin proteolysis of vesicle-bound, externally oriented reductase. Amino acid analysis of the single nonpolar peptide which remained associated with the vesicles showed that it has a molecular weight of about 6400-6500 and that 65-66% of the amino acid residues are hydrophobic. Both the intact reductase and the nonpolar peptide were blocked to sequencing. Carboxypeptidase Y digestion of the holoenzyme and of a chymotryptically generated flavopeptide, which had lost membrane-binding capacity, released 1 residue of alanine and 2 residues of phenylalanine, indicating a common carboxyl terminus. Partial sequence analysis of the reductase placed the nonpolar peptide to the NH2-terminal side of the subtilisin-generated flavopeptide domain. Thus, the membrane-binding domain of cytochrome b5 reductase is localized at the NH2-terminus of the whole protein. The nonpolar peptide bound to vesicles contains a single fluorescent tryptophan with an emission maximum characteristic of a hydrophobic environment.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002850:Chromatography, Gel; D003579:Cytochrome Reductases / Q000032:analysis*; D042966:Cytochrome-B(5) Reductase; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology; D008297:Male; D010446:Peptide Fragments / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D010446:Peptide Fragments; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase