Complete amino acid sequence of NADH-cytochrome b5 reductase purified from human erythrocytes.
The complete amino acid sequence of soluble NADH-cytochrome b5 reductase purified from human erythrocytes was determined. The enzyme, which contained 8 methionine residues, was cleaved by cyanogen bromide. The resulting nine peptides were separated by gel filtration and purified further by high-performance liquid chromatography. The purified peptides were sequenced by automated Edman degradation. Three large CNBr peptides, residues 1-101, 109-151, and 169-231, were further fragmented with trypsin, Staphylococcus aureus V8 protease or a lysyl endopeptidase of Achromobacter lyticus. The peptides obtained from the tryptic digest of citraconylated FAD-depleted apoprotein completed the alignments of the other peptides. The enzyme was composed of 275 amino acid residues. The 4 functionally important cysteine residues were located in the COOH-terminal portion. The molecular weight of the protein was calculated to be 31,260 without FAD. A prediction of the secondary structure was made by the method of Chou and Fasman. The protein was hydrophilic as a whole (43% polarity), but some regions were rich in hydrophobic residues. From the sequence homology of this enzyme with the pyridine nucleotide-binding sites of other flavoproteins, three candidates for the FAD and NADH-binding domains were suggested.
T
T
Yubisui
T
T
Miyata
S
S
Iwanaga
M
M
Tamura
M
M
Takeshita
D000595:Amino Acid Sequence; D001665:Binding Sites; D003488:Cyanogen Bromide; D003579:Cytochrome Reductases / Q000097:blood*; D042966:Cytochrome-B(5) Reductase; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D014357:Trypsin
D010446:Peptide Fragments; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D014357:Trypsin; D003488:Cyanogen Bromide