The NH2-terminal structures of human and rat liver microsomal NADH-cytochrome b5 reductases.
Detergent-solubilized NADH-cytochrome b5 reductase was purified from human liver microsomes. Both the purified enzyme and the membrane-binding domain isolated from the purified enzyme were determined to be modified at the NH2-terminal amino acid, glycine, by myristic acid in an amide form. Myristic acid was identified as a methyl ester by gas chromatography after the acid methanolysis of the purified enzyme and the NH2-terminal peptide. The NH2-terminal structure of the membrane-binding domain was determined to be CH3(CH2)12-CO-Gly-Ala-Gln-Leu-Ser-Thr-Leu-Gly-His-Met-Val-Leu-Phe-Pro-Va l- Trp-Phe-Leu-Tyr-Ser-Leu-Leu-Met-Lys. The sequence from Leu-7 to Lys-24 completely coincided with that deduced from the base sequence of complementary DNA (cDNA) from human placenta (Yubisui, T. et al. (1987) Proc. Natl. Acad. Sci. U.S. 84, 3609-3613). The NH2-terminal structure of the detergent-solubilized enzyme from rat liver microsomes was also analyzed for comparison with that of human liver microsomal enzyme. The NH2-terminal myristic acid and the first 7 amino acids of the membrane-binding domains of human, rat, and steer liver microsomal enzymes are completely conserved, and more than 70% homology was observed over the whole membrane-binding domains, implying the importance of the conserved structure as an anchor of the enzyme to the membrane.
Murakami
K
K
Department of Biochemistry, Medical College of Oita.
Yubisui
T
T
Takeshita
M
M
Miyata
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003579:Cytochrome Reductases / Q000032:analysis*; D042966:Cytochrome-B(5) Reductase; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000032:analysis; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D000432:Methanol; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010434:Pepsin A; D051381:Rats; D013045:Species Specificity; D014357:Trypsin
D000596:Amino Acids; D005227:Fatty Acids; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D014357:Trypsin; D010434:Pepsin A; D000432:Methanol