Membrane-anchoring domain of rat liver 5'-nucleotidase: identification of the COOH-terminal serine-523 covalently attached with a glycolipid.
The involvement of glycosylphosphatidylinositol (GPI) in membrane anchoring of 5'-nucleotidase was investigated by chemical analyses. 5'-Nucleotidase purified from rat liver microsomes was subjected to BrCN cleavage, hexane extraction, and high-performance liquid chromatography, resulting in the purification of a single fragment with Mr 2300. Chemical analyses revealed that the purified fragment contains the tetradecapeptide Lys-Val-Ile-Tyr-Pro-Ala-Val-Glu-Gly-Arg-Ile-Lys-Phe-Ser and characteristic components of GPI including ethanolamine, glucosamine, mannose, inositol, palmitic acid, and stearic acid. In addition, it was confirmed that digestion of 5'-nucleotidase with lysyl endopeptidase yielded a fragment containing the dipeptide Phe-Ser and the same GPI components as above. The sequences of the tetradeca- and dipeptides thus determined are identified at positions 510-523 and 522-523, respectively, in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 548, containing a hydrophobic amino acid sequence [Misumi, Y., Ogata, S., Hirose, S., & Ikehara, Y. (1990) J. Biol. Chem. 265, 2178-2183]. Taken together, these results indicate that the mature 5'-nucleotidase molecule lacks the predicted COOH-terminal peptide extension and is attached at serine-523 with GPI, which functions as the membrane anchor of 5'-nucleotidase.
Ogata
S
S
Department of Biochemistry, Fukuoka University School of Medicine, Japan.
Hayashi
Y
Y
Misumi
Y
Y
Ikehara
Y
Y
D015720:5'-Nucleotidase / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D006017:Glycolipids / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols; D008099:Liver / Q000201:enzymology*; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000378:metabolism; D012697:Serine Endopeptidases
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008567:Membranes, Artificial; D010716:Phosphatidylinositols; D012694:Serine; D015720:5'-Nucleotidase; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase