Purification and characterization of UDP-N-acetylglucosamine: alpha-6-D-mannoside beta 1-6N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase V) from a human lung cancer cell line.
A beta 1-6N-acetylglucosaminyltransferase (GnT-V) [EC 2.4.1.155] which catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to alpha-D-6-mannoside has been purified up to 20,000-fold from the cultured supernatant of the QG small lung cancer cell line with a 37% yield. The isolation procedure included chromatography on phenyl-Sepharose, hydroxylapatite, UDP-hexanolamine Sepharose, and a biantennary sugar substrate (GnGn-bi-Asn) coupled to activated CH-Sepharose 4B. Sodium dodecyl sulfate gel electrophoresis under non-reducing conditions showed a single band of 73 kDa. Under reducing conditions, however, an additional component of 60 kDa was seen. Peptide mapping analysis indicated that both of these proteins were essentially identical, indicating that the 60-kDa component is probably a proteolytically cleaved form of the 73-kDa protein. Studies on the activity of the enzyme toward a variety of pyridylaminated sugars showed that the enzyme is most active toward triantennary (GnGnGn-tri-PA) and biantennary (GnGn-bi-PA) sugars. The Km values for GnGn-bi-PA and UDP-GlcNAc were 133 microM and 3.5 mM, respectively. These studies represent the first report of the enzymatic properties of a highly purified human GnT-V.
Gu
J
J
Department of Biochemistry, Osaka University Medical School.
Nishikawa
A
A
Tsuruoka
N
N
Ohno
M
M
Yamaguchi
N
N
Kangawa
K
K
Taniguchi
N
N
D000117:Acetylglucosamine / Q000378:metabolism; D002240:Carbohydrate Sequence; D018288:Carcinoma, Small Cell / Q000201:enzymology*; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D009844:Oligosaccharides; D014537:Uridine Diphosphate N-Acetylglucosamine; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase; D000117:Acetylglucosamine