Purification and cDNA Cloning of GDP-L-Fuc:N-Acetyl-.BETA.-D-Glucosaminide:.ALPHA.l-6 Fucosyltransferase (.ALPHA.1-6 FucT) from Human Gastric Cancer MKN45 Cells.
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- Yanagidani Shusaku
- Department of Biochemistry, Osaka University Medical School
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- Uozumi Naofumi
- Department of Biochemistry, Osaka University Medical School
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- Ihara Yoshito
- Department of Biochemistry, Osaka University Medical School
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- Miyoshi Eiji
- Department of Biochemistry, Osaka University Medical School
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- Yamaguchi Nozomi
- Department of Cell Biology, Research Institute for Neural Disease and Gerontology, Kyoto Prefectural University of Medicine
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- Taniguchi Naoyuki
- Department of Biochemistry, Osaka University Medical School
抄録
GDP-L-Fuc : N-acetyl-β-D-glucosaminide : α1-6 fucosyltransferase ( α1-6 FucT), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a culture supernatant of human gastric cancer cell line MKN45. The puri-fication procedures included chromatographies on Q-Sepharose Fast Flow, synthetic GDP-hexanolamine-Sepharose, and GnGn-bi-Asn-Sepharose columns. SDS-PAGE of the puri-fied enzyme gave a major band corresponding to an apparent molecular mass of 60 kDa. The enzyme was recovered in a 12% final yield with an approximately 4, 600-fold increase in specific activity. The pH optimum was 7.5, and the enzyme was fully active in the presence of 5 mM EDTA and did not require divalent cations, Me2+ and Ca2+. Oligonucleotide primers designed from partial amino acid sequences were used to amplify and clone αl-6 FucT cDNA from a cDNA library of MKN45 cells. The cDNA encodes 575 amino acids in length, and contains the predicted N-terminal and internal amino acid sequences derived on lysyl endopeptidase digestion. The homology to porcine brain al-6 FucT is 92.2% at the nucleotide level and 95.7% at the amino acid level. No putative N-glycosylation sites were found in the predicted amino acid sequence of the human MKN45 cell enzyme or that of porcine brain. Thus, the enzyme is distinct from other fucosyltransferases which catalyze αl-2, αl-3, and αl-4 fucose addition.
収録刊行物
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- The Journal of Biochemistry
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The Journal of Biochemistry 121 (3), 626-632, 1997
The Japanese Biochemical Society
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詳細情報 詳細情報について
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- CRID
- 1571417128104366336
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- NII論文ID
- 130003533149
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- ISSN
- 0021924X
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- 本文言語コード
- en
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- データソース種別
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- CiNii Articles