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1991 Fiscal Year Final Research Report Summary

Isolation and localization of H, K-ATPase in colonic epithelial cell.

Research Project

Project/Area Number 02454114
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field General physiology
Research InstitutionYamagata University

Principal Investigator

SUZUKI Yuichi  Yamagata University School of Medicine, Dept. of Physiology, Associate professor, 医学部, 助教授 (50091707)

Co-Investigator(Kenkyū-buntansha) TERASHIMA Kazuo  Yamagata University School of Medicine, Dept. of Pathology, Associate professor, 医学部, 助教授 (70045699)
Project Period (FY) 1990 – 1991
KeywordsColon / H,K-ATPase / cDNA / Na,K-ATPase / Ouabain / ATPase / Cloning / Proton pump
Research Abstract

1)Solubilization of H,K-ATPase: Approximately 30% of the ouabain-sensitive H,K-ATPase activity in crude membrane fraction of epithelial cells from guinea pig distal colon has been solubilized with 0.05% n-Dodecyl-b-D-maltoside. 2)Histochemistry: Cerium-based cytochemical method failed to reveal the localization of the ouabainsensitive H,K-ATPase because the reaction product of ATPase activity was not blocked by ouabain. 3)cDNA cloning: cDNAs encoding the colonic H,K-ATPase were isolated and its nucleotide sequence was determined. 155 b Hpall fragment of the rat Na,K-ATPase a1 cDNA was used as a probe to screen a cDNA library constructed in l_<zap>ll from poly(A)+ RNA isolated from the mucosa of the guinea pig distal colon. The cDNA is 4242 nucleotides in length and contains 144 bp of 5'-untranslated region, 3102 bp of an open reading frame and 996 bp of 3'-untranslated region. The deduced amino acid sequence predicts a protein of 1033 residues which belongs to the P-type family of membrane-transporting ATPases and exhibits 67% amino acid identity to the rat Na,K-ATPase a1, and 64% identity to the rat gastric H,K-ATPase a subunit. In immunoblotting, a polyclonal antibody raised against a synthetic peptide (residues 10-24) identified a membrane protein of 94 kd in the distal, but not in the proximal colon. These findings suggest that the cloned cDNA represents the putative H,K-ATPase in the distal colon.

  • Research Products

    (4 results)

All Other

All Publications (4 results)

  • [Publications] 鈴木 裕一,渡辺 敏行,金子 健也: "A novel H,K-ATPase the colonic apical membarne" Japanese J Physiology.

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 渡辺 敏行他: "Primary structure of H,K-ATPase in guinea pig distal colon"

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Suzuki,T.Watanabe,K.Kaneko: "A novel H,K-ATPase the colonic apical membarne" Japanese J Physiology.

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T.Watanabe,Y.Suzuki,T,Suzuki,K,Kanako,M,Sato,T.Yoshida: "Primary structure of H,K-ATPase in guinea pig distal colon" Manuscript in preparation.

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1994-03-18  

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