Cloning of the H,K-ATPase β subunit. Tissue-specific expression, chromosomal assignment, and relationship to Na,K-ATPase β subunits

V. A. Canfield, C. T. Okamoto, D. Chow, J. Dorfman, P. Gros, J. G. Forte, R. Levenson

Research output: Contribution to journalArticle

67 Citations (Scopus)

Abstract

We have isolated cDNA clones encoding the bovine and rat gastric H,K-ATPase β subunit. A bovine abomasum λgt11 cDNA library was screened with a monoclonal antibody raised against the rabbit H,K-ATPase β subunit. A single positive phage clone containing an ~ 900-base pair cDNA insert was identified as reactive with the antibody. The identity of the cDNA was established by comparing the deduced amino acid sequence with sequences of cyanogen bromide fragments of the porcine H,K-ATPase β subunit. Polymerase chain reaction and rapid amplification of cDNA ends were used to generate a cDNA fragment encoding the carboxyl-terminal portion of the rat gastric H,K-ATPase β subunit. A rat stomach cDNA library was screened with the polymerase chain reaction product, and several full-length β subunit cDNA clones were identified. The open reading frame predicts a protein of 294 amino acids with a molecular weight of 33,689. The rat H,K-ATPase β subunit shows 41% amino acid sequence identity to the rat Na,K-ATPase β2 subunit and shares a number of structural similarities with Na,K-ATPase β subunit isoforms. By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the H,K-ATPase β subunit gene to murine chromosome 8. Northern and Western blot analysis reveals that this gene is expressed exclusively in stomach. Our results suggest that the H,K-ATPase and Na,K-ATPase β subunits evolved from a common ancestral gene and may play similar functional roles in enzyme activity.

Original languageEnglish (US)
Pages (from-to)19878-19884
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number32
StatePublished - Dec 17 1990

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Proton-Translocating ATPases
Cloning
Adenosine Triphosphatases
Organism Cloning
Complementary DNA
Tissue
Rats
H(+)-K(+)-Exchanging ATPase
Clone Cells
Gene Library
Genes
Polymerase chain reaction
Amino Acid Sequence
Amino Acids
Stomach
Abomasum
Cyanogen Bromide
Chromosomes, Human, Pair 8
Polymerase Chain Reaction
Inbred Strains Mice

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Cloning of the H,K-ATPase β subunit. Tissue-specific expression, chromosomal assignment, and relationship to Na,K-ATPase β subunits",
abstract = "We have isolated cDNA clones encoding the bovine and rat gastric H,K-ATPase β subunit. A bovine abomasum λgt11 cDNA library was screened with a monoclonal antibody raised against the rabbit H,K-ATPase β subunit. A single positive phage clone containing an ~ 900-base pair cDNA insert was identified as reactive with the antibody. The identity of the cDNA was established by comparing the deduced amino acid sequence with sequences of cyanogen bromide fragments of the porcine H,K-ATPase β subunit. Polymerase chain reaction and rapid amplification of cDNA ends were used to generate a cDNA fragment encoding the carboxyl-terminal portion of the rat gastric H,K-ATPase β subunit. A rat stomach cDNA library was screened with the polymerase chain reaction product, and several full-length β subunit cDNA clones were identified. The open reading frame predicts a protein of 294 amino acids with a molecular weight of 33,689. The rat H,K-ATPase β subunit shows 41{\%} amino acid sequence identity to the rat Na,K-ATPase β2 subunit and shares a number of structural similarities with Na,K-ATPase β subunit isoforms. By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the H,K-ATPase β subunit gene to murine chromosome 8. Northern and Western blot analysis reveals that this gene is expressed exclusively in stomach. Our results suggest that the H,K-ATPase and Na,K-ATPase β subunits evolved from a common ancestral gene and may play similar functional roles in enzyme activity.",
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Cloning of the H,K-ATPase β subunit. Tissue-specific expression, chromosomal assignment, and relationship to Na,K-ATPase β subunits. / Canfield, V. A.; Okamoto, C. T.; Chow, D.; Dorfman, J.; Gros, P.; Forte, J. G.; Levenson, R.

In: Journal of Biological Chemistry, Vol. 265, No. 32, 17.12.1990, p. 19878-19884.

Research output: Contribution to journalArticle

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T1 - Cloning of the H,K-ATPase β subunit. Tissue-specific expression, chromosomal assignment, and relationship to Na,K-ATPase β subunits

AU - Canfield, V. A.

AU - Okamoto, C. T.

AU - Chow, D.

AU - Dorfman, J.

AU - Gros, P.

AU - Forte, J. G.

AU - Levenson, R.

PY - 1990/12/17

Y1 - 1990/12/17

N2 - We have isolated cDNA clones encoding the bovine and rat gastric H,K-ATPase β subunit. A bovine abomasum λgt11 cDNA library was screened with a monoclonal antibody raised against the rabbit H,K-ATPase β subunit. A single positive phage clone containing an ~ 900-base pair cDNA insert was identified as reactive with the antibody. The identity of the cDNA was established by comparing the deduced amino acid sequence with sequences of cyanogen bromide fragments of the porcine H,K-ATPase β subunit. Polymerase chain reaction and rapid amplification of cDNA ends were used to generate a cDNA fragment encoding the carboxyl-terminal portion of the rat gastric H,K-ATPase β subunit. A rat stomach cDNA library was screened with the polymerase chain reaction product, and several full-length β subunit cDNA clones were identified. The open reading frame predicts a protein of 294 amino acids with a molecular weight of 33,689. The rat H,K-ATPase β subunit shows 41% amino acid sequence identity to the rat Na,K-ATPase β2 subunit and shares a number of structural similarities with Na,K-ATPase β subunit isoforms. By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the H,K-ATPase β subunit gene to murine chromosome 8. Northern and Western blot analysis reveals that this gene is expressed exclusively in stomach. Our results suggest that the H,K-ATPase and Na,K-ATPase β subunits evolved from a common ancestral gene and may play similar functional roles in enzyme activity.

AB - We have isolated cDNA clones encoding the bovine and rat gastric H,K-ATPase β subunit. A bovine abomasum λgt11 cDNA library was screened with a monoclonal antibody raised against the rabbit H,K-ATPase β subunit. A single positive phage clone containing an ~ 900-base pair cDNA insert was identified as reactive with the antibody. The identity of the cDNA was established by comparing the deduced amino acid sequence with sequences of cyanogen bromide fragments of the porcine H,K-ATPase β subunit. Polymerase chain reaction and rapid amplification of cDNA ends were used to generate a cDNA fragment encoding the carboxyl-terminal portion of the rat gastric H,K-ATPase β subunit. A rat stomach cDNA library was screened with the polymerase chain reaction product, and several full-length β subunit cDNA clones were identified. The open reading frame predicts a protein of 294 amino acids with a molecular weight of 33,689. The rat H,K-ATPase β subunit shows 41% amino acid sequence identity to the rat Na,K-ATPase β2 subunit and shares a number of structural similarities with Na,K-ATPase β subunit isoforms. By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the H,K-ATPase β subunit gene to murine chromosome 8. Northern and Western blot analysis reveals that this gene is expressed exclusively in stomach. Our results suggest that the H,K-ATPase and Na,K-ATPase β subunits evolved from a common ancestral gene and may play similar functional roles in enzyme activity.

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