Molecular cloning of the DNA and expression and characterization of rat testes fructose-6-phosphate,2-kinase:Fructose-2,6-bisphosphatase

Junichiro Sakata, Yumiko Abe, Kosaku Uyeda

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Abstract

We have isolated and sequenced two overlapping cDNA fragments which could encode the complete amino acid sequence of rat testis fructose-6-phosphate,2- kinase:fructose-2,6-bisphosphatase. Northern blot analysis revealed that the major 2-kilobase mRNA isolated from rat testis hybridized with a cDNA fragment. A full length cDNA, which encoded a protein of 468 amino acids, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a M(r) of 55,000 by gel electrophoresis under denaturing conditions, compared to the deduced M(r) of 54,023. Fru-6-P,2-kinase:Fru-2,6-bisphosphatase with the same M(r) 55,000 was also present in rat testis extract. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively. Various kinetic constants of the expressed fructose 6-P,2-kinase were K(m)(Fru 6-P) = 85 μM and K(m)(ATP) = 270 μM, and those of fructose 2,6-bisphosphatase were K(m)(Fru 2,6-P2) = 21 μM and K(i)(Fru 6-P) = 3.4 μM. The enzyme was phosphorylated by Fru-2,6[2-32P]P2 and also by protein kinase C, but not by cAMP-dependent protein kinase, which is in contrast to the liver and heart isozymes.

Original languageEnglish (US)
Pages (from-to)15764-15770
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number24
StatePublished - Oct 29 1991

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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