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

Research output: Contribution to journalArticle

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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 Mr of 55,000 by gel electrophoresis under denaturing conditions, compared to the deduced Mr of 54,023. Fru-6-P,2-kinase:Fru-2,6-bisphosphatase with the same Mr 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 Km Fru 6-P = 85 μM and Kum ATP = 270 μM, and those of fructose 2,6-bisphosphatase were Km Fru 2.6-pz = 21 μM and Ki 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 - 1991

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Phosphofructokinase-2
Cloning
Molecular Cloning
Testis
Rats
Complementary DNA
DNA
Enzymes
Amino Acids
Molecular sieves
Cyclic AMP-Dependent Protein Kinases
Electrophoresis
Phosphoric Monoester Hydrolases
Northern Blotting
Dimers
Liver
Escherichia coli
Protein Kinase C
Isoenzymes
Amino Acid Sequence

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular cloning of the DNA and expression and characterization of rat testes fructose-6-phosphate,2-kinase : Fructose-2,6-bisphosphatase. / Sakata, Junichiro; Abe, Yumiko; Uyeda, Kosaku.

In: Journal of Biological Chemistry, Vol. 266, No. 24, 1991, p. 15764-15770.

Research output: Contribution to journalArticle

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