Purification and characterization of rat skeletal muscle fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase

K. Kitamura, K. Uyeda, K. Kangawa, H. Matsuo

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27 Scopus citations

Abstract

Fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from rat skeletal muscle has been purified to homogeneity, and its structure and kinetic properties have been determined. The M(r) of the native enzyme was 100,000 and the subunit M(r) was 54,000. The apparent K(m) values of fructose-6-P,2-kinase for Fru-6-P and ATP were 56 and 48 μM, respectively. The apparent K(m) value for Fru-2,6-P2 of fructose-2,6-bisphosphatase was 0.4 μM, and the K(i) for Fru-6-P was 12.5 μM. The enzyme was bifunctional, and the phosphatase activity was 2.5 times higher than the kinase activity. The enzyme was not phosphorylated by cAMP-dependent protein kinase. The amino acid composition of the skeletal muscle enzyme was similar to that of the rat liver enzyme, and the carboxyl terminus sequence (His-Tyr) was the same as that of the liver enzyme. The tryptic peptides generated from the liver and skeletal muscle enzymes were identical except for two peptides. A peptide corresponding to nucleotides 14-28 of the rat liver enzyme was not detected in the skeletal muscle enzyme. A peptide whose amino acid sequence was Thr-Ala-Ser-Ile-Pro-Gln-Phe-Thr-Asn-Ser-Pro-Thr-Met-Val-Ile-Met-Val-G y-Leu-Pro-Ala-Arg was also isolated. This peptide was the same as that of rat liver enzyme (nucleotide 31-52) containing the phosphorylation site except in the muscle enzyme two amino terminus amino acids, Gly-Ser(P), have been altered to Thr-Ala. Thus, the rat skeletal muscle enzyme is very similar in structure to the rat liver enzyme except for the lack of possibly one peptide and the lack of a phosphorylation site by the substitution of the target Ser with Ala.

Original languageEnglish (US)
Pages (from-to)9799-9806
Number of pages8
JournalJournal of Biological Chemistry
Volume264
Issue number17
StatePublished - 1989

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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