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

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

Research output: Contribution to journalArticle

27 Citations (Scopus)

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

Fingerprint

Phosphofructokinase-2
Purification
Muscle
Rats
Skeletal Muscle
Enzymes
Liver
Peptides
Phosphorylation
Amino Acids
glycyl-seryl-alanine
Nucleotides
Cyclic AMP-Dependent Protein Kinases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and characterization of rat skeletal muscle fructose-6-phosphate,2-kinase : fructose-2,6-bisphosphatase. / Kitamura, K.; Uyeda, K.; Kangawa, K.; Matsuo, H.

In: Journal of Biological Chemistry, Vol. 264, No. 17, 1989, p. 9799-9806.

Research output: Contribution to journalArticle

Kitamura, K. ; Uyeda, K. ; Kangawa, K. ; Matsuo, H. / Purification and characterization of rat skeletal muscle fructose-6-phosphate,2-kinase : fructose-2,6-bisphosphatase. In: Journal of Biological Chemistry. 1989 ; Vol. 264, No. 17. pp. 9799-9806.
@article{9851ac63c56645b7bd4524342adb6a5f,
title = "Purification and characterization of rat skeletal muscle fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase",
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.",
author = "K. Kitamura and K. Uyeda and K. Kangawa and H. Matsuo",
year = "1989",
language = "English (US)",
volume = "264",
pages = "9799--9806",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "17",

}

TY - JOUR

T1 - Purification and characterization of rat skeletal muscle fructose-6-phosphate,2-kinase

T2 - fructose-2,6-bisphosphatase

AU - Kitamura, K.

AU - Uyeda, K.

AU - Kangawa, K.

AU - Matsuo, H.

PY - 1989

Y1 - 1989

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0024312314&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024312314&partnerID=8YFLogxK

M3 - Article

C2 - 2542332

AN - SCOPUS:0024312314

VL - 264

SP - 9799

EP - 9806

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 17

ER -