Purification and characterization of rat skeletal muscle fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase.
Fructose-6-phosphate,2-kinase:fructose-2,6-bis-phosphatase from rat skeletal muscle has been purified to homogeneity, and its structure and kinetic properties have been determined. The Mr of the native enzyme was 100,000 and the subunit Mr was 54,000. The apparent Km values of fructose-6-P,2-kinase for Fru-6-P and ATP were 56 and 48 microM, respectively. The apparent Km value for Fru-2,6-P2 of fructose-2,6-bis-phosphatase was 0.4 microM, and the Ki for Fru-6-P was 12.5 microM. 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-Gly-Leu-Pro - Ala-Arg was also isolated. This peptide was the same as that of rat liver enzyme (nucleotides 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.
Kitamura
K
K
Research Service of the Veterans Administration Medical Center, Dallas, Texas.
Uyeda
K
K
Kangawa
K
K
Matsuo
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009132:Muscles / Q000201:enzymology*; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
D000596:Amino Acids; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases