Purification and characterization of rat skeletal muscle fructose–6-phosphate,2-kinase: Fructose–2,6-bisphosphatase (original) (raw)
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Rabbit muscle fructose-1,6-bisphosphatase is phosphorylatedin vivo
Acta biochimica Polonica, 2003
Phosphorylated fructose-1,6-bisphosphatase (FBPase) was isolated from rabbit muscle in an SDS/PAGE homogeneous form. Its dephosphorylation with alkaline phosphatase revealed 2.8 moles of inorganic phosphate per mole of FBPase. The phosphorylated FBPase (P-FBPase) differs from the dephosphorylated enzyme in terms of its kinetic properties like K(m) and k(cat), which are two times higher for the phosphorylated FBPase, and in the affinity for aldolase, which is three times lower for the dephosphorylated enzyme. Dephosphorylated FBPase can be a substrate for protein kinase A and the amount of phosphate incorporated per FBPase monomer can reach 2-3 molecules. Since interaction of muscle aldolase with muscle FBPase results in desensitisation of the latter toward AMP inhibition (Rakus & Dzugaj, 2000, Biochem. Biophys. Res. Commun. 275, 611-616), phosphorylation may be considered as a way of muscle FBPase activity regulation.
Journal of Biological Chemistry, 1988
Fructose-6-P,2-kinase:fructose-2,6-bisphosphatase has been purified to homogeneity from beef heart. The enzyme was bifunctional and the specific activities of the kinase and the phosphatase of the pure enzyme were 60 and 30 milliunits/mg, respectively. The molecular weight of the enzyme was 118,000, consisting of two subunits of 58,000. In some preparations of the enzyme a minor protein with a subunit M, of 54,000 was present. This minor protein (54,000) was also bifunctional and showed the same immunoreactivity as the major protein. The specific activity of fructose-6-P,a-kinase of the minor component was three times higher than that of the major enzyme (58,000), but fructose-2,6-bisphosphatase activity was the same. These two forms have been separated by phosphocellulose chromatography. The tryptic peptide maps of these enzymes were very similar. The 58,000 enzyme was phosphorylated by CAMP-dependent protein kinase but the 54,000 enzyme was not. These results indicated that the minor 54,000 protein might be a proteolytically digested form of the 58,000 enzyme. The K, of the kinase for fructose-6-P and ATP was 70 PM and 260 PM, respectively for both the 58,000 and the 54,000 enzymes. K,,, for fructose-2,6-P2 and Ki for fructose-6-P of the phosphatase was approximately 40 and 11 PM, respectively. The enzyme was phosphorylated by fructose-2,6-P~ but the stoichiometry of the phosphate incorporation was 0.05 mol/mol subunit, while 0.4 mol/mol was incorporated in rat liver enzyme under the same conditions.
The Journal of Biological Chemistry, 1988
Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by CAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit M. of 58,000) was rapidly phosphorylated by both CAMPdependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by CAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit M, of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either CAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by CAMP-dependent protein kinase lowered the K, value for fructose 6-phosphate from 87 to 42 p~ without affecting the V, , , whereas the phosphorylation by protein kinase C increased the V,, value from 55 to 85 milliunits/mg without altering the K, value.
Kinetic properties of D-fructose-1,6-bisphosphate 1-phosphohydrolase isolated from human muscle
The Biochemical journal, 1995
D-Fructose-1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11) [Fru(1,6)Pase] was isolated from human muscle in an electrophoretically homogeneous form, free of aldolase contamination. The enzyme is inhibited by the substrate [fructose (1,6)-bisphosphate]. Km is 0.77 microM; Kis is 90 microM. The fructose-2,6-bisphosphate [Fru(2,6)P2], a regulator of gluconeogenesis, inhibits human muscle Fru(1,6)Pase with Ki = 0.13 microM. To determine Km, Kis and Ki the integrated method was used. AMP is an allosteric inhibitor of Fru(1,6)Pase. As with other mammalian isoenzymes, the human muscle enzyme is more strongly inhibited by AMP than is the liver isoenzyme [Dzugaj and Kochman (1980) Biochim. Biophys. Acta 614, 407-412]. Both of the inhibitors [AMP and Fru(2,6)P2] act synergistically on human muscle Fru(1,6)Pase. Ki for Fru(2,6)P2 determined in the presence of 0.4 microM AMP was 0.028 microM. The human muscle enzyme, like other mammalian Fru(1,6)Pases, requires Mg2+ for its activity. The Ka f...
Journal of Biological Chemistry
Fructose-6-P binding sites of rat liver and bovine heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated with an affinity labeling reagent, N-bromoacetylethanolamine phosphate. The rat liver enzyme was inactivated 97% by the reagent in 60 min, and the rate of inactivation followed pseudo-first order kinetics. The bovine heart enzyme was inactivated 90% within 60 min, but the inactivation rate followed pseudo-first order up to 80% inactivation and then became nonlinear. The presence of fructose-6-P retarded the extent of the inactivation to approximately 40% in 60 min. In order to determine the amino acid sequence of the fructose-6-P binding site, both enzymes were reacted with N-bromo[14C]acetylethanolamine-P and digested with trypsin; radiolabeled tryptic peptides were isolated and sequenced. A single 14C-labeled peptide was isolated from the rat liver enzyme, and the amino acid sequence of the peptide was determined as Lys-Gln-Cys-Ala-Leu-Ala-Leu-Lys. A major and two min...
Proceedings of the National Academy of Sciences, 1990
We have shown previously that bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (EC 2.7.1.105/3.1.3.46) is phosphorylated by cAMPdependent protein kinase and protein kinase C; phosphorylation results in activation of kinase. This activation of heart enzyme is in contrast to results with the liver isozyme, in which phosphorylation by cAMP-dependent protein kinase inhibits the kinase activity. As an initial step toward understanding this difference between the isozymes we have determined the DNA sequence of the heart enzyme and analyzed the amino acid sequence with special emphasis on the location of the phosphorylation site. We isolated and sequenced two overlapping cDNA fragments, which together could encode the complete amino acid sequence of bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase, a protein of 530 amino acids, with a calculated molecular weight of 60,679. Since the deduced protein contained amino acid sequences identical to the sequences of four known tryptic peptides from this enzyme we concluded that the deduced protein sequence did represent bovine heart enzyme. In addition, a cDNA fragment hybridized to a 4-kilobase mRNA from bovine heart. The phosphorylation sites of the heart enzyme were located near the C terminus, whereas the phosphorylation site of the liver isozyme is known to be located near the N terminus. These opposite locations of the phosphorylation sites may explain the contrasting effect of the covalent modification on the enzymes' activities.
The effect of natural "activation factor" and synthetic fructose-2,6-Pz on the allosteric kinetic properties of liver and muscle phosphofructokinases was investigated. Both synthetic and natural fructose-2,6-Pz show identical effects on the allosteric kinetic properties of both enzymes. Auctose-2,6-Pz counteracts inhibition by ATP and citrate and decreases the K,,, for fructose-6-P. This fructose ester also acts synergistically with AMP in releasing ATP inhibition. The K,,, values of liver and muscle phosphofructokinase for fructose-2,6-Pz in the presence of 1.26 IMI ATP are 12 milliunits/ml (or 24 l ũ r ) and 5 milliunits/ml (or 10 m), respectively. At near physiological concentrations of ATP (3 1 1 1 ) and fructose-6-P (0.2 m), however, the K,,, values for fructose-2,6-Pz are increased to 12 PM and 0.8 p t for liver and muscle enzymes, respectively. Thus, fructose-2,6-Pz is the most potent activator of the enzyme compared to other known activators such as fructose-1,6-Pz. The rates of the reaction catalyzed by the enzymes under the above conditions are nonlinear: the rates decelerate in the absence or in the presence of lower concentrations of fructose-2,6-Pz, but the rates become linear in the presence of higher concentrations of fructose-2,6-Pz. Fructose-2,6-Pz also protects phosphofructokinase against inactivation by heat. Fructose-2,6-Pz, therefore, may be the most important allosteric effector in regulation of phosphofructokinase in liver as well as in other tissues.