A comparative study on the sensitivity of Cyprinus carpio muscle and liver FBPase toward AMP and calcium.

The activity of fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) isozymes is influenced by AMP, Ca2+ and by reversible interactions with subcellular structures. In contrast to mammalian and avian isozymes, the kinetic properties of FBPases from ectothermal vertebrates are not fully described. To get some insight into mechanism of glycogen resynthesis in ectothermal vertebrates we examined the features of FBPases isolated from Cyprinus carpio skeletal muscle and liver. To investigate the evolutionary origin of the sensitivity of FBPase to effectors, we performed a phylogenetic analysis of known animal amino acids sequences of the enzyme. Based on our findings, we hypothesize that the high, mammalian-like, sensitivity of FBPase to Ca2+ is not essential for controlling the stability of glyconeogenic complex in striated muscles, instead it ensures the precise regulation of mitochondrial metabolism during prolonged Ca2+ elevation in contracting muscle fibers. Comparison of the kinetic properties of vertebrate and insect FBPases suggests that the high sensitivity of muscle isozyme to inhibitors has arisen as an adaptation enabling coordination of energy metabolism in warm-blooded animals.

Structure of E69Q mutant of human muscle fructose-1,6-bisphosphatase.

Human fructose-1,6-bisphosphatase is an allosteric enzyme that is regulated by different ligands. There are only two known isozymes in human tissues: the liver isozyme (the key enzyme of gluconeogenesis), which is regulated by fructose 2,6-bisphosphate, and its muscle counterpart (participating in glycogen synthesis), which is regulated by calcium ions. AMP, which is an allosteric inhibitor of both isozymes, inhibits the muscle isozyme with an I(0.5) that is 35-100 times lower than for the liver isozyme and the reason for this difference remains obscure. In studies aiming at an explanation of the main differences in the regulation of the two isozymes, it has been shown that only one residue, in position 69, regulates the sensitivity towards calcium ions. As a consequence of this finding, an E69Q mutant of the muscle isozyme, which is insensitive to calcium ions while retaining all other kinetic properties resembling the liver isozyme, has been prepared and crystallized. Here, two crystal structures of this mutant enzyme in complex with AMP with and without fructose 6-phosphate (the product of the catalytic reaction) are presented. The AMP binding pattern of the muscle isozyme is quite similar to that of the liver isozyme and the T conformations of the two isozymes are nearly the same.

Kinetic properties of Pelophylax esculentus muscle FBPase.

D-Fructose-1,6-bisphosphate 1-phosphohydrolase FBPase; [EC 3.1.3.11] was isolated from Pelophylax esculentus muscle in an electrophoretically homogeneous form with ca 30% yield. Its subunit molecular mass is ca 37 kDa. In this study, we determined the basic kinetic properties of the frog muscle enzyme. FBPase exhibited a maximum activity at pH 7.5. Like other FBPases the frog enzyme requires magnesium ions for its activity (K(a)=263 microM) and is activated by potassium ions (K(a)=63.6 microM). I(0.5) for calcium ion (91 microM) is 100 times higher than the corresponding value of mammalian muscle FBPase. K(s) for the substrate was 1.68 microM. Substrate excess inhibited the enzyme (K(si)=55 microM). AMP and fructose-2,6-bisphosphate (Fru-2,6P(2)) are potent inhibitors of frog muscle FBPase with I(0.5) of 0.2 microM and K(i) of 114 nM, respectively. Both inhibitors act synergistically on the frog muscle FBPase. In the presence of 0.05-0.5 microM of AMP, K(i) for Fru-2,6P(2) is 92 and 28 nM. I(0.5) for AMP for P. esculentus muscle FBPase is 55 times lower than the corresponding value for P. esculentus liver isozyme.

Glu 69 is essential for the high sensitivity of muscle fructose-1,6-bisphosphatase inhibition by calcium ions.

Muscle fructose-1,6-bisphosphatase (FBPase) is highly sensitive toward inhibition by AMP and calcium ions. In allosteric inhibition by AMP, a loop 52-72 plays a decisive role. This loop is a highly conservative region in muscle and liver FBPases. It is feasible that the same region is involved in the inhibition by calcium ions. To test this hypothesis, chemical modification, limited proteolysis and site directed mutagenesis Glu(69)/Gln were employed. The chemical modification of Lys(71-72) and the proteolytic cleavage of the loop resulted in the significant decrease of the muscle FBPase sensitivity toward inhibition by calcium ions. The mutation of Glu(69)-->Gln resulted in a 500-fold increase of muscle isozyme I(0.5) vs. calcium ions. These results demonstrate the key role that the 52-72 amino acid loop plays in determining the sensitivity of FBPase to inhibition by AMP and calcium ions.

Localization of chicken muscle FBPase in cardiomyocyte nuclei.

Fructose 1,6-bisphosphatase (FBPase; EC 3.1.3.11) localization in cardiomyocyte nuclei has recently been investigated in mammals [FEBS Lett. 539 (2003) 51]. In this study, nuclear localization of FBPase in the cardiac muscle of the chicken was studied by immunohistochemistry and other methods. A result of the electron microscopic investigation was confirmed by immunoblotting analysis. Using MALDI Q-TOF mass spectrometry and Mascot program, the nuclear FBPase was identified as muscle chicken FBPase. FBPase activity in isolated cardiomyocyte nuclei was 5.9 mU/g. Nuclear FBPase was strongly inhibited by allosteric inhibitor AMP. I(0.5) for AMP was 0.16 microM and was the same as for the purified chicken muscle enzyme.

Interaction between muscle aldolase and muscle fructose 1,6-bisphosphatase results in the substrate channeling.

Fructose 1,6-bisphosphatase (FBPase) is known to form a supramolecular complex with alpha-actinin and aldolase on both sides of the Z-line in skeletal muscle cells. It has been proposed that association of aldolase with FBPase not only desensitizes muscle FBPase toward AMP inhibition but it also might enable the channeling of intermediates between the enzymes [Rakus et al. (2003) FEBS Lett. 547, 11-14]. In the present paper, we tested the possibility of fructose 1,6-bisphosphate (F1,6-P(2)) channeling between aldolase and FBPase using the approach in which an inactive form of FBPase competed with active FBPase for binding to aldolase and thus decreased the rate of aldolase-FBPase reaction. The results showed that F1,6-P(2) is transferred directly from aldolase to FBPase without mixing with the bulk phase. Further evidence that F1,6-P(2) is channeled from aldolase to FBPase comes from the experiments investigating the inhibitory effect of a high concentration of magnesium ions on aldolase-FBPase activity. FBPase in a complex with aldolase, contrary to free muscle FBPase, was not inhibited by high Mg(2+) concentrations, which suggests that free F1,6-P(2) was not present in the assay mixture during the reaction. A real-time interaction analysis between aldolase and FBPase revealed a dual role of Mg(2+) in the regulation of the aldolase-FBPase complex stability. A physiological concentration of Mg(2+) increased the affinity of muscle FBPase to muscle aldolase, whereas higher concentrations of the cation decreased the concentration of the complex. We hypothesized that the presence of Mg(2+) stabilizes a positively charged cavity within FBPase and that it might enable an interaction with aldolase. Because magnesium decreased the binding constant (K(a)) between aldolase and FBPase in a manner similar to the decrease of K(a) caused by monovalent cations, it is postulated that electrostatic attraction might be a driving force for the complex formation. It is presumed that the biological relevance of F1,6-P(2) channeling between aldolase and FBPase is protection of this glyconeogenic, as well as glycolytic, intermediate against degradation by cytosolic aldolase, which is one of the most abundant enzyme of glycolysis.

The interaction of FBPase with aldolase: a kinetic and fluorescence investigation on chicken muscle enzymes.

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) is strongly inhibited by AMP in vitro and, therefore, at physiological concentrations of substrate and AMP, FBPase should be completely inhibited. Desensitization of rabbit muscle FBPase against AMP inhibition was previously observed in the presence of rabbit muscle aldolase. In this study, we analysed the kinetics of an FBPase catalyzed reaction and interaction between chicken muscle FBPase and chicken muscle aldolase. The initial rate of FBPase reaction vs. substrate concentration shows a maximum activity at a concentration of 20 microM Fru-1,6P2 and then decreases. Assuming rapid equilibrium kinetics, the enzyme-catalyzed reaction was described by the substrate inhibition model, with Ks approximately 5 microM and Ksi approximately 39 microM and factor beta approximately 0.2, describing change in the rate constant (k) of product formation from the ES and ESSi complexes. Based on ultracentrifugation studies, aldolase and FBPase form a hetero-complex with approximately 1:1 stoichiometry with a dissociation constant (Kd) of 3.8 microM. The FBPase-aldolase interaction was confirmed via fluorescence investigation. The aldolase-FBPase interaction results in aldolase fluorescence quenching and its maximum emission spectrum shifting from 344 to 356 nm. The Kd of the FBPase-aldolase complex, determined on the basis of fluorescence changes, is 0.4 microM at 25 degrees C with almost 1:1 stoichiometry. This interaction increases the I(0.5) for the AMP inhibition of FBPase threefold, and slightly affects FBPase affinity to magnesium ions, increasing the Ka and Hill coefficient (n). No effect of aldolase on the FBPase pH optimum was observed. Thus, the decrease in FBPase sensitivity to AMP inhibition enables FBPase to function in vivo thanks to aldolase.

The effect of high dose of cortisol on glucose-6-phosphatase and fructose-1,6-bisphosphatase activity, and glucose and fructose-2,6-bisphosphate concentration in carp tissues (Cyprinus carpio L.).

The effect of a high dose of cortisol (200 mg kg(-1) body mass) on juvenile carp was investigated. The activity of glucose-6-phosphatase in liver and of fructose-1,6-bisphosphatase in liver, kidney and muscle, the serum glucose and fructose-2,6-bisphosphate concentration as well as the serum concentration of the injected hormone were measured after 24, 72 and 216 h after intraperitoneal cortisol injection. The activities of fructose-1,6-bisphosphatase in liver and kidney and glucose-6-phosphatase in liver were elevated in comparison with the control, while the fructose-1,6-bisphosphatase activity in the muscle tissue was unchanged. After cortisol injection, the serum glucose level was nearly two times higher after 24 and 72 h and was still 50% higher after 216 h compared with controls. In contrast, the liver fructose-2,6-bisphosphate concentration was unchanged after 24 h. More than two times higher fructose-2,6-bisphosphate concentration was observed in liver after 72 h and it was still elevated after 216 h after the cortisol injection.

Rabbit muscle fructose-1,6-bisphosphatase is phosphorylatedin vivo.

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.

Different sensitivities of mutants and chimeric forms of human muscle and liver fructose-1,6-bisphosphatases towards AMP.

AMP is an allosteric inhibitor of human muscle and liver fructose-1,6-bisphosphatase (FBPase). Despite strong similarity of the nucleotide binding domains, the muscle enzyme is inhibited by AMP approximately 35 times stronger than liver FBPase: I0.5 for muscle and for liver FBPase are 0.14 microM and 4.8 microM, respectively. Chimeric human muscle (L50M288) and chimeric human liver enzymes (M50L288), in which the N-terminal residues (1-50) were derived from the human liver and human muscle FBPases, respectively, were inhibited by AMP 2-3 times stronger than the wild-type liver enzyme. An amino acid exchange within the N-terminal region of the muscle enzyme towards liver FBPase (Lys20-->Glu) resulted in 13-fold increased I0.5 values compared to the wild-type muscle enzyme. However, the opposite exchanges in the liver enzyme (Glu20-->Lys and double mutation Glu19-->Asp/Glu20-->Lys) did not change the sensitivity for AMP inhibition of the liver mutant (I0.5 value of 4.9 microM). The decrease of sensitivity for AMP of the muscle mutant Lys20-->Glu, as well as the lack of changes in the inhibition by AMP of liver mutants Glu20-->Lys and Glu19-->Asp/Glu20-->Lys, suggest a different mechanism of AMP binding to the muscle and liver enzyme.