Previously we have isolated liver FBPase from Pelophylax esc

Previously we have isolated liver FBPase from Pelophylax esculentus (former Rana esculenta, Frost et al., 2006) and characterized its kinetic properties (Dziewulska-Szwajkowska and Dzugaj, 1999) on the contrary the muscle FBPase has never been fully characterized. The primary aim of the present paper was the isolation of the P. esculentus muscle FBPase in an electrophoretically homogeneous form in order to determine the kinetic properties of the enzyme and particularly to check its sensitivity toward calcium ions. We found that like the mammalian muscle FBPases, the frog muscle isozyme was also sensitive to inhibition by calcium ions. It is feasible that in frog muscle glyconeogenesis is regulated by Fru-2,6P2 and additionally by calcium ions.
Materials and methods
Results
Discussion P. esculentus, formerly known as R. esculenta (Frost et al., 2006) is a natural hybrid between two parental species Pelophylax lessonae and Pelophylax ridibundus (Berger, 1988). Kinetic properties of P. esculentus muscle FBPase were virtually the same as mammalian and chicken muscle FBPases. The enzyme was inhibited by the substrate, allosterically inhibited by AMP, competitively by Fru-2,6P2, and the synergetic inhibition by the both inhibitors was also observed. The curve of activation by magnesium was sigmoidal indicating a cooperative interaction of frog muscle FBPase with this metal ion. It was of importance that P. esculentus muscle FBPase was also inhibited by calcium ions, although the I0.5 was 100 times higher than the corresponding value of mammalian muscle FBPase. Gluconeogenesis in mammals is hormonally regulated via fructose-2,6-bisphosphatase-phosphofructokinase-2 (FBPase-2/PFK-2) catalyzing synthesis and hydrolysis of Fru-2,6P2 the activator of phosphofructokinase-1 (PFK-1) [E.C. 2.7.1.11] and the inhibitor of FBPase. Two FBPase-2/PFK-2 isoforms have been discovered in skeletal muscle (Marsin et al., 2000, Okar et al., 2001): predominantly the muscle isozyme and a small amount of the liver isozyme, both of which are encoded by the same gene, but as a result of pre-mRNA splicing the muscle isoform is devoid of the domain containing Ser32 and thereby cannot be regulated by reversible Amikacin (Okar et al., 2001). Minatogawa and Hue reported that in rat skeletal muscle the Fru-2,6P2 level is relatively constant (Minatogawa and Hue, 1984). Whereas Wegener and Krause, presented data indicating that Fru-2,6P2 concentration in frog muscle was changing during exercise (Krause and Wegener, 1996, Wegener and Krause, 2002). It is possible that a different FBPase-2/PFK-2 muscle enzyme is present in frog muscle tissue. Since FBPase I 0.5 toward AMP is in the range 0.2–1μM the enzyme should be completely inhibited in the muscle cell in the presence of 10–20μM AMP. We found that the muscle FBPase binds muscle aldolase what results in heterologous complex formation (Rakus and Dzugaj, 2000). In the complex FBPase is utterly insensitive to inhibition by AMP or Fru-2,6P2 (Rakus et al. 2003). We also found that in skeletal muscle the FBPase colocalizes with the muscle aldolase and alfa-actinin on the Z-line (Gizak et al., 2003). Supposedly the complex: FBPase:aldolase:alfa-actinin is a part of glyconeogenic metabolom. Our investigation revealed that calcium was the strong inhibitor of the mammalian muscle FBPase (Gizak et al., 2004). The increase of the calcium ions concentration during a muscle contraction results in the breakdown of the FBPase–aldolase complex and the dissociation of FBPase from the Z-line allowing inhibition of the enzyme, on the contrary during the rest the decrease of the calcium ion concentration will stimulate the formation of FBPase:aldolase complex thus the glyconeogenesis activation. Mammalian FBPases are homotetrameric enzymes (Tillmann and Eschrich, 1998). The tertiary structure of the each liver monomer is composed of two domains: the Fru-1,6P2 domain containing the active site and the AMP binding domain with the AMP binding site. The metal binding site is located between the Fru-1,6P2 and AMP binding domains (Ke et al., 1991, Villeret et al., 1995). The mammalian liver FBPase exists at least in two conformations called R and T, depending on the relative concentrations of the enzyme effectors (Ke et al., 1991, Zhang et al., 1994). The mammalian liver FBPase consists of the upper dimer (C1 and C2) and the lower dimer (C3 and C4). It has been shown that the communication between the subunits within the dimers is possible by interaction of 7–15N-terminal residues and the loop 187–196 of C1 with the loop 52–72 of C2. A proposed mechanism for the allosteric regulation of catalysis involves three conformational states of the loop 52–72 called: engaged, disengaged and disordered. FBPase is active when the loop 52–72 oscillates between the engaged and disordered states, and inactive or less active when the loop 52–72 is stabilized in its disengaged conformation (Choe et al., 1998). Although the crystallographic study of vertebrate muscle FBPase has been reported, no details concerning the conformation of this isozyme have been released (Zhu et al., 2001). Nevertheless, it seems reasonable to assume that the vertebrate muscle isozyme also exists in conformations R and T and the similar mechanism for allosteric regulation includes three conformational states of the loop 52–72: engaged, disengaged, and disordered.