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    • It raises a question about

    2022-05-23

    It raises a question about origin of the sensitivity to Ca of both FBPase isozymes. Was the ancestral vertebrate FBPase inhibited by calcium similarly to the liver isozyme? And is the high sensitivity of FBP2 a new evolutionary feature of warm-blooded vertebrates? Additionally, since subcellular localization of FBP2 in fish striated muscles strikingly resembles that observed in mammalian muscles (Adamowicz et al., 2006), an interesting issue arises whether in ectothermal vertebrates, Ca regulates muscle glycogen resynthesis by disturbing the glyconeogenic complex, as it is observed in mammals.
    Materials and methods
    Results and discussion Calcium is a signal for both striated muscle contraction and ATP synthesis in glycolytic pathway. Simultaneously, an elevated level of calcium disrupts the glyconeogenic complex and inhibits free, cytosolic muscle FBPase (FBP2), a regulatory enzyme of glyconeogenesis. In contrast to FBP2, the liver isozyme of mammalian FBPase—FBP1, is almost insensitive to the action of Ca. Although the precise molecular mechanism of FBPase inhibition by calcium is not yet clarified, the recent findings suggest the key role of amino Enalapril Maleate residue 69 in the strong sensitivity of FBP2 to the cation. In the case of mammalian FBP2, the residue 69 is occupied by glutamic acid, whereas in FBP1 sequence, glutamine is present at this position. Mutation of mammalian FBP2 toward FBP1 (Glu69Gln) almost completely abolishes inhibition by calcium (Zarzycki et al., 2007). Phylogenetic analysis of all known animal genome-derived sequences of FBPase suggests that the residue 69 is occupied by glutamine only in FBP1 sequences. In all known FBP2 and invertebrate FBPase (where only one fbp gene exists) sequences, the position corresponding to the residue 69 is occupied by an acidic amino acid, mainly by glutamic acid, but also by aspartic acid. This may suggest that all non-liver FBPases are highly sensitive to the inhibition by calcium. Unexpectedly, Dziewulska-Szwajkowska and Dzugaj (2010) have recently found that in ectothermal vertebrates, (Pelophylax esculentus, Amphibia) FBP2 was significantly less sensitive to the action of Ca than the warm-blooded isozyme. FBPases are very sensitive to proteolytic modifications which may occur during the purification procedure. These modifications apply to the N-terminal domain of FBPase and they result in a shift of pH optimum of the enzyme, from neutral to alkaline, and in its partial desensitization toward AMP and calcium. Thus, to exclude that kinetic properties observed during the course of our study were the effect of the enzyme proteolysis, after purification of FBPases from carp muscle and liver (Table 1A, Table 1B), we tested their kinetic properties. The specific activity of FBPase was 8U/mg and 13.3U/mg for the muscle and the liver enzyme, respectively. The ratio of the activity measured at pH 9.3 to that measured at pH 7.5 was 0.15 for the enzymes isolated from both the tissues, which indicated a lack of their proteolysis. The SDS-PAGE of the liver and muscle FBPase revealed only one band corresponding to the mass of about 37kDa, indicating purity of the enzymes and a lack of proteolysis (data not shown). Kinetic properties of carp FBPases closely resembled properties of respective mammalian isozymes, except the sensitivity to Ca and AMP inhibition (Table 2, Fig. 1). Piscine FBP2 was evidently less sensitive than the mammalian one to both the inhibitors: about 3–10 times less sensitive to AMP action and about 100 times less inhibited by Ca (for mammalian FBP2 I0.5 for Ca is about 1μM, and for AMP ~ 0.1μM) (Gizak et al., 2008). However, carp FBP2 was still significantly more sensitive to Ca than FBPase isolated from carp liver (Table 2, Fig. 1), which was inhibited by calcium similarly to mammalian FBP1 (e.g. I0.5 for Ca for mammalian FBP1 is about 1mM) (Gizak et al., 2008). The inhibition of carp FBP1 by AMP was also similar to that of mammalian FBP1, while FBP2 was less sensitive to AMP action, as compared to mammalian and bird muscle isozyme (Table 2, Fig. 1).