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  • The release of FBPase and aldolase from subcellular structur

    2022-08-11

    The release of FBPase and aldolase from subcellular structures of muscle was dependent on the presence of the crowding agent imitating the physiological conditions – PEG 8000 (Table 1). The amount of both enzymes associated with structures of muscle XL184 was about 6–7 times higher in the presence of 5% PEG 8000 than in its absence (Table 1). An addition of 100μM CaCl2 to the homogenization buffer only slightly affected the amount of FBPase and aldolase associated with subcellular structures (Table 1). On the other hand, an addition of calcium to the homogenization buffer containing PEG 8000 significantly decreased the amount of FBPase associated with subcellular structures and increased the amount of the associated aldolase (Table 1). Ca2+ inhibited the activity of muscle FBPase with I0.5 equal to 0.48μM (Fig. 7) [14], whereas the inhibition of the activity of aldolase–FBPase complex was much weaker and an apparent inhibitory constant was about 24μM (Fig. 7). The relative activity of aldolase–FBPase complex in the presence of calcium ions (Fig. 7) roughly correlated with the amount of the aldolase–FBPase complex under the experimental conditions (calculated with Eq. (1)). Thus, it is possible that calcium decreases the concentration of the complex and, as a result, it can inhibit FBPase activity in a classic manner.where [AF] – concentration of aldolase–FBPase complex, [F] – total concentration of FBPase (7.15×10−7M), [A] – total concentration of aldolase (6.25×10−10M), KA – association constant between aldolase and FBPase (Fig. 5A–C).
    Discussion For years it was a common belief that lactate produced in a contracting muscle is transported via the blood stream to the liver where is converted to glucose, which subsequently is transported to the muscle again (Cori cycle). In last few years evidence has been accumulated that, in the skeletal muscle, up to 50% of lactate is converted to glycogen (glyconeogenesis) [22]. In such a pathway aldolase as well as FBPase are indispensable. Aldolase not only directly supplies substrate to FBPase [4], but also interacting with FBPase desensitizes it towards AMP inhibition [1], [3]. Glucose is the main source of energy for muscle fibres and its level therein is regulated not solely by hormones but also by intracellular calcium. Calcium is involved in the regulation of glycogenolysis by activating glycogen phosphorylase which subsequently causes the release of glucose from glycogen for ATP generation [23], [24]. Calcium also activates, in an insulin-independent manner, GLUT4-dependent entrance of glucose into muscle fibres [25] and accelerates glycolysis affecting binding of glycolytic enzymes to subcellular structures [7], [26]. On the other hand, calcium ions are presumed to inhibit glyconeogenesis: Ca2+ is the strong inhibitor of muscle FBPase and during the muscle contraction an elevated level of calcium should almost completely inhibit the enzyme [14]. Moreover, in cardiomyocytes, Ca2+ causes dissociation of FBPase from the Z-line [14] disintegrating the hypothetical glyconeogenic metabolon. In the present paper, we have reported that an increased concentration of calcium decreases both the affinity of FBPase to aldolase (Fig. 5) and to α-actinin (Fig. 6). This destabilizatory effect of Ca2+ results in the release of muscle FBPase from the multienzymatic complex located around the Z-line (Table 1, Fig. 1, Fig. 3) and free form of the enzyme is strongly inhibited both by calcium ions (Fig. 7) and AMP [1], [3]. Simultaneous to the inhibition of glyconeogenesis, the increased concentration of calcium causes the accumulation of aldolase within I-band (Fig. 2, Fig. 3), the region of the postulated glycolytic complex occurrence [6], [7], [8], [27]. The reverse regulation of the activity of glycolysis and glyconeogenesis by Ca2+ is in agreement with the data showing that calcium increases the amount of FBPase in the cytosolic extract from muscle fibres, but decreases the amount of aldolase (Table 1).