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    • The binding of DHAP to

    2022-08-04

    The binding of DHAP to aldolase resulted in a dramatic decrease of aldolase affinity to FBPase – KAapp was reduced more than 100 times. The dependence of the complex activity versus increasing DHAP concentration was biphasic (Fig. 3a). Supposedly, the first phase of the curve represents the state in which almost all aldolase molecules were bound to FBPase. The activity reported during the second phase of the curve, under high concentrations of DHAP, seems to reflect the activity mainly of an unbound form of aldolase. It is possible that within the complex the active sites of FBPase are in close vicinity to active sites of aldolase. This would enable a direct transfer, by channeling or restricted free diffusion mechanism, of F1,6-P2 from aldolase to FBPase. The synthesis of glycogen from lactate in myocytes takes place during recovery from exercise [2], [24]. The concentrations of DHAP, F6-P and F2,6-P2 in resting striated muscle Deoxycorticosterone acetate are, respectively, about 25–166 μM, 100–200 μM and below 1 μM [20], [25]. After stimulation of muscle contraction the concentration of these compounds immediately increases two to four times in the first 10 s of stimulation [20]. Thus, DHAP and F6-P might be the negative regulators of glyconeogenesis. F2,6-P2 is a commonly known inhibitor of FBPase [7], [8]; however, its role in the regulation of glycogen synthesis from lactate may be more complex. Supposedly, F2,6-P2 inhibits glyconeogenesis not only by simple competitive inhibition of FBPase but also by the destabilization of the glyconeogenic complex aldolase–FBPase. To conclude, the results presented in our report demonstrate that muscle FBPase bound with aldolase is entirely desensitized to AMP inhibition, which enables glycogen synthesis from carbohydrate precursors in myocytes. The stability of aldolase–FBPase complex is down-regulated by DHAP, F6-P and F2,6-P2 – the compounds whose concentration increases significantly and immediately during physical exercise. Since DHAP, F2,6-P2 and F6-P bind to the active sites of the enzymes, channeling or a restricted free diffusion mechanism of F1,6-P2 transfer from aldolase to FBPase may be expected.
    Acknowledgments
    Introduction Synthesis of endogenous glucose occurs mainly in the liver and kidney by gluconeogenesis from precursors such as glycerol, amino acids, and lactate [1], [2]. Fructose-1,6-bisphosphatase (FBPase, EC 3.1.3.11), a rate-limiting enzyme, catalyzes the irreversible conversion of fructose-1,6-bisphosphate (Fru-1,6-P2) to fructose-6-phosphate (F-6-P) and inorganic phosphate [3], [4], [5]. The existence of three FBPase isoenzymes: brain, muscle and liver, has been proposed considering immunological and kinetic data [3], [6], [7]. Although the rat muscle isoform displays 70% identity with the hepatic form, the specific physiological role of the muscle and brain isoenzymes remains unclear [8], [9]. The liver FBPase isoenzyme is recognized to be one of the major regulatory enzymes of gluconeogenesis [1], [2]. This isoenzyme, which has been isolated from different organisms, is a tetramer composed of identical subunits (MW 36 000–41 000) and is mainly regulated by two synergistic inhibitors: AMP and fructose-2,6-bisphosphate (F-2,6-P2) [3], [4], [5]. We recently demonstrated, by using immunohistochemical analysis in human tissues, that FBPase is expressed not only in kidney and liver, but also in a variety of organs such as small intestine, stomach, adrenal gland, testis and prostate, which might also contribute to gluconeogenesis [10]. In human and rat kidney, the specialized distribution of liver FBPase and cytosolic phosphoenolpyruvate carboxykinase (PEPCK) only in the proximal convoluted tubules of the nephron supports the idea that renal endogenous glucose production occurs mainly in this region, whereas the distal tubules specifically contribute to the glycolytic activity [10], [11]. Our results, showing aldolase B exclusively localized in the proximal tubules in the cortex of rat kidney and preferentially in hepatocytes of the periportal region of rat liver, are consistent with this idea [12]. In rat and human liver FBPase is expressed throughout the parenchyma in the hepatocytes. Similarly to kidney, FBPase expression is compartmentalized in rat liver with the periportal hepatocytes showing a higher expression level with a gradient of concentration from this region to the perivenous hepatocytes [10], [11]. Additionally, subcellular localization studies demonstrated that FBPase is located in the perinuclear region of the positive staining cells [10], [11]. The presence of glycolytic enzymes inside the nuclei has been reported [13], [14], [15]. However, due to the immunocytochemistry technique used in the analysis we were unable to distinguish between the nuclear and perinuclear location of liver FBPase. In this study, we have taken advantage of the immunofluorescence and confocal analysis to show that the liver FBPase is localized in a specialized plasma membrane compartment and inside the nuclei in rat kidney and liver cells, suggesting that the physical separation of metabolic pathway within the cell is an interesting regulatory mechanism.