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  • br Acknowledgements br Introduction Muscle FBPase

    2022-05-24


    Acknowledgements
    Introduction Muscle FBPase is very sensitive to AMP inhibition and in the presence of physiological concentrations of this effector in muscle fibres FBPase should be almost completely inactive [1], [2]. Recently, we have presented evidence that, in vitro, muscle aldolase binds to muscle FBPase and the binding results in dramatic changes in allosteric properties of the latter enzyme, desensitizing it towards AMP inhibition [2], [3] and enabling the intermediate channeling between the enzymes [4]. Investigating subcellular localization of the two enzymes we have found that, in striated muscles, FBPase and aldolase co-localize on the Z-line (with the strong binding of the former enzyme to α-actinin) [5] and we have postulated the existence of glyconeogenic metabolon around the Z-line [5]. Structural separation of glyconeogenesis and glycolysis (the glycolytic complex is known to be located on Quercetin [6], [7], [8]) may protect the muscle cell against the loss of the energy via futile cycles. Calcium is the main regulator of muscle contraction [9], [10]. It also affects the number of other cellular activities including the metabolism of carbohydrates [7], [11], [12], [13]. Quite recently, we have found that calcium is a potent inhibitor muscle FBPase and, in cardiomyocytes, causes dissociation of the enzyme from the Z-line [14]. The primary aim of the present study was to investigate the effect of calcium ions on the stability, activity and subcellular localization of aldolase–FBPase complex. In the running report, the evidence is presented that calcium decreases affinity of FBPase to aldolase and α-actinin and causes disintegration of aldolase–FBPase complex.
    Materials and methods
    Results Fluorescent labeling of the enzymes revealed that FBPase monomer bound on average three molecules of FITC, whereas two molecules of TRITC were bound with aldolase monomer. Although the labeling strongly decreased the specific activity of both enzymes, the modification did not significantly reduce the affinity of these enzymes to each other as well as of FBPase to α-actinin [5]. The protein exchange experiments revealed that FITC-FBPase is located on both sides of the Z-line (Fig. 1A) and TRITC-aldolase accumulates within the I-band and around the M-line (Fig. 2A). The same striation pattern for the both enzymes as well as for their co-localization (Fig. 3A) was achieved previously [5]. In control experiments, where muscle fibres were incubated with the respective fluorochrome alone, no staining was observed (data not shown). Co-localization of red and green signals was assessed by the co-localization analysis function in the AutoVisualise/AutoDeblur software. The degree of co-localization, expressed in Pearson’s correlation coefficient [20] was 0.73, what indicates high degree of the co-localization (Fig. 4A). The addition of 10 and 100μM calcium to the skinned muscle fibres with bound FITC-FBPase resulted in fast and complete dissociation of the enzyme from the Z-line (Fig. 1B and C). The reverse effect exerted calcium on the localization of aldolase: 10 and 100μM CaCl2 caused the slow accumulation of TRITC-aldolase in the I-band and around the M-line (Fig. 2B–G). The same effect of Ca2+ as rapid dissociation of FITC-FBPase from the Z-line and slow accumulation of TRITC-aldolase within the I-band and on the M-line was observed in the co-localization experiment (Fig. 3B and C). In the presence of calcium ions the Pearson’s correlation coefficient was −0.14, what indicates no co-localization of the proteins [20] (Fig. 4B and C). The global fitting of the sensograms revealed the binding constant for aldolase–FBPase equal to 8.56×106M−1 (Fig. 5A) and for FBPase-α-actinin equal to 1.01×107M−1 (Fig. 6A). Similar values of KA were obtained when the data was analyzed by equilibrium binding analysis (Fig. 5, Fig. 6A) and these values are in the range of KA reported previously [3], [5], [21]. The association of FBPase to aldolase and α-actinin was strongly affected by calcium ions, and 10μM Ca2+ decreased the affinity of FBPase to aldolase and to α-actinin about 4–5-fold and 10-fold, respectively (Fig. 5, Fig. 6A–C).