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  • The investigation in vitro revealed

    2022-05-16

    The investigation in vitro revealed that aldolase, as well as FBPase, has affinity to α-actinin, one of the structural protein of the Z-line (Rakus et al., 2003a, Mamczur et al., 2005). Therefore, the existence of in vivo heterologous complex, consisting of aldolase, FBPase and α-actinin has been postulated (Rakus et al., 2003a, Mamczur et al., 2005). The investigation ex vivo revealed the colocalisation of the proteins on the Z-line (Rakus et al., 2003a, Mamczur et al., 2005). Additionally, we observed substrate channeling between the muscle aldolase and the muscle FBPase, and in effect, the existence of a glyconeogenic metabolon in living RO4929097 was postulated (Rakus et al., 2004). The occurrence of glyconeogenesis in the heart muscle has not been reported. Our investigation revealed that, as in the skeletal muscle, in cardiomyocytes FBPase is localised on the Z-line. Additionally, it can be found in the nuclei as well as on intercalated discs (Gizak and Dzugaj, 2003, Gizak et al., 2004). If the glyconeogenesis occurs in cardiomyocytes, the colocalisation of FBPase and aldolase might be expected. We have studied the colocalisation of these enzymes by in situ analysis with the use of confocal and electron microscopy. The degree of colocalisation was expressed by the Pearson's correlation coefficient. Additionally, we performed the nuclear import of the fluorophore-labeled aldolase and FBPase in the presence and in the absence of lectin WGA.
    Materials and methods
    Results
    Discussion The colocalisation of FBPase and aldolase in cardiomyocytes supports the hypothesis that the glyconeogenesis occurs in the heart muscle, as well as in the skeletal muscle. For years glycolytic enzymes have been regarded simply as soluble proteins evenly distributed in the cytosolic compartment of the cell. After the discovery of the mitochondrial structure and its function, a corresponding organelle for glycolysis has been hoped to be found, but without success. Many studies indicate that glycolytic enzymes do not exist in a soluble form only, and that they also may be (reversibly) associated with cellular structures (Clarke and Masters, 1975, Masters et al., 1987, Minaschek et al., 1992, Volker et al., 1995, Ovadi and Srere, 2000). In many cases this association alters the regulatory and kinetic properties of the enzymes (Liou and Anderson, 1980). Additionally, the interactions between metabolically sequential enzymes may facilitate the channeling of substrates (Ovadi and Srere, 2000). The degree of the association seems to be variable, depending on the state of the cell. For example, the distribution of muscle hexokinase II into soluble and particulate fractions depends on insulin (Vogt et al., 1998). It has been reported that almost all glycolytic enzymes and creatine kinase bind to actin filaments within the I-band (Kraft et al., 2000, Ovadi and Srere, 2000), and probably they can function as a glycolytic multi-enzyme complex, directly providing ATP to myosin ATPase (Kraft et al., 2000, Sullivan et al., 2003). The localisation of a glyconeogenic metabolon in close proximity to a glycolytic complex raises a question concerning the compartmentalisation of metabolic pathways. The regulatory enzymes of glycolysis or glyconeogenesis, such as phosphofructokinase-1 or FBPase, are firmly bound to the structural elements of the cell (Ovadi and Srere, 2000, Gizak and Dzugaj, 2003). On the other hand, enzymes catalyzing reversible reactions, like aldolase, might create either glycolytic or glyconeogenic complexes, depending on cellular requirements (Rakus et al., 2004, Mamczur et al., 2005). In the glycogen synthesis, aldolase not only provides the substrate for FBPase but also protects it against AMP inhibition (Rakus and Dzugaj, 2000, Rakus et al., 2003b). Recently we have found that, unlike the liver isozyme, muscle FBPase is very sensitive to the inhibition by calcium ions (Gizak et al., 2004). We have also found that the aldolase–FBPase-α-actinin complex is very sensitive to the intracellular calcium concentration (Mamczur et al., 2005). The increase of the Ca2+ concentration upon the muscle contraction results in the dissociation of the aldolase–FBPase-α-actinin complex and restricts the glyconeogenesis (Mamczur et al., 2005). At the same time, the association of glycolytic enzymes with the actin-containing filaments allows the intensification of ATP production and its supply directly to the myosin (Srere and Ovadi, 1990, Ovadi and Srere, 2000, Mamczur et al., 2005).