title: Interaction between muscle aldolase and muscle fructose 1,6-bisphosphatase results in the substrate channeling.
abstract: Fructose 1,6-bisphosphatase (FBPase) is known to form a supramolecular complex with alpha-actinin and aldolase on both sides of the Z-line in skeletal muscle cells. It has been proposed that association of aldolase with FBPase not only desensitizes muscle FBPase toward AMP inhibition but it also might enable the channeling of intermediates between the enzymes [Rakus et al. (2003) FEBS Lett. 547, 11-14]. In the present paper, we tested the possibility of fructose 1,6-bisphosphate (F1,6-P(2)) channeling between aldolase and FBPase using the approach in which an inactive form of FBPase competed with active FBPase for binding to aldolase and thus decreased the rate of aldolase-FBPase reaction. The results showed that F1,6-P(2) is transferred directly from aldolase to FBPase without mixing with the bulk phase. Further evidence that F1,6-P(2) is channeled from aldolase to FBPase comes from the experiments investigating the inhibitory effect of a high concentration of magnesium ions on aldolase-FBPase activity. FBPase in a complex with aldolase, contrary to free muscle FBPase, was not inhibited by high Mg(2+) concentrations, which suggests that free F1,6-P(2) was not present in the assay mixture during the reaction. A real-time interaction analysis between aldolase and FBPase revealed a dual role of Mg(2+) in the regulation of the aldolase-FBPase complex stability. A physiological concentration of Mg(2+) increased the affinity of muscle FBPase to muscle aldolase, whereas higher concentrations of the cation decreased the concentration of the complex. We hypothesized that the presence of Mg(2+) stabilizes a positively charged cavity within FBPase and that it might enable an interaction with aldolase. Because magnesium decreased the binding constant (K(a)) between aldolase and FBPase in a manner similar to the decrease of K(a) caused by monovalent cations, it is postulated that electrostatic attraction might be a driving force for the complex formation. It is presumed that the biological relevance of F1,6-P(2) channeling between aldolase and FBPase is protection of this glyconeogenic, as well as glycolytic, intermediate against degradation by cytosolic aldolase, which is one of the most abundant enzyme of glycolysis.
authors.affiliation: Department of Animal Physiology, Zoological Institute, Wroclaw University, Cybulskiego 30, 50-205 Wroclaw, Poland.
authors.forename: Darek
authors.initials: D
authors.lastname: Rakus
authors.forename: Marta
authors.initials: M
authors.lastname: Pasek
authors.forename: Hubert
authors.initials: H
authors.lastname: Krotkiewski
authors.forename: Andrzej
authors.initials: A
authors.lastname: Dzugaj
mesh_terms: D000185:Actinin / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002414:Cations, Monovalent / Q000494:pharmacology; D003198:Computer Simulation; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000201:enzymology*; D011092:Polyethylene Glycols; D011485:Protein Binding; D011489:Protein Denaturation; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013696:Temperature; D009764:o-Phthalaldehyde / Q000494:pharmacology
chemical_list: D002414:Cations, Monovalent; D004791:Enzyme Inhibitors; D000185:Actinin; D011092:Polyethylene Glycols; D000249:Adenosine Monophosphate; D011732:Pyridoxal Phosphate; D009764:o-Phthalaldehyde; D006597:Fructose-Bisphosphatase; D005634:Fructose-Bisphosphate Aldolase; D008274:Magnesium