title: Mammalian aldolases are isomer-selective high-affinity inositol polyphosphate binders.
abstract: A search for target proteins of inositol polyphosphates in mammalian tissues revealed that fructose 1,6-bisphosphate aldolases are potent isomer-selective binders of inositol polyphosphates. Binding was measured by tryptophan fluorescence quenching, by difference spectroscopy, and, in aldolase A, by equilibrium dialysis. Among a series of inositol phosphates containing between one and six phosphates and varying in their positions, inositol 1,4,5-trisphosphate was found to be bound strongest both by aldolase A [( L]0.5 = 0.58 microM) and aldolase B [( L]0.5 = 0.83 microM). Aldolase A showed also a strong binding of inositol tetrakisphosphate [( L]0.5 = 0.83 microM), of inositol 2,4,5-trisphosphate [( L]0.5 = 1.4 microM) and of inositol 1,3,4,5,6-pentakisphosphate [( L]0.5 = 2.0 microM); in aldolase B but not in aldolase A inositol 4,5-bisphosphate was bound as strongly as inositol 1,4,5-trisphosphate [( L]0.5 = 0.95 microM) and also inositol 2,4,5-trisphosphate was tightly bound [( L]0.5 = 1.2 microM). Both in aldolase A and B, 4 mol inositol 1,4,5-trisphosphate were bound/mol tetramer, in aldolase A a total binding of 8 mol inositol 1,4-bisphosphate/mol tetramer was evaluated. Difference spectra revealed that the binding of inositol phosphates to both isoenzymes may be associated with conformational changes. The binding of all inositol phosphates led to an inhibition of the enzyme activity. In aldolase A the inhibition was purely competitive, in aldolase B a complex cooperative type of inhibition was evident with fructose 1,6-bisphosphate as a substrate whereas with fructose 1-phosphate the inhibition also was purely competitive. Model calculations based on the in vitro data indicated a significant potential of aldolase to bind preferentially inositol 1,4,5-trisphosphate also in the presence of excess fructose 1,6-bisphosphate.
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002854:Chromatography, Paper; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism*; D007536:Isomerism; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013403:Sugar Phosphates / Q000378:metabolism*; D013552:Swine
chemical_list: D007295:Inositol Phosphates; D013403:Sugar Phosphates; D005634:Fructose-Bisphosphate Aldolase