title: Interaction of myoinositoltrisphosphate-phytase complex with the receptor for intercellular Ca2+ mobilization in plants.
abstract: One of the myoinositol trisphosphates produced by the phytase-myoinositol hexakisphosphate (InsP6) reaction is Ins(2,4,5)P3. That Ins(2,4,5)P3 can elicit Ca2+ mobilization from intracellular stores in plants [Samanta, S., Dalal, B., Biswas, S., & Biswas, B.B.(1993) Biochem. Biophys. Res. Commun. 191,427] prompted us to elucidate the mechanism. The InsP3 [Ins(1,4,5)P3/Ins(2,4,5)P3]-phytase complex has been found to interact with the receptor for InsP3 in vitro forming a ternary complex, and a nanomolar concentration of InsP3 is required. For enzymatic cleavage of InsP3 by phytase, micromolar concentrations are needed, and the affinities of the phytase for different myoinositol phosphates have been found to depend upon the number of phosphate groups present in the substrate. Fraction accessibility of tryptophan residues to a neutral fluorescence quencher, acrylamide in free and myoinositol phosphates bound phytase, as determined by Stern-Volmer plot, records a progressive decrease starting from InsP6 to InsP with the notable exceptions of both Ins (1,4,5)P3 and Ins(2,4,5)P3. This deviation from the trend of change in the accessibility of tryptophan residues in myoinositol phosphate bound phytase is recorded from the fact that there is a high affinity (dissociation constant of the nanomolar order) and noncatalytic binding site in phytase for the two isomers of InsP3. In the nanomolar range of concentrations, both isomers of InsP3 bind to a second site of phytase having about 40-fold higher affinity than the normal substrate binding site. InsP3, when bound to noncatalytic site in phytase is not hydrolyzed but induces a significant change in the conformation of phytase as assayed from the relative accessibility of tryptophan residues. This conformational change in phytase is recognized by the receptor for InsP3, because in absence of InsP3 no interaction between the receptor and phytase is detected. However, InsP3-phytase complex is a better elicitor of Ca2+ efflux from microsomal/vacuolar fractions than free InsP3. This is further confirmed by the fact that when Ins(1,3,4)P3-phytase complex can elicit Ca2+ efflux from intracellular stores, Ins(1,3,4)P3 per se is minimally effective.
authors.affiliation: Department of Biophysics, Molecular Biology and Genetics, University of Calcutta, India.
authors.forename: S
authors.initials: S
authors.lastname: Dasgupta
authors.forename: D
authors.initials: D
authors.lastname: Dasgupta
authors.forename: M
authors.initials: M
authors.lastname: Sen
authors.forename: S
authors.initials: S
authors.lastname: Biswas
authors.forename: B B
authors.initials: BB
authors.lastname: Biswas
mesh_terms: D010832:6-Phytase / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D010833:Phytic Acid / Q000378:metabolism*; D010944:Plants / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D007295:Inositol Phosphates; D046911:Macromolecular Substances; D018160:Receptors, Cytoplasmic and Nuclear; C068271:inositol hexakisphosphate receptor; D010833:Phytic Acid; C062616:inositol 2,4,5-trisphosphate; D010832:6-Phytase; D002118:Calcium