INTERACTION OF MYOINOSITOLTRISPHOSPHATE-PHYTASE COMPLEX WITH THE RECEPTOR FOR INTRACELLULAR CA2+ MOBILIZATION IN PLANTS

One of the myoinositol trisphosphates produced by the phytase-myoinosi tol hexakisphosphate (InsP(6)) reaction is Ins(2,4,5)P-3. That Ins(2,4 ,5)P-3 can elicit Ca2+ mobilization from intracellular stores in plant s [Samanta, S., Dalal, B., Biswas, S., & Biswas, B. B.(1993) Biochem. Biophys. Res. Commun. 191, 427] prompted us to elucidate the mechanism . The InsP(3) [Ins(1,4,5)P-3/Ins(2,4,5)P-3]-phytase complex has been f ound to interact with the receptor for InsP(3) in vitro forming a tern ary complex, and a nanomolar concentration of InsP(3) is required. For enzymatic cleavage of InsP(3) by phytase, micromolar concentrations a re needed, and the affinities of the phytase for different myoinositol phosphates have been found to depend upon the number of phosphate gro ups present in the substrate. Fraction accessibility of tryptophan res idues to a neutral fluorescence quencher, acrylamide in free and myoin ositol phosphate bound phytase, as determined by Stern-Volmer plot, re cords a progressive decrease starting from InsP(6) to InsP with the no table exceptions of both Ins(1,4,5)P-3 and Ins(2,4,5)P-3. This deviati on from the trend of change in the accessibility of tryptophan residue s in myoinositol phosphate bound phytase is recorded from the fact tha t there is a high affinity (dissociation constant of the nanomolar ord er) and noncatalytic binding site in phytase for the two isomers of In sP(3). In the nanomolar range of concentrations, both isomers of InsP( 3) bind to a second site of phytase having about 40-fold higher affini ty than the normal substrate binding site. InsP(3), when bound to nonc atalytic site in phytase is not hydrolyzed but induces a significant c hange in the conformation of phytase as assayed from the relative acce ssibility of tryptophan residues. This conformational change in phytas e is recognized by the receptor for InsP(3), because in absence of Ins P(3) no interaction between the receptor and phytase is detected. Howe ver, InsP(3)-phytase complex is a better elicitor of Ca2+ efflux from microsomal/vacuolar fractions than free InsP(3). This is further confi rmed by the fact that when Ins(1,3,4)P-3-phytase complex can elicit Ca 2+ efflux from the intracellular stores, Ins(1,3,4)P-3 per se is minim ally effective.