assay for phosphoinositide-specific phospholipase C and the effect of pyridoxal-5-phosphate on the assay

In platelets, the major stimulatory second messengers are inositol 1, 4, 5 triphosphate [IP3] and 1,2-diacyiglycerol [DAG] produced upon the hydrolysis of phosphoinositides by phosphoinositide-speciftc phospholpase C [P1-PLC]. Pyridoxal-5-phosphate [PLP] is well known as a potent inhibitor of platelet aggregation. The mechanism[s] of its inhibitory action remains to be elucidated. P1-PLC was assayed and the effect of PLP on the assay was examined in an attempt to explain the nature of the inhibitory effect of PLP on platelet function. The assay was satisfactory and was inhibited by PLP with full inhibition at 10mMPLP

Effect of myo-inositol(1,4,5)trisphosphate on the hydrolysis of phytic acid by phytase.

Phytase is a monomeric enzyme of molecular mass 160 kDa which catalyzes the hydrolysis of phytic acid (D-myo inositol hexakisphosphate, InsP6) in a stepwise manner to myo-inositol. The enzyme-InsPn (n = 1-6) interaction at the catalytic site has a dissociation constant in the micro molar range. There also exists in the enzyme, a non-catalytic site specific for InsP3 with dissociation constant in the nano molar range. We have probed the effect of the high affinity InsP3 binding on the dissociation constant (Kd) of the phytase-InsP6 interaction and the kinetics of hydrolysis. These studies demonstrate the effect exerted by the high affinity InsP3 binding on the catalytic site of the enzyme.