Purification and biochemical properties of a high-molecular-mass inositol 1,4,5-trisphosphate 3-kinase isoenzyme in human platelets.

The phosphorylation of inositol 1,4,5-trisphosphate (InsP3) to inositol 1,3,4,5-tetrakisphosphate (InsP4) is catalysed by InsP3 3-kinase. A method is presented for a rapid purification of the enzyme from human platelets. The purified enzyme was identified as a polypeptide of M(r) 69,000-70,000 after SDS/PAGE. It had a specific activity of 1.45 +/- 0.1 mumol/min per mg, and the degree of stimulation by Ca2+/calmodulin was 17-fold at saturating calmodulin and 10 microM free Ca2+. The Km for InsP3 and for ATP was 2.0 microM and 2.5 mM respectively. Human platelet InsP3 3-kinase was not recognized by immunodetection with anti-(InsP3 3-kinase A) or anti-(InsP3 3-kinase B) antibodies. These data provide the first biochemical evidence for the existence of a novel InsP3 3-kinase isoenzyme in human platelets, which is distinct from previously reported InsP3 3-kinase A and InsP3 3-kinase B.