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 inosito l 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) 69000-70000 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 muM free Ca2+. The K(m) for I nsP3 and for ATP was 2.0 muM and 2.5 mM respectively. Human platelet I nsP3 3-kinase was not recognized by immunodetection with anti-(InsP3 3 -kinase A) or anti-(InsP3 3-kinase B) antibodies. These data provide t he first biochemical evidence for the existence of a novel InsP3 3-kin ase isoenzyme in human platelets, which is distinct from previously re ported InsP3 3-kinase A and InsP3 3-kinase B.