RAT-BRAIN INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE - CA-2+-SENSITIVITY, PURIFICATION AND ANTIBODY-PRODUCTION

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase was purified from rat brain by Blue-Sepharose, phosphocellulose and calmodulin (CaM)-Sepharose affinity chromatography. The purified enzyme was stimulated by Ca2+-CaM by 3-6-fold as compared with the activity measured in the presence of EGTA. Rat brain InsP3 3-kinase activity was associated with two silver-stained bands of about equal activity which migrated with an apparent M(r) of 50,000 on SDS/polyacrylamide gels. InsP3 3-kinase activity from rat brain could be immunoprecipitated by an antiserum against the SDS/PAGE-purified 50,000-M(r) protein doublet. InsP3 kinase activity from bovine brain and the InsP3 5-phosphatase activity from rat brain were not immunoprecipitated. On Western blot, the human crude InsP3 3-kinase reacted specifically, but less strongly than the rat brain enzyme, with the antiserum.