DETECTION AND PARTIAL-PURIFICATION OF INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE FROM PORCINE SKELETAL-MUSCLE

A myoplasmic 3-kinase was detected in porcine skeletal muscle that phosphorylated [H-3]Ins(1,4,5)P-3[D-myo-inositol(1,4,5)trisphosphate] to [H-3]Ins(1,3,3,5)P-4 [D-myo-inositol(1,3,4,5)tetrakisphosphate]. The Ins(1,4,5)P-3 3-kinase activity was ATP- and Mg2+-dependent, and was activated by Ca2+ and calmodulin. Ins(1,4,5)P-3 3-kinase activity was purified 2632-fold from soluble extracts of skeletal muscle by a combination of DEAE-Sephacel, heparin-Agarose and Ins(1,4,5)P-3 structural-analogue affinity chromatography. The highest specific activity obtained was 10.6 nmol of Ins(1,4,5)P-3 phosphorylated/min/mg protein. The partially purified enzyme had a mean K-m and V-max of 0.46 mu M and 3.15 nmol/min/mg protein for Ins(1,4,5)P-3 metabolism, respectively. After analytical gel filtration two forms of soluble Ins(1,4,5)P-3 3-kinase were observed with M(r) of 39,000 and 62,000. As in other cell types, muscle Ins(1,4,5)P-3 3-kinase was soluble, and had a higher affinity but a lower capa-city to metabolize Ins(1,4,5)P-3, in comparison to Ins(1,4,5)P-3 5-phosphatase.