Purification and characterization of D-myo-inositol (1,4,5)/(1,3,4,5)- polyphosphate 5-phosphatase from skeletal...

In this investigation we report the presence of two forms of inositol (1,4,5)P3/(1,3,4,5)P4-polyphosphate 5-phosphatase activity (types I and II) which were observed in soluble extracts of skeletal muscle after fractionation by DEAE-Sephacel chromatography. Hydrolysis of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and D-myo-inositol 1,3,4,5-tetrakisphosphate by both phosphatases was 5-phosphate-specific, Mg2+ ion-dependent and inhibited by D-2,3-bisphosphoglycerate. Soluble type I 5-phosphatase activity was purified 27,300-fold to a specific activity of 2.54 mumol of Ins(1,4,5)P3 hydrolyzed/min/mg protein after a combination of DEAE-Sephacel, Blue Sepharose, heparin-agarose and structural analogue affinity chromatography. Purified type I 5-phosphatase had an apparent mean Km of 8.9 and 1.1 microM and Vmax of 3.55 and 0.13 mumol of substrate hydrolyzed/min/mg protein for Ins(1,4,5)P3 and Ins(1,3,4,5)P4, respectively. Investigations on soluble type II 5-phosphatase after DEAE-Sephacel chromatography indicated an apparent Km of 71.4 microM Ins(1,4,5)P3 and an apparent molecular mass of 81 kDa. Soluble type I phosphatase has an apparent molecular mass of 48 kDa and an isoelectric point of 5.8. Soluble type I 5-phosphatase has kinetic constants which suggest a role in the regulation of inositol polyphosphates at physiological concentrations. These results support a role for Ins(1,4,5)P3 in the regulation of Ca2+ homeostasis in skeletal muscle.