Identification and isolation of a 75-kDa inositol polyphosphate-5-phosphatase from human platelets

We have identified, isolated, and characterized a second inositol polyphosphate-5-phosphatase enzyme from the soluble fraction of human platelets. The enzyme hydrolyzes inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) to inositol 1,4-bisphosphate (Ins(1,4)P₂) with an apparent K(m) of 24 μM and a V(max) of 25 μmol of Ins(1,4,5)P₃ hydrolyzed/min/mg of protein. The enzyme hydrolyzes inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P₄) at a rate of 1.3 μmol of Ins(1,3,4,5)P₄ hydrolyzed/min/mg of protein with an apparent K(m) of 7.5 μM. The enzyme also hydrolyzes inositol 1,2-cyclic 4,5-trisphosphate (cIns(1:2,4,5)P₃) and Ins(4,5)P₂. We purified this enzyme 2,220-fold from human platelets. The enzyme has a molecular mass of 75,000 as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and be gel filtration chromatography. The enzyme requires magnesium ions for activity and is not inhibited by calcium ions. The 75-kDa inositol polyphosphate-5-phosphatase enzyme differs from the previously identified platelet inositol polyphosphate-5-phosphatase as follows: molecular size (75 kDa versus 45 kDa), affinity for Ins(1,3,4,5)P₄ (K(m) 7.5 μM versus 0.5 μM), K(m) for Ins(1,4,5)P₃ (24 μM versus 7.5 μM), regulation by protein kinase C, wherein the 45-kDa enzyme is phosphorylated and activated while the 75-kDa enzyme is not. The 75-kDa enzyme is inhibited by lower concentrations of phosphate (IC₅₀ 2 mM versus 16 mM for the 45-kDa enzyme) and is less inhibited by Ins(1,4)P₂ than is the 45-kDa enzyme. The levels of inositol phosphates that act in calcium signalling are likely to be regulated by the interplay of these two enzymes both found in the same cell.