Inhibition of porcine brain inositol 1,3,4-trisphosphate kinase by inositol polyphosphates, other polyol phosphates, polyanions and polycations

We have partially purified an enzyme activity that phosphorylates inositol 1,3,4-trisphosphate from porcine brain, rat liver and bovine testis by FPLC chromatography on Q-Sepharose anion-exchange resin and Heparin-agarose. The products of this reaction were inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate. The same enzyme appears to be responsible for both 6-kinase and 5-kinase activities against inositol 1,3,4-trisphosphate (the 6-kinase: 5-kinase activity ratio is approximately 4 to 1), has a pH optimum of ∼ 6.8 and requires Mg²⁺ for activity. The K_m values of the enzyme for inositol 1,3,4-trisphosphate and ATP were ∼ 0.5 μM and ∼ 100 μM, respectively. Inositol 3,4,5,6-tetrakisphosphate, inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate are all competitive inhibitors with K_i values of 0.4 μM, 3 μM and 5 μM, respectively, well within their likely intracellular concentration ranges: they inhibited 6-kinase and 5-kinase activities equally. 2,3-Bisphosphoglycerate and spermine were also competitive inhibitors, with K_i values of 0.8 mM and 12 mM, respectively. Dextran sulphate was a non-competitive inhibitor with a K_i of ∼ 15 μM, and poly-l-lysine (IC₅₀ ∼ 200 μM), polyvinylsulphate (IC₅₀ ∼ 250 μM) and heparin (IC₅₀ ∼ 2 mg/ml) also inhibited. Inhibition by these compounds suggests that inositol 3,4,5,6-tetrakisphosphate (and to a lesser extent inositol 1,3,4,5-tetrakisphosphate and other naturally occurring intracellular ions) may restrict the synthesis of inositol 1,3,4,6-tetrakisphosphate and hence regulate the rate of inositol penta- and hexakisphosphate synthesis from receptorgenerated inositol phosphates.