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 approximately 6.8 and requires Mg2+ for activity. The Km values of the enzyme for inositol 1,3,4-trisphosphate and ATP were approximately 0.5 microM and approximately 100 microM, 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 microM, 3 microM and 5 microM, 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 an 12 mM, respectively. Dextran sulphate was a non-competitive inhibitor with a Ki of approximately 15 microM, and poly-L-lysine (IC50 approximately 200 microM), polyvinylsulphate (IC50 approximately 250 microM) and heparin (IC50 approximately 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 receptor-generated inositol phosphates.
Hughes
P J
PJ
Centre for Clinical Research in Immunology and Signalling, University of Birmingham, UK.
Kirk
C J
CJ
Michell
R H
RH
D000818:Animals; D000838:Anions / Q000494:pharmacology; D001921:Brain / Q000201:enzymology*; D002412:Cations / Q000494:pharmacology; D002417:Cattle; D002850:Chromatography, Gel; D004795:Enzyme Stability; D007295:Inositol Phosphates / Q000096:biosynthesis / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D011073:Polyamines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013552:Swine; D013737:Testis / Q000201:enzymology
D000838:Anions; D002412:Cations; D007295:Inositol Phosphates; D011073:Polyamines; C081652:inositol-3,4,5,6-tetrakisphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C056875:myo-inositol-trisphosphate 6-kinase; D010744:Phosphoric Monoester Hydrolases; C053153:phosphatidylinositol-3,4-bisphosphate 4-phosphatase