PHOSPHATIDYLINOSITOL PHOSPHATE 5-KINASE - PURIFICATION AND INHIBITIONSTUDIES

A membrane-associated phosphatidylinositol phosphate 5-kinase has been purified approximately 110,000-fold from sheep brains. The purificati on procedure involves : sodium chloride (1M) extraction of the membran e, 20-40% ammonium sulfate fractionation, phosphocellulose (P-ll) chro matography, a second phosphocellulose chromatography, hydroxyapatite c hromatography, heparin Sepharose chromatography, HPLC SP(SO3 polymer)- cation exchange chromatography, and HPLC gelfiltration. The purified e nzyme exhibited a final specific activity of 1750 nmole/min/mg of prot ein. The molecular mass of the enzyme was estimated to be approximatel y 60 kDa by SDS-PAGE and 130 kDa by HPLC gel filtration. Kinetic measu rements showed that the apparent Km value of phosphatidylinositol phos phate 5-kinase for the utilization of ATP is 43 mu M. The 2'(3')-0-(2, 4,6-trinitrophenyl) derivative of ATP was found to be an inhibitor of the enzyme. The mode of inhibition is competitive, with a K-i value of 55 mu M.