Phosphatidylinositol phosphate 5-kinase: purification and inhibition studies

¹Department of Applied Biology & Chemical Technology, Hong Kong Polytechnic University, Hung Hom, Hong Kong.

Preparative Biochemistry & Biotechnology

|February 1, 1996

Abstract:

A membrane-associated phosphatidylinositol phosphate 5-kinase has been purified approximately 110,000-fold from sheep brains. The purification procedure involves: sodium chloride (1M) extraction of the membrane, 20-40% ammonium sulfate fractionation, phosphocellulose (P-11) chromatography, a second phosphocellulose chromatography, hydroxyapatite chromatography, heparin Sepharose chromatography, HPLC SP(SO3- polymer)-cation exchange chromatography, and HPLC gel filtration. The purified enzyme exhibited a final specific activity of 1750 nmole/min/mg of protein. The molecular mass of the enzyme was estimated to be approximately 60 kDa by SDS-PAGE and 130 kDa by HPLC gel filtration. Kinetic measurements showed that the apparent Km value of phosphatidylinositol phosphate 5-kinase for the utilization of ATP is 43 microM. 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 Ki value of 55 microM.

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Abstract:

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