Phosphatidylinositol phosphate 5-kinase: purification and inhibition studies.
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.
Kwok
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F
Department of Applied Biology & Chemical Technology, Hong Kong Polytechnic University, Hung Hom, Hong Kong.
Humpage
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A
Cheng
C H
CH
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000645:Ammonium Sulfate; D000818:Animals; D001921:Brain / Q000201:enzymology*; D005591:Chemical Fractionation; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006493:Heparin; D007700:Kinetics; D008970:Molecular Weight; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000302:isolation & purification*; D012756:Sheep; D012965:Sodium Chloride
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D012965:Sodium Chloride; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D006493:Heparin; D017886:Durapatite; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D000645:Ammonium Sulfate