Purification and characterization of phosphatidylinositol kinase from Saccharomyces cerevisiae.
The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 8,000-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of microsomal membranes, DE-52 chromatography, hydroxylapatite chromatography, octyl-Sepharose chromatography, and two consecutive Mono Q chromatographies. The procedure resulted in the isolation of a protein with a subunit molecular weight of 35,000 that was 96% of homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidylinositol kinase activity was associated with the purified Mr 35,000 subunit. Maximum phosphatidylinositol kinase activity was dependent on magnesium ions and Triton X-100 at pH 8. The true Km values for phosphatidylinositol and MgATP were 70 microM and 0.3 mM, and the true Vmax was 4,750 nmol/min/mg. The turnover number for the enzyme was 166 min-1. Results of kinetic and isotopic exchange reactions indicated that phosphatidylinositol kinase catalyzed a sequential Bi Bi reaction mechanism. The enzyme bound to phosphatidylinositol prior to ATP and phosphatidylinositol 4-phosphate was the first product released in the reaction. The equilibrium constant for the reaction indicated that the reverse reaction was favored in vitro. The activation energy for the reaction was 31.5 kcal/mol, and the enzyme was thermally labile above 30 degrees C. Phosphatidylinositol kinase activity was inhibited by calcium ions and thioreactive agents. Various nucleotides including adenosine and S-adenosylhomocysteine did not affect phosphatidylinositol kinase activity.
Belunis
C J
CJ
Department of Food Science, Cook College, Rutgers University, New Brunswick, New Jersey 08903.
Bae-Lee
M
M
Kelley
M J
MJ
Carman
G M
GM
D019870:1-Phosphatidylinositol 4-Kinase; D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D017830:Octoxynol; D010716:Phosphatidylinositols / Q000378:metabolism; D010770:Phosphotransferases / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature
D010716:Phosphatidylinositols; D029701:Saccharomyces cerevisiae Proteins; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D017830:Octoxynol; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase; C441760:PIK1 protein, S cerevisiae; D008274:Magnesium; D002118:Calcium