Bimodal lipid substrate dependence of phosphatidylinositol kinase.
Phosphatidylinositol (PI) kinase activity was solubilized from rat liver microsomes and partially purified by chromatography on hydroxyapatite and Reactive Green 19-Superose. Examination of the ATP dependence using a mixed micellar assay gave a Km of 120 microM. The dependence of reaction rate on PI was more complicated. PI kinase bound a large amount of Triton X-100, and as expected for a micelle-associated enzyme utilizing a micelle-associated lipid substrate, the reaction rate was dependent on the micellar mole fraction, PI/(PI + Triton X-100), with a Km of 0.02 (unitless). Activity showed an additional dependence on bulk PI concentration at high micelle dilution. These results demonstrated two kinetically distinguishable steps leading to formation of a productive PI/enzyme(/ATP) complex. The rate of the first step, which probably represents exchange of PI from the bulk micellar pool into enzyme-containing micelles, depends on bulk PI concentration. The rate of the second step, association of PI with enzyme within a single micelle, depends on the micellar mole fraction of PI. Depression of the apparent Vmax at low ionic strength suggested that electrostatic repulsion between negatively charged PI/Triton X-100 mixed micelles inhibits PI exchange, consistent with a model in which intermicellar PI exchange depends on micellar collisions.
Ganong
B R
BR
Department of Biochemistry, University of Alabama, Birmingham 35294.
D019870:1-Phosphatidylinositol 4-Kinase; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D050356:Lipid Metabolism; D008862:Microsomes, Liver / Q000201:enzymology; D009994:Osmolar Concentration; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013379:Substrate Specificity
D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase