Phosphatidylinositol 4-kinase from Saccharomyces cerevisiae: Kinetic analysis using Triton X-100/phosphatidylinositol-mixed micelles

R. J. Buxeda, J. T. Nickels, C. J. Belunis, G. M. Carman

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Abstract

Phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified from Saccharomyces cerevisiae by an improved procedure over that previously reported (Belunis, C. J., Bae-Lee, M., Kelley, M. J., and Carman, G. M. (1988) J. Biol. Chem. 263, 18897-18903) for the enzyme. The molecular mass of the enzyme was 45 kDa. The 35-kDa protein previously identified as PI 4-kinase was a proteolysis product of the 45-kDa protein. A detailed kinetic analysis of the purified enzyme was performed with Triton X-100/phosphatidylinositol-mixed micelles according to the ''surface dilution'' (Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem. 250, 9013-9020) and ''dual phospholipid'' (Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5734-5739) kinetic models. Phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol at concentrations of phosphatidylinositol below 0.1 mM. Above 0.1 mM activity was only dependent on the surface concentration of phosphatidylinositol. The enzyme more closely followed the dual phospholipid model where the enzyme associated with Triton X-100 micelles when phosphatidylinositol was present. The interfacial Michaelis constant (K(m)(B)) for phosphatidylinositol was 0.0036 mol fraction and the dissociation constant (K(s)(A)) for phosphatidylinositol in the micelle surface was 0.26 mM. The results of glycerol gradient centrifugation studies showed that the enzyme was physically associated with Triton X-100/phosphatidylinositol micelles.

Original languageEnglish (US)
Pages (from-to)13859-13865
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number21
StatePublished - 1991

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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