Purification and kinetic properties of a membrane-bound phosphatidylinositol kinase of the bovine adrenal medulla.
Phosphatidylinositol (PI) kinase (EC 2.7.1.67), an integral membrane protein of chromaffin granule ghosts of the bovine adrenal medulla, was found to phosphorylate PI in the 4-position of the inositol ring. The PI kinase was purified about 200-fold from a membrane fraction containing chromaffin granules and microsomes by extraction with Triton X-114, followed by phase partition (clouding) and heparin Sepharose chromatography. The PI kinase preparation (specific activity of 5.1 nmol PIP/mg protein per min) was free from other enzymatic activities that metabolize polyphosphoinositides. Km values of 55 microM and 40 microM for ATP and PI, respectively, were estimated for the purified enzyme. Concentrations of Triton X-100 above the critical micellar concentration (0.01%, w/v) were necessary to support significant enzyme activity, which was optimal at about 0.1% (w/v). Its dependence of pH was similar to that of the membrane-bound enzyme, with a broad optimum around pH 7. Mes in the millimolar concentration range was found to strongly inhibit the activity of the purified PI kinase (I50 at about 4 mM). The enzyme was almost totally inhibited by low micromolar concentrations of free calcium, and stimulated by hydrophilic cations, e.g., Mg2+ and poly(L-lysine), with the same potencies as for the membrane-bound enzyme. The amphiphilic cation trifluoperazine, however, stimulated the activity of purified PI kinase less effectively than the membrane-bound enzyme (Husebye, E.S. and Flatmark, T. (1988) Biochem. Pharmacol. 37, 449-456), whereas the inhibitory effect of near millimolar concentrations of trifluoperazine was the same for the two forms of the enzyme. It is concluded that the membrane-bound PI kinase of this tissue is of type II according to the classification of Cantley and co-workers (Whitman et al. (1987) Biochem. J. 247, 165-174).
Husebye
E S
ES
Department of Biochemistry, University of Bergen, Norway.
Letcher
A J
AJ
Lander
D J
DJ
Flatmark
T
T
D019870:1-Phosphatidylinositol 4-Kinase; D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D002118:Calcium; D002412:Cations; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology*; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride; D010743:Phospholipids / Q000302:isolation & purification; D010770:Phosphotransferases / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000201:enzymology
D002412:Cations; D003902:Detergents; D010743:Phospholipids; D015636:Magnesium Chloride; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase; D002118:Calcium