Phosphatidylinositol 4-kinase of Torpedo californica electrocytes: physico-chemical characterization and regulation by calcium and vicinal molecules of phosphatidylinositol.
A phosphatidylinositol 4-kinase (Ptdlns 4-kinase, M(r) approximately 95,000) from the membranes of the electric organ of Torpedo californica was purified to apparent homogeneity. The Michaelis constant for ATP (KM = 280 +/- 60 microM at 20 degrees C) and the inhibition constant for adenosine (Ki = 0.4 mM at 20 degrees C) qualify the electrocyte Ptdlns 4-kinase as a type III kinase. The Ptdlns 4-kinase phosphorylates preferentially exogenous Ptdlns, added in the form of mixed Ptdlns/Triton X-100 micelles, whereas endogenously bound Ptdlns in the membrane fragments of electrocytes is a very poor substrate. It is important that the enzyme and the substrate Ptdlns are situated in different lipid bilayers. The catalytic turnover constant for exogenous Ptdlns is k = 55.3 +/- 6 min-1 at 20 degrees C and the molar Triton X-100/Ptdlns ratio of 16:1. For the substrate Ptdlns in the 'micellar solvent' Triton X-100, steady state kinetics were analysed in terms of the mole fraction X = n(Ptdlns)/[n(Ptdlns) + n(Triton X)] yielding the characteristic Michaelis mole fraction XM = 0.019 +/- 0.005 at 20 degrees C. The activity of the enzyme was enhanced about 5-fold in the presence of Triton X-114, yielding k = 277 +/- 30 min-1 at 20 degrees C. Triton X-114 has a shorter head-group, indicating that the vicinity of the Ptdlns head group in the mixed micelles should not be screened by bulky neighbours. The inhibition of the enzyme activity by Ca2+ is highly cooperative yielding the Hill inhibition constant Ki = 0.47 +/- 0.1 mM and the Hill coefficient h = 3.6 +/- 0.5. The enthalpy of activation is 100 +/- 10 kJ/mol between 0 degree C and 20 degrees C. Although the Ptdlns 4-kinase can be affinity-chromatographically copurified with the nicotinic acetylcholine (AcCho) receptor, suggesting tight association between the two proteins. AcCho does not affect the activity of the Ptdlns 4-kinase in the presence of the AcCho receptor.
Katterle
B
B
Faculty of Chemistry, University of Bielefeld, Germany.
Westerteicher
P
P
Neumann
E
E
D019870:1-Phosphatidylinositol 4-Kinase / Q000302:isolation & purification / Q000378:metabolism*; D000241:Adenosine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003902:Detergents; D004789:Enzyme Activation; D008823:Micelles; D010716:Phosphatidylinositols / Q000378:metabolism; D014101:Torpedo
D003902:Detergents; D008823:Micelles; D010716:Phosphatidylinositols; D000255:Adenosine Triphosphate; D019870:1-Phosphatidylinositol 4-Kinase; D000241:Adenosine; D002118:Calcium