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 approximate to 95 000 ) from the membranes of the electric organ of Torpedo californica was purif ied to apparent homogeneity. The Michaelis constant for ATP (K-M = 280 +/- 60 mu M at 20 degrees C) and the inhibition constant for adenosine (K-i=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 PtdIns/Triton X-100 micelles, whereas endogenou sly bound Ptdlns in the membrane fragments of electrocytes is a very poor s ubstrate. It is important that the enzyme and the substrate Ptdlns are situ ated in different lipid bilayers. The catalytic turnover constant for exoge nous Ptdlns is k = 55.3 +/- 6 min(-1) at 20 degrees C and the molar Triton X-100/PtdIns ratio of 16:1. For the substrate Ptdlns in the 'micellar solve nt' Triton X-100, steady state kinetics were analysed in terms of the mole fraction X = n(PtdIns)/[n(PtdIns)+n(Triton X)] yielding the characteristic Michaelis mole fraction X-M = 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, y ielding k = 277 +/- 30 min(-1) at 20 degrees C, Triton X-114 has a shorter head-group, indicating that the vicinity of the PtdIns head group in the mi xed micelles should not be screened by bulky neighbours. The inhibition of the enzyme activity by Ca2+ is highly cooperative yielding the Hill inhibit ion constant K-i=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 degrees C and 20 degrees C. Although the Ptdlns 4-kinase can be affinity-chromatographically copurified with the nicotinic acetylcholine (AcCho) receptor, suggesting t ight association between the two proteins. AcCho does not affect the activi ty of the PtdIns 4-kinase in the presence of the AcCho receptor.