The effect of polyphosphoinositides and phosphatidic acid on the phosphatidylinositol transfer protein from bovine brain: a kinetic study.

The phosphatidylinositol transfer protein from bovine brain (PI-TP) has lipid transfer characteristics which make it well suited to maintain phosphatidylinositol (PI) levels in intracellular membranes (Van Paridon, P.A., Gadella, Jr., T.W.J., Somerharju, P.J. and Wirtz, K.W.A. (1987) Biochim. Biophys. Acta 903, 68-77). Using a continuous fluorimetric transfer assay we have investigated in what way phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) affect the transfer activity of this protein in model systems. The effects were analysed by application of a kinetic model which yielded the association constant (K) and dissociation rate constant (k-) for the PI-TP/vesicle complex. Incorporation of PA, PIP and PIP2 into the phosphatidylcholine-containing vesicles increased the association constant solely by diminishing the dissociation rate constant. This effect could be completely accounted for by changes in the membrane surface charge density. In contrast to the inhibitory effect of PA, the inhibition caused by PIP2 was completely abolished by the addition of neomycin, in agreement with the observed preferential binding of this polyamine antibiotic to PIP2. A rise in pH from 5.5 to 8 drastically reduced the association constant for vesicles containing 16 mol% PA (e.g., from 38 to 2 mM-1), without affecting the Vmax. This effect could be mainly attributed to an increase in the negative charge on PI-TP (isoelectric point 5.5), resulting in an enhanced repulsion. Increasing the negative membrane surface charge at pH 7.4 had the opposite effect. This is interpreted to indicate that the membrane interaction site on PI-TP must be positively charged, overcoming the repulsive forces between PI-TP and the vesicle. Addition of PIP2 micelles as a third component in the transfer assay strongly inhibited PI-TP transfer activity. The extent of inhibition suggests a very high affinity of PI-TP for this lipid.