P.A. van Paridon

Binding of phospholipids to the phosphatidylinositol transfer protein from bovine brain as studied by steady-state and time-resolved fluorescence spectroscopy

The phosphatidylinositol transfer protein isolated from brain, liver, heart and platelets was found to be present in two subforms which could be distinguished on the basis of the isoelectric points. In this study we have demonstrated that the two subforms isolated from bovine brain are due to the presence of either phosphatidylinositol or phosphatidylcholine in the lipid binding site of the protein. The transfer protein accommodates one phosphatidylinositol molecule in the binding site. The binding site for the sn-2 fatty acyl chain was investigated by incorporating in the transfer protein either phosphatidylinositol or phosphatidylcholine carrying a parinaroyl-chain attached at the sn-2 position. Time-resolved fluorescence spectroscopy revealed that the sn-2 fatty acyl chains for both phospholipids in the lipid-protein complex were completely immobilized (i.e., rotational correlation times of 17.4 ns for phosphatidylcholine and 16.3 ns for phosphatidylinositol). The similarity in correlation times suggests that the sn-2 fatty acyl chains of both phospholipids are accommodated in the same hydrophobic binding site of the protein.

On the relationship between the dual specificity of the bovine brain phosphatidylinositol transfer protein and membrane phosphatidylinositol levels

The phosphatidylinositol transfer protein from bovine brain has a remarkable specificity pattern with a distinct preference for phosphatidylinositol (PI) and a low affinity for phosphatidylcholine (PC). In this study we have determined the affinity of PI-transfer protein for PI relative to that for PC by measuring the binding of the fluorescent pyrene-labeled analogs of these phospholipids. From competition binding experiments it was estimated that the transfer protein has a 16-fold higher affinity for PI than for PC. This relative affinity together with the relative abundance of PI and PC, determines what proportion of the protein contains PI (e.g. 65% of the PI-transfer protein in the case of bovine brain). From measuring lipid transfer between donor vesicles consisting of equimolar amounts of PC and PI, and an excess of acceptor vesicles consisting of various ratios of PC and PI, we have observed that the relative rates of the PI-transfer protein-mediated transfer of PI and PC varies between 5 and 20. Kinetic analysis has indicated that PI-transfer protein carrying a PI molecule has different kinetic properties than the PI-transfer protein carrying a PC molecule. It will be discussed that because of the dual specificity, PI-transfer protein is ideally suited for maintaining PI levels in intracellular membranes.

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.

Application of Fluorescent Phospholipid Analogues to Studies on Phospholipid Transfer Proteins

Fluorescence is one of the most widely used spectroscopic techniques in the research of various biological phenomena; owing to its sensitivity and specificity, it has solved problems that otherwise would have been extremely difficult to approach. Especially in membrane research, fluorescence spectroscopy has become very popular. A variety of probes have been used in this field (Lacowicz, 1983), and the fluorescent phospholipid analogues are among the most promising ones. They have been used to investigate phenomena such as lateral diffusion and dynamics of lipids (Galla and Hartman, 1980; Pugh et al., 1982; Jones and Lentz, 1986), lateral distribution of lipids (Welti and Silbert, 1982; Welti, 1982; Somerharju et al., 1985; Wiener et al.,1985; Hresko et al., 1987), transbilayer movement (Homan and Pownall, 1988), phase transitions (Sklar et al., 1977), and lipid-protein interactions (Jones and Lenz, 1986; Mustonen et al.,1987). Moreover, these fluorescent probes are very useful in studies of the spontaneous (Roseman and Thompson, 1980; Massey et al., 1982; Frank et al., 1983; Arvinte and Hildenbrand, 1984; Nichols, 1987, 1988) and protein-mediated (Somerharju et al.,1981, 1983, 1987; Somerharju and Wirtz, 1982; Nichols and Pagano, 1983; Massey et al., 1985; van Paridon et al.,1987a, 1988a) transfer of phospholipids.

Bradykinin-Induced Inositol Phosphate Metabolism in Human A431 Epidermoid Carcinoma Cells

Addition of the nonapeptide bradykinin to human A431 epidermoid carcinoma cells causes an immediate release of inositol phosphates and a rapid rise in [Ca2+]i. Half-maximal stimulation occurs at a bradykinin concentration of 4 nM. In the continuous presence of a saturating hormone concentration (1 μM) the inositol phosphate accumulation levels off within approximately 2 min; however, subsequent stimulation with histamine, another activator of phospholipase C, gives an additional increase in inositol phosphate production. Separation of the inositol phosphates by HPLC-anion exchange chromatography, reveals a rapid but transient accumulation of Ins(1,4,5)P3, followed by an increase in Ins (1,3,4,5)P4 and Ins(1,3,4)P3. Our data support a precursor/product-relationship between Ins(1,4,5)P3 and Ins(1,3,4)P3 with Ins(1,3,4,5)P4 as intermediate and argue against a messenger role of Ins(1,3,4,5)P4 and Ins(1,3,4)P3 in Ca2+ signalling.