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

Abstract

The phosphatidylinositoi 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 acyi chain was investigated by incorporating in the transfer protein either phosphatidylinositol or pbosphatidylcholine carrying a parinaroyi-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

“…This difference in affinity is caused by the interaction of the protein with the polar headgroup, since the accomodation of the acyl chains of both PI and PC on the protein is very similar (Ref. 22 and Van Paridon et al, manuscript submitted). Modification of the inositol ring by periodate oxidation and subsequent reduction by sodium borohydride, or phopshorylation at the 4-position, greatly reduced the transport by PI transfer protein [18,32].…”

“…65% of the protein was found to contain an endogenous PI molecule [22]. From the PI to PC ratio of 0.11 in a bovine brain homogenate (34) and from the Kra value of 15.6, one can calculate that 63% of the PI-transfer protein should contain an endogenous PI molecule.…”

“…Trinitrophenyl-phosphatidylethanolamine (TP-PE) was prepared from egg yolk PE and trinitrobenzene sulfonic acid [27]. PI-transfer protein was isolated from bovine brain [22], and stored in 50% glycerol at -20 o C.…”

“…PI-transfer protein has been shown to have a low affinity for PG and sphingomyelin [14,18,19], but no affinity for phosphatidic acid, phosphatidylethanolamine or phosphatidylserine [14,20]. The transfer protein acts as a soluble lipid-carrier, always containing one endogenous phospholipid molecule [18,21,22]. Because of its dual specificity, PI-transfer protein is able to deliver PI to a membrane with a low PI-content, in return for PC, resulting in a net transfer of PI [23].…”

Determination of the Stability of the Noncovalent Phospholipid Transfer Protein−Lipid Complex by Electrospray Time-of-Flight Mass Spectrometry

“…Increasing the voltage to 120 V showed that the dissociation between PG and PI-TPR, as apparent from the appearance The present measurements do not allow one to distinguish between these two residues, but do reveal that the noncovalent interaction between PG and PI-TPR in the gas phase is very strong and approaches that of a peptide bond. Since PI-TPR from mammalian cells carries either a PI or a PC molecule (5,27,28), we have analyzed these complexes as well. As shown in Figure 5, PI and PG dissociated from PI-TPR at a relatively high cv, whereas PC dissociated at a cv comparable to that observed for the PC/PC-TP complex.…”