title: The low-affinity lipid binding site of the non-specific lipid transfer protein. Implications for its mode of action.
abstract: The non-specific lipid transfer protein (nsL-TP) from bovine liver was studied by using the following fluorescent lipid analogs: phosphatidylcholine species with a sn-2-pyrenylacyl-chain of different length [Pyr(x)PC], sn-2-pyrenyldecanoyl-labelled phosphatidylinositol [Pyr(10)PI], -phosphatidylinositol 4-phosphate [Pyr(10)PIP], -phosphatidylinositol 4,5-bisphosphate [Pyr(10)PIP2] and dehydroergosterol. These analogs provided information on the effect of hydrophobicity and charge on lipid binding and transfer by nsL-TP. Binding of the Pyr(x)PC species decreased with increasing sn-2 acyl-chain length. Under equilibrium conditions, the fraction of nsL-TP that carried a PC molecule did not exceed 8%, which is consistent with a low affinity binding site. Also nsL-TP-mediated transfer of the Pyr(x)PC species decreased with increasing sn-2 acyl-chain length and was highly correlated with spontaneous transfer. Binding of the phosphoinositides increased in the order Pyr(10)PI less than Pyr(10)PIP less than Pyr(10)PIP2, indicating that an increase in lipid negative charge stimulates binding. The transfer of the phosphoinositides, however, decreased in the same order, which suggests that a high negative charge impairs the dissociation of the phospholipid from nsL-TP. Cholesterol, at concentrations up to 50 mol% in the donor membrane, hardly affected binding and transfer of Pyr(6)PC, strongly suggesting that nsL-TP has no high binding affinity for cholesterol. In agreement with this, binding of dehydroergosterol to nsL-TP was not detectable. Despite this apparently negligible affinity, nsL-TP-mediated transfer of dehydroergosterol was in the same order as that of Pyr(6)PC. The results are interpreted to indicate that transfer of lipids by nsL-TP involves the formation of a putative low-affinity lipid-protein complex. This formation is enhanced when lipid hydrophobicity decreases or lipid negative charge increases. Based on the binding and transfer data, the mode of action of nsL-TP is discussed in terms of change in free energy.
authors.affiliation: Center for Biomembranes and Lipid Enzymology (CBLE), State University of Utrecht, The Netherlands.
authors.forename: T W
authors.initials: TW
authors.lastname: Gadella
authors.forename: K W
authors.initials: KW
authors.lastname: Wirtz
mesh_terms: D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D004875:Ergosterol / Q000031:analogs & derivatives / Q000378:metabolism; D005456:Fluorescent Dyes; D050356:Lipid Metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010940:Plant Proteins; D013261:Sterols / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D005456:Fluorescent Dyes; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D010940:Plant Proteins; D013261:Sterols; C072087:sterol carrier proteins; C010905:dehydroergosterol; D002784:Cholesterol; D004875:Ergosterol