CHARACTERIZATION OF ACYL BINDING BY A PLANT LIPID-TRANSFER PROTEIN

Maize lipid-transfer protein (LTP) is a small soluble protein which is able to transfer in vitro phospholipids between membranes and to bind fatty acids or lysoderivatives. In the studies reported here, fluores cent-labelled fatty acids were used to characterise the nature of the binding site on LTP. A fluorescent analogue of 12 carbons with a pyren e moiety attached at the end, alone or in conjunction with an anthroyl oxy analogue, indicated that LTP could bind two fatty acids although w ith a marked difference in affinity. The binding capacity was strongly affected after reduction of the protein by dithiothreitol, showing th at the four S-S bonds of LTP are essential for its lipid binding prope rty. Other analogues used were 16-carbon or 18-carbon fatty acids with an anthracene moiety attached at different points of the hydrocarbon chain. Emission maxima of these molecules varied with the analogue and suggested a motional constraint for the bound fatty acid which is mor e important around the middle of the chain than at its extremities. Bi nding displacement studies were carried out with a wide range of fatty acids or fatty acyl derivatives. Fatty acids of 16 to 19 carbons were found to be the preferred ligands. The presence of one double bond di d not change appreciably the affinity of LTP, although the presence of two or three double bonds or of a hydroxyl moiety significantly reduc ed the affinity. Fatty acyl-CoA or lysoderivatives bound as well as th e corresponding fatty acid.