The binding of phosphatidylcholine to the phosphatidylcholine transfer protein: affinity and role in folding

Bovine liver phosphatidylcholine transfer protein (PC-TP) has been expresse d in Escherichia coli and purified to homogeneity from the cytosol fraction at a yield of 0.45 mg PC-TP per 10 mg total cytosolic protein. In addition , active PC-TP was obtained from inclusion bodies. An essential factor in t he activation of PC-TP was phosphatidylcholine (PC) present in the folding buffer. PC-TP from the cytosol contains phosphatidylethanolamine (PE) and p hosphatidylglycerol (PG) with a preference for the di-monounsaturated speci es over the saturated species as determined by fast atom bombardment mass s pectrometry (FAB-MS). By incubation with microsomal membranes the endogenou s PE and PG were replaced by PC. Relative to the microsomal PC species comp osition, PC-TP bound preferentially C16:0/C20:4-PC and C16:0/C18:2-PC (twof old enriched) whereas the major microsomal species C18:0/C18:1-PC and C18:0 /C18:2-PC were distinctly less bound. PC-TP is structurally homologous to t he lipid-binding domain of the steroidogenic acute regulatory protein (Nat. Struct. Biol. 7 (2000) 408). Replacement of Lys(55) present in one of the P-strands forming the lipid-binding site, with an isoleucine residue yielde d an inactive protein. This suggests that Lys(55) be involved in the bindin g of the PC molecule. (C) 2001 Elsevier Science Ireland Ltd. All rights res erved.