title: Identification of the lipid-binding site of phosphatidylcholine-transfer protein with phosphatidylcholine analogs containing photoactivable carbene precursors.
abstract: The lipid binding site of the phosphatidylcholine transfer protein from bovine liver has been investigated by use of phosphatidylcholine analogs which carry a diazirinophenoxy group linked to the omega-carbon of either the sn-2-[1-14C]hexanoyl (PC I) or sn-2-[1-14C]undecanoyl chain (PC II). Photolysis of the PC I(PC II)-transfer protein complex resulted in a covalent coupling of 30-40% of the label to the protein as shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Upon mild alkaline treatment of the photolysed complex the protein containing covalently coupled 14C-label was separated from the noncoupled 14C-label by gel permeation chromatography. The 14C-labeled protein was degraded with protease from Staphylococcus aureus, trypsin and cyanogen bromide and specific 14C-labeled peptides were sequenced by automated Edman degradation. Major sites of coupling shown by release of radioactivity were identified as Tyr54 and the peptide segment Val171-Phe-Met-Tyr-Tyr-Phe-Asp177. Both PC I and PC II coupled extensively to Tyr54 (90% and 50% of total labeling, respectively). The remainder of the radioactivity was released from the peptide Val171-Asp177 with a distinct difference in in the pattern of release depending on whether PC I or PC II were used. Thus, coupling occurred preferentially to Tyr175 and Asp177 with PC I while Val171 and Met173 were labeled preferentially with PC II. This shift in coupling is compatible with an increase of 0.6 nm for the sn-2-fatty-acyl chains of PC I and II, assuming that the peptide Val171-Asp177 has adopted the strongly predicted beta-strand configuration. These data have been interpreted in terms of the localization of phosphatidylcholine in the phosphatidylcholine transfer protein.
mesh_terms: D000595:Amino Acid Sequence; D000727:Androgen-Binding Protein; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D006838:Hydrocarbons; D050356:Lipid Metabolism; D008099:Liver / Q000378:metabolism; D008697:Methane / Q000031:analogs & derivatives*; D010446:Peptide Fragments / Q000302:isolation & purification; D010713:Phosphatidylcholines / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D010777:Photochemistry; D013050:Spectrometry, Fluorescence
chemical_list: D000727:Androgen-Binding Protein; D002352:Carrier Proteins; D006838:Hydrocarbons; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D049294:Phospholipid Transfer Proteins; C030011:carbene; D008697:Methane