Chemical modification of methionine residues of the phosphatidylcholine transfer protein from bovine liver. A spin-label study.
The role of methionine residues in the interaction of the phosphatidylcholine transfer protein from bovine liver with phospholipid vesicles was investigated by specific modification of these residues with iodoacetamide. The modified protein was digested with cyanogen bromide in order to determine which methionine residues had become resistant to this cleavage. Automated Edman degradation on the digest indicated that after 72 h of reaction, Met-1 was modified for 80%, Met-73 for 50%, Met-109 for 20%, whilst Met-173 and Met-203 were found to be unmodified. This distinct modification did not result in any loss of phosphatidylcholine transfer activity. The interaction of the phosphatidylcholine transfer protein with phospholipid vesicles was investigated by making use of electron spin resonance spectroscopy. The interaction of unmodified protein with vesicles composed of phosphatidylcholine/phosphatidic acid/spin-labeled phosphatidylethanolamine (79:16:5, mol%) or composed of phosphatidylserine/spin-labeled phosphatidylethanolamine (95:5, mol%), gave an increase of about 50% in the rotation correlation time. A similar increase was observed with the modified protein. This interaction was further investigated by labeling Met-1 and Met-73 in the transfer protein with iodoacetamidoproxyl spin-label. Spin-labeling did not inactivate the transfer protein. In addition, the electron spin resonance spectra of the spin-labeled protein were not affected upon addition of vesicles composed of phosphatidylcholine/phosphatidic acid (80:20, mol%). These experiments strongly suggest that Met-1 and Met-73 are not part of the site that interacts with the membrane.
Megli
F M
FM
van Loon
D
D
Barbuti
A A
AA
Quagliariello
E
E
Wirtz
K W
KW
D000727:Androgen-Binding Protein; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000032:analysis*; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D003488:Cyanogen Bromide; D004578:Electron Spin Resonance Spectroscopy; D005990:Glycerol; D007460:Iodoacetamide; D008099:Liver / Q000378:metabolism*; D008715:Methionine / Q000502:physiology*; D049294:Phospholipid Transfer Proteins; D013113:Spin Labels
D000727:Androgen-Binding Protein; D002352:Carrier Proteins; D049294:Phospholipid Transfer Proteins; D013113:Spin Labels; D008715:Methionine; D003488:Cyanogen Bromide; D005990:Glycerol; D007460:Iodoacetamide