Megli F M, van Loon D, Barbuti A A, Quagliariello E, Wirtz K W
Eur J Biochem. 1985 Jun 18;149(3):585-90. doi: 10.1111/j.1432-1033.1985.tb08965.x.
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.
通过用碘乙酰胺对蛋氨酸残基进行特异性修饰，研究了牛肝磷脂酰胆碱转移蛋白与磷脂囊泡相互作用中蛋氨酸残基的作用。用溴化氰消化修饰后的蛋白质，以确定哪些蛋氨酸残基对这种切割产生了抗性。对消化产物进行自动Edman降解表明，反应72小时后，Met-1有80%被修饰，Met-73有50%被修饰，Met-109有20%被修饰，而Met-173和Met-203未被修饰。这种明显的修饰并未导致磷脂酰胆碱转移活性的任何损失。利用电子自旋共振光谱研究了磷脂酰胆碱转移蛋白与磷脂囊泡的相互作用。未修饰的蛋白质与由磷脂酰胆碱/磷脂酸/自旋标记的磷脂酰乙醇胺（79:16:5，摩尔%）组成或由磷脂丝氨酸/自旋标记的磷脂酰乙醇胺（95:5，摩尔%）组成的囊泡相互作用，使旋转相关时间增加了约50%。修饰后的蛋白质也观察到类似的增加。通过用碘乙酰胺基丙氧基自旋标记对转移蛋白中的Met-1和Met-73进行标记，进一步研究了这种相互作用。自旋标记并未使转移蛋白失活。此外，加入由磷脂酰胆碱/磷脂酸（80:20，摩尔%）组成的囊泡后，自旋标记蛋白的电子自旋共振光谱不受影响。这些实验有力地表明，Met-1和Met-73不是与膜相互作用位点的一部分。
