Oomen R P, Kaplan H
Biochemistry. 1987 Jan 13;26(1):303-8. doi: 10.1021/bi00375a042.
The free monomeric form of insulin is known to adsorb strongly to many different surfaces. A question of physiological relevance for which no previous studies have been reported is whether the monomeric form of insulin binds to lipid bilayers. In order to answer this question, it is necessary to carry out studies at the very dilute concentrations (less than 10(-6) M) necessary to obtain this species. We have approached this problem by applying the method of competitive labeling [Hefford, M.A., Evans, R.M., Oda, G., & Kaplan, H. (1985) Biochemistry 24, 867-874] to study insulin at concentrations as low as 3 X 10(-8) M, in the presence and absence of large unilamellar liposomes. With 1-fluoro-2,4-dinitrobenzene as the labeling reagent, the relative chemical reactivities of the functional groups of insulin were found to decrease markedly when insulin was incubated with liposomes consisting of egg lecithin and cholesterol (2:1 mol/mol) in 1.0 M KCl, pH 7.5 at 37 degrees C. The decrease for each functional group was found to directly correlate with its proximity to the dimer-forming surface of the monomer. It is concluded that insulin binds to lipid bilayers in a specific orientation, with the dimer-forming surface interacting with the bilayer. These results demonstrate the feasibility of applying competitive labeling to obtain structure-function relationships of membrane-interactive proteins in general and monomeric insulin in particular.
已知胰岛素的游离单体形式能强烈吸附于许多不同表面。此前尚无相关研究报道的一个具有生理学意义的问题是，胰岛素单体形式是否会与脂质双层结合。为回答这个问题，有必要在获得该物种所需的极低浓度（低于10^(-6) M）下开展研究。我们通过应用竞争性标记法[赫福德，M.A.，埃文斯，R.M.，小田，G.，& 卡普兰，H.（1985年）《生物化学》24卷，867 - 874页]来解决这个问题，以研究浓度低至3×10^(-8) M的胰岛素，研究过程中存在和不存在大单层脂质体。以1 - 氟 - 2,4 - 二硝基苯作为标记试剂，发现在37℃、pH 7.5的1.0 M KCl溶液中，当胰岛素与由蛋黄卵磷脂和胆固醇（摩尔比2:1）组成的脂质体一起孵育时，胰岛素官能团的相对化学反应活性显著降低。发现每个官能团的降低与它靠近单体形成二聚体的表面直接相关。得出的结论是，胰岛素以特定方向与脂质双层结合，形成二聚体的表面与双层相互作用。这些结果证明了应用竞争性标记来获得一般膜相互作用蛋白特别是单体胰岛素的结构 - 功能关系的可行性。
