Suchi R, Stern-Bach Y, Gabay T, Schuldiner S
Institute of Life Sciences, The Hebrew University of Jerusalem, Israel.
Biochemistry. 1991 Jul 2;30(26):6490-4. doi: 10.1021/bi00240a020.
N,N'-Dicyclohexylcarbodiimide (DCC) has been previously shown to inhibit the amine transporter from chromaffin granules [Gasnier, B., Scherman, D., & Henry, J.P. (1985) Biochemistry 24, 3660-3667]. A study of the mechanism of inhibition is presented together with the demonstration of covalent modification of the protein. DCC inhibits binding of R1 (reserpine) and R2 (tetrabenazine) types of ligands to the transporter as well as transport. Ligands of the R2 type, but not those of the R1 type, protect against inhibition of all the reactions by DCC, i.e., accumulation of serotonin, binding if reserpine (R1 ligand), and binding of ketanserine (R2 ligand). The ability of a given R2 ligand to protect the transporter correlates well with its binding constant. Water-soluble carbodiimides, such as 1-ethyl-3-[3-(diethylamino)propyl]carbodiimide (EDC), do not have any effect on the catalytic activity of the transporter. A fluorescent hydrophobic analogue of DCC, N-cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]carbodiimide (NCD-4), inhibits at about the same concentration range as DCC. [14C]DCC labels several polypeptides in the chromaffin granule membranes. Labeling of a polypeptide with an apparent Mr of 80K is inhibited in the presence of R2 ligands. The labeled polypeptide copurifies with the recently identified and isolated transporter [Stern-Bach, Y., Greenberg-Ofrath, N., Flechner, I., & Schuldiner, S. (1990) J. Biol. Chem. 256, 3961-3966].
N,N'-二环己基碳二亚胺（DCC）先前已被证明可抑制嗜铬颗粒中的胺转运体[加斯尼尔，B.，舍尔曼，D.，& 亨利，J.P.（1985年）《生物化学》24卷，3660 - 3667页]。本文介绍了对其抑制机制的研究以及蛋白质共价修饰的证明。DCC抑制R1（利血平）和R2（丁苯那嗪）类型配体与转运体的结合以及转运。R2类型的配体，而非R1类型的配体，可保护转运体免受DCC对所有反应的抑制，即5-羟色胺的积累、利血平（R1配体）的结合以及酮色林（R2配体）的结合。给定R2配体保护转运体的能力与其结合常数密切相关。水溶性碳二亚胺，如1-乙基-3-[3-(二乙氨基)丙基]碳二亚胺（EDC），对转运体的催化活性没有任何影响。DCC的一种荧光疏水类似物，N-环己基-N'-[4-(二甲基氨基)-α-萘基]碳二亚胺（NCD-4），在与DCC大致相同的浓度范围内具有抑制作用。[14C]DCC标记嗜铬颗粒膜中的几种多肽。在R2配体存在的情况下，一种表观分子量为80K的多肽的标记受到抑制。标记的多肽与最近鉴定和分离出的转运体[斯特恩 - 巴赫，Y.，格林伯格 - 奥弗拉特，N.，弗莱克纳，I.，& 舒尔迪纳，S.（1990年）《生物化学杂志》256卷，3961 - 3966页]共纯化。
