De Pinto V, al Jamal J A, Benz R, Palmieri F
Dipartimento Farmaco-Biologico, Università di Bari, Italy.
FEBS Lett. 1990 Nov 12;274(1-2):122-6. doi: 10.1016/0014-5793(90)81345-o.
The role of positive charges located on the hydrophilic surface of the mitochondrial outer membrane channel was investigated by studying the interaction between LDAO-solubilized porin and a cation-exchanger column. The binding of porin to the column material was inhibited when the elution buffer had a pH of 9 or when 2 mM dextran sulfate was added to the buffer at neutral pH. Interestingly, the addition of a synthetic copolymer of methacrylate, maleate and styrene known as a potent modulator of the voltage-dependence, did not influence the interaction between column material and porin. Incubation of porin with fluorescein isothiocyanate (FITC) resulted in the isolation of a porin fraction in which on average two lysines located on the surface of the pore-forming complex per 35 kDa polypeptide were modified. The voltage-dependence of the fluorescein isothiocyanate modified porin was strongly decreased as compared with the unmodified porin. The experiments presented here give the first biochemical evidence that positively charged lysine residues located on the surface of the channel-forming complex are responsible for the gating of the mitochondrial porin-channel.
通过研究月桂酰二甲基胺乙内酯（LDAO）增溶的孔蛋白与阳离子交换柱之间的相互作用，对位于线粒体外膜通道亲水性表面的正电荷的作用进行了研究。当洗脱缓冲液的pH值为9时，或者在中性pH值的缓冲液中加入2 mM硫酸葡聚糖时，孔蛋白与柱材料的结合受到抑制。有趣的是，添加一种被称为电压依赖性强效调节剂的甲基丙烯酸酯、马来酸酯和苯乙烯的合成共聚物，并不影响柱材料与孔蛋白之间的相互作用。用异硫氰酸荧光素（FITC）孵育孔蛋白，得到了一个孔蛋白组分，其中每35 kDa多肽平均有两个位于成孔复合物表面的赖氨酸被修饰。与未修饰的孔蛋白相比，异硫氰酸荧光素修饰的孔蛋白的电压依赖性大大降低。此处给出的实验提供了首个生化证据，即位于通道形成复合物表面的带正电荷的赖氨酸残基负责线粒体孔蛋白通道的门控。
