Inhibition of the reconstituted mitochondrial oxoglutarate carrier by arginine-specific reagents
Inhibition of the reconstituted mitochondrial oxoglutarate carrier by arginine-specific reagents
复制标题
DOI：
10.1006/abbi.1996.0281
复制
发表时间：
1996-07-01
影响因子：
3.9
通讯作者：
Palmieri, F
中科院分区：
文献类型：
--
作者：
Stipani, I;Mangiullo, G;Palmieri, F
研究了精氨酸特异性试剂对纯化重组的氧戊二酸载体蛋白线粒体膜功能的影响。二羰基试剂2,3-丁二酮、2,3-戊二酮、2,3-和3,4-己二酮、1-苯基-1,2-丙二酮、苯乙二醛和苯乙二醛衍生物对氧戊二酸盐转运的抑制作用呈浓度依赖性，2,3-己二酮的IC50为0.05 mM，4-羟基-3-硝基苯乙二醛的IC50为0.08 mM, 2,3-戊二酮的IC50为0.17 mM。pH从6.0到8.0，抑制作用增强，说明反应基团的pK较高。已知分别与半胱氨酸残基和赖氨酸残基发生特异性可逆反应的Mersalyl和pyridoxal 5'-phosphate（或4,4'-二硝基二苯乙烯-2,2'-二磺酸盐）无法保护氧戊二酸载体免受-二羰基试剂的抑制。其他不与精氨酸残基发生反应的二酮类化合物对氧戊二酸运输活性没有显著影响。氧戊二酸和l -苹果酸可有效保护氧戊二酸载体免受精氨酸特异性试剂的失活；其他非载体底物的二羧酸没有保护作用。在半饱和的外部结合位点观察到50%的底物保护。这些结果表明，本研究中使用的精氨酸特异性试剂与氧戊二酸载体在精氨酸残基水平上相互作用，精氨酸残基对于底物的结合和/或易位至关重要，并且可能位于底物结合位点内或附近。(C) 1996学术出版社，Inc.
The effect of arginine-specific reagents on the function of the purified and reconstituted oxoglutarate carrier protein of the inner mitochondrial membrane has been investigated. The alpha-dicarbonyl reagents 2,3-butanedione, 2,3-pentanedione, 2,3- and 3,4-hexanedione, 1-phenyl-1,2-propanedione, phenylglyoxal, and phenylglyoxal derivatives caused a concentration-dependent inhibition of oxoglutarate transport with an IC50 of 0.05 mM for 2,3-hexanedione, 0.08 mM for 4-hydroxy-3-nitrophenylglyoxal, and 0.17 mM for 2,3-pentanedione. The inhibition increased with pH from 6.0 to 8.0, indicating that the pK of the reacting group(s) is rather high. Mersalyl and pyridoxal 5'-phosphate (or 4,4'-dinitrostilbene-2,2'-disulfonate), which are known to react specifically and reversibly with cysteine residues and lysine residues, respectively, were unable to protect the oxoglutarate carrier against inhibition by alpha-dicarbonyl reagents. Other diketone compounds, which do not react with arginine residues, had no significant effect on the oxoglutarate transport activity. Oxoglutarate and L-malate effectively protected the oxoglutarate carrier against inactivation caused by arginine-specific reagents; other dicarboxylates, which are not substrates of the carrier, had no protective effect. A 50% substrate protection was observed at half-saturation of the external binding site. These results indicate that the arginine-specific reagents used in this investigation interact with the oxoglutarate carrier at the level of an arginine residue(s), which is essential for binding and/or translocation of substrates and which may be localized in, or near, the substrate-binding site. (C) 1996 Academic Press, Inc.