SUBUNIT STRUCTURE, EXPRESSION, AND FUNCTION OF NAD(H)-SPECIFIC ISOCITRATE DEHYDROGENASE IN SACCHAROMYCES-CEREVISIAE
SUBUNIT STRUCTURE, EXPRESSION, AND FUNCTION OF NAD(H)-SPECIFIC ISOCITRATE DEHYDROGENASE IN SACCHAROMYCES-CEREVISIAE
复制标题
DOI：
10.1128/jb.172.8.4280-4287.1990
复制
发表时间：
1990-08-01
影响因子：
3.2
通讯作者：
MCALISTERHENN, L
中科院分区：
文献类型：
--
作者：
KEYS, DA;MCALISTERHENN, L
从酿酒酵母中纯化线粒体NAD(H)特异性异柠檬酸脱氢酶，分析其亚基结构和表达。该酶的两个亚基具有不同的分子量（39,000和40,000）和略有不同的等电点，通过变性电泳技术进行了分离。对纯化亚基的序列分析表明，这些多肽具有不同的氨基末端。利用兔体内制备的天然酶的抗血清，通过亲和纯化获得亚单位特异性免疫球蛋白G，表明亚单位在免疫化学上也是不同的。研究发现，在不同条件下生长的酵母细胞中，NAD(H)特异性异柠檬酸脱氢酶活性和免疫反应性水平与第二种三羧酸循环酶苹果酸脱氢酶的水平密切相关。通过筛选具有醋酸盐阴性生长表型的酵母突变体，鉴定出具有NAD(H)特异性异柠檬酸脱氢酶缺陷的酿酒酵母突变体。免疫化学分析表明，一个突变株缺乏40,000分子量亚基（IDH1），另一个突变株缺乏39,000分子量亚基（IDH2）。从IDH1和IDH2突变体中分离的线粒体显示出利用异柠檬酸盐或柠檬酸盐消耗呼吸氧气的能力明显降低。这证实了NAD(H)特异性异柠檬酸脱氢酶在三羧酸循环氧化功能中的重要作用。
Mitochondrial NAD(H)-specific isocitrate dehydrogenase was purified from Saccharomyces cerevisiae for analyses of subunit structure and expression. Two subunits of the enzyme with different molecular weights (39,000 and 40,000) and slightly different isoelectric points were resolved by denaturing electrophoretic techniques. Sequence analysis of the purified subunits showed that the polypeptides have different amino termini. By using an antiserum to the native enzyme prepared in rabbits, subunit-specific immunoglobulin G fractions were obtained by affinity purification, indicating that the subunits are also immunochemically distinct. The levels of NAD(H)-specific isocitrate dehydrogenase activity and immunoreactivity were found to correlate closely with those of a second tricarboxylic acid cycle enzyme, malate dehydrogenase, in yeast cells grown under a variety of conditions. S. cerevisiae mutants with defects in NAD(H)-specific isocitrate dehydrogenase were identified by screening a collection of yeast mutants with acetate-negative growth phenotypes. Immunochemical assays were used to demonstrate that one mutant strain lacks the 40,000-molecular-weight subunit (IDH1) and that a second strain lacks the 39,000-molecular-weight subunit (IDH2). Mitochondria isolated from the IDH1 and IDH2 mutants exhibited a markedly reduced capacity for utilization of either isocitrate or citrate for respiratory O2 consumption. This confirms an essential role for NAD(H)-specific isocitrate dehydrogenase in oxidative functions in the tricarboxylic acid cycle.