Functional analysis of human P5, a protein disulfide isomerase homologue
Functional analysis of human P5, a protein disulfide isomerase homologue
复制标题
DOI：
10.1093/oxfordjournals.jbchem.a003242
复制
发表时间：
2002-09-01
影响因子：
2.7
通讯作者：
Tsujimoto, Y
中科院分区：
文献类型：
--
作者：
Kikuchi, M;Doi, E;Tsujimoto, Y
人P5（hP5）在大肠杆菌pET系统中表达，并通过顺序Ni2+-螯合树脂柱层析纯化。纯化的hP5的表征表明它同时具有异构酶和分子伴侣活性，但两种活性均低于人蛋白二硫键异构酶(PDI)。此外，观察到hP5具有肽结合能力，并且其伴侣活性以硫氰酸酶和柠檬酸合酶为底物得到证实，但以D-甘油醛-3-磷酸脱氢酶为底物则没有得到证实，这表明hP5对于伴侣活性具有底物特异性。 hP5 中两个硫氧还蛋白相关基序的突变表明，第一个基序对于异构酶活性比第二个基序更重要，并且每个基序中的第一个半胱氨酸对于异构酶活性是必需的。由于缺乏异构酶活性的硫氧还蛋白基序突变体保留了底物柠檬酸合酶的伴侣活性，因此 hP5 的异构酶和伴侣活性可能是独立的，如 PDI 所示。
Human P5 (hP5) was expressed in the Escherichia coli pET system and purified by sequential Ni2+-chelating resin column chromatography. Characterization of purified hP5 indicated that it has both isomerase and chaperone activities, but both activities are lower than those of human protein disulfide isomerase (PDI). Moreover, hP5 was observed to have peptide-binding ability, and its chaperone activity was confirmed with rhodanese and citrate synthase as substrates, but not with D-glyceraldehyde-3-phosphate dehydrogenase, showing that hP5 has substrate specificity with respect to chaperone activity. Mutation of two thioredoxin-related motifs in hP5 revealed that the first motif is more important than the second for isomerase activity and that the first cysteine in each motif is necessary for isomerase activity. Since thioredoxin motif mutants lacking isomerase activity retain chaperone activity with the substrate citrate synthase, the isomerase and chaperone activities of hP5 are probably independent, as was shown for PDI.