Matsuda Y, Higashiyama S, Kijima Y, Suzuki K, Kawano K, Akiyama M, Kawata S, Tarui S, Deutsch H F, Taniguchi N
Department of Biochemistry, Osaka University Medical School, Japan.
Eur J Biochem. 1990 Dec 27;194(3):713-20. doi: 10.1111/j.1432-1033.1990.tb19461.x.
Manganese superoxide dismutase (Mn-SOD) has been purified with a high yield (320 mg) from human liver (2 kg) and crystallized. Low-angle laser light scattering of the enzyme has shown that native enzyme is a tetrametic form. Four of the eight cysteine residues in the tetramer reacted with 5,5'-dithiobis(2-nitrobenzoic acid) or with iodoacetamide. The others were only reactive in protein heated with SDS or urea after reduction with dithiothreitol or 2-mercaptoethanol. The reactive sulfhydryl group was found to be located at Cys196 by amino acid sequence analysis of Nbs2-reactive peptides isolated by activated thiol-Sepharose covalent chromatography. Incubation of Mn-SOD in 1% SDS for 2 or 3 days at 25 degrees C or 5 min at 100 degrees C gave material showing two prominent components on polyacrylamide gel electrophoresis in the presence of 0.1% SDS. The major component had a molecular mass of 23 kDa; the other, 25 kDa. Reduction of the protein by dithiothreitol or 2-mercaptoethanol heated in SDS produced only the 25-kDa monomer species. Essentially, no thiol groups were detected in the 23-kDa form, in which two cysteine residues appear to have been oxidized to form an intrasubunit disulfide. This indicates that Cys196 has a reactive sulfhydryl and appears to be a likely candidate for a mixed disulfide formation in vivo.
锰超氧化物歧化酶（Mn-SOD）已从2千克人肝脏中高产量（320毫克）纯化并结晶。该酶的低角度激光光散射表明天然酶为四聚体形式。四聚体中八个半胱氨酸残基中的四个与5,5'-二硫代双（2-硝基苯甲酸）或碘乙酰胺反应。其他残基只有在用二硫苏糖醇或2-巯基乙醇还原后，在经SDS或尿素加热的蛋白质中才具有反应活性。通过对经活化硫醇-琼脂糖共价色谱分离的Nbs2反应性肽段进行氨基酸序列分析，发现反应性巯基位于Cys196处。在25℃下将Mn-SOD在1% SDS中孵育2或3天，或在100℃下孵育5分钟，在含有0.1% SDS的聚丙烯酰胺凝胶电泳上得到显示两个主要条带的物质。主要条带的分子量为23 kDa；另一条带为25 kDa。在SDS中加热的蛋白质用二硫苏糖醇或2-巯基乙醇还原后仅产生25 kDa的单体形式。基本上，在23 kDa形式中未检测到巯基，其中两个半胱氨酸残基似乎已被氧化形成亚基内二硫键。这表明Cys196具有反应性巯基，似乎是体内形成混合二硫键的可能候选者。
