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植物过氧化物还原蛋白BAS1是巯基依赖的过氧化物酶,通过催化的Cys残基还原过氧化氢,依赖NADPH的叶绿体硫氧还蛋白还原酶保持BAS1的还原态。玉米含有两种BAS1:2-CysPrxA和2-CysPrxB。利用RT-PCR方法从玉米幼叶中克隆了编码成熟2-CysPrxA的基因,并将蛋白Cys34残基突变成Ser34。SDS-PAGE显示纯化的野生型和突变体蛋白为一条主带,分子量约为23kDa;体外蛋白结合实验表明纯化的叶绿体硫氧还蛋白还原酶通过分子间二硫键结合纯化的2-CysPrxA的C34S突变体,非还原SDS-PAGE显示纯化的野生型2-CysPrxA含有分子间二硫键组成的二体,而纯化的C34S突变体呈现单体,巯基专一性标记化合物AMS修饰及活性分析表明纯化的BAS1还原态是催化还原过氧化氢所所必须的,它由硫氧还蛋白还原酶及其辅酶NADPH所催化。
The plant peroxiredoxin, BAS1, is a thiol-dependent peroxidase that reduces hydrogen peroxide through catalytic Cys residues, while NADPH-dependent chloroplast thioredoxin reductase retains the reduced state of BAS1. Corn contains two BAS1: 2-CysPrxA and 2-CysPrxB. The gene encoding mature 2-CysPrxA was cloned from young leaves of corn by RT-PCR and the protein Cys34 residue was mutated to Ser34. SDS-PAGE showed that the purified wild-type and mutant proteins were a main band with a molecular weight of about 23 kDa. In vitro protein binding experiments showed that the purified chloroplast thioredoxin reductase bound to purified C34S of 2-CysPrxA through intermolecular disulfide bond The mutant, non-reducing SDS-PAGE showed that the purified wild-type 2-CysPrxA contained intermolecular disulfide bonds, whereas the purified C34S mutant showed monomers. AMS modification and activity analysis of the sulfhydryl specific marker compound showed that purification The BAS1 reduced state is necessary for the catalytic reduction of hydrogen peroxide, which is catalyzed by thioredoxin reductase and its coenzyme NADPH.