Garza-Ramos G, Tuena de Gómez-Puyou M, Gómez-Puyou A, Gracy R W
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México.
Eur J Biochem. 1992 Sep 1;208(2):389-95. doi: 10.1111/j.1432-1033.1992.tb17199.x.
The reactivation of the homodimeric enzyme triosephosphate isomerase (TPI) was studied in reverse micelles. The enzyme was denatured in conventional aqueous mixtures with guanidine hydrochloride and transferred to reverse micelles formed with cetyltrimethylammonium bromide, hexanol, n-octane and water. In the transfer step, denatured TPI monomers distributed in single micelles, and guanidine hydrochloride was diluted more than 100 times. Under optimal reactivation conditions, 100% of the enzyme activity could be recovered. The rate of appearance of the catalytic activity increased with the concentration of protein, which indicated that catalysis required the formation of the dimer. The rate of TPI reactivation also increased with increasing protein concentration in the system with denatured TPI covalently derivatized at the catalytic site with the substrate analogue 3-chloroacetol phosphate. Thus, reactivation could take place via the formation of dimers composed of an inactive and an active subunit. Reactivation critically depended on the amount of water in the reverse micelles. The plot of the extent of reactivation versus the amount of water (2.5-7.0%) was markedly sigmoidal. Less than 20% reactivation took place with water concentrations below 3.5%, due to the formation (in less than 30 s) of stable inactive structures. The results indicate that reverse micelles provide a useful system to probe the events involved in the transformation of unfolded monomers to polymeric enzymes.
在反胶束中研究了同二聚体酶磷酸丙糖异构酶（TPI）的再活化。该酶在含有盐酸胍的传统水性混合物中变性，然后转移到由十六烷基三甲基溴化铵、己醇、正辛烷和水形成的反胶束中。在转移步骤中，变性的TPI单体分布在单个胶束中，盐酸胍被稀释了100多倍。在最佳再活化条件下，可恢复100%的酶活性。催化活性的出现速率随蛋白质浓度的增加而增加，这表明催化作用需要二聚体的形成。在催化位点用底物类似物3-氯乙酰磷酸共价衍生化的变性TPI体系中，TPI的再活化速率也随蛋白质浓度的增加而增加。因此，再活化可以通过由一个无活性亚基和一个活性亚基组成的二聚体的形成来实现。再活化关键取决于反胶束中的水量。再活化程度与水量（2.5 - 7.0%）的关系图呈明显的S形。当水浓度低于3.5%时，由于（在不到30秒内）形成了稳定的无活性结构，再活化率低于20%。结果表明，反胶束为探究未折叠单体向聚合酶转变过程中涉及的事件提供了一个有用的体系。
