Cardoso Carmen Lúcia, de Moraes Marcela Cristina, Guido Rafael Victorio Carvalho, Oliva Glaucius, Andricopulo Adriano Defini, Wainer Irving William, Cass Quezia Bezerra
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, São Paulo, Brazil.
Analyst. 2008 Jan;133(1):93-9. doi: 10.1039/b711145b. Epub 2007 Oct 8.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi, and an immobilized enzyme reactor (IMER) has been developed for use in the on-line screening for GAPDH inhibitors. An IMER containing human GAPDH has been previously reported; however, these conditions produced a T. cruzi GAPDH-IMER with poor activity and stability. The factors affecting the stability of the human and T. cruzi GAPDHs in the immobilization process and the influence of pH and buffer type on the stability and activity of the IMERs have been investigated. The resulting T. cruzi GAPDH-IMER was coupled to an analytical octyl column, which was used to achieve chromatographic separation of NAD(+) from NADH. The production of NADH stimulated by d-glyceraldehyde-3-phosphate was used to investigate the activity and kinetic parameters of the immobilized T. cruzi GAPDH. The Michaelis-Menten constant (K(m)) values determined for d-glyceraldehyde-3-phosphate and NAD(+) were K(m) = 0.5 +/- 0.05 mM and 0.648 +/- 0.08 mM, respectively, which were consistent with the values obtained using the non-immobilized enzyme.
甘油醛-3-磷酸脱氢酶（GAPDH）在克氏锥虫的生命周期中起着重要作用，并且已经开发出一种固定化酶反应器（IMER）用于在线筛选GAPDH抑制剂。先前已经报道了一种含有人类GAPDH的IMER；然而，这些条件产生的克氏锥虫GAPDH-IMER活性和稳定性较差。研究了固定化过程中影响人类和克氏锥虫GAPDH稳定性的因素以及pH和缓冲液类型对IMERs稳定性和活性的影响。所得的克氏锥虫GAPDH-IMER与分析型辛基柱相连，用于实现NAD(+)与NADH的色谱分离。由d-甘油醛-3-磷酸刺激产生的NADH用于研究固定化克氏锥虫GAPDH的活性和动力学参数。测定的d-甘油醛-3-磷酸和NAD(+)的米氏常数（K(m)）值分别为K(m) = 0.5 +/- 0.05 mM和0.648 +/- 0.08 mM，这与使用非固定化酶获得的值一致。
