RESUMO
The geometry of the channel formed by nontoxic derivative of diphtheria toxin CRM197 in lipid bilayer was determined using the dependence of single-channel conductance upon the hydrodynamic radii of different nonelectrolytes. It was found that the cis entrance of CRM197 channel on the side of membrane to which the toxoid was added at pH 4.8 and the trans entrance on the opposite side at pH 6.0 had effective radii of 3.90 and 3.48 Å, respectively. The 3-alkyloxycarbonylmethyl-5-(2-hydroxyethyl)-4-methyl-1,3-thiazolium salts reversibly reduced current via CRM197 channels. The potency of the blockers increased with increasing length of alkyl chain at symmetric pH 6.0 and remained high and stable at pH 4.8 on the cis side. Comparative analysis of CRM197 and amphotericin B pore size with the inhibitory action of thiazolium salts revealed a significant increase in CRM197 pore dimension at pH 6.0. Addition of thiazolium salt with nine carbons alkyl tail increased by â¼30% the viability of human carcinoma cells A431 treated with diphtheria toxin.
Assuntos
Canais Iônicos , Sais , Proteínas de Bactérias/metabolismo , Toxoide Diftérico , Humanos , Potenciais da MembranaRESUMO
Apoptosis signal-regulating kinase 1 (ASK1) is a mediator of the MAPK signaling cascade, which regulates different cellular processes including apoptosis, cell survival, and differentiation. The increased activity of ASK1 is associated with a number of human diseases and this protein kinase is considered as promising therapeutic target. In the present study, the kinase domain of human ASK1 was expressed in Escherichia coli (E. coli) in soluble form. The expression level of ASK1 was around 0.3-0.47 g per 1 L after using auto-induction protocol or IPTG induction. A one-step on column method for the efficient purification of recombinant ASK1 was performed. Our approach yields sufficient amount of recombinant ASK1, which can be used for inhibitor screening assays and different crystallographic studies.