Villa Héctor, Pérez-Pertejo Yolanda, García-Estrada Carlos, Reguera Rosa M, Requena José María, Tekwani Babu L, Balaña-Fouce Rafael, Ordóñez David
Departamento de Farmacología y Toxicología (INTOXCAL), Facultad de Veterinaria, Universidad de León, Spain.
Eur J Biochem. 2003 Nov;270(21):4339-47. doi: 10.1046/j.1432-1033.2003.03826.x.
ATP-regenerating enzymes may have an important role in maintaining ATP levels in mitochondria-like kinetoplast organelle and glycosomes in parasitic protozoa. Adenylate kinase (AK) (ATP:AMP phosphotransferase) catalyses the reversible transfer of the gamma-phosphate group from ATP to AMP, releasing two molecules of ADP. This study describes cloning and functional characterization of the gene encoding AK2 from a genomic library of Leishmania donovani and also its expression in leishmania promastigote cultures. AK2 was localized on an approximately 1.9-Mb chromosomal band as a single copy gene. L. donovani AK2 gene is expressed as a single 1.9-kb mRNA transcript that is developmentally regulated and accumulated during the early log phase. The overexpression of L. donovani AKgene in Escherichia coli yielded a 26-kDa polypeptide that could be refolded to a functional protein with AK activity. The recombinant protein was purified to apparent homogeneity. Kinetic analysis of purified L. donovani AK showed hyperbolic behaviour for both ATP and AMP, with Km values of 104 and 74 microM, respectively. The maximum enzyme activity (Vmax) was 0.18 micromol.min(-1).mg(-1) protein. P1,P5-(bis adenosine)-5'-pentaphosphate (Ap5A), the specific inhibitor of AK, competitively inhibited activity of the recombinant enzymes with estimated Ki values of 190 nM and 160 nM for ATP and AMP, respectively. Ap5A also inhibited the growth of L. donovani promastigotes in vitro which could be only partially reversed by the addition of ADP. Thus, presence of a highly regulated AK2, which may have role in maintenance of ADP/ATP levels in L. donovani, has been demonstrated.
ATP再生酶可能在维持寄生原生动物中线粒体样动基体细胞器和糖体中的ATP水平方面发挥重要作用。腺苷酸激酶（AK）（ATP：AMP磷酸转移酶）催化γ-磷酸基团从ATP可逆地转移到AMP，释放出两分子ADP。本研究描述了从杜氏利什曼原虫基因组文库中克隆编码AK2的基因及其功能特性，以及它在利什曼原虫前鞭毛体培养物中的表达。AK2作为单拷贝基因定位在大约1.9-Mb的染色体带上。杜氏利什曼原虫AK2基因表达为单一的1.9-kb mRNA转录本，其表达受发育调控并在对数早期积累。杜氏利什曼原虫AK基因在大肠杆菌中的过表达产生了一种26-kDa的多肽，该多肽可以重折叠成具有AK活性的功能蛋白。重组蛋白被纯化至表观均一性。对纯化的杜氏利什曼原虫AK的动力学分析表明，其对ATP和AMP均表现出双曲线行为，Km值分别为104和74μM。最大酶活性（Vmax）为0.18μmol·min⁻¹·mg⁻¹蛋白。AK的特异性抑制剂P1，P5-（双腺苷）-5'-五磷酸（Ap5A）竞争性抑制重组酶的活性，对ATP和AMP的估计Ki值分别为190 nM和160 nM。Ap5A也抑制杜氏利什曼原虫前鞭毛体在体外的生长，添加ADP只能部分逆转这种抑制作用。因此，已证明存在高度调控的AK2，其可能在维持杜氏利什曼原虫中的ADP/ATP水平方面发挥作用。
