Phosphoglycerate kinase (PGK) is a key enzyme of glycolysis which also acts as a mediator of DNA replication and repair in the nucleus. We have cloned and expressed PGK in Brugia malayi. The rBmPGK was found to be 415 amino acid residues long having 45 kDa subunit molecular weight. This enzyme was also identified in different life stages of bovine filarial parasite Setaria cervi. The enzyme activity was highest in microfilarial stage followed by adult female and male as also shown by real time PCR in the present study. Further using BmPGK primers the cDNA prepared from S. cervi was amplified and sequenced which showed 100% homology with Brugia malayi PGK. B. malayi and S. cervi, PGK consists of conserved calmodulin binding domain (CaMBD) having 21 amino acids. In the present study we have shown the CaMBD binds to calcium-calmodulin and regulates its activity. The binding of calmodulin (CaM) with CaMBD was confirmed using calmodulin agarose binding pull down assay, which showed that the rBmPGK binds to CaM agarose-calcium dependent manner. The effect of CaM-Ca2+on the activity of rBmPGK was studied at different concentration of CaM (0.01-5.0 μM) and calcium chloride (0.01-100 μM). The rBmPGK was activated up to 85% in the presence of CaM at 1 μM and 10 μM concentration of CaCl2. Interestingly this activation was abrogated by metal chelator EDTA. Similar results were shown in case of Setaria cervi PGK. A significant increase (90 ± 10) % in ScPGK activity was observed in the presence of CaM and CaCl2 at 1.0 μM and 1.0 mM respectively, further increase in the conc. of CaCl2, the activity of ScPGK was found to be decreased like rBmPGK. Bioinformatics studies have also confirmed the interaction between CaMBD and CaM which showed CaM interacted to Phe 206, Gln 220, Arg 223 and Asn 224 of rBmPGK CaM binding domain. On the basis of these findings, it has been suggested that the activity of filarial PGK could be regulated in cells by Ca2+-CaM depending upon the concentration of calcium. To the best of our knowledge this is first report in filarial parasite.

中文翻译：

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丝状磷酸甘油酸激酶的表征。

磷酸甘油酸激酶（PGK）是糖酵解的关键酶，也可作为细胞核内DNA复制和修复的介体。我们已经在马来亚布鲁加（Brugia malayi）中克隆并表达了PGK。发现rBmPGK是415个氨基酸残基，具有45kDa亚基分子量。在牛丝状寄生虫Setaria cervi的不同生命阶段也发现了这种酶。酶活性在微丝期最高，其次是成年雌性和雄性，如本研究中实时PCR所示。进一步使用BmPGK引物，扩增并测序了从宫颈葡萄球菌制备的cDNA，该cDNA显示出与马来亚布鲁吉氏PGK的100％同源性。B. malayi和S. cervi，PGK由具有21个氨基酸的保守钙调蛋白结合域（CaMBD）组成。在本研究中，我们显示了CaMBD与钙调钙蛋白结合并调节其活性。钙调蛋白琼脂糖结合下拉实验证实了钙调蛋白（CaM）与CaMBD的结合，这表明rBmPGK以CaM琼脂糖钙依赖性的方式结合。研究了不同浓度的CaM（0.01-5.0μM）和氯化钙（0.01-100μM）下CaM-Ca2 +对rBmPGK活性的影响。在存在1μMCaM和10μMCaCl2浓度的CaM的情况下，将rBmPGK激活至85％。有趣的是，这种活化被金属螯合剂EDTA废除了。Setaria cervi PGK的结果相似。在分别存在1.0μM和1.0 mM的CaM和CaCl2的情况下，观察到ScPGK活性显着提高（90±10）％，浓度进一步提高。的CaCl2，发现ScPGK的活性像rBmPGK一样降低。生物信息学研究还证实了CaMBD和CaM之间的相互作用，表明CaM与rBmPGK CaM结合域的Phe 206，Gln 220，Arg 223和Asn 224相互作用。基于这些发现，已经提出，取决于钙的浓度，Ca2 + -CaM可以调节细胞中丝状PGK的活性。据我们所知，这是丝虫寄生虫的首次报道。已经提出，取决于钙的浓度，Ca2 + -CaM可以调节细胞中丝状PGK的活性。据我们所知，这是丝虫寄生虫的首次报道。已经提出，取决于钙的浓度，Ca2 + -CaM可以调节细胞中丝状PGK的活性。据我们所知，这是丝虫寄生虫的首次报道。