Iqbal Junaid, Panjwani Shamvil, Siddiqui Ruqaiyyah, Khan Naveed Ahmed
Department of Biological and Biomedical Sciences, Aga Khan University, Stadium Road, Karachi, Pakistan.
Parasitol Res. 2015 Feb;114(2):457-63. doi: 10.1007/s00436-014-4203-3. Epub 2014 Oct 31.
The deoxyribonuclease (DNase) activities of Acanthamoeba castellanii belonging to the T4 genotype were investigated. Using zymographic assays, the DNase activities had approximate molecular masses of 25 and 35 kDa. A. castellanii DNases exhibited activity at wide-ranging temperature of up to 60 °C and at pH ranging from 4 to 9. The DNases activities were unaffected by proteinase-K treatment, divalent cations such as Ca(++), Cu(++), Mg(++), and Zn(++), or divalent cation chelating agent ethylenediaminetetraacetic acid (EDTA) or sodium dodecyl sulfate (SDS). The non-reliance on divalent cations and homology data suggests that A. castellanii DNases belong to the class of eukaryotic lysosomal DNase II but exhibit robust properties. The DNases activity in A. castellanii interfered with the genomic DNA extraction. Extraction methods involving EDTA, SDS, and proteinase-K resulted in low yield of genomic DNA. On the other hand, these methods resulted in high yield of genomic DNA from human cells suggesting the robust nature of A. castellanii DNases that are unaffected by reagents normally used in blocking eukaryotic DNases. In contrast, the use of chaotropic agent such as guanidine thiocyanate improved the yield of genomic DNA from A. castellanii cells significantly. Further purification and characterization of Acanthamoeba DNases is needed to study their non-classic distinct properties and to determine their role in the biology, cellular differentiation, cell cycle progression, and arrest of Acanthamoeba.
对属于T4基因型的卡氏棘阿米巴的脱氧核糖核酸酶（DNase）活性进行了研究。使用酶谱分析，DNase活性的近似分子量为25 kDa和35 kDa。卡氏棘阿米巴DNase在高达60°C的广泛温度范围以及pH值为4至9的条件下均表现出活性。DNase活性不受蛋白酶K处理、二价阳离子如Ca(++)、Cu(++)、Mg(++)和Zn(++)、或二价阳离子螯合剂乙二胺四乙酸（EDTA）或十二烷基硫酸钠（SDS）的影响。对二价阳离子的不依赖性和同源性数据表明，卡氏棘阿米巴DNase属于真核溶酶体DNase II类，但具有强大的特性。卡氏棘阿米巴中的DNase活性干扰了基因组DNA的提取。涉及EDTA、SDS和蛋白酶K的提取方法导致基因组DNA产量较低。另一方面，这些方法从人类细胞中获得了高产的基因组DNA，这表明卡氏棘阿米巴DNase具有强大的性质，不受通常用于抑制真核DNase的试剂的影响。相比之下，使用诸如硫氰酸胍等离液剂可显著提高卡氏棘阿米巴细胞基因组DNA的产量。需要对棘阿米巴DNase进行进一步的纯化和表征，以研究其非经典的独特性质，并确定它们在棘阿米巴的生物学、细胞分化、细胞周期进程和停滞中的作用。
