Papadaki Amalia, Boleti Haralabia
Intracellular Parasitism Laboratory, Microbiology Department, Hellenic Pasteur Institute, Athens 11521, GREECE.
Light Microscopy Unit, Hellenic Pasteur Institute, Athens 11521, GREECE.
Bio Protoc. 2019 Oct 5;9(19):e3384. doi: 10.21769/BioProtoc.3384.
Acid ecto-phosphatases are enzymes that hydrolyze phosphomonoesters in the acidic pH range with their active sites facing the extacellular medium. Their activities can be measured in living cells. In bacteria and protozoan pathogens, acid ecto-phosphatases have been associated with the survival of intracellular pathogens within phagocytes through inhibition of the respiratory burst, suggesting that they act as virulence factors. Extracellular acid phosphatase activity in has been associated with the degree of promastigote virulence/infectivity. The levels of acid ecto-phosphatase activity in different sp or even strains of the same species vary and this has been linked to their virulence. It may also be related to their ability to survive and multiply in the insect host. Acid phosphatase enzymatic activity can be measured in crude membrane fractions and in membrane fractions enriched in plasma membrane, however, in these cases, the intracellular acid phosphatases, mainly localized in lysosomes, contribute to the final result. Therefore, measuring phosphatase activity at the surface of live cells in acidic pH range is the only accurate way to measure acid ecto-phosphatase activity. This assay is performed at 25 °C or 37 °C for 30 min using as substrate the generic phosphatase substrate p-nitrophenyl phosphate (NPP), in a citrate buffer, with or without sodium tartrate (L(+)-tartaric acid), as histidine acid phosphatases are classified according to their sensitivity to tartate inhibition. The steps of the protocol consist of pelleting cells in suspension, in this case promastigotes, washing twice with HEPES buffer, resuspending the cells in the substrate reaction mixture and terminating the reaction by the addition of 0.5 N NaOH. The cells are removed by centrifugation and the absorbance of the reaction product (-nitrophenolate=NP) in the supernatant is measured at 405 nm. The enzymatic activity (A values) is normalized for the mean number of cells/ml used for each independent experiment.
酸性外磷酸酶是一类酶，它们在酸性pH范围内水解磷酸单酯，其活性位点面向细胞外介质。它们的活性可以在活细胞中进行测量。在细菌和原生动物病原体中，酸性外磷酸酶通过抑制呼吸爆发与吞噬细胞内细胞内病原体的存活相关，这表明它们作为毒力因子发挥作用。[此处原文缺失具体物种名称]中的细胞外酸性磷酸酶活性与前鞭毛体的毒力/感染性程度相关。不同物种甚至同一物种的不同菌株中酸性外磷酸酶活性水平各不相同，这与它们的毒力有关。这也可能与它们在昆虫宿主中存活和繁殖的能力有关。酸性磷酸酶的酶活性可以在粗膜组分和富含质膜的膜组分中进行测量，然而，在这些情况下，主要定位于溶酶体的细胞内酸性磷酸酶会对最终结果产生影响。因此，在酸性pH范围内测量活细胞表面的磷酸酶活性是测量酸性外磷酸酶活性的唯一准确方法。该测定在25℃或37℃下进行30分钟，使用通用磷酸酶底物对硝基苯磷酸酯（NPP）作为底物，在柠檬酸盐缓冲液中，添加或不添加酒石酸钠（L(+)-酒石酸），因为组氨酸酸性磷酸酶根据它们对酒石酸盐抑制的敏感性进行分类。该实验方案的步骤包括将悬浮液中的细胞（在这种情况下是前鞭毛体）沉淀，用HEPES缓冲液洗涤两次，将细胞重悬于底物反应混合物中，并通过加入0.5N NaOH终止反应。通过离心去除细胞，并在405nm处测量上清液中反应产物（对硝基苯酚盐=NP）的吸光度。酶活性（A值）针对每个独立实验中使用的每毫升细胞平均数进行标准化。
