Partial purification and characterization of particulate acid phosphatase of Leishmania donovani promastigotes.
Partial purification and characterization of particulate acid phosphatase of Leishmania donovani promastigotes.
复制标题
杜氏利什曼原虫前鞭毛体颗粒酸性磷酸酶的部分纯化和表征。
DOI：
10.1016/0305-0491(82)90510-7
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
D. Katsoulis
中科院分区：
文献类型：
--
作者：
R. Glew;M. Czuczman;W. Diven;R. L. Berens;M. Pope;D. Katsoulis
1. 超过90%的总酸性磷酸酶活性的超声多诺瓦氏L. promastigotes包含在一个颗粒部分（200,000 X g 30分钟）。通过DEAE-Sephadex、QAE-Sephadex、Sepharose 4B、concanavalin-A Sepharose等层析层析，该酶可被定量提取并溶解于Triton X-100 （0.2 g/100 ml）中，纯度超过200倍，得率达54%。磷酸酶是一种真正的酸性水解酶（pH最适，5.0-5.5），具有相当广泛的底物特异性；它能催化水解4-甲基伞形草酰磷酸、二磷酸胸腺酞、磷酸吡哆醛、果糖1,6 -二磷酸、葡萄糖6-磷酸、葡萄糖1-磷酸、ADP和AMP。它是一种大的（在Triton X-100存在下170,000道尔顿），稳定的酸性酶（pI= 4.1），在酸性（pH 4.3）聚丙烯酰胺凝胶中具有零型或1型同工酶的电泳迁移率。4. 该酶被氟化钠、2-巯基乙醇和一定摩尔量的多阴离子钼和重金属配合物抑制，这些配合物包括：[C (NH2) 3] 4 [(C3H7O3PO3) 2Mo5O15] X 3H2O、[C (NH2) 3] 2 [(C6H5) 2AsMo4O15H] X H2O、（NH4） 4 [SiMo12O40] X H2O和（NH4） 6 [P2Mo18O62] X 9H2O。5. L. donovani promastigotes含有非常低水平的其他10酸性pH最佳水解酶，除了适度水平的α -聚焦酶。
1. More than 90% of the total acid phosphatase activity in a sonicate of L. donovani promastigotes is contained in a particulate fraction (200,000 X g 30 min). The enzyme can be quantitatively extracted and solubilized with the aid of Triton X-100 (0.2 g/100 ml) and purified over 200-fold with 54% yield by chromatography on DEAE-Sephadex, QAE-Sephadex, Sepharose 4B and concanavalin-A Sepharose. 2. The phosphatase is a true acid hydrolase (pH optimum, 5.0-5.5) and has a rather broad substrate specificity; it will catalyze the hydrolysis of 4-methylumbelliferylphosphate, thymolphthalein diphosphate, pyridoxal phosphate, fructose 1, 6-diphosphate, glucose 6-phosphate, glucose 1-phosphate, ADP and AMP. 3. It is a large (170,000 daltons in the presence of Triton X-100), stable and acidic enzyme (pI= 4.1) that has the electrophoretic mobility of a type zero or type 1 isoenzyme in acid (pH 4.3) polyacrylamide gels. 4. The enzyme is inhibited by sodium fluoride, 2-mercaptoethanol and mumolar amounts of a number of polyanionic molybdenum and heavy metal complexes that include the following:[C (NH2) 3] 4 [(C3H7O3PO3) 2Mo5O15] X 3H2O,[C (NH2) 3] 2 [(C6H5) 2AsMo4O15H] X H2O,(NH4) 4 [SiMo12O40] X H2O and (NH4) 6 [P2Mo18O62] X 9H2O. 5. L. donovani promastigotes contain very low levels of 10 other acid pH optimum hydrolytic enzymes, with the exception of modest levels of alpha-fucosidase.