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首页> 外文期刊>Infection and immunity >Purification and Properties of the Cathepsin D Type Proteinase from Beef and Rabbit Lung and Its Identification in Macrophages
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Purification and Properties of the Cathepsin D Type Proteinase from Beef and Rabbit Lung and Its Identification in Macrophages

机译:牛和兔肺组织蛋白酶D型蛋白酶的纯化,性质及其在巨噬细胞中的鉴定

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The acid-acting proteinase, cathepsin D (EC 3.4.4.23), was purified from extracts of homogenized rabbit lung and beef lung by autolysis at acid pH, acetone and ammonium sulfate fractionation, column chromatography, and isoelectric focusing. Four isoenzymes were obtained from each source. With acid hemoglobin as the substrate, the proteinase from rabbit lung had a pH optimum of 3.0 and that from beef lung had a pH optimum of 3.6. Their activity was not affected by thiol reagents or by Fe2+, Mn2+, or Mg2+. One isoenzyme from rabbit lung was used to immunize a goat, and one from beef lung was used to immunize a rabbit. In immunoelectrophoresis, each resulting antiserum formed a single precipitin line with its homologous enzyme. They cross-reacted with the other three isoenzymes from the same species, but not with any isoenzyme from the other species. At high concentrations, each antiserum completely inhibited the proteolytic activity of its homologous enzyme. The antiserum against rabbit lung cathepsin D also inhibited the proteolytic activity of rabbit peritoneal and pulmonary macrophages. In limited quantities, this antiserum has now been made commercially available and is being used with labeled antibody techniques to identify under a microscope the presence of cathepsin D in macrophages and to study its role in the pathogenesis of tuberculosis.
机译:通过在酸性pH下自溶,丙酮和硫酸铵分级分离,柱色谱和等电聚焦,从均质的兔肺和牛肺的提取物中纯化出酸性作用蛋白酶组织蛋白酶D(EC 3.4.4.23)。从每种来源获得了四种同工酶。以酸性血红蛋白为底物,来自兔肺的蛋白酶的最适pH为3.0,而来自牛肺的蛋白酶的最适pH为3.6。它们的活性不受硫醇试剂或Fe 2 + ,Mn 2 + 或Mg 2 + 的影响。来自兔肺的一种同工酶用于免疫山羊,来自牛肺的一种同工酶用于免疫兔。在免疫电泳中,每个产生的抗血清与其同源酶形成一个沉淀素系。它们与同一物种的其他三种同工酶发生交叉反应,但与其他物种的任何同工酶均未发生交叉反应。在高浓度下,每种抗血清都完全抑制其同源酶的蛋白水解活性。抗兔肺组织蛋白酶D的抗血清也抑制了兔腹膜和肺巨噬细胞的蛋白水解活性。该抗血清现已限量出售,已与标记抗体技术一起用于在显微镜下鉴定巨噬细胞中组织蛋白酶D的存在并研究其在结核病发病机理中的作用。

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