Activation mechanism of sialidase by protease

酸性唾液酸酶在37ﾟC孵育时被激活，这种激活具有时间和温度依赖性，在37ﾟC下，pH在4.3%到5.2%之间时，激活效果最好。考察了不同的蛋白水解酶抑制剂对其活性的影响。在被测试的蛋白酶抑制剂中，氨基肽酶A的抑制剂阿司他丁显著抑制激活。对人胎盘氨基肽酶的分离进行了研究。Sephadex G-200凝胶过滤糖蛋白组分，分离出两种氨基肽酶。两种酶的最适pH均为7，与唾液酸酶的最适pH(5.0)不同。人胎盘氨基肽酶活性低、稳定性差，纯化难度较大。我们还没有成功地建立酶的纯化和特性。为了探讨唾液酸酶激活的其他因素的可能性，缓冲液、阳离子、阴离子对…的影响在各种影响因素中，卤素离子，尤其是氯离子对唾液酸酶活性有明显的刺激作用。氯离子的激活具有时间和浓度依赖性。沿着这条路线的进一步工作现在正在进行中。猫氨酸A，也就是所谓的保护蛋白，作为一种与溶酶体β-半乳糖苷酶(β-Gal)的酶复合体存在，并参与溶酶体唾液酸酶的稳定酶表达。我们研究了牛β-半乳糖复合体中的儿茶素A的酶性质，以及它是如何参与β-半乳糖和唾液酸酶复合体的分子多样性的。牛保护性蛋白与人蛋白同源物在SDS-PAGE上的相对分子质量为48 kDa，儿茶素A活性最适pH为6.0。使用抗β-Gal抗体的免疫沉淀表明，儿茶素A是唾液酸酶和β-Gal复合体的组成部分。超过700 kDa的唾液酸酶复合体在pH 7.5下短暂解聚，唾液酸酶通过形成具有酶活性的较小种类的唾液酸酶而不可逆失活。669 kDaβ-Gal复合体可逆地解离为120 kDa的β-Gal和170 kDa的儿茶素A。加热和DIFP处理使儿茶素A失活，但不影响其形成活性。在解离条件下，β-Gal和儿茶素A的活性都不稳定，这表明它不仅在生理上稳定了β-Gal，而且通过形成复合体而稳定了自身。鸡唾液酸酶还与β-半乳糖和儿茶素A形成络合物。在pH 7.5时，该络合物可逆解离为120 kDa的β-半乳糖二聚体和100 kDa的儿茶素A二聚体，但解聚过程中唾液酸酶不可逆地失活。鸡唾液酸酶似乎是以多酶复合体的形式存在的，通过这种复合体可以稳定唾液酸酶的活性。较少

An acid sialidase was activated by incubation at 37ﾟC.This activation showed both time and temperature dependencies, with the most effective activation observed at 37ﾟC in the pH range between 4.3 and 5.2. The influence of various protease inhibitors on its activation was investigated. Among the protease inhibitors tested, amastatin, an inhibitor of aminopeptidase A,significantly inhibited activation. Isolation of the aminopeptidase from human placenta was examined. Sephadex G-200 gel filtration of the glycoprotein fraction separated the two aminopeptidases. Both enzymes had pH optimum of 7, which was different from the optimum pH (5.0) for activation of sialidase. It was very difficult to purify human placental aminipeptidase, because of its lability and low activity. We do not succeed yet to establish the purification and characterization of the enzymes. To inquire the possibility that other factor is responsible for activation of the sialidase, the effects of buffer, cation, anion a … More nd pH were investigated Among the verious factors tested, halogen ions, especially chloride ion, significantly stimulated the activation of sialidase. The activation by chloride ion showed both time and concentration dependencies. Further work along this line is now in progress.Catepsine A,so called protective protein, occurs as an enzyme complex with lysosomal beta-galactosidase (beta-Gal) and is involved in the stable enzyme exprssion of lysosomal sialidase. We investigated the enzymatic properties of catepsin A in the bovine beta-Gal complex and how it is involved in the molecular multiplicities of the beta-Gal and sialidase complexes. Bovine protective protein homologus to the human protein had a molecular weight of 48kDa on SDS-PAGE and catepsin A activity optimum about pH 6.0. Immunoprecipitation using an anti beta-Gal antibody demonstrated that catepsin A is a component of both the sialidase and beta-Gal complexes. The over 700kDa sialidase complex depolymerized by a brief inicubation at pH 7.5 and the sialidase was inactivated irreversibly via formation of an enzyme active smaller species of sialidase. The 669kDa beta-Gal complex dissociated reversibly into a 120kDa beta-Gal and a 170 kDa catepsin A.Inactivation of catepsin A By heat and DIFP treatment sdid not affect its complex forming activity. Both beta-Gal and catepsin A activities were labile under the dissociated condition, indicating that it physiologically stabilizes not only beta-Gal but also itself by forming the complex. Chicken sialidase also occurs as a complex with beta-Gal and catepsin A.The complex reversibly dissociated into 120kDa beta-Gal dimer and 100kDa catepsin A dimer at pH 7.5, but the sialidase irreversibly inactivated during the depolymerization. Chicken sialidase seems to exist as a multienzyme complex, by which the sialidase activity appears to be stabilized. Less