Activation mechanism of sialidase by protease

酸性唾液酸酶通过在37 ° C下孵育而被激活。这种激活显示出时间和温度依赖性，在37 ° C下在4.3和5.2之间的pH范围内观察到最有效的激活。研究了不同蛋白酶抑制剂对其活性的影响。在测试的蛋白酶抑制剂中，氨肽酶A的抑制剂amastatin显著抑制活化。从人胎盘中分离氨肽酶。Sephadex G-200凝胶过滤的糖蛋白馏分分离的两个氨基肽酶。两种酶的最适pH值均为7，这与唾液酸酶的最适pH值（5.0）不同。人胎盘氨肽酶活性低，不稳定，纯化困难。我们还没有成功地建立酶的纯化和表征。为探讨其他因素对唾液酸酶活性的影响，研究了缓冲液、阳离子、阴离子和 关于我们 在所测试的各种因素中，卤素离子，尤其是氯离子，对唾液酸酶的激活有显著的促进作用。氯离子的激活表现出时间和浓度依赖性。Catepsine A是一种保护性蛋白，与溶酶体β-半乳糖苷酶（β-Gal）形成复合物，参与溶酶体唾液酸酶的稳定表达。我们研究了牛β-Gal复合物中catepsin A的酶性质以及它如何参与β-Gal和唾液酸酶复合物的分子多样性。牛保护蛋白与人保护蛋白同源，在SDS-PAGE上分子量为48 kDa，catepsin A活性最适pH为6.0。使用抗β-Gal抗体的免疫沉淀证明，catepsin A是唾液酸酶和β-Gal复合物的组分。超过700 kDa的唾液酸酶复合物通过在pH 7.5下的短暂接种而解聚，并且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地失活。669 kDa的β-Gal复合物可逆地解离成120 kDa的β-Gal和170 kDa的catepsin A。β-Gal和catepsin A活性在解离条件下都不稳定，表明它不仅在生理上稳定β-Gal，而且通过形成复合物本身稳定。鸡唾液酸酶也与β-Gal和catepsin A形成复合物。在pH 7.5时，复合物可逆地解离成120 kDa的β-Gal二聚体和100 kDa的catepsin A二聚体，但唾液酸酶在解聚过程中不可逆地失活。鸡唾液酸酶似乎以多酶复合物的形式存在，通过该复合物，唾液酸酶活性似乎是稳定的。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