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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
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.
(comp.Biochem.Physiol.,115B,5411996,Biochim,Biophys.Acta 1997 impress) Less