Rabbit antiserum was raised against sialidase preparation and the antiserum precipitated sialidase activity effectively. By the screening of bovine brain cDNA library with the anti-sialidase antibody, six clones which react with the antibody was obtained. Now, nucleotide sequence analysis of the cDNA is in progress.
An acid sialidase, partially purified from human placenta, was activated by incubaton 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 The partially purified enzyme preparation contained aminopeptidase activity, which was inhibited by amastatin. Zinc ions inhibited either the activation of sialidase or the aminopeptidase activity in the enzyme preparatin. These results su
ggest the possibility of participation of aminopeptidase function in the activation process of sialidase.
It has been known that beta-gal exists as an enzyme complex with not only carboxypeptidase(CP) but also sialidase. CP protein is likely to be an essential factor to stabilize beta-gal as well as to express sialidase activity by forming the complex in lysosome. In order to clarify the function of CP,we have characterized CP in the purified beta-gal/CP complex from bovine liver. CP activity was optimum at pH 6 and activated by divalent cations, but strongly inhibited by DIFP,a serine protease inhibitor. By affinity labelling with ^3[H]-DIFP,the catalytic site of CP activity was demonstrated on the 30K protein. Two forms of beta-gal/CP complex(700K,100K)were separated by gelfiltration. Although 700K complex were de-polymerized to 110K one at pH 7, it was associated to 700K complex at pH 4.5. This conversion was observed even when CP activity was inactivated. 30K and/or 20K proteins, which are components of CP,may be necessary for forming high molecular weight beta-gal complex to stabilize beta-gal in lysosome, however, it seems that CP activity is not needed to form beta-gal/CP cpmlex.
In order to be clarified the active component of lysosomal sialidase complex, synthesis of the sialic acid derivatives which inhibits sialidase activity was tried. Among the newly synthesized compounds tested, aminophenyl or nitrophenyl thio sialosides significantly inhibited sialidase. Now, synthesis of the derivatives of sialic acid containing photoreactive functional groups is in progress. Less