GM3ガングリオシドシアリダーゼに関する研究

STUDIES ON GM3 GANGLIOSIDE SIALIDASE

　抄録　GM₃ガングリオシドの分解酵素であるGM₃ガングリオシドシアリダーゼの欠損がmucolipidosisIVで報告されている。この疾患の診断を目的として白血球でこの酵素測定法を検討した。

　白血球をデキストラン沈澱法で分離後ホモゲナイズして酵素溶液とした。GM₃ガングリオシドは牛副腎より精製し，トリチュームラベルして基質とした。本酵素の測定は，反応後，遊離する³H-lactosylceramideをTLCで分離し，シンチレーションカウンターで測定した。　

GM₃ ganglioside (hematoside) is the major gan-glioside of the extraneural tissues, the neural gan-gliosides being of longer carbohydrate chain length. However, this ganglioside was reported to be ac-cumulated in the nervous tissues of a patient withHurler's syndrome and Mucopolysaccharidosis III and sialidosis with partialβ-galactosidase deficiency. Recently, Bach et al. found storage of GM₃ and GD₃ gangliosides in mucolipidosis IV (ML-IV) and and this disorder as a deficiency of ganglioside sialidase.

Therefore, studies on the assay condition for GM₃ ganglioside sialidase and on the properties of this enzyme are required to clarify the mechanism of the nervous involvement in these disorders and to detect patients with ML-IV.

GM₃ ganglioside was purified from bovine adrenal medulla and tritium labelled in the sphingosine moiety.

The pH optimum was 4.8. The reaction was linear up to 8 hours. The amount of substrate hydrolyzed was proportional to the amount of the enzyme source. up to 80μg of leukocyte protein per tube. Among bile salts tested, only sodium taurocholate was an effective activator of GM₃ ganglioside sialidase. The optimal concentration of sodium taurocholate was 1.2mg/ml. The ap-parent km value for GM₃ ganglioside sialidase in leukocyte was 3.1×10^-5M.

Compared to sialyllactitol sialidase, GM₃ ganglio-side sialidase in leukocytes was more stable to heating, sonication or freezing and thawing.

On the basis of these findings, we devised the optimal assay condition for this enzyme in human leukocytes.