PROCESS FOR ISOLATION AND PURIFICATION OF A NEW NEUTRAL SPHINGOMYELINASE DERIVED FROM MAMMAL CELL

摘要

PURPOSE: An active coenzyme solution having Mg¬2¬+ free neutral Sphingomyelinase is provided which is used as an activator of ceramide production. CONSTITUTION: 300 g of bovine brain and 1,500 g of homogenized buffer solution are mixed and homogenized by Polytron homogenizer. The homogenized solution is centrifuged at 40°C for 30 minutes, followed by mixing a culture fluid containing 2.5 micrometer £N-methyl-14C|SM. The culture solution contains 5 mM EDTA, 25 micrometer £N-methyl-14C|Sphingomyelin, 2mM sodium deoxycholate, and 100 mM tris-HCl buffer solution. The solution is cultured at 37°C for 30 minutes, followed by extraction of £N-methyl-14C|phosphocholine by Bligh and Dyer's method. The neutral Sphingomyelinase is useful as an active material to form ceramide.