HYDROPHOBIC NATURE OF MAMMALIAN CERAMIDE GLYCANASES - PURIFIED FROM RABBIT AND RAT MAMMARY TISSUES

The ceramide glycanase (CGase) activities, which cleave the intact oli gosaccharide chain and the ceramide moiety of a glycosphingolipid, hav e been characterized from two mammalian sources. The enzymatic activit ies are almost comparable in rabbit and rat mammary tissues. The major ity of the activities has been concentrated in the soluble fraction wh ich could be partially purified using hydrophobic columns. The rabbit mammary ceramide glycanase activity has been purified up to 1438-fold using ion exchange and hydrophobic columns in tandem. The purified pro tein exhibited a molecular mass of 54 kDa which could be immunostained on the Western blot with clam anti-CGase polyclonal antibody. In addi tion, a 98 kDa protein also exhibited positive immunostain in a succes sive purified fraction with that antibody and is under investigation. The requirement for the optimal enzymatic activities are similar for b oth rabbit and rat CGase activities. The CGase activity requires the p resence of detergent for optimal activity but is not dependent on the presence of any divalent cations. However, Hg2+, Zn2+, and Cu2+ are in hibitory to the enzymatic activities. It has been observed that rat as well as rabbit CGases are inhibited by both D- and L-PDMP (1-phenyl-2 -deeanoylamino-3-morpholino-1 . HCl) and its higher analogue (1-phenyl -2-palmitoylamino-3-morpholino-1-propanol . HCl). Alkyl amines contain ing C-12 and higher chains are also found to inhibit both rat and rabb it CGase activities. Substantial levels of CGase activities have also been observed in various human tumor cells as well as in developing av ian brains. These observations are significant in view of the recent f indings that ceramide, which is one of the enzymatic reaction products of CGase activity, is mediating different cellular events like signal transduction and apoptosis. The role of this enzyme in development, m etastasis and cellular regulation are anticipated.