title: Hydrophobic nature of mammalian ceramide glycanases: purified from rabbit and rat mammary tissues.
abstract: The ceramide glycanase (CGase) activities, which cleave the intact oligosaccharide chain and the ceramide moiety of a glycosphingolipid, have been characterized from two mammalian sources. The enzymatic activities are almost comparable in rabbit and rat mammary tissues. The majority of the activities has been concentrated in the soluble fraction which 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 protein exhibited a molecular mass of 54 kDa which could be immunostained on the Western blot with clam anti-CGase polyclonal antibody. In addition, a 98 kDa protein also exhibited positive immunostain in a successive purified fraction with that antibody and is under investigation. The requirement for the optimal enzymatic activities are similar for both rabbit and rat CGase activities. The CGase activity requires the presence of detergent for optimal activity but is not dependent on the presence of any divalent cations. However, Hg2+, Zn2+, and Cu2+ are inhibitory 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-decanoylamino-3-morpholino-1-propanol.HCl) and its higher analogue PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol.HCl). Alkyl amines containing C12 and higher chains are also found to inhibit both rat and rabbit CGase activities. Substantial levels of CGase activities have also been observed in various human tumor cells as well as in developing avian brains. These observations are significant in view of the recent findings 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, metastasis and cellular regulation are anticipated.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556, USA.
authors.forename: M
authors.initials: M
authors.lastname: Basu
authors.forename: S
authors.initials: S
authors.lastname: Dastgheib
authors.forename: M A
authors.initials: MA
authors.lastname: Girzadas
authors.forename: P H
authors.initials: PH
authors.lastname: O'Donnell
authors.forename: C W
authors.initials: CW
authors.lastname: Westervelt
authors.forename: Z
authors.initials: Z
authors.lastname: Li
authors.forename: J
authors.initials: J
authors.lastname: Inokuchi
authors.forename: S
authors.initials: S
authors.lastname: Basu
mesh_terms: D000818:Animals; D005260:Female; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008321:Mammary Glands, Animal / Q000201:enzymology*; D011817:Rabbits; D051381:Rats; D013379:Substrate Specificity
chemical_list: D006026:Glycoside Hydrolases; C051111:ceramide glycanase