title: Isolation and characterization of ceramide glycanase from the leech, Macrobdella decora.
abstract: We have devised a simple method for achieving 890-fold purification of ceramide glycanase with 17% recovery from a North American leech, Macrobdella decora. The method includes water extraction, ammonium sulfate fractionation, and chromatography on octyl-Sepharose, Matrex gel blue A, and Bio-Gel A-0.5m columns. The final preparation showed one major protein band at 54 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By using Bio-Gel A-0.5m filtration, the native enzyme was found to have a molecular mass of 330 kDa. With GM1 as substrate, the optimum pH of this enzyme was determined to be 5.0; the enzyme was stable between pH 4.5 and 8.5. Zn2+ at 5 mM and Cu2+, Ag+, and Hg2+ at 1 mM strongly inhibited the hydrolysis of GM1 by ceramide glycanase. The ceramide glycanase released the intact glycan chain from various glycosphingolipids in which the glycan chain is linked to the ceramide through a beta-glucosyl linkage. This enzyme also cleaved lyso-glycosphingolipids such as lyso-GM1 and lyso-LacCer and synthetic alkyl beta-lactosides. Among seven alkyl beta-lactosides tested, the enzyme only hydrolyzed the ones with an alkyl chain length of four or more carbons. The enzyme also hydrolyzed 2-(octadecylthio)ethyl O-beta-lactoside and 2-(2-carbomethoxyethylthio)ethyl O-beta-lactoside. p-Nitrophenyl, benzyl, and phytyl beta-lactosides, on the other hand, were not hydrolyzed. These results suggest that the enzyme can recognize the hydrophobic portion of glycolipid substrates. The fact that 2-(2-carbomethoxyethylthio)ethyl O-beta-N-acetyllactosaminide and DiGalCer were refractory to the enzyme indicated that in the substrate the first sugar attached to the hydrophobic chain cannot be N-acetylglucosamine and galactose. Furthermore, dodecyl maltoside, Gal alpha 1----6Glc beta Cer, and the LacCer in which the --CH2OH of the galactose was converted into --CHO were also resistant to the enzyme, and Man beta 1----4 Glc beta Cer was hydrolyzed at a much slower rate than LacCer. These results indicate that the nature and the linkage of the sugar attached to the glucose have a profound effect on the action of this enzyme. The hydrolysis of glycosphingolipids by ceramide glycanase is stimulated by bile salts. Among various bile salts tested, sodium cholate at a concentration of 1 microgram/microliter was found to be most effective in stimulating the hydrolysis of various glycosphingolipids with the exception of LacCer. For LacCer, sodium taurodeoxycholate at a concentration of 2-3 micrograms/microliters was most effective. Tween 20, Nonidet P-40, and Triton X-100 did not stimulate the hydrolysis of GM1.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112.
authors.forename: B
authors.initials: B
authors.lastname: Zhou
authors.forename: S C
authors.initials: SC
authors.lastname: Li
authors.forename: R A
authors.initials: RA
authors.lastname: Laine
authors.forename: R T
authors.initials: RT
authors.lastname: Huang
authors.forename: Y T
authors.initials: YT
authors.lastname: Li
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D006028:Glycosphingolipids / Q000378:metabolism; D007202:Indicators and Reagents; D007700:Kinetics; D007865:Leeches / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity
chemical_list: D003902:Detergents; D006028:Glycosphingolipids; D007202:Indicators and Reagents; D006026:Glycoside Hydrolases; C051111:ceramide glycanase