Partial purification and characterization of beta-galactosidase from rat brain hydrolyzing glycosphingolipids.
Adult rat brain beta-galactosidase was partially purified with the use of lactosylceramide, galactosyl-N-acetylgalactosaminyl-galactosyl-glucosylceramide, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)galactosyl-glucosylceramide, and 4-methylumbelliferyl theta-galactoside as substrates. Approximately 50-fold purification was achieved by solubilization, ammonium sulfate fractionation, Sephadex G-200 and DEAE-cellulose column chromatography. Activities toward the above four substrates behaved essentially identically throughout the pruification procedure. Considerable differences were noted between the apparent properties determined with whole homogenate and those of the purer enzyme preparations. Based on these studies, assay procedures for the purified preparation for the three glycosphingolipid substrates were standardized. Inhibition studies with the use of varieties of simple sugars, oligosaccharide chains prepared from glycosphingolipids, and intact sphingolipids suggested that the enzyme which cleaves lactosylceramide may be different from the enzyme(s) which is active toward the other two glycosphingolipids. The oligosaccharide chains of the glycosphingolipids were much poorer inhibitors for the respective glycosphingolipid beta-galactosidases than the original intact glycosphingolipids or ceramide, and in some instances, even unrelated sphingolipids. These findings indicated the importance of the lipophilic groups and perhaps of the entire molecular configuration of glycosphingolipids in determining the specificity of these glycosphingolipid theta-galactosidases.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002518:Ceramides; D002845:Chromatography; D004355:Drug Stability; D005696:Galactosidases / Q000302:isolation & purification / Q000378:metabolism*; D006017:Glycolipids; D006358:Hot Temperature; D009439:Neuraminidase; D009844:Oligosaccharides / Q000032:analysis; D051381:Rats; D013107:Sphingolipids; D013329:Structure-Activity Relationship; D013656:Taurocholic Acid / Q000494:pharmacology
D002518:Ceramides; D006017:Glycolipids; D009844:Oligosaccharides; D013107:Sphingolipids; D013656:Taurocholic Acid; D005696:Galactosidases; D009439:Neuraminidase