Acid beta-galactosidase from human fibroblasts. A microscale purification method monitored by a highly sensitive enzyme assay.
A highly sensitive microassay method and a microscale purification system were developed to isolate the residual acid beta-galactosidase in GM1-gangliosidosis fibroblasts. The sensitivity of the microassay system, composed of a 96-well microplate and a microplate fluorometer, was 100-fold higher than that of the conventional system and the response was linear in the pmole range. Acid beta-galactosidase was characterized as a thiol enzyme which was inactivated by a mercuric compound. This enzyme was completely adsorbed on an Hg-agarose column and was easily eluted from the column by 10 mM 2-mercaptoethanol. The microscale purification system using Con A-Sepharose, PAT-Sepharose, and Hg-agarose column chromatography achieved 565- and 7,970-fold purifications of acid beta-galactosidase with an overall yields of 44% and 45% from normal and GM1-gangliosidosis fibroblasts, respectively. The purified enzyme fractions did not contain any other lysosomal enzyme activities except for a small amount of beta-N-acetylhexosaminidase activity.
D002847:Chromatography, Agarose; D005347:Fibroblasts / Q000201:enzymology*; D005677:G(M1) Ganglioside; D005696:Galactosidases / Q000302:isolation & purification*; D005733:Gangliosidoses / Q000201:enzymology; D006801:Humans; D008628:Mercury; D008832:Microchemistry; D001616:beta-Galactosidase / Q000302:isolation & purification*
D005677:G(M1) Ganglioside; D005696:Galactosidases; D001616:beta-Galactosidase; D008628:Mercury