Purification and immunological quantification of rat liver lysosomal glycosidases.
Although lysosomal enzyme activities are known to vary in response to numerous physiological and pharmacological stimuli, the relationship between lysosomal enzyme activity and enzyme concentration has not been systematically studied. Therefore we developed radioimmunoassays for two lysosomal glycosidases in order to determine lysosomal enzyme concentration. beta-Galactosidase and beta-glucuronidase were purified from rat liver 2780-fold and 1280-fold respectively, by using differential centrifugation, affinity chromatography, ion-exchange chromatography and molecular-sieve chromatography. Polyclonal antibodies to these enzymes were raised in rabbits, and two radioimmunoassays were established. Antibody specificity was shown by: (i) selective immunoprecipitation of enzyme activity; (ii) identical bands of purified enzyme on SDS/polyacrylamide-gel electrophoresis and immunoelectrophoresis; (iii) single immunoreactive peaks in molecular-sieve chromatography experiments. Sensitivities of the assays were such that 15 ng of beta-galactosidase and 45 ng of beta-glucuronidase decreased the ratio of bound to free radiolabel by 50%; minimal detectable amounts of immunoreactive enzymes were 2 ng and 10 ng respectively. The assays were initially used to assess the effects of physiological perturbations (i.e. fasting and age) on enzyme concentrations in rat liver; these experiments showed that changes in enzyme concentrations do not always correlate with changes in enzyme activities. This represents the first report of radioimmunoassays for lysosomal glycosidases. The results suggest that these radioimmunoassays provide useful technology for the study of regulatory control mechanisms of the concentrations of lysosomal glycosidases in mammalian tissues.
de Groen
P C
PC
Gastroenterology Research Unit, Mayo Medical School, MN 55905.
LeSage
G D
GD
Tietz
P S
PS
LaRusso
N F
NF
D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D005696:Galactosidases / Q000302:isolation & purification*; D005966:Glucuronidase / Q000276:immunology / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity; D001616:beta-Galactosidase / Q000276:immunology / Q000302:isolation & purification*
D005696:Galactosidases; D001616:beta-Galactosidase; D005966:Glucuronidase