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. 1989 Nov 15;264(1):115–123. doi: 10.1042/bj2640115

Purification and immunological quantification of rat liver lysosomal glycosidases.

P C de Groen 1, G D LeSage 1, P S Tietz 1, N F LaRusso 1
PMCID: PMC1133554  PMID: 2513804

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alpers D. H. Separation and isolation of rat and human intestinal beta-galactosidases. J Biol Chem. 1969 Mar 10;244(5):1238–1246. [PubMed] [Google Scholar]
  2. Berger F. G., Lusis A. J. Relationship between genetic variation in thermal stability and electrophoretic mobility of mouse beta-galactosidase. Biochem Genet. 1978 Oct;16(9-10):1007–1014. doi: 10.1007/BF00483750. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Chisolm G. M., 3rd, Sila C. A., Hmiel S. P. Measurements of the degradation products of radioiodinated proteins. Anal Biochem. 1981 Mar 1;111(2):212–219. doi: 10.1016/0003-2697(81)90556-x. [DOI] [PubMed] [Google Scholar]
  6. D'Azzo A., Hoogeveen A., Reuser A. J., Robinson D., Galjaard H. Molecular defect in combined beta-galactosidase and neuraminidase deficiency in man. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4535–4539. doi: 10.1073/pnas.79.15.4535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dean R. T. Rabbit beta-glucuronidase. Purification and properties, and the existence of multiple forms. Biochem J. 1974 Mar;138(3):395–405. doi: 10.1042/bj1380395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Diment S., Dean M. F. Receptor-mediated endocytosis of fibroblast beta-glucuronidase by peritoneal macrophages. Biochim Biophys Acta. 1983 Apr 5;762(2):165–174. doi: 10.1016/0167-4889(83)90068-x. [DOI] [PubMed] [Google Scholar]
  9. Frost R. G., Holmes E. W., Norden A. G., O'Brien J. S. Characterization of purified human liver acid beta-D-galactosidases A2 and A3. Biochem J. 1978 Oct 1;175(1):181–188. doi: 10.1042/bj1750181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. GREENWOOD F. C., HUNTER W. M., GLOVER J. S. THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. Biochem J. 1963 Oct;89:114–123. doi: 10.1042/bj0890114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Galjart N. J., Gillemans N., Harris A., van der Horst G. T., Verheijen F. W., Galjaard H., d'Azzo A. Expression of cDNA encoding the human "protective protein" associated with lysosomal beta-galactosidase and neuraminidase: homology to yeast proteases. Cell. 1988 Sep 9;54(6):755–764. doi: 10.1016/s0092-8674(88)90999-3. [DOI] [PubMed] [Google Scholar]
  12. Hansen J. B., Veneziale C. M. Intracellular concentration of skeletal and cardiac muscle phosphofructokinase in diabetic and normal animals. J Lab Clin Med. 1980 Jan;95(1):133–143. [PubMed] [Google Scholar]
  13. Haven M. C., Orsulak P. J., Arnold L. L., Crowley G. Data-reduction methods for immunoradiometric assays of thyrotropin compared. Clin Chem. 1987 Jul;33(7):1207–1210. [PubMed] [Google Scholar]
  14. Himeno Mnishimura Y., Tsuji H., Kato K. Purification and characterization of microsomal and lysosomal beta-glucuronidase from rat liver by use of immunoaffinity chromatography. Eur J Biochem. 1976 Nov 15;70(2):349–359. doi: 10.1111/j.1432-1033.1976.tb11024.x. [DOI] [PubMed] [Google Scholar]
  15. Himeno M., Nishimura Y., Takahashi K., Kato K. The synthesis of rat liver lysosomes. III. Chemical composition of microsomal and lysosomal beta-glucuronidases purified from rat liver. J Biochem. 1978 Feb;83(2):511–518. doi: 10.1093/oxfordjournals.jbchem.a131938. [DOI] [PubMed] [Google Scholar]
  16. Holmes E. W., O'Brien J. S. Purification and properties of acid beta-galactosidase from feline liver. Biochemistry. 1979 Mar 20;18(6):952–958. doi: 10.1021/bi00573a003. [DOI] [PubMed] [Google Scholar]
  17. Johnson M. L., Veneziale C. M. Hormonal regulation of liver pyruvate kinase concentration and activity. Biochemistry. 1980 May 13;19(10):2191–2195. doi: 10.1021/bi00551a030. [DOI] [PubMed] [Google Scholar]
  18. LaRusso N. F., Fowler S. Coordinate secretion of acid hydrolases in rat bile. J Clin Invest. 1979 Oct;64(4):948–954. doi: 10.1172/JCI109561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lusis A. J., Paigen K. Relationships between levels of membrane-bound glucuronidase and the associated protein egasyn in mouse tissues. J Cell Biol. 1977 Jun;73(3):728–735. doi: 10.1083/jcb.73.3.728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lusis A. J., Paigen K. The large scale isolation of mouse beta-glucuronidase and comparison of allozymes. J Biol Chem. 1978 Oct 25;253(20):7336–7345. [PubMed] [Google Scholar]
  22. Miller A. L., Frost R. G., O'Brien J. S. Purified human liver acid beta-D-galactosidases possessing activity towards G(M1)-ganglioside and lactosylceramide. Biochem J. 1977 Sep 1;165(3):591–594. doi: 10.1042/bj1650591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Novikoff P. M., La Russo N. F., Novikoff A. B., Stockert R. J., Yam A., Le Sage G. D. Immunocytochemical localization of lysosomal beta-galactosidase in rat liver. J Cell Biol. 1983 Nov;97(5 Pt 1):1559–1565. doi: 10.1083/jcb.97.5.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ohishi I., Shioya A. Rabbit liver beta-glucuronidase. II. Immunological properties and examination of some species difference. Jpn J Pharmacol. 1971 Aug;21(4):551–556. [PubMed] [Google Scholar]
  25. Plapp B. V., Cole R. D. Purification and characterization of bovine liver beta-glucuronidase. Arch Biochem Biophys. 1966 Sep 26;116(1):193–206. doi: 10.1016/0003-9861(66)90027-0. [DOI] [PubMed] [Google Scholar]
  26. Skudlarek M. D., Swank R. T. Biosynthesis of two lysosomal enzymes in macrophages. Evidence for a precursor of beta-galactosidase. J Biol Chem. 1979 Oct 25;254(20):9939–9942. [PubMed] [Google Scholar]
  27. Stahl P. D., Touster O. Beta-glucuronidase of rat liver lysosomes. Purification, properties, subunits. J Biol Chem. 1971 Sep 10;246(17):5398–5406. [PubMed] [Google Scholar]
  28. Tomino S., Meisler M. Biochemical and immunological studies of purified mouse beta-galactosidase. J Biol Chem. 1975 Oct 10;250(19):7752–7758. [PubMed] [Google Scholar]
  29. Tomino S., Paigen K., Tulsiani D. R., Touster O. Purification and chemical properities of mouse liver lysosomal (L form) beta-glucuronidase. J Biol Chem. 1975 Nov 10;250(21):8503–8509. [PubMed] [Google Scholar]
  30. Tulsiani D. R., Six H., Touster O. Rat liver microsomal and lysosomal beta-glucuronidases differ in both carbohydrate and amino acid compositions. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3080–3084. doi: 10.1073/pnas.75.7.3080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Warburton M. J., Wynn C. H. The turnover of hamster fibroblast lysosomal beta-D-glucuronidase. Biochem J. 1977 Jan 15;162(1):201–203. doi: 10.1042/bj1620201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wiederanders B., Oelke B. Accumulation of inactive cathepsin D in old rats. Mech Ageing Dev. 1984 Mar;24(3):265–271. doi: 10.1016/0047-6374(84)90112-x. [DOI] [PubMed] [Google Scholar]

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