Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1978 Dec 1;175(3):1069–1077. doi: 10.1042/bj1751069

alpha-Galactosidases II, III and IV from seeds of Trifolium repens. Purification, physicochemical properties and mode of galactomannan hydrolysis in vitro.

J Williams, H Villarroya, F Petek
PMCID: PMC1186169  PMID: 33663

Abstract

Five alpha-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) were identified by chromatography and by their different electrophoretic mobilities, in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidases II, III and IV were purified to homogeneity, with increases in specific activity of approx. 4600-, 4900- and 2800-fold respectively. The enzymes were purified by a procedure that included (NH4)2SO4 precipitation, hydroxyapatite, Sephadex G-75 and DEAE-cellulose chromatography, and preparative polyacrylamide-gel disc electrophoresis. The purified enzymes showed a single protein band, corresponding to the alpha-galactosidase activity, when examined by polyacrylamide-gel electrophoresis. The pH optimum was determined with o-nitrophenyl alpha-D-galactoside and the galactomannan of T. repens To as substrate. All three enzymes are highly thermolabile. Hydrolysis of oligosaccharides and galactomannans was examined, including two galactomannans from the germinated seed of T. repens (T24 and T36). By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the mol.wts. of the multiple forms of enzyme were found to be identical (41 000).

Full text

PDF
1069

Selected References

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

  1. COURTOIS J. E., LEDIZET P. RECHERCHES SUR LES GALACTOMANNANES. III. SUR QUELQUES PROPRI'ET'ES DES GALACTOMANNANES DES GRAINES DE GLEDITSCHIA FEROX. Bull Soc Chim Biol (Paris) 1963 Aug 31;45:731–741. [PubMed] [Google Scholar]
  2. COURTOIS J. E., PETEK F., TO-DONG [Separation of 2alpha-galactosidases with distinct properties from the seeds of Plantago ovata]. Bull Soc Chim Biol (Paris) 1963;45:95–100. [PubMed] [Google Scholar]
  3. Dey P. M., Dixon M. Separation and properties of alpha-galactosidase and beta-galactosidase from Cajanus indicus. Biochim Biophys Acta. 1974 Nov 25;370(1):269–275. doi: 10.1016/0005-2744(74)90051-5. [DOI] [PubMed] [Google Scholar]
  4. Dey P. M., Pridham J. B. Biochemistry of -galactosidases. Adv Enzymol Relat Areas Mol Biol. 1972;36:91–130. doi: 10.1002/9780470122815.ch3. [DOI] [PubMed] [Google Scholar]
  5. Dey P. M., Wallenfels K. Isolation and characterization of alpha-galactosidase from Lens esculanta. Eur J Biochem. 1974 Dec 16;50(1):106–112. doi: 10.1111/j.1432-1033.1974.tb03877.x. [DOI] [PubMed] [Google Scholar]
  6. Doly J., Petek F. Polyacrylamide gradient electrophoresis for protein purification on the milligram scale. J Chromatogr. 1977 Jul 11;137(1):69–81. doi: 10.1016/s0021-9673(00)89242-0. [DOI] [PubMed] [Google Scholar]
  7. HJERTEN S., LEVIN O., TISELIUS A. Protein chromatography on calcium phosphate columns. Arch Biochem Biophys. 1956 Nov;65(1):132–155. doi: 10.1016/0003-9861(56)90183-7. [DOI] [PubMed] [Google Scholar]
  8. HYLIN J. W., SAWAI K. THE ENZYMATIC HYDROLYSIS OF LEUCAENA GLAUCA GALACTOMANNAN. ISOLATION OF CRYSTALLINE GALACTOMANNAN DEPOLYMERASE. J Biol Chem. 1964 Apr;239:990–992. [PubMed] [Google Scholar]
  9. Hedrick J. L., Smith A. J. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys. 1968 Jul;126(1):155–164. doi: 10.1016/0003-9861(68)90569-9. [DOI] [PubMed] [Google Scholar]
  10. Lee Y. C., Wacek V. Galactosidases from Aspergillus niger. Arch Biochem Biophys. 1970 May;138(1):264–271. doi: 10.1016/0003-9861(70)90307-3. [DOI] [PubMed] [Google Scholar]
  11. PETEK F., DONG T. [Separation and study of 2 alpha-galactosidases from coffee beans]. Enzymologia. 1961 Jun 30;23:133–142. [PubMed] [Google Scholar]
  12. Payne D. M., Porter D. W., Gracy R. W. Evidence against the occurrence of tissue-specific variants and isoenzymes of phosphoglucose isomerase. Arch Biochem Biophys. 1972 Jul;151(1):122–127. doi: 10.1016/0003-9861(72)90480-8. [DOI] [PubMed] [Google Scholar]
  13. Petek F., Villarroya E., Courtois J. E. Purification et propriétés de l'alpha-galactosidase des graines germées de Vicia sativa. Eur J Biochem. 1969 Apr;8(3):395–402. doi: 10.1111/j.1432-1033.1969.tb00540.x. [DOI] [PubMed] [Google Scholar]
  14. Suzuki H., Li S. C., Li Y. T. Alpha-galactosidase from Mortierella vinacea. Crystallization and properties. J Biol Chem. 1970 Feb 25;245(4):781–786. [PubMed] [Google Scholar]
  15. Villarroya H., Petek F. Purification and properties of a beta-mannanase from alfalfa seeds. Biochim Biophys Acta. 1976 Jun 7;438(1):200–211. doi: 10.1016/0005-2744(76)90236-9. [DOI] [PubMed] [Google Scholar]
  16. Villarroya H., Williams J., Dey P., Villarroya S., Petek F. Purification and properties of beta-mannanases I and II from the germinated seeds of Trifolium repens. Mode of galactomannan degradation in vitro. Biochem J. 1978 Dec 1;175(3):1079–1087. doi: 10.1042/bj1751079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. WALLENFELS K., MALHOTRA O. P. Galactosidases. Adv Carbohydr Chem. 1961;16:239–298. doi: 10.1016/s0096-5332(08)60264-7. [DOI] [PubMed] [Google Scholar]
  18. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  19. Williams J., Villarroya H., Petek F. Purification and properties of an alpha-D-galactoside galactohydrolase from the seeds of Trifolium repens (white clover). Biochem J. 1977 Mar 1;161(3):509–515. doi: 10.1042/bj1610509. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES