Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 1988 May;87(1):95–103. doi: 10.1104/pp.87.1.95

Purification and Characterization of Gibberellic Acid-Induced Cysteine Endoproteases in Barley Aleurone Layers 1

Susan Koehler 1,2, Tuan-Hua David Ho 1,2
PMCID: PMC1054705  PMID: 16666134

Abstract

Using in series ammonium sulfate precipitation, gel filtration, and DEAE anion exchange high performance liquid chromatography, we have purified to homogeneity a protease of Mr 37,000 secreted from barley (Hordeum vulgare L. cv Himalaya) embryoless half-seeds. This protease exists in three isozymic forms whose synthesis and secretion from barley aleurone layers was shown to be a gibberellic acid (GA3)-dependent process (R Hammerton, T-HD Ho 1986 Plant Physiol 80: 692-697). This protease constitutes a major portion of the protease activity secreted from half-seeds between 72 to 96 hours of incubation in the presence of GA3 as detected on activity gels containing hemoglobin as the substrate. Analysis of digestion products by urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that this protease is an endoprotease, therefore it is designated as barley endoprotease-A (EP-A). Inhibitor studies demonstrated that EP-A belongs to the cysteine class of endoproteases. The optimum pH for EP-A activity was 5.0, and the temperature optimum was 45°C. Comparison of cyanogen bromide generated peptide fragments and NH2-terminal sequence analyses of the three individual EP-A isozymes demonstrates that they are very similar to each other. The NH2-terminal sequence shows extensive sequence homology to the NH2-terminal sequence of papain and several other cysteine proteinases. We also provide evidence that EP-A is not `aleurain,' a putative cysteine proteinase encoded by a GA3-induced barley cDNA clone (JC Rogers, D Dean, GR Heck 1985 Proc Natl Acad Sci USA 82:6512-6516).

Full text

PDF
95

Images in this article

97Fig. 1
on p.97
98Fig. 2
on p.98
99Fig. 5
on p.99
100Fig. 6
on p.100
101Fig. 9
on p.101

Selected References

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

  1. Barrett A. J., Kembhavi A. A., Brown M. A., Kirschke H., Knight C. G., Tamai M., Hanada K. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J. 1982 Jan 1;201(1):189–198. doi: 10.1042/bj2010189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baumgartner B., Chrispeels M. J. Purification and characterization of vicilin peptidohydrolase, the major endopeptidase in the cotyledons of mung-bean seedlings. Eur J Biochem. 1977 Jul 15;77(2):223–233. doi: 10.1111/j.1432-1033.1977.tb11661.x. [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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Brown P. H., Ho T. H. Barley aleurone layers secrete a nuclease in response to gibberellic Acid : purification and partial characterization of the associated ribonuclease, deoxyribonuclease, and 3'-nucleotidase activities. Plant Physiol. 1986 Nov;82(3):801–806. doi: 10.1104/pp.82.3.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chrispeels M. J., Varner J. E. Gibberellic Acid-enhanced synthesis and release of alpha-amylase and ribonuclease by isolated barley and aleurone layers. Plant Physiol. 1967 Mar;42(3):398–406. doi: 10.1104/pp.42.3.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Enari T. M., Mikola J. Peptidases in germinating barley grain: properties, localization and possible functions. Ciba Found Symp. 1977;(50):335–352. [PubMed] [Google Scholar]
  8. Gribskov M., Burgess R. R. Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins. Nucleic Acids Res. 1986 Aug 26;14(16):6745–6763. doi: 10.1093/nar/14.16.6745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hammerton R. W., Ho T. H. Hormonal regulation of the development of protease and carboxypeptidase activities in barley aleurone layers. Plant Physiol. 1986 Mar;80(3):692–697. doi: 10.1104/pp.80.3.692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jacobsen J. V., Varner J. E. Gibberellic Acid-induced synthesis of protease by isolated aleurone layers of barley. Plant Physiol. 1967 Nov;42(11):1596–1600. doi: 10.1104/pp.42.11.1596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  13. Miller B. L., Huffaker R. C. Partial purification and characterization of endoproteinases from senescing barley leaves. Plant Physiol. 1981 Oct;68(4):930–936. doi: 10.1104/pp.68.4.930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  15. Needleman S. B., Wunsch C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 1970 Mar;48(3):443–453. doi: 10.1016/0022-2836(70)90057-4. [DOI] [PubMed] [Google Scholar]
  16. Nikodem V., Fresco J. R. Protein fingerprinting by SDS-gel electrophoresis after partial fragmentation with CNBr. Anal Biochem. 1979 Sep 1;97(2):382–386. doi: 10.1016/0003-2697(79)90089-7. [DOI] [PubMed] [Google Scholar]
  17. Rastogi V., Oaks A. Hydrolysis of storage proteins in barley endosperms : analysis of soluble products. Plant Physiol. 1986 Jul;81(3):901–906. doi: 10.1104/pp.81.3.901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rogers J. C., Dean D., Heck G. R. Aleurain: a barley thiol protease closely related to mammalian cathepsin H. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6512–6516. doi: 10.1073/pnas.82.19.6512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Stuart I. M., Loi L., Fincher G. B. Development of (1-->3,1-->4)-beta-d-Glucan Endohydrolase Isoenzymes in Isolated Scutella and Aleurone Layers of Barley (Hordeum vulgare). Plant Physiol. 1986 Feb;80(2):310–314. doi: 10.1104/pp.80.2.310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Swank R. T., Munkres K. D. Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal Biochem. 1971 Feb;39(2):462–477. doi: 10.1016/0003-2697(71)90436-2. [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES