Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 1981 Oct;68(4):930–936. doi: 10.1104/pp.68.4.930

Partial Purification and Characterization of Endoproteinases from Senescing Barley Leaves 1

Bruce L Miller 1, Ray C Huffaker 1
PMCID: PMC426015  PMID: 16662028

Abstract

Two major endoproteinases were purified from senescing primary barley leaves. The major enzyme (EP1) appeared to be a thiol proteinase and accounted for about 85% of the total proteolytic activity measured in vitro. This proteinase was purified 5,800-fold and had a molecular weight of 28,300. It was highly unstable in the absence of dithiothreitol or at a pH greater than 7.5. Leupeptin, at a concentration of 10 micromolar, inhibited this enzyme 100%. A second proteinase (EP2) was purified approximately 50-fold and had a molecular weight of 67,000. It was inhibited 20% by 1 millimolar dithiothreitol and 50% by 1 millimolar phenylmethyl sulfonylfluoride. EP2 contributed about 15% of the total proteolytic activity measured in vitro. Both proteinases hydrolyzed a variety of artificial and protein substrates, and both had pH optima of 5.5 to 5.7 when either azocasein or [14C]ribulose-1,5-bisphosphate carboxylase ([14C]RuBPCase) was the substrate. The thiol endoproteinase hydrolyzed azocasein linearly but hydrolyzed [14C]RuBPCase biphasically. A third endoproteinase (EP3), not detected by standard proteolytic assays, was observed when [14C]RuBPCase was the substrate.

Full text

PDF
935

Images in this article

933Image
on p.933
933Image
on p.933

Selected References

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

  1. Anderson J. W., Rowan K. S. Activity of peptidase in tobacco-leaf tissue in relation to senescence. Biochem J. 1965 Dec;97(3):741–746. doi: 10.1042/bj0970741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barrett A. J. The many forms and functions of cellular proteinases. Fed Proc. 1980 Jan;39(1):9–14. [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. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  5. Drivdahl R. H., Thimann K. V. Proteases of Senescing Oat Leaves: II. Reaction to Substrates and Inhibitors. Plant Physiol. 1978 Apr;61(4):501–505. doi: 10.1104/pp.61.4.501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  7. Feller U. K., Soong T. S., Hageman R. H. Leaf Proteolytic Activities and Senescence during Grain Development of Field-grown Corn (Zea mays L.). Plant Physiol. 1977 Feb;59(2):290–294. doi: 10.1104/pp.59.2.290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hampp R., De Filippis L. F. Plastid Protease Activity and Prolamellar Body Transformation during Greening. Plant Physiol. 1980 Apr;65(4):663–668. doi: 10.1104/pp.65.4.663. [DOI] [PMC free article] [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. Kleinkopf G. E., Huffaker R. C., Matheson A. A simplified purification and some properties of ribulose 1,5-diphosphate carboxylase from barley. Plant Physiol. 1970 Aug;46(2):204–207. doi: 10.1104/pp.46.2.204. [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. Martin C., Thimann K. V. The role of protein synthesis in the senescence of leaves: I. The formation of protease. Plant Physiol. 1972 Jan;49(1):64–71. doi: 10.1104/pp.49.1.64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Moore S. Amino acid analysis: aqueous dimethyl sulfoxide as solvent for the ninhydrin reaction. J Biol Chem. 1968 Dec 10;243(23):6281–6283. [PubMed] [Google Scholar]
  14. ORNSTEIN L. DISC ELECTROPHORESIS. I. BACKGROUND AND THEORY. Ann N Y Acad Sci. 1964 Dec 28;121:321–349. doi: 10.1111/j.1749-6632.1964.tb14207.x. [DOI] [PubMed] [Google Scholar]
  15. Pike C. S., Briggs W. R. Partial Purification and Characterization of a Phytochrome-degrading Neutral Protease from Etiolated Oat Shoots. Plant Physiol. 1972 Apr;49(4):521–530. doi: 10.1104/pp.49.4.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ragster L. V., Chrispeels M. J. Azocoll-digesting Proteinases in Soybean Leaves: Characteristics and Changes during Leaf Maturation and Senescence. Plant Physiol. 1979 Nov;64(5):857–862. doi: 10.1104/pp.64.5.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Santarius K., Ryan C. Radial diffusion as a sensitive method for screening endopeptidase activity in plant extracts. Anal Biochem. 1977 Jan;77(1):1–9. doi: 10.1016/0003-2697(77)90283-4. [DOI] [PubMed] [Google Scholar]
  18. Vavreinová S., Turková J. SH-proteinase from bean Phaseolus vulgaris var. Perlicka. Biochim Biophys Acta. 1975 Oct 22;403(2):506–513. doi: 10.1016/0005-2744(75)90078-9. [DOI] [PubMed] [Google Scholar]
  19. Wittenbach V. A. Breakdown of Ribulose Bisphosphate Carboxylase and Change in Proteolytic Activity during Dark-induced Senescence of Wheat Seedlings. Plant Physiol. 1978 Oct;62(4):604–608. doi: 10.1104/pp.62.4.604. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES