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Electrophoretic analysis of plant cysteine and serine proteinases using gelatin-containing polyacrylamide gels and class-specific proteinase inhibitors.
Electrophoresis. 1993 Jan-Feb; 14(1-2):94-8.E

Abstract

Inclusion of gelatin in polyacrylamide gels provides a sensitive way of detecting multiple proteolytic activities in crude extracts from any source. The present study describes a method allowing discrimination between cysteine and serine proteinases in plant extracts, using gelatin-containing gels in combination with class-specific proteinase inhibitors. Preincubation of extracts with 4 mM phenylmethylsulfonyl fluoride, a serine proteinase inhibitor, or with 25 microM L-trans-epoxysuccinyl-L-leucylamido(4-guanidino) butane, a cysteine proteinase inhibitor, allowed the identification of enzymes from both classes in extracts of tomato fruit and papaya latex. The efficiency of the two low molecular weight inhibitors used was very high, and the irreversibility of the inhibiting effect was maintained during electrophoresis conducted in the presence of sodium dodecyl sulfate. The analytic procedure described here, with a detection threshold of less than 100 pg enzyme, is the first that allows quick and accurate discrimination of plant cysteine and serine proteinases separated in electrophoretic gels. This simple and rapid technique could be of interest for studying the evolution of class-specific proteinases in plant extracts during various developmental, physiological, and pathogenic processes. It is also potentially applicable to the majority of eucaryotic and procaryotic systems.

Authors+Show Affiliations

Département de Phytologie, FSAA, Université Laval, Québec, Canada.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

8462522

Citation

Michaud, D, et al. "Electrophoretic Analysis of Plant Cysteine and Serine Proteinases Using Gelatin-containing Polyacrylamide Gels and Class-specific Proteinase Inhibitors." Electrophoresis, vol. 14, no. 1-2, 1993, pp. 94-8.
Michaud D, Faye L, Yelle S. Electrophoretic analysis of plant cysteine and serine proteinases using gelatin-containing polyacrylamide gels and class-specific proteinase inhibitors. Electrophoresis. 1993;14(1-2):94-8.
Michaud, D., Faye, L., & Yelle, S. (1993). Electrophoretic analysis of plant cysteine and serine proteinases using gelatin-containing polyacrylamide gels and class-specific proteinase inhibitors. Electrophoresis, 14(1-2), 94-8.
Michaud D, Faye L, Yelle S. Electrophoretic Analysis of Plant Cysteine and Serine Proteinases Using Gelatin-containing Polyacrylamide Gels and Class-specific Proteinase Inhibitors. Electrophoresis. 1993 Jan-Feb;14(1-2):94-8. PubMed PMID: 8462522.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Electrophoretic analysis of plant cysteine and serine proteinases using gelatin-containing polyacrylamide gels and class-specific proteinase inhibitors. AU - Michaud,D, AU - Faye,L, AU - Yelle,S, PY - 1993/1/1/pubmed PY - 1993/1/1/medline PY - 1993/1/1/entrez SP - 94 EP - 8 JF - Electrophoresis JO - Electrophoresis VL - 14 IS - 1-2 N2 - Inclusion of gelatin in polyacrylamide gels provides a sensitive way of detecting multiple proteolytic activities in crude extracts from any source. The present study describes a method allowing discrimination between cysteine and serine proteinases in plant extracts, using gelatin-containing gels in combination with class-specific proteinase inhibitors. Preincubation of extracts with 4 mM phenylmethylsulfonyl fluoride, a serine proteinase inhibitor, or with 25 microM L-trans-epoxysuccinyl-L-leucylamido(4-guanidino) butane, a cysteine proteinase inhibitor, allowed the identification of enzymes from both classes in extracts of tomato fruit and papaya latex. The efficiency of the two low molecular weight inhibitors used was very high, and the irreversibility of the inhibiting effect was maintained during electrophoresis conducted in the presence of sodium dodecyl sulfate. The analytic procedure described here, with a detection threshold of less than 100 pg enzyme, is the first that allows quick and accurate discrimination of plant cysteine and serine proteinases separated in electrophoretic gels. This simple and rapid technique could be of interest for studying the evolution of class-specific proteinases in plant extracts during various developmental, physiological, and pathogenic processes. It is also potentially applicable to the majority of eucaryotic and procaryotic systems. SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/8462522/Electrophoretic_analysis_of_plant_cysteine_and_serine_proteinases_using_gelatin_containing_polyacrylamide_gels_and_class_specific_proteinase_inhibitors_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0173-0835&date=1993&volume=14&issue=1-2&spage=94 DB - PRIME DP - Unbound Medicine ER -