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Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis.
Electrophoresis. 1996 Jan; 17(1):74-9.E

Abstract

A method for assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis (gelatin-PAGE) is described. As suggested by the use of well-known cystatins (human stefins A and B, and oryzacystatins I and II) and the plant cysteine proteinase papain, the ability of cystatin/cysteine proteinase complexes to remain stable during electrophoresis is associated with the degree of affinity between the enzyme and the inhibitor (and inversely associated with the Ki values), at least with the disulfide bond-lacking cystatins. Complexes with Ki values > or = 10(-8) M (weak interactions) are partly or completely dissociated under the conditions used, while those with lower Ki values (strong interactions) remain stable. As shown by the differential effects of two plant cystatins, oryzacystatins I and II, against a cysteine proteinase present in crude (complex) extracts from a plant pest -- the two-spotted spider mite (Tetranychus urticae Koch), the gelatin-PAGE procedure is suitable for studying the ability of cystatins to form highly stable complexes with cysteine proteinases, without the need for prior purification steps. Considering the well-recognized potential of proteinase inhibitors for pest and pathogen control, this analytical approach will be useful for rapidly assessing the respective potential of various cystatins for protection of plants, animals, and humans.

Authors+Show Affiliations

Michaud D
Pacific Agriculture Research Centre, Agriculture and Agri-Food Canada, Vancouver.
Cantin L
No affiliation info available
Raworth DA
No affiliation info available
Vrain TC
No affiliation info available

MeSH

AnimalsCystatinsCysteine EndopeptidasesCysteine Proteinase InhibitorsElectrophoresis, Polyacrylamide GelEnzyme StabilityEvaluation Studies as TopicGelatinMitesProtein Denaturation

Pub Type(s)

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

Language

eng

PubMed ID

8907521

Citation

Michaud, D, et al. "Assessing the Stability of Cystatin/cysteine Proteinase Complexes Using Mildly-denaturing Gelatin-polyacrylamide Gel Electrophoresis." Electrophoresis, vol. 17, no. 1, 1996, pp. 74-9.
Michaud D, Cantin L, Raworth DA, et al. Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis. Electrophoresis. 1996;17(1):74-9.
Michaud, D., Cantin, L., Raworth, D. A., & Vrain, T. C. (1996). Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis. Electrophoresis, 17(1), 74-9.
Michaud D, et al. Assessing the Stability of Cystatin/cysteine Proteinase Complexes Using Mildly-denaturing Gelatin-polyacrylamide Gel Electrophoresis. Electrophoresis. 1996;17(1):74-9. PubMed PMID: 8907521.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis. AU - Michaud,D, AU - Cantin,L, AU - Raworth,D A, AU - Vrain,T C, PY - 1996/1/1/pubmed PY - 1996/1/1/medline PY - 1996/1/1/entrez SP - 74 EP - 9 JF - Electrophoresis JO - Electrophoresis VL - 17 IS - 1 N2 - A method for assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis (gelatin-PAGE) is described. As suggested by the use of well-known cystatins (human stefins A and B, and oryzacystatins I and II) and the plant cysteine proteinase papain, the ability of cystatin/cysteine proteinase complexes to remain stable during electrophoresis is associated with the degree of affinity between the enzyme and the inhibitor (and inversely associated with the Ki values), at least with the disulfide bond-lacking cystatins. Complexes with Ki values > or = 10(-8) M (weak interactions) are partly or completely dissociated under the conditions used, while those with lower Ki values (strong interactions) remain stable. As shown by the differential effects of two plant cystatins, oryzacystatins I and II, against a cysteine proteinase present in crude (complex) extracts from a plant pest -- the two-spotted spider mite (Tetranychus urticae Koch), the gelatin-PAGE procedure is suitable for studying the ability of cystatins to form highly stable complexes with cysteine proteinases, without the need for prior purification steps. Considering the well-recognized potential of proteinase inhibitors for pest and pathogen control, this analytical approach will be useful for rapidly assessing the respective potential of various cystatins for protection of plants, animals, and humans. SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/8907521/Assessing_the_stability_of_cystatin/cysteine_proteinase_complexes_using_mildly_denaturing_gelatin_polyacrylamide_gel_electrophoresis_ L2 - https://doi.org/10.1002/elps.1150170113 DB - PRIME DP - Unbound Medicine ER -