Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis.
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.
Pacific Agriculture Research Centre, Agriculture and Agri-Food Canada, Vancouver.
D
D
Michaud
L
L
Cantin
D A
DA
Raworth
T C
TC
Vrain
D000818:Animals; D015891:Cystatins / Q000737:chemistry* / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004795:Enzyme Stability; D005069:Evaluation Studies as Topic; D005780:Gelatin; D008925:Mites / Q000201:enzymology; D011489:Protein Denaturation
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D005780:Gelatin; D003546:Cysteine Endopeptidases