Assessing the stability of cystatin/cysteine proteinase complexes using mildly‐denaturing gelatin‐polyacrylamide gel electrophoresis

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.

Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases

“…Michaud et al (1996) had previously shown that protease activity in extracts from T. urticae was almost totally explained by cysteine-protease type in both in vitro assays and in zymograms. Likewise, proteolytic activities from T. urticae extracts were inhibited by E-64 and pepstatin A indicating cysteine-and aspartic-protease activities, respectively, in this mite (Nisbet and Billingsley 2000).…”

“…In contrast, an almost complete inhibition of the gelatinase bands from the B. chilensis extraets were detected after being preincubated with the HvCPI-6 cystatin. These differences could be determined by the stability Table 2 Specific activities as in Table 2 of each cystatin/cysteine-protease complex (Michaud et al 1996;Kiggundu et al 2010), which in the case of T. urticae may favour dissociation during the electrophoretic process. According to Michaud et al (1996), it is considered a weak complex stability when Ki valúes are higher than 10~8 M. In our hands the HvCPI-6 Kis were in the 10" 6 -10" 7 M range against commercial cathepsin B-and H-like proteases, respectively, but its Ki valúes against most cathepsin L-like tested was 10~9 M (Abraham et al 2006;Martínez et al 2009).…”

“…Mite species that feed on plants rely mostly on cysteine peptidase activities for the digestión of dietary proteins (Nisbet and Billingsley 2000). Particularly, protease activity in extracts from T. urticae was almost totally explained by cysteine type protease activity in vitro assays and in zymograms (Michaud et al 1996). Therefore, a control strategy based on the use of phytocystatins (PhyCys) would provide new tools to protect crops against spider mites.…”

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

“…The mixture was incubated on ice for 30 min, and then centrifuged at 12000 g for 10 min to remove cellular debris and insoluble material. The supernatant was collected and the protein content adjusted to 1 mg/ml with extraction solution.Insect material for gelatinase zymography (see below) was homogenized directly in 100 μL of gelatin-PAGE sample loading buffer (62.5 mM Tris-HCl (pH 8.0), 2% (w/v) sucrose and 0.001% (w/v) bromophenol blue) (Michaud et al, 1996a). Protein concentration in the extracts was determined using the Bio-Rad Protein Assay Kit™ (Bio-Rad, Mississauga ON, Canada), with bovine serum albumin as a protein standard.…”

“…The insect extracts (3 μL, for 3 μg of protein) were incubated with excess amounts of protein inhibitors (25 μg of inhibitor in 5 μL), or with 5 μL of 50 mM Tris-HCl, pH 8.0 (non-inhibited control) for 10 min at 37˚C before electrophoresis. Protease (gelatinase) forms were visualized as clear bands against a Coomassie blue-stained background following electrophoresis (Michaud et al, 1996a). …”