An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels

Abstract

A two-step gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE) procedure was devised for the detection of proteinases and the study of proteinase/inhibitor interactions in complex biological extracts. The proteins are first resolved by sodium dodecyl sulfate (SDS)-PAGE under reducing or nonreducing conditions, and electrotransferred into a 0.75 mm-thick accompanying polyacrylamide slab gel containing 0.1% w/v porcine gelatin. The active proteinase bands are developed by a gelatin proteolysis step in the accompanying gel in the presence or absence of diagnostic proteinase inhibitors, allowing the assessment of proteinase classes and the visual discrimination of inhibitor-'sensitive' and -'insensitive' proteinases in complex extracts. Alternatively, protein extracts are preincubated with specific reversible inhibitors before electrophoresis, allowing a rapid discrimination of strong and weak interactions implicating proteinases and reversible inhibitors. In comparison with the standard gelatin/PAGE procedure, that involves copolymerization of gelatin with acrylamide in the resolving gel, this new procedure simplifies proteinase patterns, avoids overestimation of proteinase numbers in complex extracts, and allows in certain conditions the estimation of proteinase molecular weights. Stem bromelain (EC 3.4.22.32), bovine trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), and the extracellular (digestive) cysteine proteinases of five herbivorous pests are used as model enzymes to illustrate the usefulness of this approach in detecting proteinases and in studying their interactions with specific proteinaceous inhibitors potentially useful in biotechnology.

Analysis of green kiwi fruit (Actinidia deliciosa cv. Hayward) proteinases by two‐dimensional zymography and direct identification of zymographic spots by mass spectrometry

“…The over‐estimation of the molecular masses observed when gelatin was incorporated into the gel might be due to the interaction of actinidin with gelatin during the electrophoretic run and to the consequent reduction of the migration rate of proteins. This hypothesis is in accord with reports by other authors indicating that the inclusion of gelatin in polyacrylamide gels reduces the migration rate of proteins by 15–20% 22…”

A multicomponent, elicitor‐inducible cystatin complex in tomato, Solanum lycopersicum

“…The three cystatins were expressed in and purified from E. coli using the GST gene fusion system (Michaud et al ., 1994), cleaved from the GST moiety (Fig. 5a), then assayed against papain, the herbivorous insect digestive protease Ldp30 and the major extracellular cysteine proteases of two root parasitic nematodes, Mhp1 and Mip1 (Visal‐Shah et al ., 2001). As shown in Table 2, the native form of Sl CYS9 showed weak activity against papain and Ldp30, giving K i (app) values in the micromolar range.…”

“…Trans ‐epoxysuccinyl‐ l ‐leucylamido‐(4‐guanidino) butane (E‐64), papain (from papaya latex, EC 3.4.22.2), phenylmethylsulfonyl fluoride (PMSF), ethylenediamine tetraacetic acid (EDTA) and pepstatin A were purchased from Sigma (Oakville, ON, Canada). LdP30, a digestive cystatin‐sensitive protease from the coleopteran insect Colorado potato beetle ( Leptinotarsa decemlineata Say), was purified by affinity chromatography from third‐instar larvae reared on potato plants, using oryzacystatin as an affinity ligand (Visal‐Shah et al ., 2001). The secreted cysteine proteases Mhp1 and Mip1, from the root‐parasitic nematodes Meloidogyne hapla and Meloidogyne incognita , were prepared from preparasitic J 2 larvae as described earlier (Michaud et al ., 1996).…”

Characterization of Siam Tulip (Curcuma alismatifolia) Cystatin (CaCPI)

“…Several lines of evidence suggest that plant cystatins are responsive to abiotic stresses such as drought, salt, abscisic acid and cold treatment, helping plants better tolerate stresses (Gaddour et al, 2001;Van der Vyver et al, 2003;Diop et al, 2004;Massonneau et al, 2005;Christova et al, 2006). In addition, plant cystatins have a significant role in plant defense mechanisms, where they have been shown to inhibit the activity of digestive cysteine proteases of herbivorous arthropods, field slugs and parasitic nematodes (Zhao et al, 1996;Walker et al, 1999;Visal-Shah et al, 2001;Arai et al, 2002). Moreover, some plant cystatins show detrimental effects against pathogenic fungi (Pernas et al, 1999;Soares-Costa et al, 2002;Martinez et al, 2003;Martinez et al, 2005a;Yang and Yeh, 2005;Christova et al, 2006) Siam tulip is a popular ornamental plant exported from Thailand.…”