Jean Trudel

Detection of chitinase activity after polyacrylamide gel electrophoresis

Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.

Diversity of cucumber chitinase isoforms and characterization of one seed basic chitinase with lysozyme activity

Abstract Up to six bands with chitinase activity could be detected in seeds or young seedlings after native polyacrylamide gel electrophoretic (PAGE) separation of proteins at pH 8.9 in the Davis system. Two root-specific acidic chitinases were found between 2 and 11 days after germination. Up to eight bands with chitinase activity were observed after native PAGE separation of proteins at pH 4.3 in the Reisfeld system. Since one chitinase activity migrated with the lowest electrophoretic mobility in both polyacrylamide gel systems, a total of 13 chitinases could be found. Seeds (0, 1 and 2 days after germination) were the best source of basic chitinase activities. The strongest activity had a relative mobility ( R m ) value of 0.80 when compared to the mobility of purified hen egg white lysozyme set at 1.00. This activity ( R m 0.80) was partially purified by ammonium sulfate fractionation, ion-exchange chromatography and preparative PAGE. The enzyme had an estimated molecular weight of 27 000 after sodium dodecyl sulfate (SDS)-PAGE. It exhibited lysozyme activity as determined by lysis of Micrococcus cells. Its lysozyme activity was optimal at pH 4.5 and at low ionic strength. Its chitinase activity was typical of an endochitinase type as estimated by the relative efficiency of hydrolysis of 4-methylumbelliferyl-chitotriose versus 4-methylumbelliferyl-chitobiose Cucumber seeds thus contain a highly basic endochitinase/lysozyme which disappears almost completely 3 days after germination. The role of this seed hydrolase has yet to be determined.

Detection of chitin deacetylase activity after polyacrylamide gel electrophoresis

Mucor racemosus and Rhizopus nigricans were used as sources of chitin deacetylases. Crude protein extracts were subjected to polyacrylamide gel electrophoresis at pH 8.9 (Davis system) or 4.3 (Reisfeld system) under native conditions. After electrophoresis, an overlay gel containing 0.1% (w/v) glycol chitin as substrate was incubated in contact with the separation gel. Chitin deacetylase activity was revealed by uv illumination with a transilluminator after staining for 5 min in 0.01% (w/v) Calcofluor white M2R. Chitosan (deacetylated chitin) generated by chitin deacetylases appeared more fluorescent than the intact chitin embedded in the overlay gel. Chitosan in a separate overlay gel was also subjected to a nitrous acid treatment which specifically depolymerizes chitosan while leaving chitin intact. Hydrolysis of chitosan by nitrous acid followed by Calcofluor staining yielded dark (nonfluorescent) bands (chitin deacetylase activities) in the fluorescent chitin-containing gel. Both assays revealed the presence of several chitin deacetylases from Zygomycetes. The same assays were performed after denaturing electrophoresis in 12% (w/v) polyacrylamide gels containing 0.1% (w/v) glycol chitin. Enzymes were renatured in buffered 1% (v/v) purified Triton X-100. Chitin deacetylases with estimated molecular weights between 26,000 and 64,000 were detected after Calcofluor staining. The assays were also performed in two-dimensional gel electrophoretic systems. Chitin deacetylases can be rapidly revealed by using the assay involving the nitrous acid treatment. However, both assays (with and without nitrous acid treatment) should be run to conclusively demonstrate chitin deacetylase activity after polyacrylamide gel electrophoresis.

Secreted hen lysozyme in transgenic tobacco: recovery of bound enzyme and in vitro growth inhibition of plant pathogens

Abstract Less than 1% of secreted hen egg white lysozyme (HEWL) was isolated in intercellular fluid neutral phosphate buffer extracts of transgenic tobacco ( Nicotiana tabacum L.). When foliar tissue was infiltrated with 50 mM calcium chloride or 1% histamine, 10–20% of HEWL was isolated when compared to HEWL obtained in tissue homogenates boiled in the presence of sodium dodecyl sulfate. The presence of mature HEWL was shown by N-terminal amino acid microsequencing of the major electrophoretic form (14.4 kDa). A minor electrophoretic form (17 kDa) having the same N-terminus end (KVFGRC) as the major mature from was also found. Tissue imprinting of transgenic tobacco cut stem, petiole and leaf tissue for detecting lysis of Micrococcus luteus cells embedded as lysozyme substrate in polyacrylamide gels could be performed in the presence of histamine for release of HEWL. Despite tight binding of HEWL in transgenic tobacco, some HEWL activity could be detected around growing transgenic tobacco seedling rootlets. Moreover, HEWL recovered from transgenic tobacco extracellular extracts was shown to inhibit the growth of some bacterial and fungal plant pathogens.

Expression of active hen egg white lysozyme in transgenic tobacco

A full-length complementary DNA clone of hen egg white lysozyme (HEWL) was inserted into pBI121 vector lacking the β-glucuronidase (GUS) gene and used to transform tobacco. The HEWL gene had its own 18-amino acid signal peptide. HEWL activity was detected using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay. The enzyme was active against Micrococcus luteus cells and migrated at the same molecular mass as purified mature HEWL. The protein was also found by immunodetection of HEWL after SDS-PAGE. Twenty-seven independent transgenic tobacco plants were analyzed for the expression of HEWL by a modified lysoplate assay for quantifying HEWL activity. Twenty-five plants exhibited HEWL activity up to 30 ng of HEWL per mg of leaf tissue. When using successive intercellular fluid (IF) extracts to study the localization of HEWL, it was found that no more than 10% of HEWL could be recovered in IF extracts. This is contrary to several extracellular proteins, such as the pathogenesis-related proteins. However, purified HEWL was recovered with the same yield as in transgenic tobacco leaves when it was injected into normal leaf tissue. Transgenic tobacco plants did not exhibit a phenotype change due to the expression of HEWL. The HEWL gene could be a useful reporter gene because the activity of the protein can be quantified by a simple assay.

A new lectin-gold complex for ultrastructural localization of galacturonic acids.

We report the development of a cytochemical affinity technique for detection of galacturonic acids at the ultrastructural level. The highly purified gonad lectin from Aplysia depilans (AGL) was tagged with colloidal gold particles and used for labeling carbohydrates in resin-embedded sections of various plant and fungal tissues. Patterns of AGL binding sites were compared to those obtained with a D-galactose-specific lectin, Ricinus communis agglutinin I. Differences in labeling patterns were noted, indicating that the lectins exhibited differential carbohydrate binding. In addition, the considerable loss of labeling over isolated wheat coleoptile walls treated for removal of pectin, after incubation with the AGL-gold complex, strongly suggested an affinity of AGL for pectic substances. A series of cytochemical controls, including sugar inhibition tests, has proven the specificity of the technique and the high affinity of AGL towards galacturonic acids. The potential value of this new lectin for ultrastructural studies on cell wall pectic substances in plant biology and pathology is demonstrated.

Purification and characterization of a lysozome from wheat germ

Abstract Several proteins of wheat germ were able to lyse Micrococcus luteus cells. One lysozyme, named W1A, was purified by ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and preparative polyacrylamide gel electrophoresis (PAGE) under native conditions. The enzyme had a molecular weight of 25 400 as determined by sodium dodecyl sulfate (SDS)-PAGE. The reducing groups released from the lysis of Micrococcus cell walls by W1A lysozyme were N- acetylmuramic acid residues as for hen egg white lysozyme (HEWL). Chitin substrates were hydrolyzed to some extent by this enzyme. With Micrococcus cells as substrate, the pH optimum for W1A lysozyme was 6.0 at an optimal ionic strength of 0.05. Under these conditions, the K m value was 166 mg/l with purified Micrococcus cell walls and the Vmax value was 0.56 A540 unit/min at 22°C. W1A lysozyme exhibited the highest lytic activity at 60°C whereas the enzyme was inactive above 90°C. W1A lysozyme was strongly inhibited by poly- l -lysine and glycol chitosan. This is the first report of the presence of multiple electrophoretic forms of plant lysozyme activity as determined by native PAGE.