4): 0, 75, 100, 125, 150, 175, 200 and then 300 mM. The eluted fractions corresponding to maximum protein peaks were then 20-fold reconcentrated (second step of ultrafiltration; cut-off 10 kDa), and assayed in the well test to determine the killer fraction. The resulting positive (for killer activity) fraction was subsequently examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (38 : 2 ratio of acrylamide : bis-acrylamide of 12% solution; 2 h of run under a constant voltage,
150 mV). The proteins were stained with a silver staining kit (Sigma, St. Louis, MO). The molecular mass of the purified killer toxin was estimated by comparing the purified fractions with a known marker protein (Molecular weight marker SDS6H2; Sigma). Kwkt activity was determined according to Somers & Bevan (1969). Briefly, 70-μL toxin samples Selumetinib in vivo were filter-sterilized through 0.45-μm pore-size membrane filters (Millipore) and put into wells (7 mm diameter), cut in the malt agar plates that had previously been seeded with 105 cells mL−1 of the sensitive indicator
yeast strain. The killing activity was measured as the diameter of the inhibition halo around the well after incubation for 48 h at 25 °C, and is defined as the mean zone of inhibition across replicate wells. The linear relationship observed between the logarithm of the killer toxin concentration and the diameter of the inhibition halo assayed using this well-established method was used to define the Kwkt activity, as arbitrary
units (AU), with 1 AU defined as the toxin concentration selleck products that resulted in an inhibition halo of 8 mm (actual diameter, 1 mm, considering the 7.0 mm diameter of the well) (Ciani & Fatichenti, 2001). One AU corresponds to about 1.0-μg killer protein. Kwkt was treated with endoglycosidase H (45 IU mg−1 protein; Nitroxoline ICN Biomedicals). The assay was performed following the procedure described by Elgersma et al. (1997). Briefly, 10 μL endoglycosidase H (0.01 IU μL−1) was added to 50 μL of purified Kwkt (350 AU) and 140 μL buffer (150 mM sodium citrate, pH 5.5, 1 mM PMSF, 10 μM pepstatin, 5 mM sodium azide, 643 μL H2O). The samples were incubated at 37 °C for 48 h with gentle agitation, and examined by SDS-PAGE, as described above. Four trials were prepared in must, with the proliferation of D. bruxellensis monitored as follows: positive control without Kwkt and without SO2 addition; negative control with 60 mg L−1 SO2; two samples with 40 and 80 mg L−1 purified Kwkt (12 and 24 AU mL−1, respectively). In each trial, D. bruxellensis cells from a 72-h preculture were inoculated into 250 mL sterile grape juice, to a final concentration of 103 cells mL−1, together with an inoculum of the S. cerevisiae starter strain of 2 × 106 cells mL−1. The zymocidial activity of Kwkt on D. bruxellensis was monitored by viable plate counts on WL nutrient agar (Oxoid).
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