Journal: The Journal of Biological Chemistry

Article Title: A novel subtilase inhibitor in plants shows structural and functional similarities to protease propeptides

doi: 10.1074/jbc.M117.775445

Figure Lengend Snippet: Expression of Arabidopsis SBTs and inhibition by SPI-1. A, purification of SPI-1 and SBT4.13. Two micrograms of recombinant SPI-1 (left) and SBT4.13 (right) purified form E. coli extracts (SPI-1) and apoplastic washes of agroinfiltrated N. benthamiana plants (SBT4.13) were separated by 15 or 12% SDS-PAGE, respectively. Gels were stained with Coomassie Brilliant Blue R-250. The molecular mass of the marker proteins is indicated. B, SDS-PAGE and Western blotting analysis of transiently expressed Arabidopsis subtilases. The Coomassie-stained gel is shown (top) as a control for protein loading. Only the most highly expressed SBTs (SBT1.7, -4.13, and -5.2) are visualized by Coomassie staining. To detect SBTs on the immunoblot, a mixture of polyclonal antisera directed against tomato SBTs 1–4 (1:10,000) was used in combination with a peroxidase-conjugated secondary antibody (1:10,000; Calbiochem) with enhanced chemiluminescence detection. C, activity of Arabidopsis SBTs and inhibition by SPI-1. SBT activity was assayed with fluorescence-labeled casein as a universal substrate (Pierce) in the presence of 0, 0.2, or 5 μm SPI-1. Activity was normalized against protein concentration assayed using the Bradford procedure, and it is shown relative to background activity in extracts of empty vector-infiltrated control plants (background in the presence of 5 μg of SPI-1 was set at 0). Data represent the mean of three experiments using independent inhibitor preparations; error bars indicate standard deviation. Two-way analysis of variance in combination with Tukey's multiple-comparisons test (GraphPad Prism 6) was performed, and significant differences are shown between samples without and with 0.2 μm SPI-1 at p ≤ 0.001 (*) and p ≤ 0.0001 (**), respectively. D, activity of SPI-1 against SBT4.13, α-chymotrypsin, and subtilisin A. Protease activity was assayed with 1 mm N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide as the substrate for chymotrypsin and subtilisin A and with an internally quenched fluorigenic peptide substrate (25 μm) for SBT4.13. Assays were performed at room temperature with increasing concentrations of SPI-1. Activity is expressed as percentage of the respective protease activity without inhibitor. Error bars indicate standard deviation.

Article Snippet: SBT4.13 enzyme kinetics A fluorigenic peptide substrate, Abz-YLPA↓GVPI-Tyr(NO 2 ) (PepMic, Suzhou, China), was used for kinetic measurements of SBT4.13 activity under steady-state conditions.

Techniques: Expressing, Inhibition, Purification, Recombinant, SDS Page, Staining, Marker, Western Blot, Control, Activity Assay, Fluorescence, Labeling, Protein Concentration, Plasmid Preparation, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: A novel subtilase inhibitor in plants shows structural and functional similarities to protease propeptides

doi: 10.1074/jbc.M117.775445

Figure Lengend Snippet: Interaction of SBT4.13 with SPI-1. A, apparent inhibition constant. Purified SBT4.13 (1.5 nm) was incubated with two serial dilutions (1:2) of SPI-1 starting at 100 and 200 nm, respectively. Activity was recorded over 10 min at room temperature using a fluorigenic peptide substrate at 25 μm. An apparent inhibition constant (Ki(app)) of 123 ± 48 pm was calculated using the Morrison equation (39, 40) in GraphPad Prism 6 (R2 = 0.979). Results represent the mean of three biological replicates with independent SPI-1 preparations. B, dissociation constant analyzed by MST. Labeled SBT4.13 (250 pm) was titrated against a serial dilution (1:1) of unlabeled SPI-1 starting at 12.5 nm. The baseline-corrected normalized fluorescence (Fnorm) is plotted against the SPI-1 concentration. A Kd of 546 ± 155 pm was calculated with GraphPad Prism 6 (R2 = 0.911). Results represent the mean ± S.E. (error bars) of three biological replicates using independent SPI-1 preparations, all run in technical triplicates.

Article Snippet: SBT4.13 enzyme kinetics A fluorigenic peptide substrate, Abz-YLPA↓GVPI-Tyr(NO 2 ) (PepMic, Suzhou, China), was used for kinetic measurements of SBT4.13 activity under steady-state conditions.

Techniques: Inhibition, Purification, Incubation, Activity Assay, Labeling, Serial Dilution, Fluorescence, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: A novel subtilase inhibitor in plants shows structural and functional similarities to protease propeptides

doi: 10.1074/jbc.M117.775445

Figure Lengend Snippet: Purification of the SBT4.13·SPI-1 complex. SBT4.13 and the SBT4.13·SPI-1 complex were separated by gel filtration. Fractions were analyzed by SDS-PAGE, and SBT4.13 activity was assayed using a fluorigenic peptide substrate at 25 μm. A, gel filtration of SBT4.13 (100 μg) monitored at 280 nm (gray line) and collected in 200-μl fractions. SBT4.13 activity was assayed in 0.5-μl aliquots (black dots) in two technical replicates. Fifteen-microliter aliquots of the same fractions were separated by 15% SDS-PAGE and Coomassie-stained as shown below the chromatogram. B, SBT4.13 (100 μg) incubated for 10 min in the presence of a 3-fold molar excess of SPI-1 was separated by gel filtration, and fractions were analyzed as described for A. mAU, milliabsorbance units.

Article Snippet: SBT4.13 enzyme kinetics A fluorigenic peptide substrate, Abz-YLPA↓GVPI-Tyr(NO 2 ) (PepMic, Suzhou, China), was used for kinetic measurements of SBT4.13 activity under steady-state conditions.

Techniques: Purification, Filtration, SDS Page, Activity Assay, Staining, Incubation