Dual Function of Novel Pollen Coat (Surface) Proteins: IgE-binding Capacity and Proteolytic Activity Disrupting the Airway Epithelial Barrier
Figure 5
Total proteinase activity of pollen coat proteins from Bermuda, Timothy, and Johnson grass assessed by gelatin and casein zymography.
Bands with proteinase activity were visualized as white clear or light lytic zones against a dark background of Coomassie-blue-stained gel (A, gelatin) and (B, casein). On the left, molecular mass markers in kDa are indicated. (C) Pollen coat proteins from Bermuda grass. MW markers were on the left lane, and crude extracts (Ext), concentrated with 80% Acetone (Ac) and with 70% ammonium sulfate (Am), pure cysteine protease (pure, 5 and 7.5 µg/lane) and trypsin as a positive control are shown. The gelatin zymography gives a representative image from 4 separate studies that yielded similar results. (D) Protease activities in pollen surface proteins from Bermuda, Johnson, and Timothy grass corresponding to the lanes in (A) and (B) in the upper panels. The protease activities of pollen surface proteins, equalized for protein concentration, were examined for proteinase activity by use of a chromogenic substrate. The activity was recorded and expressed as units of enzyme activity per milligram of total protein. The cysteine protease colorimetric assay shows significantly greater proteinase activity in the proteins extracted from the Bermuda grass pollen surface compared with proteins extracted from Johnson grass or Timothy grass pollen surface. Bars represent the mean of proteinase activity units of four replicates.