N-Terminal Deletion of Peptide:N-Glycanase Results in Enhanced Deglycosylation Activity
Figure 1
Enzymatic properties of Png1p-Rad23p complex.
(A) Comparison of deglycosylation activity between Png1p and Png1p-Rad23p complex. Png1p and Png1p-Rad23p complex were incubated with RNase B (0.5 mg/ml) in 30 µl of 50 mM Hepes buffer, pH 7.0, and 5 mM DTT at 30°C. (B) Influence of temperature on Png1p-Rad23p complex activity. Png1p-Rad23p complex was incubated with denatured RNase B (0.5 mg/ml) in 30 µl of 50 mM Hepes buffer, pH 7.0 at different temperature for 1 h. (C) Influence of pH on Png1p-Rad23p complex activity. Png1p-Rad23p complex was incubated with denatured RNase B (0.5 mg/ml) in 30 µl buffer with different pH at 30°C for 1 h. (pH 5.0–8.0, Na2HPO4- Citric Acid; pH 9.0 and pH 10.0, Gly-NaOH). All proteins used in the assay were purified at the same time, following the same protocol. The molar ratio of enzyme to substrate was 1∶30 in each reaction. Samples were taken at the indicated time points and subjected to SDS–PAGE followed by Coomassie staining. The zero time point was taken prior to addition of Png1p-Rad23p complex. The resulting Coomassie stained gels were quantified by densitometry with Image J program.