I have extracted protease from culture filtrate of Fusarium isolates (n=14) grown in PDB media. For partial characterization, I checked activity at different pH, temperature and in presence of inhibitor. Surprisingly, the activity of proteases in some isolates increased in presence of 0.5mM PMSF inhibitor (inhibitor of serine protease). Later, I increased concentration to 5mM (ten fold increase) but did not see reduction in activity. The same was observed in 0.1% gelatin zymography. However, inhibition of activity is seen with EDTA and Pepstatin A inhibitors. But I could not find possible explanation of increase in activity in presence of PMSF. Looking forward for some logical information. Thank you so much.
Pepstatin A is an aspartyl protease inhibitor, so maybe that explains why your proteases were not inhibited by PMSF. Your increase of activity was significant? What order of magnitude? Was your PMSF solution new or had already been prepared and stocked?
The increase in activity of your protease may have been due to the solvent that the PMSF was dissolved in. Did you perform a solvent control?
May be your fungal protease is a metallo protease as it's activity declined with EDTA. PMSF is a functional inhibitor of serine protease. Does SDS also inhibits the fungal protease?
Swati Madhu have you tried any other serine protease inhibitor? because increase in activity in the presence of PMSF but a decrease in activity in the presence of EDTA suggests the fungal enzyme belongs to another class of protease, perhaps, Metalloprotease or an Aspartyl protease since it was inhibited by Pepstatin.
Jorgaq Pata thank you so much for reply. There was significant increase in activity in presence of PMSF. In one isolate, the activity increased 2 fold. for eg: without inhibitor activity was 50 units/mg, in presence of PMSF, it increased to 101 units/mg. I used freshly prepared PMSF (i made it by dissolving it in Isopropanol, and stored at -20 °C). It might be possible that my fungal isolates are not producing serine protease but how does twofold increased activity i am getting repeatedly? Is there anything that PMSF is doing which causes increase in activity compared to control?
Adam B Shapiro Thank you for reply. I dissolved inhibitor in Isopropanol and i did not perform solvent control. May be i can do that. Thank you for suggestion.
Shamsher S. Kanwar Thank you sir. Ya it might be possible that it is not serine protease. But how can PMSF cause two fold increase compared to control? I did try SDS, but these proteases were not inhibited by it.
Adeyemi Oluwadare Ayodeji Thank you so much for reply. I did not tried other serine protease inhibitor but i tried increasing inhibitor concentration upto 10 fold. But still I found increases activity compared to control.
Swati Madhu probably isopropanol is doing that, carry on proper controls as it was mentioned before to check whether your buffer is stimulating protease activity. Just add your buffer without PMSF to see if there is any activity change.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides