I used inhibitors with different concentration EDTA and PMSF of 1 and 5mM, in the case of EDTA no activity was detected at 1 and 5mM, while in case of PMSF at 1mM activity was detected and no activity was detected at 5mM.
I know that the working concentration of PMSF is 1mM, but I just want to confirm that 5mM PMSF cannot be used?
PMSF inhibits serine proteases (like trypsin, etc.); EDTA metalloproteases. It is quite common practice to include EDTA at 5 mM and PMSF at 0.5 mM, NOT because there is a protease that requires both, but because there are many different proteases present in crude tissue brei. (See e.g. http://www.biosynth.com/index.asp?topic_id=194&g=19&m=226)
For any inhibitor or activator working concentration will be there, if it is excess it wil inhibit. wen it is inhibited by EDTA ur enzyme is metalloprotease.
If the enzyme is inhibited by EDTA it NOT mandatory the have the metall ion in the active site (a metalloprotease). For example, for Proteinase K it is published that this serine protease is inhibited by EDTA, because it has two Calcium binding sites. The stronger binding site stabilizes the tertiary structure of the protease. Without bound Ca-ion the protease becomes meta-stable.
PMSF inhibits serine proteases (like trypsin, etc.); EDTA metalloproteases. It is quite common practice to include EDTA at 5 mM and PMSF at 0.5 mM, NOT because there is a protease that requires both, but because there are many different proteases present in crude tissue brei. (See e.g. http://www.biosynth.com/index.asp?topic_id=194&g=19&m=226)
EDTA chelate cations and PMSF acts on serine residue and block , so your protease could be a metalloprotease with a serine residue near active site .So the PMSF inhibit this protease
There are Serin proteases which activity also depends of calcium. So in your case I think it is a Serin protease because is inhibit by PMSF but also binds calcium important for its activity. To test and eliminate the posibility (I doubt that it is) that is a metalloprotease test it with an specific inhibitor specific for metalloprotease as 1,10-phenanthroline (soluble in methanol when you prepare the stock solution) and if you can test it also with other kind serin-proteases inhibitors like benzamidine, aprotinin, TPCK, TLCK will be better. Although EDTA is considered an inhibitor of metalloproteases it is not very specific.
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