Most Peculiar Problem with an enzyme activity - 4 -years of nothing (Jan/20/2005 )
I am chasing a decarboxylase. It is measured by release of radioactive carbondioxide trapped on suitable filters. The bacterial system I am using- I never got any activity from cell extracts, supernatant, membrane fraction, cell free supernatants - nothing. The end product is there which means the enzyme must be there too.
One day I used intact cells and boom! There was activity. Lots of activity. I fractionated the whole cell batch - membrane/cell wall/CFP/cytosol/ everything. The activity disappeared once again. What is happening? where and how is the decarboxylase functioning from and why is it going away just like that?
The system is prokaryotic Mycobacterium. I need your imagination, not your expertise, so please feel free to post at will.
Maybe during the extraction process you are inactivating the enzyme.
do you think it will be located at places such as mitocondia or peroxisome? i guess that the traditional extraction methods are often so crude that you won't get your enzyme. not to mention that the activity could be lost during the process.
have you used any gfp or staining to check the localization of your enzyme? have you also used western blot to detect if there is any target in your extract? it could be that you didn't get the protein in your extract at all. if you did get your protein, then it is the question how to not let it become latent.