I am investigating the function of ACC synthase isolated from various vegetables such as pumpkin, melon, and cucumber. So, I initially isolated ACC synthase genes and purified ACC synthase proteins from E. coli. After that, I checked the ACC synthase activity in accordance with the method described in the papers (Boualem et al., PLoS One, 2009). But, I failed to get the meaningful data from my pilot experiments; I didn't find the difference of activity between CsACS2 and mutated CsACS2.
The important difference between my and Boualem's protocols is that I didn't use Aspergillus adenosine deaminase, because there was not available from Sigma. So, I used other adenosine deaminase Streptococcus thermophilus. Also, I didn't partial purification of this enzyme, because I didn't know the activity of adenosine deaminase that I bought. Instead of purification, I increased the concentrations of this adenosine deaminase in the reaction.
So, I am wondering how to check the activity of ACC synthase using the substitute of Aspergillus adenosine deaminase and some important tips to check the activity of ACC synthase.
If anyone have a experience in checking ACS activity in vitro, please let me know.
Thank you for your help in advance.
I speculate that you are seeing only background activity due to the use of an impure coupling enzyme (adenosine deaminase, ADA) to convert the methylthioadenosine (MTA) product (derived from S-adenosylmethionine, SAM) to an inosine derivative, with resulting UV absorbance change. In other words, you are seeing only the activity of some enzyme contaminant in the ADA.
Another way to test for the activity of purified ACS would be to leave out the ADA, and directly measure the conversion of SAM to MTA using a reverse-phase HPLC system to separate and quantify them by their UV absorbance at 254 nm. If that works out, you may then decide it is worth the trouble to purify the ADA.
Dear Dr. Shapiro,
I will try to check ACS activity without addition of ADA.
Thank you for your kind comment.
Dear Dr. Shapiro,
As you suggested, I did check MTA level using HPLC system. I successfully detected MTA level. The problem is AFA.
Thanks!
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