Can anyone help me get the exact readings using a standardized protocol for an AGPase enzyme assay in seeds of wheat at my laboratory?

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Konstantin Yu. Kulichikhin

We used spectrophotometric assay of AGPase activity. You do not need radioactive substances for it.

Ravi Mehndiratta

We are performing the enzyme assay at 4 C and at 4 minute interval using ice cold condtions. There is question mark that Dont u think the enzyme will loose activity at 30C in the protocol suggested by you and is the protocol valid for wheat too ?

This protocol is for wheat. We adjusted it for wheat and we used it for wheat. Enzyme activity is measured usually at +25 or at +30 (it depends on the enzyme). At +4 you will never get the activity: temperature is too low. Extraction should be at +4 in order to avoid heat inactivation of enzyme.

We are doing the experiment at 4 minute time frame at 15 second interval. Does your reading come in increasing order with the time frame ? Whats the role of ppi in this experiment? We are using PGA instead of ppi? Should we change it ?

Could you, please, send me your protocol?

PPi is a substrate of enzymatic reaction.

AGPase catalyse the reaction

ADPG + PPi = ATP + Glc-1-P

Sir please see the enzyme assay protocol attached ...

Thank you, sir, I got it. Could you, please, write me full reference of Kleczkowski et al. (1993)? If you have pdf file of this article that would be the best. I will be back in few hours..

I found two manuscripts by Kleczkowski et al 1993. The files are attached. Please, give me some more time to write you back..

Sure sir . Th first pdf file you have mailed is the protocol we are following .

I added some brief comments to your protocol. Please find the file attached. If you have any additional questions, please write me back.

Sure sir . Thanks for your time . It is really appreciated . I will read the protocol in detail and any further queries would be readdressed to you .

Just a little correction and remarks to the protocol (new and corrected text is highlighted by red) and original manuscript of Nakamura et al. 1989/..