The protein was actually extracted and purified from the cells and used in the solution.
Perhaps there is an enzyme in vivo that reverses the reaction? The substrate for your enzyme might also be the substrate for an in vivo enzyme and the two compete. Or maybe your substrate is not crossing the cell membrane. If you could give more details about the reaction, it would help in finding a solution.
The reason for this disappearance in activity of your protein is due to metabolic reactions taking place in the living animal. Some metabolic reactions or pathways interfere with the activity of your protein.
Metabolic breakdown is more persistent in Vivo. Complex the protein with some stabilizing agent.
Check up this, it may help;
https://doi.org/10.1074/jbc.M117.792119
https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.life.illinois.edu/biochem/455/Lab%2520exercises/B-gal/enzymology.pdf&ved=2ahUKEwjHmMrctKDxAhUByYUKHZ-fDf0QFjAMegQIJhAC&usg=AOvVaw0-gTPGCSfC8xndgYvSusgL&cshid=1623993226281
The loss of specific activity of the Protein in vivo might be due to the binding of any modulator or other molecules to the protein of interest which is blocking the active site of the enzyme (protein).
I think in the in-vivo condition only, we can pull down the protein of interest to undertake whether any other modulator is attached with it or not. And to accomplish this, we can use an antibody against the protein of interest and then pull down.
Then perform centrifugation and get the precipitate for further studies.
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