I am having a problem by assaying ATP-citrate lyase (ACL) continously. I had the validation with and without cell free extract (CFE) in my reaction mixture. The result is that in the absence of CFE, the exogenous activity is 0, but after its addition, the gradient drops abruptly and very steeply. So I supposed my CFE has some enzymes that interfere with my assay.

In fact, in my assay, there is also azide. I try to remove azide but the result are same as the assay with azide. So I supposed azide is nonfunctional in my assay.

After done some readings, I felt that the problem came from the enzyme that actively consume NADH, which is the complex I of the respiratory chain (NADH-CoQ oxidoreductase). Since azide only inhibits the complex IV which does not consume NADH, I felt that the addition of azide is not helpful.

I found out that rotenone and amytal inhibit the transfer of electron from FMN to FeS in complex I but not the transfer of electron from NADH to FMN. That is why i am kind of insecure whether the addition of those inhibitors is helpful to eliminate consumption of NADH. This is to allow only ACL to consume NADH so that my assay can be conducted.