In EROD assay is it mandatory to isolate the microsomal part for fluorometric determination ?

Famous Koshland theory was developed for water-soluble enzymes (99% of all)/ Some vital enzymes, like cytochrome P450s (CYPs), are embedded into phospholipid matrix of microsomal (endoplasmic reticulum) membranes. By definition, hydrophobic substrates of CYP (mainly, medicinal drugs), has to reach CYP active center thru lipid barrier and plasticity of CYP is still a question to answer.

From the practical experience I have observed, when I have added extra amount of tissue homogenate (more than recommended) to study any enzyme activity (for low expressed enzyme assay), the initial absorbance starts with very high value (some times nearer to 1) and never gets satisfactory result. Moreover, excessive amount of tissue homogenate makes the rection mixture colored (similar to tissue homogenate color).

So, what should the balanced amount of homogenate should I use for any enzyme activity assay?

How do the excessive amount of homogenate affect the reaction?

Thanks in advance.

I made some mouse liver microsomes in aliquots (each aliquot eas designed for one EROD assay, no reuse here) and stored them in -80 last year.  Their EROD activities were tested a few days after I froze them (two separate experiments, two aliquots, with good consistent results between these two).  Recently I took another aliquot out and re-tested it.  Surprisingly the activity was quite off (less than half of what I got last year).  I checked the instrument, the substrate (aliquoted and stored at -20, only for one time use), NADPH (freshly made NADPH was used all the time).  Can't find anything suspicious.  Tested a few of them and they're all lower than what they were last year, so I think it's really unlikely due to insufficient mixing during the process of making aliquots.

Was suggested to use a NADPH-generating system, instead of NADPH.  Ingredients tried:  4 mM NADP sodium salt hydrate, 4 units/mL G6DPH from yeast (Type VII, ammonium sulfate suspension, G7877 Sigma), 0.1 M G6P sodium salt (G7879 Sigma).  This is a recipe for 10X solution.  Aliquoted, stored in -80C, only took out in dark.  Didn't work at all (no reaction).

Does anyone know what may go wrong?  For both the EROD assay and the NADPH-generating system.  Thanks a lot!

I'm setting up GDH activity by a coupled enzyme assay in which glutamate is consumed by GDH generating NADH, which reacts with a probe generating a colorimetric (450 nm).

How do you prepare mammalian cells for enzyme assay? I used RIPA buffer for protein extraction but I did not find any GDH activity. Is it possible that GDH may be sensitive to the components of the RIPA Buffer?

Protocol for the reverse reaction. 

Hello everyone!

I have done an experiment in enzyme assay of alpha glucosidase by using pNPG ( p- nitrophenol-a-D-glucopyranoside ) as a substrate. This is my mixture reaction: 0.9ml phosphate buffer, 0.1 ml enzyme solution. After 10 min of reaction, I add 1ml Na2CO3 1M to stop the reaction and measure the absorbance of p-nitrophenol released by employing spectrophotometer (Abs 400nm). I got the OD values but how can I use these figures to calculate enzyme activit (Unit - U)?

Thank you for reading my question and I really appreciate if you can help me!!!

 i have transformed construct of  gene in Schizosaccharomyces pombe,now for enzyme activity i have to  isolate microsomes, as my protein is membrane bound.

 I know sigma protocol but don't know how to process the sample and deal with the product or the substrate already present in the sample. Also, is it necessary to purify the microsomal part to measure this enzymes activity or to use whole cell lysate?