Hello,
I'm trying to determined the Vmax, Km, etc...for a membrane bound short chain dehydrogenase. I am planning on using cell lysates as a source of the enzyme. Do you know of any protocol to carry out this experiment or have any other advice in these methods? I am new to enzymology!
Thank you,
Hi Gregory, in most cases the concentration of the enzyme [E] is not needed for determining Km, Vmax, etc. as long as the substrate concentration [S] is >> than [E], if the reaction is simply E+S -> E +P. (P being product)
But, you are working with a DH'ase, which probably requires cofactors.
Also, the problem with using impure enzyme preparations, like cell lysates, is that you have to know that you are measuring the activity of 1 enzyme and that is not influenced by inhibitors, allosteric modulators, cofactors and other enzymes in the cell lysates that bind/catalyze or modify your substrate/cofactors/enzyme.
In short, you can have an in-house assay for isolating the enzyme from cell lysates, but it will be very difficult to publish.
If you are using an extract of cells that you have transfected (or transformed if you're using bacteria) with the gene for the enzyme, then you can use an extract of the untransfected cells to measure the background activity from enzymes other than the one you transfected.
A potential difficulty with measuring the Michaelis constant (Km) of fatty acids is that the local concentration at the membrane, where the enzyme is, may not be the same as the overall concentration because the fatty acid can bind to the membrane.
Hi there,
Using a crude cell lysate extract as enzyme source for enzymatic assays will only allow you to determine apparent catalytic parameters which might be very different from the ones you would obtain with fully purified enzyme.
Thank you for the answers. I just want to titrate in amounts of crude lysate from WT cells and cells that have a dehydrogenase knocked out to see if there is a decrease in overall activity - apparent Km will be fine in this case. Then I want to add pure enzyme back into the reactions with the enzyme knocked out.
Hi Gregory, according to your aim, you will treat you samples complexly. So, you need to set up enough controls, e.g. knock out control, protein control. It is very difficult to determinate the Km value in a cell lysate system.
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