Enzyme Activity - efficiency of inhibition (Jan/05/2008 )

hallo,

i work with reversible inhibitor of proteasomes.

how can i be sure that proteasome is still effective inactivated when i prepare my cell lysate?

can lysis somehow interfere with my inhibition?

how will i know how effective my inhibition really is?

any suggestions are helpful

thanks

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What kind of lysis-buffer do you use ? what kind of downstream-applications are you gonna do ?

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commercial protease inhibition kits are designed and tested for efficiency (I hope); if you have doubts, you have to check protease activity with specific tests (by zymographs f.i.)

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It is no commercial kit. i use a drug used in cancer therapy, which mechanism of action is to inhibit the chymotryptic activity of the proteasome.

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homogenization/lysis buffer: (180mM KCl, 5mM MOPS, 2mM EDTA) pH7,25

Application: test the inhibition efficiency by cleavage of substrate (measurement 405nm)

For all further analysis i have to know the inhibition efficiency!

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to determine the inhibition efficiency you need to perform a competitive assay. then you will know what concentration of inhibitor you will need to inhibit in the presence of the concentration of substrate you will have in the preparation.

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it´s an in vivo application of inhibition. inhibitor concentration for in vivo application is limited (can not be varied).
i only want to test proteasome activity in different organs of non treated animals (100%) compared to treated ones. given as substrate cleavage/protein [nM/µgprotein].

Q is, how lysis and further treatment of tissue interferes with reversible inhibition?

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as long as inhibitor is present inhibition will continue. if you remove inhibitor in later stages then inhibition will end and enzyme will become fully active.

in other words, reversible inhibitors don't bind to the enzyme permanently.

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thank you mdfenko,

OK understand, and remember i heared it time ago. enzymekinetiks and so...

which means that in the case my inhibitor is present in the homogenate the inhibition takes place. but not when inhibitor is destroyed somehow.

another problem takes place during measurement of substrate cleavage: when i measure the same plate twice i get differences in my values. could this be due to temperature changes? could i somehow quench reaction to get reproduceable values? in some publications they quench in others they don´t.

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reactions tend to continue unless stopped (or quenched). you may want to use one of the methods you found in the literature.

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