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Enzyme activity assay


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#1 Carpetto

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Posted 07 October 2010 - 03:00 AM

Dear everyone,

I hope this topic is right here. If not, please let me know.
I want to apply HMG-CoA Reductase inhibitors (statins) to cells (non hepatic). To study successful treatment and inhibition of the enzyme, I would like to analyse the activity of the HMG-CoA Reductase. I found this kit on the sigma homepage (see attachment), but it is for a purified HMG-CoA Reductase enzyme, only - so it may does not help.

HMG-CoA Reductase Assay Kit
Catalog: CS1090

There are also some assays that use 14C-HMG-CoA (HMG-CoA - the substrate) in cells. But, I cannot apply a radioactive method. This method includes a ultra-centrigation step to enrich the transmembrane proteins such the enzyme of interest. My idea is, according to the assay of sigma, to use the sample preparation of the radioactive method, but instead of radiolabelled HMG-CoA; I use normal HMG-CoA and measure the reduction of NADPH via A340. But, does this give a specific signal? There are many other proteins using NADPH as well. I guess because auf the sample preparation process they lack of they substrate. So then I add HMG-CoA as the substrate and NADPH, another protein is not using up NADPH? Or are there any proteins just using NADPH without any other substrate? Has anybody else other ideas/protocols to measure the HMG-CoA-reductase activity?

I very appreciate your suggestions, help or any comments!

Thanks a lot!

Carpetto

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#2 Paraboxa

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Posted 08 October 2010 - 08:35 AM

I imagine the manufacturer's wont support use of their kits in non-standardised methods. This may have a knock-on effect if you want to publish this work. The rest of the research community has to be able to repeat what you have done. That aside, if you use positive and negative controls in your experiment, you will be able to analyse "background" NADPH changes due to proteins other than yours.



Dear everyone,

I hope this topic is right here. If not, please let me know.
I want to apply HMG-CoA Reductase inhibitors (statins) to cells (non hepatic). To study successful treatment and inhibition of the enzyme, I would like to analyse the activity of the HMG-CoA Reductase. I found this kit on the sigma homepage (see attachment), but it is for a purified HMG-CoA Reductase enzyme, only - so it may does not help.

HMG-CoA Reductase Assay Kit
Catalog: CS1090

There are also some assays that use 14C-HMG-CoA (HMG-CoA - the substrate) in cells. But, I cannot apply a radioactive method. This method includes a ultra-centrigation step to enrich the transmembrane proteins such the enzyme of interest. My idea is, according to the assay of sigma, to use the sample preparation of the radioactive method, but instead of radiolabelled HMG-CoA; I use normal HMG-CoA and measure the reduction of NADPH via A340. But, does this give a specific signal? There are many other proteins using NADPH as well. I guess because auf the sample preparation process they lack of they substrate. So then I add HMG-CoA as the substrate and NADPH, another protein is not using up NADPH? Or are there any proteins just using NADPH without any other substrate? Has anybody else other ideas/protocols to measure the HMG-CoA-reductase activity?

I very appreciate your suggestions, help or any comments!

Thanks a lot!

Carpetto






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