detecting enzyme activity/presence

beachbum · Post by **beachbum** » Wed Mar 01, 2006 4:59 pm

Hi, I am conducting an experiment where I'm trying to determine if caspases are present in this type of yeast. I don't know how to measure the presence of caspases though and all I have seen so far is by flow cytometry. Are there simpler ways to detect caspase activity without using any kits or could you analyze caspase activity by measuring growth on a medium? If there aren't any other ways, how could I do the experiment by using flow cytometry? Thankyou.

carolinethorn · Post by **carolinethorn** » Thu Mar 02, 2006 2:17 pm

Hi,

Lets review a couple of things:

Caspases are a type of enzyme that cleave or cut proteins (proteases). Often they cleave other caspases as part of the cascade of programmed cell death or apoptosis. So the "substrate" for the reaction can be a caspase or another protein that looks like a caspase.

A common way to measure an enzyme reaction is to make a crude cell extract, by "lysing" your yeast cells which just means breaking the outer membranes but leaving everything inside working in the "lysate". The lysate contains your enyzme you can add a substrate and measure the products formed. Often we use a sustrate that has a coloured or fluorescent tag that can be measured using a spectrophotometer (can see quantity of reaction - quantitative test) or by the appearance of colour in the reaction tube (can see if reaction has occured or not - qualitative test). Its more difficult to measure products that do not have a tag but it is possible but you would need a lab equipped to do western blotting. So the most straightforward way would probably be to use a kit with a tagged substrate.

You could also use an inhibitor of caspases to show that the reaction is occuring because of caspases in your cell extract and not another kind of protease enzyme. There is a well known capsase inhibitor called zVAD.

As far as i know there is no way to measure this using growth on a medium but I am not an expert on caspases just someone who teaches biochemistry and the general process of apoptosis.
If you can find a local university or college lab who study apoptosis they would have some of the substrates that you would need and might be able to help you on this.

best of luck,
Caroline

beachbum · Post by **beachbum** » Thu Mar 02, 2006 2:25 pm

Thanks so much for your help Caroline. It really did help alot! Thanks again.

beachbum · Post by **beachbum** » Thu Mar 02, 2006 3:58 pm

I actually have another question. First of all, is a crude cell extract the same as a lysate buffer? Secondly, if I added the crude cell extract and lysed the cell, would the cell still function as normal?

I am also adding a section of my study where I am to promote nitric oxide (NO) synthesis in the yeast cells. Currently, the known NO inducers are a wide variety of cytokines (which aren't present in yeast, or at least not discovered), and lipopolysaccharide (LPS). LPS seems like the best bet to use, however, it is highly unlikely that LPS can penetrate through the yeast cell wall and no articles have been published regarding this topic. If I lysed the cells and only analyzed the cell lysates, which would eliminate the cell wall pentration factor, would it work? Even if I did lyse the cells, would yeast demonstrate senstivity towards the addition of LPS or do you know of any other NO inducers that can be used in yeast cells?

Thankyou so much for your time.

carolinethorn · Post by **carolinethorn** » Thu Mar 02, 2006 4:19 pm

crude cell extract is not the same as lysate buffer. a buffer is a solution that helps stabilise a solution by balencing ions, so lysate buffer is a buffer solution that can be used to stabilise a lysate. so lets say you have a flask of yeast cells in liquid culture medium. you would prepare the crude cell extract by centrifuging your suspension of yeast cells gently and removing the growth medium then adding the lysate buffer. the lysate buffer might have detergent in it to break open the cells or you might shear them open with passing them up and down through a needle or using a sonicator machine.

the cell extract will not function in exactly the same way as a whole cell but it can do some limited things - like enzyme reactions - for a short period of time if you keep it on ice so it doesnt degrade.

the way that cytokines and LPS induce NO in mammalian cells is by increasing the activity of the nitric oxide synthase gene. I had a quick look at the Yeast database and they don't seem to have a nitric oxide synthase gene. http://www.yeastgenome.org/
Also in mammalian cells, this occurs via a signalling cascade that starts from the cytokine or LPS receptor at the cell membrane so may not work if the cell is not in tact.

Can you explain why you are trying to generate NO to see if we can find another way around this?
Also some more general aims of your project would be useful,

best of luck,
Caroline

beachbum · Post by **beachbum** » Thu Mar 02, 2006 5:16 pm

Thanks. The main objective of my project is to identify the role of NO in PCD. Although the Saccharomyces genomic database doesn't list an NOS gene, a constitutive NOS (cNOS) gene has been identified http://www.ncbi.nlm.nih.gov/entrez/quer ... t=Abstract

The experiment has worked, I used a nonspecific NOS inhibitor and a NO scavenger to inhibit NO generation and no cell death occured. My regional science fair is already over but I have qualified to the next round and a judge suggested determining the effects of inducing NO in PCD, but what I am having trouble now is with finding a NO inducer.

carolinethorn · Post by **carolinethorn** » Thu Mar 02, 2006 5:36 pm

What a great project! Thats really cool that you got results with the inhibitor and the scavenger. Now I understand why you want to induce the nitric oxide synthase.
The paper you linked describes the yeast NOS as a constitutive enzyme which suggests that it is active all of the time and may not be induced by anything.
However I did find an article that talks about an inducible yeast NOS
http://www.ncbi.nlm.nih.gov/entrez/quer ... med_docsum
They used pressure to induce it - don't know if thats an option for you?

Congratulations on getting to next round of the science fair.
-Caroline

beachbum · Post by **beachbum** » Thu Mar 02, 2006 6:33 pm

Caroline,
Thanks so much for all your help and the links you have provided. I really didn't expect so much information! Thanks for all your kind comments and its been really nice of you for spending all the time answering my questions. Thanks again!

Amy.