Catalase and superoxide dismutase regulation

[cfmendez]

Hi everybody,

 

I'm new to this forum so I briefly introduce myself. I am a researcher at the Department of Biochemistry of the School of Medicine at Buenos Aires, working in the area of electrolyte transport in renal proximal tubule cells.

 

My project deals mainly with the effect of dopamine (DA) that acts as a physiological regulator of Na,K-ATPase activity in the proximal tubule. Now, I have confirmed that DA increases the concentration of reactive oxygen species (ROS) even at minute concentrations, a result that moved me to explore both catalase (CAT) and superoxide dismutase (SOD) enzyme activities. I have measured CAT and SOD in cells exposed to DA and found a time-dependent decrease for CAT and increase for SOD. I would have no problems with those results, except that the time frame is minutes of DA exposure. Let´s get this clear I see significant increases and decreases in SOD and CAT activities respectively already after 15 min.

 

I searched for articles in hope of similar results only to find descriptions of CAT and SOD regulation at transcriptional levels and after several hours or even days of stimulation. I may have miss the point, but the only reference I could find of use is one paper describing SOD regulation by phosphorylation in Listeria. I asked around and got a definite no for answer. Neither CAT nor SOD are subjected to activity regulation I've been told, and people see effects at mRNA level at much longer times than the ones I use.

 

One last detail. The times employed in my experiments are not arbitrary of course. DA regulation of NKA activity is a very fast process. Already after 2 minutes you see an effect, therefore all my experiments are done using that time frame.

 

The obvious question is: do my results make sense? I'll be most glad to read you comments and opinions.

 

Thanks in advance

 

Carlos

[CharonY]

You may want to check whether there papers out there that have time-course experiments on oxidative stress. I cannot point you to a particular paper off the top of my head, but I know that I read some years ago.

 

From a biological point of view expression changes of proteins within minutes is quite reasonable time for protein turnover. It depends on the specific stimulus and the respective underlying regulatory mechanism, of course.

 

However, from my own experiments (again, years ago) I saw mRNA level changes in bacteria during oxidative stresses well below one minute (basically below time resolution). The effect thereof on protein levels is cumulative.

 

The question that I have is in which time frame you expect DA exposure to result in oxidative stress (and by which mechanism). Direct exposure to oxidative stress (e.g. using paraquat or H2O2) only takes minutes to result in observable expression changes and does not require activity attenuation for that time frame.

[cfmendez]

Hi CharonY,

thank you very much for your reply. I'll try to answer your questions and comments.

 

From a biological point of view expression changes of proteins within minutes is quite reasonable time for protein turnover. It depends on the specific stimulus and the respective underlying regulatory mechanism, of course.

I agree although I believe that's certainly true for proteins exhibiting a high turnover rate. Is this the case of CAT and SOD?

 

The question that I have is in which time frame you expect DA exposure to result in oxidative stress (and by which mechanism). Direct exposure to oxidative stress (e.g. using paraquat or H2O2) only takes minutes to result in observable expression changes and does not require activity attenuation for that time frame.

As judged from experiments where I measured ROS levels using DCHF and by ERK1/2 activation the answer is 5 minutes or less. I think the mechanism behind oxidative stress involves DA biotransformation to DA-quinone and aminochrome. But, again, is it reasonable to observe changes in activity? What would happen to CAT and SOD during the initial phase of oxidative stress? At the end of the day I feel that is the true question here.

[CharonY]

Regarding the turnover: yes the regulation of oxidative stress response is usually fast. However, while it explains the increase of SOD activity, it would not explain catalase decrease. Usually enzymes like this are allowed to linger around before being degraded.

If you really want to figure out whether there is some kind of modulation of activity as opposed to enzyme abundance change, I would advise you to quantify the SOD and catalase content directly. The easiest ways is mass spectrometrically, if you got access, or via Western. qPCR would be even faster but it is not trivial to translate mRNA changes into actual protein abundance changes.

Also, as a matter of habit re-validate the assays, and see how significant the differences really are.