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Enzymes kinetic'd

In addition to finishing my first quilted ant, today I set up my first enzyme kinetics assay. We have a whole list of different enzymes we want to examine, but we have to start somewhere, so I figured we'd start with what should be the easiest, citrate synthase.

In preparing for this assay, I came across a paper from 2001 where a group of exercise physiologists set out to look at the effects of acute exercise on citrate synthase activity in trained vs. untrained human skeletal muscle. Here's how the study worked, in a nutshell: they took a set of six guys (yeah, these things are always on guys, right?) who were initially characterized as "sedentary." In the initial phase, they had the participants perform an "acute knee-extensor exercise bout with their left leg," and then right after that they took muscle samples from both legs and measured citrate synthase activity. Then they had these participants complete an individualized 8-week left leg training regimen with their knee extensor machine (1-hour sessions, three times a week). Then they repeated the acute exercise bout and took another set of muscle samples.

I am going to refer to this paper as "the skateboarder study" because I once read about a guy who skateboarded across Australia and wound up with one leg/calf much stronger than the other. I hope those guys were compensated well for the whole procedure.

Anyway. My mentor from Nebraska gave us a ton of advice for how to validate our enzyme assays, but it's one thing to read through the methods for these things and a completely different thing to actually sit down and go through the procedure. Basically, as soon as you go from living tissue in an animal to a sample in a test tube, you have to be very careful about how you're handling things to ensure the sample remains representative of what happens in the living animal. In my case, that meant assembling a game plan for: dissecting crickets, quickly homogenizing tissues to extract enzymes, mixing up the reaction cocktail, and measuring the reaction under precise conditions. Oh, also, all of the reagents involved are expensive and come in tiny quantities, so you have to know how to work with small masses of things without royally screwing up. No pressure, eh?

Long story short, my first attempt was about as successful as it gets. I ran both a negative control that stayed negative (no reaction occurred in the absence of the enzyme), and a positive control that was positive (reasonably good reaction when I added a known quantity of expensive purified enzyme). In addition, I ran some samples from two crickets. The cricket flight muscles behaved beautifully. The cricket fat body samples did not behave, nor did the leg muscle samples. Puzzling, but not insurmountable.



Apr. 15th, 2016 06:15 am (UTC)
Re: The skateboarder study, that sort of change can be long lasting, too. My sisters one butt cheek is still much bigger than her other one, because for 6 months or so, it was the muscle she used to move that leg. This was almost 10 years ago, now.

(Yes, I make fun of it occasionally. Because I'm a proper big sister.)

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