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Things went smoothly with last night's and this morning's timepoints, overall. So now that's a wrap and I can focus more fully on other projects, like data analysis, woo!

It's both interesting and frustrating to recognize common effects of sleep deprivation. I lay down on the couch at 2:30 am, but didn't really sleep properly because I was pretty wired from the experiment. I got up at 4 am to take food away from a box of crickets, and then got up again at around 6:30 because I wasn't really sleeping anyway. The morning's experiment ran from 7:30-10:30.

I would have liked to have dug in more on some data analysis projects last night, and I did manage to get some stuff done, but I'm still doing too much task-switching these days. Add on tiredness and/or sleep deprivation, and trying to get work done is like trying to sludge my way through molasses.

That said, here are some interesting things I've been learning:

In conjunction with our measurements of nutrient oxidation rates, we've been working on quantifying the activities of some cherry-picked key metabolic enzymes. For example, way back in the day when you learned about glycolysis in high school biology, you might remember learning the names of the enzymes that catalyze the steps of glycolysis. In crickets where we see an increase in glucose oxidation rates, we'd like to know how increase oxidation is accomplished, under the idea that an increase in glucose oxidation indicates an increase in rates of flux through glycolysis. One method used by many organisms is to increase the activity of enzymes associated with that metabolic pathway, but in theory that end point could be accomplished in no small number of ways. Still, the first enzyme of glycolysis, hexokinase, is a fairly straightforward place to start.

So, how do you quantify an enzyme's activity? It's not particularly simple. You need to figure out a method that lets you quantify either the disappearance of a substrate or the appearance of a product, ideally over time. Fortunately, for common enzymes like hexokinase, lots of people have figured out clever ways to do this, and it's even possible to buy kits that take the guesswork out of figuring out the ideal reaction parameters.

Even with that out of the way, you still need to think about ways to standardize your measurements - hexokinase activity relative to what? The generally accepted standard method involves quantifying the total protein in your tissue sample. Dividing your rate by the protein concentration then gives you what's known as the enzyme's "specific activity." Note that you might get a very different result if you standardized based on your tissue sample's mass - more on that below.

"Specific activity" only gives you enzyme activity relative to the protein concentration. The next level is to multiply the specific activity by the total mass of the tissue being assayed, to quantify the total enzyme activity within that tissue.

Anyway, I've learned some interesting things in the process of quantifying hexokinase activity in cricket fatbody. Interesting thing 1: the protein concentration of reproductive (short-winged) crickets' fatbody is much higher than the protein concentration of the dispersal (long-winged) crickets' fatbody. If you think about this for a minute, it can make sense when you realize that the dispersal morph uses its fatbody as a storage depot for the lipids that are accumulated for endurance flight. If there are more lipids present per unit of fatbody, there will be a lower protein concentration.

But what about total activity? Yesterday, I finished collecting data for the second piece of the enzyme analysis: quantification of the total mass of the fatbody in each morph. That led to interesting thing 2: The dispersal morph has a larger total fatbody mass than the reproductive morph. Okay, maybe that's not too surprising if it's doing double-duty with its fatbody, using it for metabolic purposes AND using it to store lipids. Something else to think about, though, is that unlike mammals, insects have limited storage capacity within their non-stretchy exoskeletons. So this is where the rubber hits the road in terms of a flight-reproduction trade-off: if your fatbody is bigger (and the flight muscles are, too), something else is probably smaller. And that something else is the ovaries.

Anyway, with regards to hexokinase, there's a trend towards higher hexokinase specific activity in the dispersal morph. So given that the dispersal morph also has a larger fatbody, I expect the difference between the morphs to be exaggerated when considering the total hexokinase activity in the fatbody. That aligns nicely with our glucose tracer oxidation results, where there's a trend towards generally higher rates of glucose oxidation in the dispersal morph and a larger-amplitude change in oxidation rates across the circadian cycle.

It's not quite a smoking gun, however, because the specific activity of hexokinase doesn't appear to change much across the circadian cycle. So having constitutively higher hexokinase specific activity may help facilitate big changes in glucose flux, but it isn't modulating rates of flux.

So we'll proceed on to our next candidate.



( 4 remarks — Remark )
Mar. 23rd, 2017 05:26 am (UTC)
Having hexokinase isozymes would also help, or having a hexokinase that has -- what's the opposite of a noncompetitive inhibitor? A molecule that increases enzyme activity through conformational changes? much the way of positive feedback cycles, where the product of a cascade stimulates the first enzyme in the cascade. Trying to assess specific activity with those in play seems like it would be a headache.
Mar. 23rd, 2017 04:53 pm (UTC)
Hmm, the psychopharmacology equivalent would be an "indirect agonist."

In this case, we don't necessarily want/need to know the precise mode of action, and overall we have a stronger case for modifications happening at phosphofructokinase, so I think we're going to chase that rabbit next, instead. :^)
Mar. 24th, 2017 01:33 am (UTC)
How do you home in on rate-limiting steps? Competitive inhibitors?
Mar. 23rd, 2017 11:13 pm (UTC)
And of course, now that I'm doing some related reading: allosteric modulation, generally.
( 4 remarks — Remark )

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