LDH Isoform in Rat species; an outlier in gel electrophersis determination of phylogeny

A small portion of a project of mine requires I analyze the distribution of LDH isoforms obtained from gel electrophoresis. Gel contained samples from numerous animals, including cow, sheep, goat, horse, human, chicken and rat. While the distribution of LDH isoforms between the numerous animal species were mostly consistent, varying mainly in concentration of specific isozymes according to species, and hardly changing in relative position, Rat serum resulted in a pattern very different others. Lane of gel corresponding to rat showed LDH isoform positions shifted drastically higher than might have been expected; this was especially noticeable in LDH5 isoform, which was fairly high above the injection well as it traveled towards the cathode.

Assuming LDH isoforms are distributed relative to other species according to phylogeny, we’d assume that human and rat should have the closest gel patterns; our results don’t show this at all.

I’m not looking for a thorough analysis of data (I certainly didn’t provide enough information to allow for such). I merely am stumped as to where to find an explanation for this variability. Scholarly databases don’t seem to have the specific information relevant to me. I’m hoping there may be some wise biologist types that have experience with this obscure subject. I’m rather frustrated with my efforts so far.

How many times did you run the gel?

We actually only ran one gel; however, amongst the other groups rat was consistently found to have higher LDH5 positions compared to other animals. Professor also verified the validity of this result, but did not clue us in onto why. Like I said, I haven’t been able to find any literature that explains this specific result.

One (entirely made up) solution may be that the LDH5 in rat is somehow more positive than in other species, resulting in it traveling higher towards the cathode. However, as far as I know the LDH5 isoform has a consistent structure amongst the mammalian species; I don’t understand why it would have a different charge / migration pattern.

This is a tragically specific question, I’m not expecting much.

It could be, for some reason, that there’s been a big evolutionary shift of that particular protein specifically along the rat lineage. Have you looked at the DNA alignments? It actually wouldn’t take a whole lot to change it - just a few charged amino acids one way or the other.

Edit: The UCSC Genome Browser is a great tool for looking at evolutionary conservation between species: genome.ucsc.edu. I don’t know the exact name of the gene you’re looking at, or I’d check it myself.

It’s a pain in the butt, however I would BLAST the rat and one or two other LDH isoforms and perform an alignment. If the rat isoforms have many more amino acids, it will show up in the alignment and explain the upward shift. If not, you may have carrier proteins or dimerization occurring. I assume you ran a denatured gel, and that you stained the gel to see the bands, as opposed to blotting and probing with an antibody (which could have funky cross-reactivity between species).

My first guess’d be some post-translational mod in the rat that adds a few charged groups. That’s a lot simpler than mutation across an entire gene family.

You’re running isozymes? That’s so 1995…

Lots of things can give funky patterns for isozymes - maybe the rat has a charged amino acid that is changing the banding pattern.

Why not use the DNA sequence? Isozymes are crap for phylogenetics in any case.

Since you are separating isoforms of LDH and refer to LDH5 I am assuming you are running native PAGE or something similar, as a denaturing process would only resolve the M and H isoforms from one another. If everything is working as it should, the simplest explanation is that differences in the sequence for one or both LDH subunit types affect migration. A quick look at the protein sequences for the M subunit (encoded by LDH-A gene) shows that both human and rat forms have similar isoelectric points, so that is unlikely to be the reason. Perhaps the simplest thing (other than re-running the gel) would be to do mass spec on the various bands and see if they are indeed what you think they are. This would also allow you to confirm that their molecular masses are not different due to some posttranslational modification, which strikes me as unlikely but not impossible. By far the most common posttranslational modification affecting migration in my experience is glycosylation, which generally doesn’t occur for intracellular proteins.

Sorry I can’t offer anything more definitive. If I were in your shoes the first thing I’d do would be to repeat the experiment, followed by mass spec if the results are the same as before. Western blotting against M and H isoforms separately may also be useful if you have the necessary antibodies available.

Right you are, and I think the tid bit of info regarding similar isoelectric points for both Rat and Human is actually consistent with our results. We actually weren’t able to visually detect any LDH5 from our human sample; this may be attributed to a lower concentration of LDH5 in humans, especially in serum. So that would mean that, while human and rat have similar Isoelectric points for M subunits, we weren’t able to make this correlation because human LDH5 was not in high enough concentration for comparison.

Impressive you were able to help me with so little information. Much respect.

Any chance you can run mass spec on the sample and see what you’ve actually got in there? LDH isn’t too large, it could be reasonably analyzed Top Down.