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Zymography Question

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Zymography Question

Postby RastaTings » Sun Apr 08, 2007 8:39 pm

Ok, so I think this would apply to cell biology (it's from a cell biology class). I recieved this questions on an assignment

Ductal elongation in the mammary gland is dependant upon the action of MMPs. In order to identify if MMP3 is active during secondary branching, you prepared extracts from mouse mammary glands undergoing secondary branching morphogenesis. You then resolved the proteins on collagen-based polyacrylamide gel and performed full zymogrpahy.

intact collagen 540 kDa
cleaved collagen fragments 340 kDa and 200kDa

Pro-MMP3 57 kDa
MMP3 45 kDa

A) What size bands were visible in the gel?
B) WhY?

Ok so, if I remember correctly zymography is where the gel is the substrate. So in this case I'm pretty sure it's collagen. So because of this MMP3 should be cleaving the gel, so for A) in the answer is 340 and 200 kDa bands? This seems way too simple of an answer. I checked over my course text book and there is nothing on zymogen in there. If someone could point me in the right direction it would be greatly appreciated, thanks for your time!
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Postby blcr11 » Mon Apr 09, 2007 9:47 am

A zymograph is a gel which includes substrate, not that the gel itself is the substrate for an enzyme. After running the gel to separate the different sized proteins in the sample, the running buffer is replaced by a suitable buffer for enzyme activity and allowed to react for some period of time. The gel is then stained for protein. Light bands against a dark background show up in regions of the gel where there was enzyme activity during the incubation.

A zymogen is simply the "inactive" precursor of an enzyme, or the proenzyme; for example, chymostrypsinogen is the proenzyme of chymotrypsin; chymotrypsinogen is activated by proteolysis to yield the active enzyme. I say "inactive" because some proenzymes can self-activate--others require an external factor of one sort or another.
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