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Zymography Question

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Zymography Question

Postby RastaTings » Mon Apr 09, 2007 3:24 am

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 karthik38 » Thu Oct 04, 2007 10:20 am

This is Nice question, But I found out the enzyme wil eat the substrate, So in that place enzyme (MMP) only wil be present. So the molecular weight is either 57 or 45 Kda. By auto proteolytic cleavage the enzyme is activated during the incubation with developing buffer.
Refered wikipedia "A zymogen (or proenzyme) is an inactive enzyme precursor. A zymogen requires a biochemical change (such as a hydrolysis reaction revealing the active site, or changing the configuration to reveal the active site) for it to become an active enzyme. The biochemical change usually occurs in a lysosome where a specific part of the precursor enzyme is cleaved in order to activate it. The amino acid chain that is released upon activation is called the activation peptide".

So u wil see 45 Kda enzyme (activated) in the gel

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