Just to clarify, from what I have read, leupeptin should not inhibit your MMPs in the gelatin zymography assay? You just wondering what it is doing to case multiple bands?
Is it possible that leupeptin is inhibiting another protease in your system that is involved in processing the MMPs? In the reference below, it mentions a system where they see multiple forms of MMP2. This might explain why you are seeing larger bands, however, it doesn't really explain the presence of the smaller bands.
Is it possible the smaller bands are a result of incorrect processing by yet a another protease or maybe an autocatalytic event that only occurs when the correct protease is inhibited by leupeptin?
Do antibodies exist that would allow you to do a Western to test whether the four bands are just different forms of the same MMP? You could compare the MW weights with those observed from the gelatin zymography.
If so, you could see what happens when you add leupeptin and EDTA or combine leupeptin with another protease inhibitor. Under these conditions, are all four bands still present by Western blotting? If you only see one band with leupeptin and EDTA, maybe the MMP has autocatalytic activity when incorrectly processed.
reference: Functional Interplay between Type I Collagen and Cell Surface Matrix Metalloproteinase Activity. J Biol Chem. 2001 Jul 6;276(27):24833-42. Epub 2001 Apr 30.
let me know what you think.
Date: Thursday, March 17, 2005 5:35:42 PM
I am looking for MMP-1,2 and 9.First we used a different set of protease inhibitors to see if there were MMP's and if has multiple protease activities. Leupeptin is a cysteine-containing proteases inhibitor. But since this particular bands show up we would like to know what could be the reasons. Related to our MMP of interest has been checked with the inhibition by EDTA (chelator).
Date: Thursday, March 17, 2005 5:21:45 PM
Which MMP are you looking for? Have you compared this to a zymogram of the MMP without inhibitor?
Many MMPs will have more than one band on a zymogram because they form dimers and degrade to truncated versions that are still active. You may need to incubate longer to inhibit the activity of your MMP completely.
Also, are you sure leupeptin inhibits the activity of the MMP you are looking for? I'm not certian that leupeptin acts on metalloproteinases.
Date: Tuesday, March 15, 2005 10:22:07 AM
When using leupeptin as an inhibitor for matrix metalloproteinases in a gelatin zymography, I obtained four(4) bands instead of one (uninhibited)or none(inhibition). Two bands above the expected position and two bands below the expected position. What could be the behavior of leupeptin in this case?