I have repeatedly attempted to activate the proenzyme form of MMP-1 and MMP-8 by incubating them in 1 mM APMA, but to no success. According to the standard protocol, MMP-1 and MMP-8 are activated by incubating them at 37°C in 1mM APMA for 3 hr and 1 hr, respectively. The APMA (Sigma-Aldrich, A9563) stock was made at a concentration of 10 mM using 0.6 M NaOH solution and added to the MMP stock solutions, diluting the APMA to the desired concentration. However, upon running SDS-PAGE we are unable to see any cleavage of the proenzymes. Am I missing something? Any suggestions? Thank you in advance.
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Are you concerned that the final pH might be extremely high due to dissolving the APMA in 0.6 M NaOH. The enzymes might be denatured by the high pH. I recommend trying again, but instead dissolving the APMA in buffer or DMSO.
I agree with that approach. I use 1 mM APMA to activate 1 micromolar of proMMPs at 37 degrees, 1 hour from a 100 mM APMA stock in 100% DMSO.
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