The selectivity of galardin and an azasugar-based hydroxamate compound for human matrix metalloproteases and bacterial metalloproteases
Fig 2
Purification and activation of proMMP-9.
(A) Imperial stained SDS-PAGE showing the purity of purified recombinant human full length proMMP-9 expressed in Sf9 cells (rproMMP-9) and of proMMP-9 purified from THP-1 cells (proMMP-9) as described in the Materials and Methods section. PT is the pass through fraction from Gelatin-Sepharose Chromatography of the recombinant enzyme, and 4 times more protein was loaded to the gel in the lanes labelled PT(2) compared to the lanes labelled PT(1). Std. 1 is the molecular size marker SpectraTM Mulitcolor High Range Protein Ladder and sb is sample buffer. Prior to electrophoresis, samples were either treated (+) or not treated (-) with DTT. Gelatin (B-D) and real-time gelatin (E) zymography of purified proMMP-9, trypsin activated (MMP-9) proMMP-9 from THP-1 cells, purified rproMMP-9, AMPA (rMMP-9(A)), trypsin (rMMP-9(T)) and MMP-3 (rMMP-9(M3)) activated recombinant proMMP-9. Std.2 in (B-E) is a mixture of proMMP-9 from THP-1 cells and proMMP-2 from human skin fibroblasts. Std. 3 is the 37 kDa catalytic domain of human MMP-9.