Modulation of 72-Kilodalton Type IV Collagenase: MATERIALS AND METHODS(2)
Vitamin C Quantitation
Ascorbic acid was quantitated at different times (0-4 h) to ensure that vitamin C was present under experimental conditions. Medium and cells were precipitated with 0.35 M perchloric acid, and their filtrates were processed for HPLC. Chromatography was carried out as suggested by Lee et al.. Intracellular ascorbic acid was normalized to 50 ^g DNA. Total DNA per well was quantitated according to Burton.
Metalloproteinase Mixed Activity
To evaluate MMP activity in vitamin C-stimulated cells, gelatinolytic activity was quantified using thermally denaturalized radioactively labeled collagen type I/III as a substrate according to Terato et al.. Each sample was assayed in basal conditions and after being treated with 1.0 mM aminophenol mercuric acetate, an MMP activator. MMP activity was calculated taking into account the EDTA-inhibitable activity and was expressed as specific activity (^g degraded gelatin/^g incubated protein per 12 h at 37°C). buy asthma inhaler
Gel-Substrate Gelatinolytic Activity
Ten micrograms of protein from vitamin C-conditioned media was applied per lane to a gelatin-containing polyacrylamide gel in small format (Bio-Rad, Richmond, CA), prepared according to a previously described method. At the end of the process, gels were stained with Coomassie R-250 blue. Molecular weight markers (M 14-200) were included on each run. To evaluate the direct effect of vitamin C on MMP activity, pro-MMP-2 was purified as suggested by Murphy and Crabbe.