Modulation of 72-Kilodalton Type IV Collagenase: RESULTS(1)
Media from WISH cells incubated in the absence of vitamin C showed a gelatinase-specific activity (mean ± SD, n = 8) of 124.3 ± 4.9 ^g degraded gelatin/^g protein. Gelatinolytic-specific activity decreased progressively with 10 ^g of vitamin C to 82.1 ± 9.7, with 29 ^g to 63.0 ± 7.8, with 50 ^g to 52.6 ± 9.6, with 100 ^g to 47.8 ± 6.1, and with 200 ^g to 37.2 ± 6.1. All concentrations of vitamin C significantly (ANOVA, p < 0.01) diminished the gelatinolytic activity considering the absence of vitamin C as the basal production. A 4-h incubation in the presence of vitamin C was enough to obtain a maximum effect (data not shown). Concentration of intracellular vitamin C during this time was assessed. Most of the ascorbic acid (86%, range from 83.0 to 88.3, n = 8) remained in the extracellular space under experimental conditions. buy asthma inhalers
Gel-substrate assays of the WISH media showed the presence of a main lysis band with an estimated molecular mass of 71 kDa. This band corresponded to the MMP-2 or gelatinase A, according to the relative migration of the purified standard. This lysis band decreased its relative intensity as the vitamin C dose increased (Fig. 1A).
FIG. 1. Gelatin substrate gels. Lanes 1-5 correspond to the gelatinolytic activity in media of cells stimulated with 0, 10, 29, 100, and 200 ^g/ml of vitamin C (A) or equimolar amounts of glutathione (B). Purified pro-MMP-2 standard is in lane 6. Progressive decrease of activity was detected in vitamin C-treated cells. Purified pro-MMP-2 was loaded in five lanes; once run, individual lanes were excised and incubated in a buffer to which vitamin C had been added in the same concentrations as mentioned above. No direct effect of vitamin C on enzymatic activity was documented (C).