552 REACTION OF 4-METHYXUMBELLIFERYLGUANIDINOBENZOATE (MUGB) WITH SKIN FIBROBLASTS FROM PATIENTS WITH CYS-ITIC FIBROSIS (CF)

Abstract

Plasma of patients with CF has been shown to have reduced protease levels as measured by rate assays(Pediat.Res.9:739,1975) and by “titration” with MUGB(Am.J.Hum.Genet.29:111A,1977). In order to study whether the reduction in protease activity is a generalized phenomenon in CF, we have quantitated proteases in cultivated skin fibroblasts derived from patients with CF. Fibroblasts were grown in MEM and fetal calf serum and harvested by scraping. The cells were homogenized in 0.01M veronal HCl and 0.15M NaCl, pH 8.3 and centrifuged at low speed. The supernatant was assayed for reactivity towards MUGB by following the liberation of 4-methylumbelliferone(4-MU). The activity was particulate, inhibited by benzamidine, and not inhibited by p-hydrok ymecuribenzoate. The mean “titre” values(nmoles 4-MU released per mg protein) corrected for nonspecific hydrolysis of MUGB were: The differences were significant for N:H and N:CF(p< 0.001) and H:CF(p< 0.01). Cultivated normal human amniotic fluid cells gave values in the range of those observed for control skin fibroblasts. These data suggest that the reduction in protease levels in patients with CF is a generalized phenomenon.