SIMPLE MEASUREMENT OF GLYCOSAMINOGLYCAN PRODUCED BY CULTURED FIBROBLASTS USING 4-METHYLUMBELLIFERYL BETA-D-XYLOSIDE

A simple and rapid method was devised for measurement of glycosaminogl ycan produced by cultured cells. 4-Methylumbelliferyl-beta-D-xyloside was added to the medium of the cultured cells. After incubation, glyco saminoglycan, which was produced from 4-methylumbelliferyl-beta-D-xylo side as a primer and secreted into the medium, was separated by protei nase digestion, trichloroacetic acid treatment and ethanol precipitati on. The glycosaminoglycan, bearing a fluorescent moiety at the reducin g terminal, was electrophoresed on cellulose acetate membrane, and the n the fluorescent band visible on the membrane was extracted. The fluo rescence of the band was measured, and from this the amount of glycosa minoglycan was estimated. Using this method, it was possible to quanti fy a very small amount of glycosaminoglycan with relatively high sensi tivity without employing a radioisotope. This method was applied for d etermination of glycosaminoglycan produced by cultured fibroblasts fro m human uterine cervix, and also the effect of a hormone on glycosamin oglycan production. It was found that uterine cervical fibroblasts pro duced twice as much glycosaminoglycan as skin fibroblasts.