Journal of Biochemical and Biophysical Methods 1993-07-01

Simple measurement of glycosaminoglycan produced by cultured fibroblasts using 4-methylumbelliferyl beta-D-xyloside.

A Tanaka, A Morikawa, Y Saito, S Tamura, T Nakamura, K Takagaki, M Endo

Index: J. Biochem. Biophys. Methods 26(4) , 309-16, (1993)

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Abstract

A simple and rapid method was devised for measurement of glycosaminoglycan produced by cultured cells. 4-Methylumbelliferyl-beta-D-xyloside was added to the medium of the cultured cells. After incubation, glycosaminoglycan, which was produced from 4-methylumbelliferyl-beta-D-xyloside as a primer and secreted into the medium, was separated by proteinase digestion, trichloroacetic acid treatment and ethanol precipitation. The glycosaminoglycan, bearing a fluorescent moiety at the reducing terminal, was electrophoresed on cellulose acetate membrane, and then the fluorescent band visible on the membrane was extracted. The fluorescence of the band was measured, and from this the amount of glycosaminoglycan was estimated. Using this method, it was possible to quantify a very small amount of glycosaminoglycan with relatively high sensitivity without employing a radioisotope. This method was applied for determination of glycosaminoglycan produced by cultured fibroblasts from human uterine cervix, and also the effect of a hormone on glycosaminoglycan production. It was found that uterine cervical fibroblasts produced twice as much glycosaminoglycan as skin fibroblasts.