Effect of β-D-xyloside on the glomerular proteoglycans. I. Biochemical studies

The effect of p-nitrophenyl-β-D-xylopyranoside on glomerular extracellular matrices (glomerular basement membrane and mesangial matrix) proteoglycans was studied. The proteoglycans of rat kidneys were labeled with [³⁵S]sulfate in the presence or absence of β-xyloside (2.5 mM) by using an isolated organ perfusion system. The proteoglycans from the glomeruli and perfusion medium were isolated and characterized by Sepharose CL-6B chromatography and by their behavior in CsCl density gradients. With xyloside treatment there was a twofold decrease in ³⁵S-labeled macromolecules in the tissues but a twofold increase in those recovered in the medium as compared with the control. The labeled proteoglycans extracted from control kidneys eluted as a single peak with K(av) = 0.25 (M(r) = ~130,000), and ~95% of the radioactivity was associated with heparan sulfate proteoglycan (HS-PG), the remainder with chondroitin (or dermatan) sulfate proteoglycan (CS-PG). In the xyloside-treated kidneys, the proteoglycans extracted from the tissue eluted as two peaks, K(av) = 0.25 (M(r) = ~130,000) and 0.41 (M(r) = ~46,000), which contained ~40 and ~60% of the total radioactivity, respectively. The first peak contained mostly the HS-PG (~90%) while the second peak had a mixture of HS-PG (~70%) and CS-PG (~30%). In controls, ~90% of the radioactivity, mostly HS-PG, was confined to high density fractions of a CsCl density gradient. In contrast, in xyloside experiments, both HS-PG and CS-PG were distributed in variable proportions throughout the gradient. The incorporated ³⁵S activity in the medium of xyloside-treated kidneys was twice that of the controls and had three to four times the amount of free chondroitin (or dermatan) sulfate glycosaminoglycan chains. The data suggest that β-xyloside inhibits the addition of de novo synthesized glycosaminoglycan chains onto the core protein of proteoglycans and at the same time stimulates the synthesis of chondroitin or dermatan sulfate chains which are mainly discharged into the perfusion medium.