Quantitative analysis of glomerular basement membrane glycosaminoglycans and evidence for their binding to adjacent cell membrane lipids.
Glycosaminoglycans (GAG) were isolated from rat renal glomerular basement membranes subjected to extraction with detergents, and were quantitatively analyzed using a recently described competitive binding assay that is specific for and sensitive to microgram amounts of chondroitin and heparan sulfate. Whereas crude membranes prepared by osmotic lysis contained only 6 micrograms/mg dry wt, subsequent treatment with Triton X-100 or deoxycholate (DOC) increased measureable GAG to about 17 and 34 micrograms/mg, respectively. Repeated freezing and thawing of isolated glomeruli also augmented measurable GAG content in subsequently osmotically lysed membranes to levels observed in Triton-treated membranes. DOC solubilized approximately equal to 15-20% of membrane-associated GAG. Chondroitin sulfate comprised approximately equal to 30% of total GAG, and all of the chondroitin sulfate but only 10% of the heparan sulfate was extracted from the insoluble matrix by DOC. The findings indicate that GAG content of glomerular basement membrane is several-fold higher than previously estimated, and that a substantial portion is bound to cell membrane lipids. The results further suggest two populations of GAG in basement membrane; one that is intercalated with adjacent cell membranes, and another that remains as an integral component of the insoluble matrix after detergent extraction.
D000818:Animals; D001485:Basement Membrane / Q000032:analysis; D001667:Binding, Competitive; D003840:Deoxycholic Acid; D003902:Detergents; D005615:Freezing; D006025:Glycosaminoglycans / Q000032:analysis* / Q000378:metabolism; D006497:Heparan Sulfate; D007678:Kidney Glomerulus / Q000032:analysis*; D008297:Male; D008563:Membrane Lipids / Q000378:metabolism*; D017830:Octoxynol; D009994:Osmolar Concentration; D011092:Polyethylene Glycols; D011485:Protein Binding; D051381:Rats
D003840:Deoxycholic Acid; D003902:Detergents; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D008563:Membrane Lipids; D017830:Octoxynol; D011092:Polyethylene Glycols