Glycosaminoglycans were isolated from purified fractions of Isolation of Glomeruli. Kidneys were obtained from decapitated rats (150-200 g, both male and female), frozen at -20°C in normal saline, and stored for 24 hr to several weeks. Twelve to fifteen kidneys were removed at a time, and glomeruli were isolated therefrom by the technique of Krakower and Greenspon (6), carried out at 4°C. The efficiency of the glomerular isolation was monitored by examining a droplet of the suspension under a dissecting microscope. Any preparation found to contain tubular or interstitial fragments was resuspended in normal saline and centrifuged at 75 X g for 1-2 min, and the sediment was reexamined for contaminating tissue fragments. This process was repeated until all detectable contaminants were removed and the preparation consisted of virtually 100% glomeruli of which -u85% were free of Bowman's capsule and t15% were still encapsulated (Bowman's capsule present). The glomeruli thus isolated were pooled and stored at -20°C in normal saline.Isolation of GBM. Basement membrane fractions were prepared from the isolated glomeruli by the method of Meezan et al. (7) with minor modifications. Briefly, the glomeruli were hypotonically lysed in 0.05% sodium azide for 2 hr, digested with deoxyribonuclease (100 units/ml in 1 M NaCl) for 2 hr, and subsequently treated with 1% deoxycholate for 3 hr, all procedures being carried out at 4°C. The GBM fractions thus obtained were washed twice with distilled water and once with 0.15 M NaCl. An aliquot of each fraction was processed for electron microscopic examination in order to check for contamination by non-GBM (cell) components and to assess the preservation of the anionic sites and of their characteristic distribution pattern by using cationized ferritin (4). The remainder of each GBM fraction was lyophilized.Extraction of GAG from GBM. In general, the method followed was that used for the isolation of GAG from bovine lung by Linker and Hovingh (8,9). Isolated GBM (-50-100 mg) were suspended in 50 ml of 0.1 M acetate buffer (pH 5.5), containing 1 mM EDTA and cysteine. Crystalline papain (10 mg, 10.8 units/mg) was added, and the suspension was incubated at 60°C for 24 hr. The pH was then raised to 7.3 with a few crystals of Tris base, Pronase (50 mg, 89,600 PUK/g) was added, and the digestion was continued for another 24 hr at 370C. The suspension was then centrifuged at 10,000 X g for 15 min. The sediment was saved for electron microscopic exAbbreviations: GAG, glycosaminoglycans; GBM, glomerular basement membrane(s).
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