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Characterization of glycopeptides obtained on alkaline hydrolysis and on extensive collagenase and pronase digestion of the intestinal basement membrane showed the existence of two distinctly different carbohydrate units. One of these is a disaccharide, composed of glucose and galactose, linked to hydroxylysine. It was shown to be identical to the unit (2-O-D-glucopyranosyl-O-D-galactopyranosylhydroxylyasine) present in vertebrate basement membranes, as determined from stability to alkaline hydrolysis, retention time on amino acid analyzer, chemical composition, graded acid hydrolysis, methylation analysis, and periodate oxidation. Direct quantitation after alkaline hydrolysis showed the presence of 9.71 disaccharide units/1000 amino acid residues, indicating that 89% of the hydroxylysine residues of the intestinal membrane are glycosylated. The other unit, consisting of the remaining monosaccharides of the membrane, was separated from the disaccharide unit by gel filtration and ion exchange chromatography of collagenase/pronase digests. Chemical analyses and molecular weight determination by thin layer gel filtration chromatography of purified glycopeptides indicated that this unit is an oligosaccharide which is composed of fucose, galactose, mannose, galactosamine, and glucosamine in a mole ratio of 1:1:1:1:2, respectively. The amount of this unit was calculated to be 2.6 units/1000 amino acid residues.
Renal tubules from rabbit kidneys were isolated from thin shavings of the kidney surface. Basement membrane was then prepared following sonication of the isolated tubules. To insure preservation of the integrity of the basement membrane polypeptides, the protease inhibitors, diisopropyl fluorophosphate, ethylenediaminetetraacetic acid, N-ethylmaleimide, and epsilon-amino-caproic acid were used at all stages of the preparations. The optimal conditions of sonication and centrifugation were established and the chemical composition of basement membrane prepared under these conditions was examined in detail. Glycine, hydroxyproline, and hydroxylysine were found in concentrations of 206, 65, and 18 residues per thousand, respectively, in basement membrane from young kidneys. About 38% of the basement membrane was found to be soluble in sodium dodecyl sulfate upon incubation at 90 degrees C, and to possess relatively low amounts of the amino acids characteristic of collagen. Electrophoretic analysis of this fraction revealed that the major subunits ranged in approximate molecular weight from 18,500 to greater than 10(6). When analyzed with disulfide bonds reduced, a molecular weight range from 31,000 to 275,000 was observed for this fraction. The sodium dodecyl sulfate-insoluble fraction could be dissolved upon reduction and alkylation and its composition was enriched in the amino acids characteristic of collagen. Polypeptides from this fraction were analyzed by electrophoresis in agarose and in agarose-acrylamide gels. The approximate molecular weight of the smallest component was 164,000. Additional polypeptides were observed whose molecular weights occurred in multimers of this component, up to 1.1 x 10(6), possibly indicating covalent cross-linked multimers of a basic collagen-like polypeptide(s).
A rapid, sensitive, and reproducible assay to determine the adhesion of platelets to collagen has been developed. Collagen fibers and adherent platelets are retained on polycarbonate membrane filters. Chemical modification of collagen by acetylation and of platelets by treatment with chymotrypsin markedly reduces adhesion. The role of fibronectin in the collagen-platelet interaction has been examined. Treatment of platelets with purified antibody or Fab' fragments to fibronectin only slightly reduces adhesion. Preincubation of platelets with high concentrations of gelatin reduces adhesion by only 22% but fails to inhibit aggregation. Thus, fibronectin has only a limited role in the adhesion of platelets to collagen and is either not involved in the adhesion that leads to aggregation or is only one of several adhesion mechanisms, any of which alone can initiate aggregation.