Bovine Glomerular Basement Membrane

The collagenous domain of bovine glomerular basement membrane was excised in soluble form by limited pepsin digestion and characterized. The domain amounts to approximately 25% of the whole membrane by weight. The constituent polypeptides were studied using several electrophoresis systems and amino acid analysis. The electrophoresis systems used were a combination of agarose and polyacrylamide which enabled an examination over a molecular weight range from 20,000 to >log. A 0.1% sodium dodecylsulfate, 2.5% agarose gel system resolves the nonreduced collagenous domain into 13 components which vary in molecular weight from 85,000 to 5 million. Upon reduction, the majority of this material is converted to a 165,000 molecular weight component(s) and cross-linked (aldehyde-derived) multimers of this component(s) containing as many as six cross-linked monomers. Two-dimensional sodium dodecyl sulfate electrophoresis, with the first dimension run in 2.5% agarose and the second dimension in 5% polyacrylamide, indicates that the 165,000 molecular weight component(s) is actually composed of several polypeptides ranging in molecular weight from 126,000 to 189,000 which are not resolved on the agarose gel. A second pepsin digestion was performed after the original digestion product was reduced and alkylated under nondenaturing conditions. This resulted in a conversion of the larger polypeptides to three lower molecular weight peptides. Two of these peptides exhibit an electrophoretic migration identical with a1 and a2 chains of collagen. Concurrent with these conversions is an increase in the glycine content suggesting an increase in the collagenous nature of the second digestion product. The results indicate that the collagenous domain of the glomerular basement membrane consists of various size collagen molecules connected by disulfide bonds and aldehyde-derived cross-links to form high molecular weight aggregates containing as many as 30 of these polypeptides, and that the larger collagenous polypeptides contain a size segments within their structure.