Biochemical and immunochemical characterization and internal alignment of pepsin-derived collagenous fragments of the alpha 1(IV) chain from bovine kidney cortices.

This communication describes the immunochemical and biochemical characterization of three polypeptide chains, alpha 1(IV)130K, alpha 1(IV)110K, and alpha 1(IV)75K belonging to the alpha 1(IV) chain of basement membrane collagen isolated from a pepsin digest of bovine kidney cortices. From the CNBr digests of the mixture of these chain fragments three peptides, a major and two minor peptides with an apparent Mr = 32,000, 24,000 and 13,000, respectively, were purified and characterized. The data presented show that CNBr peptides 24K and 13K are generated from CNBr peptide 32K by pepsin cleavage in the native molecule at the NH2-terminal end. Antisera were raised in rabbits against peptide CB32K. Inhibition assays using enzyme-linked immunoadsorbant assays (ELISA) showed cross-reactivity with alpha 1(IV)130K, alpha 1(IV)110K and alpha 1(IV)75K fragments. Peptides CB24K and 13K also inhibited the antiserum. Antiserum was not active when tested against alpha 1(IV)95K, alpha 1(IV)55K, alpha 2(IV)120K, and alpha 2(IV)95K fragments as inhibitors. These studies provide further evidence that alpha 1(IV)130K, 110K, and 75K are derived from the same parent chain. The pepsin cleavage sites resulting in the formation of these fragments and their internal alignment are described.