Isolation and characterization of type VIII collagen synthesized by cultured rabbit corneal endothelial cells. A conventional structure replaces the interrupted-helix model.
Radioactive proline-labeled type VIII collagen was biosynthesized in the presence of beta-aminoproprionitrile by rabbit corneal endothelial cells and isolated from the culture medium. Type VIII was purified in the presence of protease inhibitors and at neutral pH by ultrafiltration, precipitation with 3.9 M NaCl, sedimentation in sucrose gradients, and DEAE-Sephacel chromatography. The major components of this collagen, VIII-1, -2, and -3, exhibited apparent molecular weights of greater than 194,000, 124,000, and 61,000, respectively, and were shown to contain identical CNBr peptides. Following separation of VIII-1, -2, and -3 from each other and any residual proteases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exposure to acetic acid led to the conversion of VIII-1 to VIII-2 and VIII-3. Thus, VIII-1 is not a continuous single peptide chain, and the preliminary interrupted-helix model of the type VIII structure (Benya, P. D. (1980) Renal Physiol. 3, 30-35) was revised. VIII-3 appears to be the parent alpha 1 (VIII)-chain, with VIII-2 and VIII-1 representing beta- and gamma-chain configurations stabilized by strong noncovalent acid-labile interactions and beta-aminoproprionitrile-insensitive covalent cross-links. Based on two-dimensional CNBr peptide mapping, the alpha-chain is composed of six peptides. Mr 5,300-19,600. The terminal peptides are pepsin sensitive and correlate with two noncollagenous domains, NC1 (Mr 14,700) and NC2 (Mr 4-5,000). NC1 contains the site of acid-labile chain association.
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D000818:Animals; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D003094:Collagen / Q000302:isolation & purification*; D003315:Cornea / Q000166:cytology*; D003488:Cyanogen Bromide; D004727:Endothelium / Q000032:analysis*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D014462:Ultrafiltration
D046911:Macromolecular Substances; D010446:Peptide Fragments; D003094:Collagen; D010434:Pepsin A; D003488:Cyanogen Bromide