Heterogeneity of collagens in rabbit cornea: type VI collagen.
Normal adult rabbit corneas were digested with 5% pepsin and their collagens extracted with acetic acid. Collagen extracts were fractionated by differential salt precipitation. The 2.5 M NaCl fraction was then redissolved with tris buffer and precipitated with sodium acetate. The precipitate contained a high-molecular-weight disulfide-bonded aggregate which, upon reduction with mercaptoethanol, was converted into three distinct polypeptides having molecular weights between 45 and 66 Kd. These physical characteristics, together with the susceptibility of these polypeptides to collagenase and their amino acid composition, identified the high molecular weight aggregate as type VI collagen. Corneas from neonate rabbits and adult corneas containing 2-week-old scars were organ cultured in the presence of [14C] glycine to incorporate radiolabel into collagen. Tissues were digested with 0.02% pepsin and their collagens extracted with formic acid. The total radioactivity of the extracts and tissue residues was determined before the collagens were separated by SDS-polyacrylamide slab gel electrophoresis. Radioactive collagen polypeptides bands were then stained with Coomassie blue, processed for fluorography, and analyzed by densitometry. The results show that: (1) type VI collagen is synthesized by neonate corneas and healing adult corneas; (2) it is not readily solubilized from either corneal tissue by 0.02% pepsin digestion and formic acid extraction; and (3) the proportion of type VI collagen deposited in scar tissue is markedly lower than that found in neonate corneas.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002250:Carbon Radioisotopes / Q000378:metabolism; D002921:Cicatrix / Q000378:metabolism; D003094:Collagen / Q000032:analysis* / Q000096:biosynthesis / Q000145:classification; D003315:Cornea / Q000032:analysis* / Q000166:cytology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000378:metabolism; D008970:Molecular Weight; D009924:Organ Culture Techniques; D010434:Pepsin A / Q000378:metabolism; D011817:Rabbits; D014945:Wound Healing
D000596:Amino Acids; D002250:Carbon Radioisotopes; D003094:Collagen; D010434:Pepsin A; D005998:Glycine