Nereis cuticle collagen. Isolation and properties of a large fragment resistant to proteolysis by bacterial collagenase.
Native cuticle collagen, obtained from Nereis virens, was incubated with purified bacterial collagenase (EC 3.4.4.19). The kinetics of proteolysis were monitored by viscometry, in parallel with similar digestions of calf skin collagen. Comparison of the kinetics of digestion of the two collagens, at similar enzyme to substrate ratios (w/w), showed that the native cuticle collagen was relatively refractory to digestion by bacterial collagenase. Characterization of the cuticle collagen digest by sodium dodecyl sulfate-polyacrylamide electrophoresis and agarose gel filtration in CaCl2 showed a large polypeptide, of about 300,000 daltons, to be a major product. The native form of this product, a unique fragment, was isolated from the digest by ethanol precipitation. It was found to have an intrinsic viscosity of 120 dl/g, to have an optical rotary dispersion curve characteristic of collagen, to undergo a typical collagenous thermal transition with a Tm of 23.2 degrees, and to have a calculated molar mass of 900,000 g with molecular dimensions of 9,000 X 13 A. It had an amino acid composition which was similar, but not identical with the native cuticle collagen. Although the original substrate contained two dissimilar chains, A and B, in a molar ratio of 1:2, the collagenase-resistant product appeared to be composed of only one type of polypeptide fragment. Possibly, the original subunits contain similar, if not identical collagenase-resistant regions.
D000818:Animals; D002417:Cattle; D003013:Clostridium / Q000201:enzymology; D003094:Collagen / Q000302:isolation & purification*; D003720:Densitometry; D003012:Microbial Collagenase; D011077:Polychaeta; D012867:Skin; D014783:Viscosity
D003094:Collagen; D003012:Microbial Collagenase