title: Structural and immunological characterization of type IV collagen isolated from chicken tissues.
abstract: Previously, a type IV collagen fraction was isolated from chicken gizzard and further fractionated into three components called F1, F2 and F3 [Mayne, R. and Zettergren, J.G. (1980) Biochemistry, 19, 4065-4072]. F1 and F2 were consistently isolated in a 2:1 proportion, and the existence of a small native fragment of structure (F1)2F2 was proposed. In the present series of experiments, a type IV collagen fraction was isolated from the chicken kidney and shown to consist almost entirely of F1 and F2 which were again present in a 2:1 proportion. Identical one-dimensional peptide maps for F1 and F2 from both sources were obtained by polyacrylamide gel electrophoresis of peptides obtained after cleavage with cyanogen bromide or Staphylococcus aureus V8 protease. The denaturation temperature of a preparation containing F1 and F2 in native form was determined by optical rotatory dispersion and a single melting curve was observed with a melting temperature of 33 degrees C. This result provides further supportive evidence that F1 and F2 exist as a native fragment (F1)2F2. Antibodies were prepared in rabbits against a type IV collagen fraction isolated from chicken gizzard, and immunofluorescent staining of a wide variety of basement membranes was demonstrated. Experiments were performed in which various type IV collagen fractions were observed in the electron microscope after rotary shadowing. The lengths of (F1)2F2 and F3 were 147 nm and 174 nm respectively, the sum of these lengths (321 nm) corresponding closely to the length of the major triple-helical domain of type IV collagen (326-328 nm). A specific cleavage site was located at a distance of 215 nm from the 7-S domain which, together with the length of (F1)2F2, gives a total length of 362 nm. This value corresponds closely to the maximum length of the arms which originate from the 7-S domain (355 nm) when type IV collagen was solubilized with a low concentration of pepsin. The results suggest that (a) type IV collagen isolated from the chicken gizzard is closely related, if not identical, to type IV collagen isolated from other tissues; (b) there is a single type IV collagen molecule of chain organization[alpha 1(IV)]2 alpha2(IV); (c) the order of the pepsin-resistant fragments within a type IV molecule is 7S-F3-(F1)2F2.
mesh_terms: D000818:Animals; D001485:Basement Membrane / Q000648:ultrastructure; D002645:Chickens; D003094:Collagen / Q000302:isolation & purification*; D005895:Gizzard, Avian / Q000378:metabolism* / Q000648:ultrastructure; D007120:Immunochemistry; D007668:Kidney / Q000378:metabolism; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000302:isolation & purification; D013194:Staining and Labeling; D013329:Structure-Activity Relationship
chemical_list: D010446:Peptide Fragments; D003094:Collagen