Isolation of bovine type X collagen and immunolocalization in growth-plate cartilage.
Type X collagen was extracted with 1 M-NaCl and 10 mM-dithiothreitol at neutral pH from fetal-bovine growth cartilage and purified to homogeneity by using f.p.l.c. gel filtration on a Superose 12 column, followed by ion-exchange chromatography on a Mono Q column. The purified protein migrates in SDS/polyacrylamide gels with an apparent Mr of 58,000 under reducing conditions and as a high-Mr oligomer in its unreduced form. The amino acid composition is similar to the published composition of chick type X collagen. Pepsin digestion at 4 degrees C decreases the Mr of the monomer to 43,000; purified bacterial collagenase digests most of the molecule, leaving a non-collagenous domain of apparent Mr 15,000, which probably represents the C-terminal globular domain. The IgG fraction from a rabbit antiserum raised against purified bovine type X collagen was specific for this collagen by the criteria of e.l.i.s.a. and immunoblotting after immunoabsorption with collagen types I, II, IX and XI. Immunofluorescence localization of type X collagen in sections of fetal-bovine and human cartilage was possible after acetone fixation of sections and hyaluronidase treatment. Type X collagen was restricted to the zone of hypertrophic and calcified cartilage inside the bone spicules of the growth plate.
Max-Planck-Geselischaft, Klinische Arbeitsgruppen für Rheumatologie, Erlangen, Federal Republic of Germany.
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003094:Collagen / Q000032:analysis / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006132:Growth Plate / Q000032:analysis / Q000166:cytology*; D007106:Immune Sera; D015151:Immunoblotting; D007074:Immunoglobulin G; D008970:Molecular Weight
D000596:Amino Acids; D007106:Immune Sera; D007074:Immunoglobulin G; D003094:Collagen