ISOLATION OF HUMAN TYPE-X COLLAGEN AND IMMUNOLOCALIZATION IN FETAL HUMAN CARTILAGE

Type X collagen was extracted with 1 M NaCl and 10 mM dithiothreitol at neutral pH from fetal human growth plate cartilage and purified to homogeneity by gel filtration and anion-exchange chromatography. The purified protein migrates in SDS/polyacrylamide gels with an apparent M(r) of 66 000 under reducing conditions, and as a high-M(r) oligomer under non-reducing conditions. Purified collagenase digests most of the molecule; pepsin digestion at 4-degrees-C decreases the M(r) of the monomer to 53 000. A rabbit antiserum was raised against purified human type X collagen; the IgG fraction was specific for this collagen by criteria of ELISA and immunoblotting after absorption with collagen types I, II, VI, IX and XI. Immunohistological studies localized type X collagen exclusively in the zone of hypertrophic and calcifying cartilage.