Type X collagen does not bind to matrix vesicles.
Cultured chick embryo tibial hypertrophic chondrocytes released matrix vesicles and Type X collagen into the culture medium. When the culture medium was filtered through a 0.1 micron nitrocellulose filter, both the matrix vesicles, measured as alkaline phosphatase, and the Type X collagen were retained quantitatively. None of the other collagen types in the culture medium was retained on the filter. Dissolution of the matrix vesicles on the filter in detergent solutions resulted in quantitative solubilization of the Type X collagen also. These results suggested that the Type X collagen was intimately associated with the matrix vesicles. However, when membrane filters that were composed of materials other than nitrocellulose, and that had a range of pore sizes, were used to filter the culture medium, the ratios of total matrix vesicles to total Type X collagen retained on the filters ranged from 53 (polysulfone membranes) to 0.3 (nitrocellulose-cellulose acetate membranes). Thus it is concluded that the quantitative retention of matrix vesicles and Type X collagen on 0.1 micron nitrocellulose filters was due to true filtration of the matrix vesicles and to selective adsorption of the Type X collagen. Removal of the noncollagenous extensions from the Type X collagen by brief pepsin or trypsin treatment converted the Type X collagen to its 45 kDa collagenous domain, which was no longer retained on nitrocellulose filters, suggesting that the adsorption of the Type X collagen on the filters was through one or both of its noncollagenous extensions. When the culture medium was subjected to ultracentrifugation to pellet the matrix vesicles, 98% of the membrane-associated alkaline phosphatase (matrix vesicles) was pelleted but only 15-20% of the Type X collagen was recovered in the pellet. These results indicate that matrix vesicles and Type X collagen are not the associated products of hypertrophic chondrocyte.
Department of Orthopaedic Surgery, Asahikawa Medical College, Japan.
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Inao
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D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002356:Cartilage / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D003094:Collagen / Q000302:isolation & purification / Q000378:metabolism*; D003470:Culture Media; D016326:Extracellular Matrix Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D014461:Ultracentrifugation
D003470:Culture Media; D016326:Extracellular Matrix Proteins; D003094:Collagen; D000469:Alkaline Phosphatase