Identification and purification of collagen-synthesizing polysomes with anti-collagen antibodies.
Antibodies against embryonic chick bone collagen were prepared in rabbits and were purified by affinity and ion exchange chromatography until collagen-specific and RNase-free. 125I-anti-collagen antibodies were used to locate the collagen-synthesizing polysomes of 8-day chick embryo wings and legs on sucrose gradients by measuring the polysome associated radioactivity. The 125I-anti-collagen antibodies bound predominantly to polysomes in the heavy region of sucrose gradients. These binding sites could only be saturated with homologous anti-collagen antibodies. Further evidence for the specificity of this reaction was provided by a correlation of the amount of anti-collagen antibodies bound in the heavy regions of sucrose gradients with the amount of collagen being synthesized by a particular tissue. The validity of this immunochemical method was confirmed by localizing collagen-synthesizing polysomes by an independent method which utilizes their ability to incorporate [3H]proline into collagen peptides in a cell-free system. The collagen-synthesizing polysomes are found in a single, rather broad peak in these gradients. The results of shortening the centrifugation time indicate that larger species of collagen-synthesizing polysomes are not present in these tissues. Partial purification of the collagen-synthesizing polysomes may be achieved by specifically sedimenting them after treatment with anti-collagen antibodies followed by goat anti-rabbit antibodies.
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D001666:Binding Sites, Antibody; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D003094:Collagen / Q000096:biosynthesis / Q000276:immunology*; D005121:Extremities / Q000196:embryology; D006909:Hydroxyproline / Q000378:metabolism; D011132:Polyribosomes / Q000276:immunology / Q000378:metabolism*; D011392:Proline / Q000378:metabolism; D011817:Rabbits / Q000276:immunology; D014921:Wings, Animal / Q000196:embryology
D000906:Antibodies; D003094:Collagen; D011392:Proline; D006909:Hydroxyproline