Kinetics of reduction of the intersubunit disulfides of the carboxyl propeptide of type I procollagen.
The carboxyl propeptide produced by proteolysis during maturation of type I procollagen to collagen was purified to homogeneity from the medium of cultured chick embryo calvaria by a new method. The propeptide was identified as such by its amino acid composition and migration pattern through sodium dodecyl sulfate-polyacrylamide gels in the absence and presence of dithiothreitol. Reduction of the intersubunit disulfides, which covalently join the two C1 and one C2 polypeptides of the carboxyl propeptide, was studied by incubating the propeptide in the presence of dithiothreitol for various times under nondenaturing conditions at pH 8.2. The reduction process was characterized by the appearance of disulfide-linked dimers. The appearance of dimers correlated with the disappearance of the carboxyl propeptide. Monomers, retaining intrasubunit disulfides, appeared concomitant with dimer formation. Reduction of the intersubunit disulfides of the dimers followed; intrasubunit disulfides were retained. The rate of the first process, trimer to dimer plus monomer, was an order of magnitude larger than the rate for the second process, dimer to monomers. The dimeric intermediates were composed of approximately equivalent amounts of (C1)2 and (C1, C2). The kinetics of formation and reduction of (C1)2 and (C1, C2) could not be differentiated by the techniques used. The relative amounts of intermediates found were not those expected if quasi-equivalent intersubunit disulfides were reduced in a random fashion. A possible model for reduction of the intersubunit disulfides of the propeptide has been proposed, and implications for the intersubunit polypeptide surface contacts have been discussed.
D000818:Animals; D001842:Bone and Bones / Q000378:metabolism; D002021:Buffers; D002642:Chick Embryo; D004220:Disulfides; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D009924:Organ Culture Techniques; D010084:Oxidation-Reduction; D011347:Procollagen / Q000302:isolation & purification
D002021:Buffers; D004220:Disulfides; D046911:Macromolecular Substances; D011347:Procollagen; D004229:Dithiothreitol