Thyroxine-rich iodopeptides as fragment of reduced guinea pig thyroglobulin.

Full reduction of guinea pig thyroglobulin with mercaptoethanol followed by polyacrylamide gel electrophoresis reveals the presence besides the well known three polypeptide chains, of small amounts of iodopeptides with a molecular weight of 3 000 to 46 000 daltons. One of these peptides with a molecular weight of roughly 10 000 daltons has a much higher efficiency to couple iodotyrosines into thyroxine than any other fragment of thyroglobulin. Despite its small quantity this peptide contributes more than one third to the total thyroxine synthesis from a given iodide shot at any time up to five days after its injection. Iodopeptides akin to that found in guinea pigs were recently described by several authors in different species. They probably represent the preferential domains for hormone synthesis within the intact thyroglobulin molecule.

Changes in the polypeptide assembly of guinea pig thyroglobulin induced by thyrotropin-regulated thyroid activity.

We have previously reported that the relative proportion of three polypeptide chains in guinea pig thyroglobulin is closely related to the iodine content of the protein. The present work demonstrates that it is not the iodine content per se but, rather, TSH-regulated thyroid activity which modulates the substructure of thyroglobulin. In a first set of experiments, the impact of TSH stimulation on sodium dodecyl sulfate (SDS)-induced dissociation of 19S thyroglobulin into 12S subunits was compared to that of iodination. While in control animals the ratio of 12S to 19S thyroglobulin was 48:52, it changed to 35:65 in glands strongly stimulated with TSH and blocked with MMI. This rise in the relative proportion of 19S thyroglobulin occurred despite a simultaneous drop of iodine content from 0.6% to 0.24%. It was only after TSH suppression that the well known inverse correlation between the level of iodination and dissociability reappeared. In a second set of experiments, SDS-treated thyroglobulin was fully reduced by splitting disulfide bonds with mercaptoethanol. In addition to the previously described three polypeptide chains, A, B, and C, a hitherto neglected nonreducible fraction comigrated with 19S thyroglobulin on polyacrylamide gels. Native thyroglobulin with widely varying iodine contents was obtained from unstimulated glands and from glands strongly stimulated with TSH. Drastic changes in the polypeptide chain assembly, depending on the degree of TSH stimulation but entirely independent of iodination, were observed. There was a strong negative correlation between the nonreducible 19S thyroglobulin fraction and both the B and C polypeptide chains with all experimental manipulations. We conclude that thyroglobulin substructure is highly dependent on the degree of TSH stimulation of the thyroid. TSH, through stimulation of unknown metabolic pathways, is a more important determinant of thyroglobulin substructure than the degree of iodination of the protein.

Fractionation of individual, biologically active factor VIII multimers.

We have designed an electrophoretic system for the fractionation of individual, biologically active multimers of factor VIII. Human factor VIII, purified by gel filtration on Sepharose CL-2B from plasma cryoprecipitate, was submitted to electrophoresis without SDS on 2.0% polyacrylamide gels in 0.04 M Tris/0.06 M Tes buffer, pH 7.5. Staining with Coomassie blue revealed a series of protein bands. Measurement of electrophoretic mobility showed constant size intervals between adjacent bands. Electrophoresis in a second dimension, in the presence of SDS, resulted in an identical order of mobilities, suggesting that the different migration rates of factor VIII proteins in the first electrophoretic system were size- and not charge-dependent. After electrophoresis in the absence of SDS both factor VIII coagulant and ristocetin cofactor activities as well as factor VIII-related antigen were recovered by elution from gel slices. The distribution of activity peaks resembled that of Coomassie-stained factor VIII proteins found in control gels. We thus demonstrate that an electrophoretic fractionation of factor VIII multimers is possible even at neutral pH where factor VIII activities are retained.