The disulfide bond pattern between fragments obtained by the limited proteolysis of bovine thyroglobulin.
The comparative analysis of the products of the limited proteolysis of bovine thyroglobulin with trypsin by SDS-polyacrylamide gel electrophoresis in non-reducing and reducing conditions revealed the presence of disulfide linkages between some of the fragments. In order to define the disulfide bond pattern between the proteolytic fragments of thyroglobulin, these were isolated by SDS-polyacrylamide gel electrophoresis in non-reducing conditions and electrophoretic transfer onto polyvinylidene difluoride membranes. Individual bands were desorbed from the membranes and re-analyzed by SDS-polyacrylamide gel electrophoresis in reducing conditions. The resulting peptides were identified by comparison with the peptides directly obtained by SDS-electrophoresis in reducing conditions, and characterized by amino-terminal peptide sequencing either in this study or in a previous investigation (Gentile F., Salvatore G., Eur. J. Biochem. 218 (1993) 603-621). The analysis revealed that several fragments, produced by cleavages within the context of various cysteine-rich repeats of type 1 and within cysteine-rich repeat 3b.1, did not separate in the absence of reduction. On the other hand, the products of the cleavages at the carboxy-terminal extremity of the linker between type 2 and type 3 cysteine-rich repeats, and in the middle of the acetylcholinesterase-similar domain of thyroglobulin separated freely, with no need for reduction. On the base of these data, a model is presented in which distinct subsets of cysteine-rich repeats and the carboxy-terminal, acetylcholinesterase-similar domain of thyroglobulin form sequentially aligned subdomains with internal disulfide linkages.
Centro di Endocrinologia e Oncologia Sperimentale del Consiglio Nazionale delle Ricerche and Dipartimento di Biologia e Patologia Cellulare e Molecolare, University Federico II, Naples, Italy.
B M
BM
Veneziani
F
F
Giallauria
F
F
Gentile
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate; D013954:Thyroglobulin / Q000032:analysis*; D014357:Trypsin
D004220:Disulfides; D010446:Peptide Fragments; D012967:Sodium Dodecyl Sulfate; D013954:Thyroglobulin; D014357:Trypsin