Identification of hormonogenic tyrosines in fragment 1218-1591 of bovine thyroglobulin by mass spectrometry. Hormonogenic acceptor TYR-12donor TYR-1375.
A fragment of bovine thyroglobulin encompassing residues 1218-1591 was prepared by limited proteolysis with thermolysin and continuous-elution polyacrylamide gel electrophoresis in SDS. The reduced and carboxymethylated peptide was digested with endoproteinase Asp-N and fractionated by reverse-phase high performance liquid chromatography. The fractions were analyzed by electrospray and fast atom bombardment mass spectrometry in combination with Edman degradation. The post-translational modifications of all seven tyrosyl residues of the fragment were characterized at an unprecedented level of definition. The analysis revealed the formation of: 1) monoiodotyrosine from tyrosine 1234; 2) monoiodotyrosine, diiodotyrosine, triiodothyronine (T3), and tetraiodothyronine (thyroxine, T4) from tyrosine 1291; and 3) monoiodotyrosine, diiodotyrosine, and dehydroalanine from tyrosine 1375. Iodothyronine formation from tyrosine 1291 accounted for 10% of total T4 of thyroglobulin (0.30 mol of T4/mol of 660-kDa thyroglobulin), and 8% of total T3 (0.08 mol of T3/mol of thyroglobulin). This is the first documentation of the hormonogenic nature of tyrosine 1291 of bovine thyroglobulin, as thyroxine formation at a corresponding site was so far reported only in rabbit, guinea pig, and turtle thyroglobulin. This is also the first direct identification of tyrosine 1375 of bovine thyroglobulin as a donor residue. It is suggested that tyrosyl residues 1291 and 1375 may support together the function of an independent hormonogenic domain in the mid-portion of the polypeptide chain of thyroglobulin.
Gentile
F
F
Centro di Endocrinologia e Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare, University of Naples Federico II, Italy.
Ferranti
P
P
Mamone
G
G
Malorni
A
A
Salvatore
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D013820:Thermolysin; D013954:Thyroglobulin / Q000737:chemistry*; D013974:Thyroxine / Q000737:chemistry*; D014284:Triiodothyronine / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
D010446:Peptide Fragments; D014284:Triiodothyronine; D014443:Tyrosine; D013954:Thyroglobulin; D013820:Thermolysin; D013974:Thyroxine