Tyrosine 130 Is an Important Outer Ring Donor for Thyroxine Formation in Thyroglobulin (1998)

The thyroid couples two iodotyrosine molecules to produce thyroid hormone at the acceptor site in thyroglobulin, leaving dehydroalanine or pyruvate at the donor position. Previous work has located the acceptors but not the principal iodotyrosine donors. We incorporated [14C]tyrosine into beef thyroid slices, isolated and iodinated the [14C]thyroglobulin (Tg I), separated its N-terminal ∼22-kDa hormone-rich peptide, and digested the latter with trypsin and endoproteinase Glu-C (EC3.4.21.19). Nonlabeled thyroglobulin (Tg II) was isolated from the same glands and processed similarly, without iodination in vitro. Tg I was used to initially recognize pyruvate in peptide fractions, and Tg II was used to then identify its location in the thyroglobulin polypeptide chain. Sequencing of a tryptic peptide by mass spectrometry and Edman degradation showed a cleavage after Val129. An endoproteinase Glu-C-generated peptide had the predicted molecular mass of a fragment containing residues 130–146 with Tyr130 replaced by pyruvate; the identification of this peptide was supported by obtaining the expected shortened fragment after tryptic digestion. 14C-labeled pyruvate was identified in the same fraction as this peptide. We conclude that Tyr130 is an important donor of the outer iodothyronine ring. Its likely acceptor is Tyr5, the most important hormonogenic site of thyroglobulin, because Tyr5 and Tyr130 are proximate, because they are the most prominent early iodination sites in this part of thyroglobulin, and because the N-terminal region was previously found capable of forming T4 by itself. The thyroid couples two iodotyrosine molecules to produce thyroid hormone at the acceptor site in thyroglobulin, leaving dehydroalanine or pyruvate at the donor position. Previous work has located the acceptors but not the principal iodotyrosine donors. We incorporated [14C]tyrosine into beef thyroid slices, isolated and iodinated the [14C]thyroglobulin (Tg I), separated its N-terminal ∼22-kDa hormone-rich peptide, and digested the latter with trypsin and endoproteinase Glu-C (EC3.4.21.19). Nonlabeled thyroglobulin (Tg II) was isolated from the same glands and processed similarly, without iodination in vitro. Tg I was used to initially recognize pyruvate in peptide fractions, and Tg II was used to then identify its location in the thyroglobulin polypeptide chain. Sequencing of a tryptic peptide by mass spectrometry and Edman degradation showed a cleavage after Val129. An endoproteinase Glu-C-generated peptide had the predicted molecular mass of a fragment containing residues 130–146 with Tyr130 replaced by pyruvate; the identification of this peptide was supported by obtaining the expected shortened fragment after tryptic digestion. 14C-labeled pyruvate was identified in the same fraction as this peptide. We conclude that Tyr130 is an important donor of the outer iodothyronine ring. Its likely acceptor is Tyr5, the most important hormonogenic site of thyroglobulin, because Tyr5 and Tyr130 are proximate, because they are the most prominent early iodination sites in this part of thyroglobulin, and because the N-terminal region was previously found capable of forming T4 by itself. thyroglobulin human and bovine Tg, respectively thyroxine 3,5,3′-triiodothyronine 3,5-diiodotyrosine 3-iodotyrosine pyruvate dehydroalanine endoproteinase Glu-C collisionally activated dissociation mass spectrometry polyacrylamide gel electrophoresis high performance liquid chromatography. Thyroid hormone synthesis involves a two step modification of tyrosyl residues within the 2750-residue polypeptide chain of bovine Tg1 (1Taurog A. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid. 7th Ed. Lippincott-Raven Publishers, Philadelphia, PA1996: 47-81Google Scholar). First, certain tyrosyls are iodinated to form the hormone precursors MIT and DIT, and then two iodinated tyrosyls couple to form the biologically active iodothyronines T3 and T4. In this latter reaction, a tyrosyl donates its iodinated phenyl group to become the outer ring of the iodothyronine residue at an acceptor site, leaving DHA or its derivative at the donor position (2Gavaret J-M. Cahnmann H.J. Nunez J. J. Biol. Chem. 1979; 254: 11218-11222Abstract Full Text PDF PubMed Google Scholar, 3Gavaret J-M. Nunez J. Cahnmann H.J. J. Biol. Chem. 1980; 255: 5281-5285Abstract Full Text PDF PubMed Google Scholar). Both iodination and subsequent coupling are mediated by a thyroperoxidase at the apical membrane of the cell (1Taurog A. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid. 7th Ed. Lippincott-Raven Publishers, Philadelphia, PA1996: 47-81Google Scholar). The most important T4 forming site in all vertebrate species examined is at Tyr5. It appears in a 20–26-kDa N-terminal peptide released by reduction of mature Tg (4Dunn J.T. Kim P.S. Dunn A.D. J. Biol. Chem. 1982; 257: 88-94Abstract Full Text PDF PubMed Google Scholar, 5Rawitch A.B. Chernoff S.B. Litwer M.R. Rouse J.B. Hamilton J.W. J. Biol. Chem. 1983; 258: 2079-2082Abstract Full Text PDF PubMed Google Scholar, 6Rawitch A.B. Litwer M.R. Gregg J. Turner C.D. Rouse J.B. Hamilton J.W. Biochem. Biophys. Res. Commun. 1984; 118: 423-429Crossref PubMed Scopus (25) Google Scholar, 7Dunn J.T. Anderson P.C. Fox F.W. Fassler C.A. Dunn A.D. Hite L.A. Moore R.C. J. Biol. Chem. 1987; 262: 16948-16952Abstract Full Text PDF PubMed Google Scholar, 8Fassler C.A. Dunn J.T. Anderson P.C. Fox J.W. Dunn A.D. Hite L.A. Moore R.C. Kim P.S. J. Biol. Chem. 1988; 263: 17366-17371Abstract Full Text PDF PubMed Google Scholar, 9Lamas L. Anderson P.C. Fox J.W. Dunn J.T. J. Biol. Chem. 1989; 264: 13541-13545Abstract Full Text PDF PubMed Google Scholar). Two additional hormonogenic sites occur in the C-terminal region at Tyr2553 and Tyr2746 (human numbering) (7Dunn J.T. Anderson P.C. Fox F.W. Fassler C.A. Dunn A.D. Hite L.A. Moore R.C. J. Biol. Chem. 1987; 262: 16948-16952Abstract Full Text PDF PubMed Google Scholar, 8Fassler C.A. Dunn J.T. Anderson P.C. Fox J.W. Dunn A.D. Hite L.A. Moore R.C. Kim P.S. J. Biol. Chem. 1988; 263: 17366-17371Abstract Full Text PDF PubMed Google Scholar, 9Lamas L. Anderson P.C. Fox J.W. Dunn J.T. J. Biol. Chem. 1989; 264: 13541-13545Abstract Full Text PDF PubMed Google Scholar, 10Marriq C. Rolland M. Lissitzky S. EMBO J. 1982; 1: 397-401Crossref PubMed Scopus (49) Google Scholar, 11Marriq C. Rolland M. Lissitzky S. Biochem. Biophys. Res. Commun. 1983; 112: 206-213Crossref PubMed Scopus (32) Google Scholar) as well as at a mid-chain site at Tyr1290 (7Dunn J.T. Anderson P.C. Fox F.W. Fassler C.A. Dunn A.D. Hite L.A. Moore R.C. J. Biol. Chem. 1987; 262: 16948-16952Abstract Full Text PDF PubMed Google Scholar, 8Fassler C.A. Dunn J.T. Anderson P.C. Fox J.W. Dunn A.D. Hite L.A. Moore R.C. Kim P.S. J. Biol. Chem. 1988; 263: 17366-17371Abstract Full Text PDF PubMed Google Scholar, 12Gentile F. Ferranti P. Mamone G. Malorni A. Salvatore G. J. Biol. Chem. 1997; 272: 639-646Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Priority for hormonogenesis at these sites varies among species (13Roe M.T. Anderson P.C. Dunn A.D. Dunn J.T. Endocrinology. 1989; 124: 1327-1332Crossref PubMed Scopus (12) Google Scholar) and under different physiological conditions, including iodine availability and TSH stimulation (8Fassler C.A. Dunn J.T. Anderson P.C. Fox J.W. Dunn A.D. Hite L.A. Moore R.C. Kim P.S. J. Biol. Chem. 1988; 263: 17366-17371Abstract Full Text PDF PubMed Google Scholar, 9Lamas L. Anderson P.C. Fox J.W. Dunn J.T. J. Biol. Chem. 1989; 264: 13541-13545Abstract Full Text PDF PubMed Google Scholar). Far less is known about the tyrosyls that contribute the outer iodophenyl ring of the iodothyronines. Gavaret et al. (3Gavaret J-M. Nunez J. Cahnmann H.J. J. Biol. Chem. 1980; 255: 5281-5285Abstract Full Text PDF PubMed Google Scholar), using14C-labeled Tg from pig thyroid slices, concluded that the residual side chain of the donor existed as DHA while in peptide linkage and was converted to Pyr after its release from Tg by enzymatic digestion, or to acetate after acid hydrolysis. Locating the origins of these donors within Tg has been difficult. Proposed sites, all resting on indirect evidence, have included the Tyr2469 or Tyr2522 of bTg, based on the conversion of DHA either to labeled Ala by boro[3H]hydride reduction or to labeled Asp by Na14CN (14Palumbo G. J. Biol. Chem. 1987; 262: 17182-17188Abstract Full Text PDF PubMed Google Scholar), and Tyr residues 5, 926, 986, 1008, and 1375 of bTg, from 4-aminothiophenol modification of DHA residues (15Ohmiya Y. Hayashi H. Kondo T. Kondo Y. J. Biol. Chem. 1990; 265: 9066-9071Abstract Full Text PDF PubMed Google Scholar). Marriq et al. (16Marriq C. Lejeune P-J. Venot N. Vinet L. Mol. Cell. Endocrinol. 1991; 81: 155-164Crossref PubMed Scopus (35) Google Scholar) iodinated in vitro a CNBr-derived peptide from low iodine hTg and concluded that Tyr130 was converted to DHA with donation of an iodophenyl ring for iodothyronine formation at Tyr5. However, Xiaoet al. (17Xiao S. Pollock H.G. Taurog A. Rawitch A.B. Arch. Biochem. Biophys. 1995; 320: 96-105Crossref PubMed Scopus (9) Google Scholar), using the same substrate under similar experimental conditions, found that Tyr130 served as acceptor rather than donor, suggesting that the CNBr peptide is not a good model for hormonogenesis in intact Tg. Using a different approach based on graded iodination in vitro of low iodine hTg, we identified three potential donors, at Tyr residues 130, 847, and 1487 in hTg (9Lamas L. Anderson P.C. Fox J.W. Dunn J.T. J. Biol. Chem. 1989; 264: 13541-13545Abstract Full Text PDF PubMed Google Scholar). These represented sites that were iodinated early but did not form hormone with further iodination. More recently, Gentileet al. (12Gentile F. Ferranti P. Mamone G. Malorni A. Salvatore G. J. Biol. Chem. 1997; 272: 639-646Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) suggested from mass spectrometry of a peptide of bTg that Tyr1375 is an outer ring donor, probably to a neighboring minor hormonogenic site at Tyr1290. In the present paper we looked for an outer ring donor in the N-terminal part of Tg, the region of its most important hormonogenic site Tyr5, and offer evidence that Tyr130 is the dominant donor site. Fresh bovine thyroids were obtained from a local abattoir, transported to the laboratory in iced Earle's medium, and used within 1–3 h of death. We made two Tg preparations: Tg I, from thyroid slices incubated with [14C]Tyr and subsequently iodinated in vitro; and Tg II, isolated directly from the same beef thyroid without [14C]Tyr incorporation and without subsequent iodination. For Tg I, 2 g of thyroid slices were incubated in 10 ml of a tyrosine-free medium (Life Technologies, Inc., Selectamine) containing 500 μCi of uniformly labeled [14C]Tyr (500 μCi/μmol, NEN Life Science Products) at 37 oC in a water-saturated atmosphere of air-CO2 (95:5) for 20 h, then rinsed and briefly homogenized in 0.05 m sodium phosphate buffer, pH 7.4, followed by centrifugation at 105,000 ×g for 1 h. Thyroglobulin was isolated from the resultant supernatant fraction at 4 oC on a Sephacryl S300 column (2.5 × 100 cm) equilibrated in the same buffer. To obtain Tg II, we processed 5 g of thyroid tissue in the same manner without prior incubation. The protein content of isolated Tg was determined using bovine serum albumin as standard (18Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) and its iodine content by a modification of the Sandell-Kolthoff method (19Dunn J.T. Crutchfield H.E. Gutekunst R. Dunn A.D. Thyroid. 1993; 3: 119-123Crossref PubMed Scopus (284) Google Scholar). Iodination was carried out by exposure of Tg to iodine at a ratio of 20 atoms of iodine/molecule of 660-kDa Tg. We incubated 3.5 mg (5.3 nmol) of 14C-labeled Tg in 4 ml of 0.025 m sodium phosphate buffer, pH 7.0, containing: 1.6 mg of glucose (Calbiochem); 15 μg of glucose oxidase, 296 units/mg (Calbiochem); 17.6 μg (106 nmol) of KI; and 162 μg of lactoperoxidase (Sigma). The reaction was started by the addition of glucose oxidase. Carrier-free K125I (0.05 μCi) was added to an aliquot (50 μl) of this mixture at the beginning of the incubation. Iodination, with and without added 125I, was carried out at 37 oC for 1 h and the reaction stopped by the addition of 0.02% sodium azide. The 125I-labeled sample was used directly to determine protein bound 125I and 125I iodoamino acid distribution after Pronase digestion by paper chromatography (4Dunn J.T. Kim P.S. Dunn A.D. J. Biol. Chem. 1982; 257: 88-94Abstract Full Text PDF PubMed Google Scholar). Excess reagents and unreacted iodine in the non-125I-labeled sample were removed by chromatography on a column (1.5 × 100 cm) of Sephacryl S300 in sodium phosphate buffer, pH 7.4, containing 0.02% sodium azide. The peptide composition of 14C-labeled Tg (Tg I) following iodination and of nonlabeled Tg (Tg II) was analyzed by SDS-PAGE on 4–20% gradient gels under reducing conditions (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Gels were stained with Coomassie Brilliant Blue and 14C-labeled gels subjected to autoradiography after treatment with EN3HANCETM (NEN Life Science Products). Molecular mass markers were myosin (200 kDa), galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21 kDa), and lysozyme (14 kDa). Samples of Tg I and Tg II were reduced with 2-mercaptoethanol at a 40-fold molar excess in 8m urea at pH 8.5 and alkylated with acrylonitrile as described previously (21Dunn A.D. Myers H.E. Dunn J.T. Endocrinology. 1996; 137: 3279-3285Crossref PubMed Scopus (33) Google Scholar). The resultant peptides were separated on a Sephacryl S200 column in 0.1 m sodium phosphate buffer, pH 7.4, with 6 m urea and 0.02% sodium azide. Isolation of the 22-kDa N-terminal peptide is described under "Results." The 22-kDa peptide fractions from Tg I and from Tg II were each digested with l-1-tosylamide 2-phenylethyl chloromethyl ketone-treated trypsin (Sigma) at an enzyme to substrate ratio of 1:50 (w/w) in 0.1 mNH4HCO3, 0.1 mm CaCl2for 1 h at 37 oC and the reaction stopped with soybean trypsin inhibitor (Sigma). Tryptic peptides were first fractionated on a column (1.5 × 60 cm) of Bio-Gel P-6 (Bio-Rad), and selected fractions were further separated by reverse-phase HPLC on a C8 column in a 0.1 mNH4HCO3:acetonitrile gradient. The 22-kDa peptide fractions from Tg I and from Tg II were also separately digested with endo-Glu-C (sequencing grade, Promega), enzyme to substrate ratio 1:50 (w/w), in 0.1 mNH4HCO3 for 2 h at 37 oC, the reaction was stopped with 1.3-dichloroisocoumarin, and the resultant peptides were separated by HPLC under conditions similar to those for tryptic peptides. Samples from Tg I were digested with Pronase, substrate:enzyme ratio 1:10, in 50 μl of 0.1 mNH4HCO3 for 16 h at 37 °C, and the products were passed through a Centricon 10 membrane with several rinses of 0.1% trifluoroacetic acid. 14C-labeled compounds from the digestion were separated by reverse-phase HPLC using a Sephasil C8 column (Amersham Pharmacia Biotech) and a gradient of 0–90% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 0.5 ml/min. Standards used were Tyr, MIT, DIT, T3, T4, and Pyr or 14C-labeled Pyr (16 μCi/μmol, NEN Life Science Products). The fraction corresponding to Pyr was then eluted isocratically on an ion exclusion column (Aminex HPX-87H, Bio-Rad) with 0.0025 N H2SO4 at a flow rate of 0.6 ml/min. Oxalic, citric, malic, succinic, formic, and acetic acids (Bio-Rad) were used as standards in addition to Pyr and 14C-labeled Pyr. Detection was by 14C-label content orA 210. Pyruvate eluted with a retention time of 7.70 min, between citrate (7.50 min) and malate (9.04 min). Tg I was used to identify [14C]Pyr-containing peptides. The same fractions isolated in parallel from Tg II were then used for further analysis by mass spectrometry. The molecular masses of selected peptides were determined in a Finnigan-MAT TSQ7000 system with an electron spray ion source interfaced to a 10 cm x 75 μm id POROS 10RC reversed-phase capillary column. Peptides were eluted from the column by 0.1 macetic acid-acetonitrile gradient at a flow rate of 0.6 μl/min. Sequences of peptides were determined by CAD using electron spray-tandem mass spectrometry with argon as the collision gas and in some cases by automated Edman degradation on an Applied Biosystems 470A Protein Sequencer as described previously (7Dunn J.T. Anderson P.C. Fox F.W. Fassler C.A. Dunn A.D. Hite L.A. Moore R.C. J. Biol. Chem. 1987; 262: 16948-16952Abstract Full Text PDF PubMed Google Scholar). We obtained 9 mg of Tg I (1.2 million dpm/mg of protein) from thyroid slices incubated with [14C]Tyr. Twenty-three percent of 14C in the soluble fraction of labeled slices was recovered in Tg (Fig. 1 A). The of Tg II was mg (Fig. 1 We iodinated mg of Tg I using 20 atoms of iodine/molecule 660-kDa Tg. The in 1 the on gel of Tg I (Fig. 1 after iodination and of Tg II aliquot of Tg I, iodinated in the same but with added showed incorporation into including as T4 and as Tg I of of protein in vitro iodination and of protein with of protein in Tg 2 and I two the distribution after Pronase digestion in Tg I iodination with that after iodination. Two are in Tg I (Fig. 2 the corresponding to Tyr and a minor in of iodinated Tg I (Fig. 2 released MIT, DIT, and thyroid as well as an additional 14C-labeled early on HPLC with a retention time corresponding to that of Pyr. was in Tg We the of the in this by in a subsequent step by ion exclusion chromatography that with of compounds in intact Tg I and its Tg I was reduced and alkylated and then separated into fractions on an S200 column (Fig. Tg I was iodinated at 20 atoms of iodine/molecule of Tg as described under in the fraction of recovered from the S200 column. in a Tg I was reduced and alkylated and then separated into fractions on an S200 column (Fig. 14C-labeled Tg I was iodinated at 20 atoms of iodine/molecule of Tg as described under in the fraction of recovered from the S200 column. Tg I and Tg II were reduced and and then each was separated into fractions by exclusion chromatography in for Tg I and II peptides of (Fig. and several of a of and several of is based on its on appears the same as the described by Gregg et C. Rouse J. Hamilton J.W. Rawitch A.B. J. Biol. Chem. 1988; 263: Full Text PDF PubMed Google Scholar), that mass from its We identified the as an N-terminal peptide released from Tg by on the of its its high hormone content I), and from some of its tryptic peptides a of with of and 20 Its high content suggested this fraction also N-terminal peptides of Tg. Both fractions and had a high Pyr content after Pronase digestion from Tg I and Tg II were selected for further separately but in was digested with and the resultant peptides were separated into three fractions on a column (Fig. 4 for Tg fraction Tg I) had the content with in fractions and and on HPLC into at 20 peptides (Fig. 5 and (Fig. 5 corresponding to each of the 14C-labeled were analyzed by or including the distribution in Tg I) and by Edman degradation Tg I) or by mass spectrometry Tg II) and were to the tryptic peptides of N-terminal bTg, in The high content of suggested that included a peptide. It was well separated from a Pyr standard in this HPLC Edman degradation identified several peptides in this but had Tyr in the of bTg, suggesting that the donor peptide have been at its N and (Fig. was identified in form residue by Edman degradation of from Tg I and by mass spectrometry of this peptide from Tg The molecular mass of the latter was 0.5 to the mass of residues with the N-terminal residue converted to a an expected of the conditions used the The CAD for this peptide from Tg II its and the of residues after of tryptic peptides of from Tg I (Fig. on HPLC using a C8 column and an gradient. rate was 1 with peptide of tryptic peptides from (Fig. of (Tg residues (Tg I), (Tg (Tg distribution (Tg tryptic peptides as in corresponding to 14C were analyzed by or including 14C distribution after Pronase digestion and by Edman degradation or by The Tg used is in of its 14C as MIT in a of N-terminal bTg L. S. M. G. Nature. PubMed Scopus Google Scholar). The of and 10 are (21Dunn A.D. Myers H.E. Dunn J.T. Endocrinology. 1996; 137: 3279-3285Crossref PubMed Scopus (33) Google Scholar). tryptic peptides are tryptic peptides as in corresponding to 14C were analyzed by or including 14C distribution after Pronase digestion and by Edman degradation or by The Tg used is in of its 14C as MIT Samples of fraction (Fig. from Tg I and Tg II were also digested with and the resultant peptides were separated on HPLC into or fractions (Fig. for Tg II) and (Fig. for Tg The distribution of after Pronase digestion of each from Tg I is in had a high capillary from Tg II separated into peptide (Fig. and the molecular masses of in were determined by mass spectrometry. The molecular mass of peptide 1 was to the mass of a endo-Glu-C peptide of bTg (Fig. 9 with the expected of from the cleavage described and the of Pyr or DHA at Tyr130 (Fig. 9 Pyr is likely than DHA because the mass a form of the peptide (Fig. corresponding to a of with of the group of Pyr. Pyr at residue to the donor peptide in of the tryptic because of the group at the N-terminal Edman The CAD for peptide 1 (Fig. was not because of its expected from its high content To the identification of this peptide we digested further with The resultant fragment had a mass of to the of the C-terminal of the endo-Glu-C peptide, an expected of trypsin digestion (Fig. 9 with a mass of The mass for this peptide also showed in form with a (Fig. was identified by molecular mass and as Tg residues an expected of endo-Glu-C digestion. 2 and 4 did not peptides and 5 was not of in peptides of from Tg I (Fig. as percent of in each in a of fraction from Tg capillary HPLC spectrometry of peptide 1 from A. The molecular mass of the intact peptide is fragment of this peptide is also a of peptide residues and its peptide C. Rouse J. Hamilton J.W. Rawitch A.B. J. Biol. Chem. 1988; 263: Full Text PDF PubMed Google peptide obtained after trypsin digestion, and its C-terminal We a approach to the of a iodothyronine outer ring donor sites within the 2750-residue polypeptide chain of Tg. we isolated peptide fractions containing after in vitro iodination of Tg labeled with [14C]Tyr. to the identification of peptides from iodinated Tg isolated in parallel to the 14C-labeled Tg, using mass spectrometry and to the L. S. M. G. Nature. PubMed Scopus Google Scholar). The Pyr is a derivative of DHA (2Gavaret J-M. Cahnmann H.J. Nunez J. J. Biol. Chem. 1979; 254: 11218-11222Abstract Full Text PDF PubMed Google Scholar, 3Gavaret J-M. Nunez J. Cahnmann H.J. J. Biol. Chem. 1980; 255: 5281-5285Abstract Full Text PDF PubMed Google Scholar). identify the Tyr130 of bTg as donor of an outer iodothyronine ring. We this on the a tryptic peptide residue 130, as identified in Tg I by Edman degradation and in Tg II by suggested an modification of this an endo-Glu-C peptide from Tg II had the predicted molecular mass of a peptide containing residues with Tyr130 replaced by tryptic digestion shortened this peptide to a predicted from the of a of residues and the same HPLC fraction (Fig. from endo-Glu-C digested of Tg I identification of Tyr130 as donor with made from indirect evidence by Marriq et al. (16Marriq C. Lejeune P-J. Venot N. Vinet L. Mol. Cell. Endocrinol. 1991; 81: 155-164Crossref PubMed Scopus (35) Google Scholar) and by (9Lamas L. Anderson P.C. Fox J.W. Dunn J.T. J. Biol. Chem. 1989; 264: 13541-13545Abstract Full Text PDF PubMed Google Scholar). for the physiological of this donor site from a described by et al. T. P. M. J. G. J. 1991; PubMed Scopus Google its 4 of Tg, for a including suggesting that Tyr130 was for of thyroid spectrometry and Edman degradation of tryptic peptide of bTg (Fig. showed the expected through residue suggesting peptide cleavage between and residue The for this cleavage and its to iodination are not of iodinated Tg has hormone-rich N-terminal peptides of in all species Gregg et C. Rouse J. Hamilton J.W. Rawitch A.B. J. Biol. Chem. 1988; 263: Full Text PDF PubMed Google Scholar) have these in reduced bTg to residues and either or The cleavage we following appears different in and al. C. Lejeune P-J. Venot N. Vinet L. 1989; PubMed Scopus (12) Google Scholar) also a cleavage between residues and in a fragment of we not the that the we with coupling at that site, we likely that the formation of DHA with the of the phenyl ring at the time of coupling the to subsequent experimental We not have evidence that this cleavage in the thyroid in An N-terminal peptide of residues has not been in reduced Tg after of iodination. Gavaret et al. (2Gavaret J-M. Cahnmann H.J. Nunez J. J. Biol. Chem. 1979; 254: 11218-11222Abstract Full Text PDF PubMed Google Scholar) that of the peptide with the DHA residue to its cleavage and the of DHA to Pyr at the N of the resultant peptide. mass the of Pyr rather than DHA in the donor at by the time was isolated and to the tryptic peptide containing residues with the donor at 130, is with Pyr the Edman The work of et al. (12Gentile F. Ferranti P. Mamone G. Malorni A. Salvatore G. J. Biol. Chem. 1997; 272: 639-646Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) that cleavage of the peptide and the of DHA to Pyr at the donor site are not of the coupling reaction because they intact DHA at residue 1375 in bTg within a peptide residues as identified by mass spectrometry. In this we on the N-terminal region of bTg because the most important hormonogenic site of Tg and isolated by reduction after iodination. of the N was in fractions and (Fig. and I), similar of the of Tg and of its labeled We did not the in fraction but at some was at residue because this fraction of on (Fig. also expected to a residual peptide residues after cleavage of the to produce an as in 6 C. Rouse J. Hamilton J.W. Rawitch A.B. J. Biol. Chem. 1988; 263: Full Text PDF PubMed Google Scholar). showed Tyr5 and Tyr130 to the important early iodination sites in the first residues of hTg (9Lamas L. Anderson P.C. Fox J.W. Dunn J.T. J. Biol. Chem. 1989; 264: 13541-13545Abstract Full Text PDF PubMed Google Scholar), is to bTg, al. S. Rawitch A.B. Taurog A. Arch. Biochem. Biophys. 1996; PubMed Scopus Google Scholar) did not early iodination at Tyr130 in in vitro that the N is by to form for with a to of Tg at residue were to hormone for Endocrinology. 1993; PubMed Scopus Google Scholar). In Tyr130 and Tyr5 are in hormone synthesis in several experimental (16Marriq C. Lejeune P-J. Venot N. Vinet L. Mol. Cell. Endocrinol. 1991; 81: 155-164Crossref PubMed Scopus (35) Google Scholar, S. Pollock H.G. Taurog A. Rawitch A.B. Arch. Biochem. Biophys. 1995; 320: 96-105Crossref PubMed Scopus (9) Google Scholar, M.T. C. J. Endocrinol. 1995; PubMed Scopus Google Scholar) the to in is We these Tyr5 the most likely acceptor for the from position of hormone formation in the of the Tg is to donors and acceptors We and Fox from the of for and in peptide and and for in the