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.

Thyroglobulin (Tg), 1 a homodimeric glycoprotein with a molecular mass of 660 kDa, is the site of the biosynthesis of 3,5,3Ј-triiodothyronine (T 3 ) and 3,5,3Ј,5Ј-tetraiodothyronine (thyroxine, T 4 ) (reviewed in Ref. 1). T 3 and T 4 are synthesized via the iodination and coupling of a small subset of tyrosyl residues within the polypeptide chains of Tg. The coupling reaction takes place by the transfer of an iodophenyl group from a donor 3-monoiodotyrosine or 3,5-diiodotyrosine to an acceptor 3,5-diiodotyrosine. This causes the formation of T 3 or T 4 , respectively, at the acceptor site and dehydroalanine at the donor site (2,3). Both reactions are catalyzed by thyroid peroxidase. Different tyrosyl residues have different reactivities toward iodine, so that iodination proceeds in a sequential order, which is controlled by the native structure of Tg (4,5). Early iodinated tyrosyl residues are preferentially involved in iodothyronine synthesis (6); the coupling of iodotyrosines, in turn, has stringent steric requirements (7). In fact, out of 72 tyrosyl residues per bovine Tg monomer, only 15 are iodinated and a maximum of 6 -8 of them undergo coupling to form T 3 and T 4 (8,9). So far, four major hormonogenic tyrosines have been identified, by the isolation and sequencing of hormone-rich peptides from Tgs of various animal species and comparison of their sequences with the cDNA-deduced sequences of bovine (10) and human Tg (11). Tyr-5 was the most favored site for T 4 formation in most species studied, including humans (12), calf (13), sheep, hog (14), rabbit (15), and guinea pig (16). In hog (17), rabbit (15), guinea pig (16), and human Tg subjected in vitro to low-level iodination (18), Tyr-2553 (human Tg numbering) was the second most efficient T 4 -forming residue, whereas Tyr-2746 was a site of preferential synthesis of T 3 (15,16,18,19). Another T 4 -forming site found in rabbit and guinea pig Tg corresponded to human Tyr-1290: in those species this site was third in ranking order of hormonogenic efficiency and its function was greatly enhanced by TSH (15,16). Nevertheless, so far it has received little attention in the bovine and human species. Tyrosines reported as possible donor sites include Tyr-5, -926, -986 or -1008, -1375 (20), -2469 and/or -2522 of bovine Tg (21), and Tyr-130 of human Tg (22).
The main goal of this work was to establish whether Tyr-1291 of bovine Tg is also a site of T 4 formation. To this purpose, a preparation of bovine Tg containing 1.05% iodine by mass was subjected to limited proteolysis with thermolysin and the products were separated by preparative SDS-PAGE. A thorough mass spectrometric analysis of a peptide spanning residues 1218 -1591, together with an analysis of its iodine and iodoamino acid content, were performed. Post-translational modifications of three out of seven tyrosyl residues were documented at an unprecedented level of definition: in particular, we report the first direct evidence of the entire spectrum of modifications typical of a hormonogenic acceptor and a hormonogenic donor site at residues 1291 and 1375, respectively, of bovine Tg. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Preparation of Tg-Bovine Tg was prepared from fresh bovine thyroids from the local abbattoir. The tissue was finely minced with scissors and Tg extracted briefly on ice in 0.1 M sodium phosphate, pH 7.2, and purified by fractional precipitation with 1.4 -1.8 M ammonium sulfate, 50 mM Tris/HCl, pH 7.2, and gel filtration on Sephacryl S-300 HR in 130 mM NaCl, 50 mM Tris/HCl, pH 7.2, at 4°C.
Limited Proteolysis of Tg-Limited proteolysis of Tg with thermolysin was carried out as described previously (23). Tg at the concentration of 1 mg/ml in 130 mM NaCl, 50 mM Tris/HCl, pH 8.0, was incubated with thermolysin at the enzyme/substrate ratio of 1/1000 or 1/100 (w/w) at 30°C for the time indicated. The digestion was stopped by adding EDTA to a final concentration of 10 mM and concentrated SDS-PAGE sample buffer to a concentration of 10 mM Tris/HCl, pH 6.8, 1% SDS, 5% ␤-mercaptoethanol, 1.36 M glycerol, 0.0025% bromphenol blue, and by heating the samples in a boiling water bath for 1.5 min.
Separation and Identification of the Products of the Limited Proteolysis of Tg-Analytical SDS-PAGE in reducing conditions of the digestion products was performed according to Laemmli (24) on 4 -16% total acrylamide gradient gels polymerized on GelBond PAG plastic backing (FMC BioProducts). The gels were stained with 0.1% Coomassie Brilliant Blue R-250 in 25% 2-propanol (v/v), 10% acetic acid (v/v), destained in 25% methanol (v/v), 10% acetic acid, soaked in 0.7 M glycerol, and air dried. The main peptides produced were identified on the basis of their mobility, according to detailed characterization of their NH 2 -terminal peptide sequences provided in a previous study (23).
Purification of Peptide b6 TL -Bovine Tg was digested for 80 min with thermolysin at the enzyme/substrate ratio of 1/100 (w/w) and the digestion stopped as described above. The fragments were precipitated in chloroform/methanol (25), redissolved in SDS-PAGE sample buffer, and separated by preparative continuous-elution SDS-PAGE, using an electrophoresis chamber Bio-Rad model 491. A discontinuous gel with an annular cross-section was prepared according to Laemmli (24) in a cylindrical assembly having a diameter of 3.5 cm, whose center was occupied by a cooling core having a diameter of 1.5 cm. The 50-ml separating gel contained 12% total acrylamide and was 6.3 cm high; it was topped with a 10-ml 1.3-cm stacking gel containing 3.75% total acrylamide. The products of digestion of 25 mg of Tg with thermolysin were loaded onto a single gel. The electrode buffer contained 0.025 M Tris, 0.19 M glycine, 0.1% SDS, pH 8.2. The apparatus was designed so that, as soon as the migrating bands reached the lower extremity of the gel, they were conveyed by a stream of electrode buffer, aspirated by a peristaltic pump from a reservoir, to a fraction collector. Electrophoresis was carried at 20 mA. Collection was started as soon as the tracking dye began to exit from the gel (in 16 h); 4 fractions per hour were collected, with the pump flow rate set at 20 ml/h, for 24 h. The fractions were analyzed by SDS-PAGE. Those of interest were pooled and the pool was concentrated by lyophilization, freed from Tris/HCl, and glycine by filtration through PD-10 Sephadex G-25 cartridges (Pharmacia Biotech) in distilled water, and from SDS by filtration through Extractigel resin (Pierce) (1 ml of resin every 50 ml of the original pool) in distilled water. The sample was finally lyophilized and stored at Ϫ20°C.
Reduction and Carboxymethylation of Peptide b6 TL -Purified peptide b6 TL was dissolved in 300 l of 0.3 M Tris/HCl, pH 8.0, containing 6 M guanidine/HCl, 1 mM EDTA, and treated with dithiothreitol (10/1 molar excess with respect to cysteinyl residues) at 37°C for 2 h. The reduced peptide was carboxymethylated by reaction with a 5/1 molar excess of iodoacetic acid, with respect to total -SH groups, at pH 8.0 at room temperature for 30 min in the dark. The sample was freed from low molecular weight compounds by filtration through a PD-10 G-25 column in 50 mM ammonium bicarbonate, pH 8.5, and lyophilized.
Enzymatic Digests-The reduced and carboxymethylated peptide b6 TL was hydrolyzed with endoproteinase Asp-N at the enzyme/substrate ratio of 1/100 (w/w) in 50 mM ammonium bicarbonate, 10% (v/v) acetonitrile, pH 8.5, at 37°C for 18 h. Hydrolyses of HPLC-purified peptides with trypsin and endoproteinase Lys-C were carried out in 50 mM ammonium bicarbonate, pH 8.5, at 37°C, using an enzyme/substrate ratio of 1/50 (w/w), for 4 and 20 h, respectively. All the reactions were immediately followed by lyophilization.
Separation of Peptides Obtained by Hydrolysis with Endoproteinase Asp-N-The peptides obtained by hydrolysis of 0.5 mg of peptide b6 TL with endoproteinase Asp-N were fractionated by HPLC with a Vydac C-18 column (250 ϫ 4.6 mm, 5 m) equilibrated in 0.1% (v/v) trifluoroacetic acid in water (solvent A), containing 4% of 0.07% trifluoroacetic acid in acetonitrile (solvent B). After 5 min at 4% of solvent B, elution was performed by a two-step linear gradient of solvent B percentage from 4 to 25% over 25 min, and from 25 to 60% over the following 45 min. The flow rate was 1 ml/min.
Electrospray Mass Spectrometry (ES/MS)-ES mass spectra of the peptides produced by hydrolysis of peptide b6 TL with endoproteinase Asp-N were recorded with a PLATFORM mass spectrometer (Fisons, Manchester, United Kingdom) equipped with an electrospray ion source. Samples from the HPLC separation (10 l, 50 pmol) were injected into the ion source at a flow rate of 10 l/min; the spectra were scanned from 2000 to 400 at the speed of 10 s/scan. Mass calibration was carried out using the multiple charged ions from a separate introduction of horse heart myoglobin (average molecular mass 16, 950.5 Da). The quantitative analysis was performed by integration of the multiple charged ions of the single species. Molecular masses are reported as average values.
Fast Atom Bombardment Mass Spectrometry (FAB/MS)-FAB mass spectra were recorded with a VG Analytical ZAB-2SE double-focusing mass spectrometer fitted with a VG caesium gun operating at 25 kV. Samples (0.1 nmol) were dissolved in 5% acetic acid and loaded onto a glycerol-coated probe tip; thioglycerol was added to the matrix just before introducing the probe into the ion source. The amplification of the electric signal was reduced during the magnet scan, according to the intensity of the mass signals observed on the oscilloscope. The values correspond to the monoisotopic masses of the protonated molecular ions of the peptides and are reported as integer numbers.
Peptide Recognition-The mass signals recorded in the spectra were associated with the corresponding peptides, on the basis of the expected molecular masses, using a computer program (26). Edman degradation steps were performed on HPLC-purified peptides, and were followed by the mass spectrometric analysis of the truncated peptides, in order to confirm the assignments, as already described (27).
Analytical Techniques-Iodine determinations were performed as described (28). The concentration of Tg was estimated by the absorbance at 280 nm, using a percentual extinction coefficient of 10.5 (29). The concentration of peptide samples was assayed using a bicinchoninic acid Protein Assay Reagent (Pierce) and bovine Tg as the standard. For the analysis of iodoamino acids, triplicate samples were hydrolyzed by a modification of a method already described (30): 0.4-mg aliquots of Tg and purified peptide b6 TL were incubated at 37°C with Pronase at the enzyme/substrate weight ratio of 1/1 in 0.5 ml of 0.1 M Tris/HCl, 50 mM 2-mercapto-1-methylimidazole, pH 8.0, to which 10 l of toluene were added; after 24 h, aminopeptidase M at the enzyme/substrate ratio of 1/10 was added and digestion prolonged for another 24 h at 37°C. Iodoamino acids were separated by reverse-phase HPLC in a Kontron HPLC equipped with a Brownlee C-8 column (250 ϫ 4.6 mm, 5 m), as already described (31). Iodoamino acid peaks were identified by comparison with iodoamino acid standards: the contents of iodoamino acids were calculated from the iodine contents of the respective peaks. Since 2-mercapto-1-methylimidazole co-eluted with MIT and interfered in the iodine assay, its contribution was determined in triplicate samples of bovine serum albumin which were subjected to the identical treatment.

RESULTS
Limited Proteolysis of Bovine Tg and Purification of Proteolysis Products-A detailed analysis of the products of the limited proteolysis of bovine Tg with thermolysin has been reported (23). Typical time courses and a flow-diagram of the proteolysis at pH 8.0 at 30°C are shown in panels A and B, respectively, of Fig. 1. The proteolytic peptides corresponded exactly to those which were previously observed and characterized by amino-terminal sequencing (23). Therefore, in the present work the proteolytic peptides were identified according to their electrophoretic mobilities, on the basis of the data already reported (23).
For the preparation of peptide b6 TL , five 25-mg aliquots of a bovine Tg containing 1.05% iodine by mass were hydrolyzed with thermolysin at the enzyme/substrate ratio of 1/100 at pH 8.0 at 30°C for 80 min. The fragments were separated by preparative continuous-elution SDS-PAGE, concentrated, further purified, and lyophilized as described under "Experimental Procedures." The analysis by SDS-PAGE of the fractions of a typical preparation is shown in panels C and D of Fig. 1. In the end, 2.2 mg of pure peptide were obtained (Fig. 1, panel E). Because peptide b6 TL represented 10% of the peptides detected by densitometry of the gel (Fig. 1, panel A) (23) and these were 80% of the starting protein material, the yield of the purification procedure was 22%.
Analysis of Peptide b6 TL by Mass Spectrometry-A 50-kDa peptide starting at residue 1291 (peptide b6 TL ) (Fig. 1, panels A and B) was reduced and carboxymethylated, digested with endoproteinase Asp-N, and the digest was fractionated by reverse-phase HPLC on a Vydac C-18 column (250 ϫ 4.6 mm, 5 m). The chromatogram is shown in Fig. 2. All fractions were directly analyzed by ES/MS, and some were freeze-dried and analyzed also by FAB/MS. The results of the analysis by ES/MS are reported in Table I. The mass signals in the spectra were associated with the corresponding peptides along the sequence of bovine Tg, between residues 1200 and 1630, using a suitable computer program (26) (Fig. 3). Several cleavage sites were only partially hydrolyzed during the digestion, which yielded several overlapping peptides. A few aspecific cleavages occurred at the amino side of glutamic acid residues. However, the data permitted verification of the entire amino acid sequence of peptide b6 TL , which was identical to the cDNA-derived sequence (10). Ala-1591 was identified as the COOH-terminal residue of peptide b6 TL . In fact, two peptides, spanning residues 1567-1591 and 1580 -1591, both ended at Ala-1591 and, therefore, were not expected on the basis of the enzymatic specificity of endoproteinase Asp-N. Moreover, no peptide was detected whose sequence matched Tg sequence beyond Ala-1591. The mass spectrometric analysis of the HPLC fractions of peptide b6 TL (Table I) (Table  I and Fig. 4). These assignments were confirmed by submitting the above fractions to FAB/MS followed by two manual Edman degradation steps, after which the m/z values of the truncated peptides were measured again by FAB/MS (Fig. 5) Tyr-1375 Is a Hormonogenic Donor Residue-The chromatogram of Fig. 2 contained four peaks (13, 19, 26, and 27), whose analysis by ES/MS revealed mass signals related to peptide 1366 -1381, containing one Tyr residue at position 1375 (Table  I and   a Numbers refer to the peaks of the chromatogram shown in Fig. 2. b Average molecular masses in Da (mean Ϯ S.D.) obtained by integrating the multiple peaks corresponding to each molecular species, differing only in the total number of charges, measured by ES/MS. c Numbers indicate the amino acid residues at the extremities of each peptide.
d Masses calculated on the basis of the cDNA-derived sequence of bovine Tg (10), taking into account the modifications of tyrosyl residues indicated. addition of one and two iodine atoms to Tyr-1375, respectively. These identifications were confirmed by incubating the four fractions with endoproteinase Lys-C, to cleave peptide 1366 -1381 into peptides 1366 -1374 and 1375-1381. When analyzed by FAB/MS (Fig. 6), the four digests had in common the MH ϩ ion at m/z ϭ 931 predicted for peptide 1366 -1374 DVEEAL-AGK, whereas they differed in the MH ϩ ions corresponding to peptide 1375-1381. In fact, the spectrum of fraction 19 showed the MH ϩ ion at m/z ϭ 797 predicted for the unmodified peptide 1375-1381 YLAGRFA, while the spectra of fractions 26, 27, and 13 showed MH ϩ ions at m/z ϭ 923, 1049, and 703, corresponding to peptide 1375-1381, in which Tyr-1375 had been converted to MIT, DIT, and DHA, respectively. The analysis by FAB/MS, after one step of Edman degradation (Fig. 6), showed the expected mass shifts, by which all four peptides moved to m/z ϭ 634, due to the loss of Tyr (fraction 19, ⌬m ϭ Ϫ163), MIT (fraction 26, ⌬m ϭ Ϫ289), DIT (fraction 27, ⌬m ϭ Ϫ415), and DHA (fraction 13, ⌬m ϭ Ϫ69). These data proved that Tyr-1375 is an iodophenyl donor residue. Mass signals corresponding to various forms of peptide 1355-1393, in which Tyr-1375 was present as such or had been modified to MIT, DIT, and DHA were detected also in fraction 16 (Table I).
Asn-1346 Is Not Glycosylated-The sole putative site of N-linked glycosylation of peptide b6 TL , corresponding to Asn-1346 (within the consensus sequence Asn-Ile-Thr) (10), was unmodified. In fact, peptides 1330 -1354 (fraction 18) and 1336 -1354 (fraction 10) had mass values typical of the non-glycosylated species (Table I), and no evidence was found of glycosylated forms of the above peptides. Table II indicate that the iodine content of peptide b6 TL (1.11% by mass) exceeded slightly the average iodine content of the parent bovine Tg (1.05% by mass). Thus, the fraction of total Tg iodine contained in 2 mol of peptide b6 TL /mol of Tg dimer (0.16) was only slightly higher than the fraction of Tg mass that they accounted for (0.15). In particular, 13% of total iodine in peptide b6 TL was found in T 4 and 3% in T 3 , as opposed to 21 and 5%, respectively, in bovine Tg. Tyr-1291 contributed 10% of the T 4 and 8% of the T 3 content of Tg. The relative amounts of iodine incorporated into iodothyronines and iodotyrosines were 1 versus 5 in peptide b6 TL , and 1 versus 3 in Tg. On the basis of the moles of iodoamino acids formed per mole of Tg, the overall extent of modification of Tyr-1234, -1291, and -1375 appeared to be quite large, considering that the other 4 Tyr residues were unmodified (see Table I (Table I)  peptide b6 TL , more than 0.5 mol had to be formed in correspondence of Tyr-1291 and -1375, and less than 2.0 by the iodination of the 2 mol of Tyr-1234 per mole of Tg dimer, as the ES/MS spectrum of peak 23 revealed the presence of some unmodified Tyr-1234 (see Table I). This makes it probable that the amount of DHA formed at Tyr-1375 was of the same order of magnitude of the amount of iodothyronines formed at Tyr-1291 and leaves room for only a small amount of unmodified Tyr at positions 1291 and 1375. In fact, no unmodified Tyr-1291 was found in the mass spectra. DISCUSSION We report a detailed analysis of the post-translational modifications of seven tyrosyl residues comprised in fragment 1218 -1591 of bovine thyroglobulin. In particular, we demonstrate the formation of MIT, DIT, T 3 , and T 4 from Tyr-1291, and of MIT, DIT, and DHA from Tyr-1375. Modification of Tyr-1234 was restricted to formation of MIT, while Tyr-1450, -1464, -1484, and -1512 were unmodified.

Efficiency of Tyr-1375 as a T 4 -and T 3 -forming Site-The data of
Mass spectrometry is widely employed for the analysis of post-translational modifications of proteins (32). However, it has been used here for the first time to identify iodinated tyrosyl residues in Tg, and has proved extremely valuable as a source of primary structure data not available from earlier use of Edman degradation. In the past, the identification of hormonogenic sites by the sequencing of hormone-rich peptides of Tg was not always as direct. The only iodotyrosines and iodothyronines directly identified, by the manual method of sequencing with dimethylaminoazobenzeneisothiocyanate (35, 36), were those located at positions 2553, 2567, and 2746 of hog Tg (human Tg numbering) (17,19), and 5 of human Tg (12, 33, 34).
On the other hand, the phenylthiohydantoin-derivatives of iodoamino acids, in iodopeptides subjected to automated sequencing, were generally not identified by comparison with proper standards. The localization of hormonogenic sites in the NH 2 -terminal peptides of calf (13), sheep and hog Tg (14), in the tryptic peptides of rabbit (15) and guinea pig Tg labeled in vivo with 125 I (16), and in Tg from human goiters subjected to low-level iodination in vitro with 125 I (18), was based on the monitoring of the contents of 127 I or 125 I in the automated sequencing cycles, and the determination of the distribution of iodoamino acids. In this regard, although 125 I labeling provides an easy way to trace Tg iodopeptides and iodination sites and study hormonal turnover, it is not suited for the study of physiologically iodinated Tg of humans and other large animals.
The identification of donor tyrosyl residues was also indirect in all cases reported so far. In one study of bovine Tg, in which the separation of dehydroalanine-containing peptides exploited the conversion of dehydroalanine to S-(4-aminophenyl)cysteine, the presence of the latter at positions 5, 926, 986 or 1008, and 1375 was inferred from the lack of known phenylthiohydantoin-derivatives in sequencing cycles where tyrosine was expected, and from differences between the actual and expected tyrosine content of the peptides (20). In another study, the labeling of dehydroalanyl residues of bovine Tg with NaB 3 H 4 and their conversion to labeled aspartic acid with Na 14  residues (21). Finally, the proposal that alanine recovered at position 130 of peptide 1-171 of human Tg derived, in fact, from the conversion of dehydroalanine was largely based on speculation (22).
On the other hand, in the present study, mass spectrometry allowed the direct, unambiguous characterization of the entire spectrum of modifications of every tyrosyl residue within a large fragment of Tg. Not only the identification of T 4 and T 3 in correspondence of Tyr-1291 of bovine Tg is unprecedented, but also the localization of a donor site at position 1375 cannot be considered merely confirmatory, as it is based, for the first time, on a direct demonstration. By using the combination of limited proteolysis, preparative electrophoresis and mass spectrometry employed here, we project to extend our analysis to other still unsettled aspects of hormonogenesis in Tg, including: 1) the localization of the hormonogenic donor tyrosines of human Tg; 2) the identification of acceptor tyrosines other than tyrosine number 5 in physiologically iodinated human Tg; 3) the resolution of the uncertainties about donor sites at positions 986, 1008 (20), 2469 and 2522 (21) of bovine Tg.
The formation of T 4 at a site corresponding to residue 1291 of bovine Tg was already reported in rabbit (15,16) and guinea pig Tg (16), in which it contributed 17 and 11%, respectively, of Tg's T 4 . From the data reported in Table II, it appears that also in bovine Tg, Tyr-1291 contributed an appreciable amount of T 4 , together with a small amount of T 3 . It also appears that both Tyr-1291 and Tyr-1375 were to a large extent modified, mostly to DIT; however, only in one-fifth of the cases modification proceeded and iodothyronines were formed. On one hand, this probably reflects a high degree of accessibility of both residues. In this regard, the hydrophilicity plot of this region was not particularly informative (not shown); however, it is noteworthy that Tyr-1291 is located 70 residues apart from a cluster of protease-sensitive sites encompassing residues 1142, 1184, and 1218 (23). On the other hand, the prevalence of iodotyrosines at these sites raises interesting questions concerning the factors of steric hindrance that may limit the efficiency of a hormonogenic site, and the structural requirements that must be satisfied for efficient coupling to occur. In rabbit and guinea pig Tg labeled in vivo with 125 I, Tyr-1290 (human Tg numbering) contributed greatly to Tg's flexibility in meeting varying demands for hormone formation, as TSH enhanced T 4 formation at residue 1290, at the expense of T 4 formation at residue number 5, while increasing T 3 formation at residue 2746 (16). Under TSH stimulation, the percentage of T 4 neosynthesized at residue 1290 changed from 10 to 14% in rabbit and from 13 to 24% in guinea pig. In guinea pig, tyrosine 1290 was the most active site for new T 4 formation even in the presence of basal TSH levels (16). It is possible that also in bovine Tg the formation of T 4 at Tyr-1291 increase under TSH stimulation, e.g. as a consequence of iodide shortage. In this regard, it would be interesting to measure the share of Tg's total T 4 formed at Tyr-1291 at increasing levels of Tg iodination. On the other hand, in turtle Tg labeled in vivo with 125 I, only 5% of T 4 and 11% of T 3 were newly formed at Tyr-1290 (human Tg numbering) (37), while in human Tg iodinated in vitro with 7.8 atoms of iodine/Tg molecule, only traces of iodothyronines were found at residue 1290 (18). Only further work may establish whether this reflected the low level of Tg iodination, or the low efficiency of Tyr-1290 in human Tg. Interestingly, in human Tg, aspartic acid substitutes for tyrosine at position 1375. In addition, human Tyr-1447 was proposed to be a possible donor site, because it was iodinated early but did not provide inner iodothyronyl rings upon further iodination (18), whereas the corresponding Tyr-1450 of bovine Tg was unmodified in the present study. Although there is no indication that acceptor and donor residues need to be contiguous in the Tg sequence, the apparently low hormonogenic potential of human Tyr-1290 might indicate that, in bovine Tg, T 4 formation at Tyr-1291 depends on the presence of donor Tyr-1375, whereas, in human Tg, Tyr-1447 is not as good a donor site.
It was proposed that different hormonogenic sites of Tg evolved independently, and may also function independently from each other and the rest of the Tg molecule (38). Various observations support this hypothesis. Thyroid hormone formation within truncated NH 2 -terminal Tg fragments, derived from the abortive translation of normal-sized mRNAs, was probably responsible for the correction of hypothyroidism, by iodide supplementation, in a strain of Dutch goats with congenital goiter (39 -41), and for euthyroidism in Afrikander cattle (42)(43)(44). Efficient T 4 formation was demonstrated in isolated fragment 1-171 of human Tg (22,34). Thyroid hormones were also formed upon in vitro iodination of a fragment comprising the 224 COOH-terminal amino acids of rat Tg (45). It would be interesting to test the ability of peptide b6 TL , isolated from low-iodine bovine Tg, to sustain T 4 (and T 3 ) formation at Tyr-1291 upon peroxidase-catalyzed iodination in vitro. Should T 4 be formed, peptide b6 TL could represent an interesting model for the study of the minimal structural requirements of the hormonogenic function. Iodine and protein determinations and digestions with Pronase and aminopeptidase M were performed as described under "Experimental Procedures." Iodoamino acids were separated by reverse-phase HPLC with a Brownlee C-8 column (250 ϫ 4.6 mm, 5 m), as reported (31). Iodoamino acid contents were calculated from the iodine contents of the respective peaks.