From the Department of Biochemistry, University of Kansas Medical Center, Kansas City, Kansas 66103. Upon reduction and alkylation, 19 S bovine thyro-.
THEJOURNAL
OF
BXOMGICAL CHEMISTRY
Vol. 256, No. 18. Issue of September 25, pp. 9425-9430.1981
Printed in U.S.A.
Thyroglobulin Structure-Function ISOLATION AND CHARACTERIZATION OF A THYROXINE-CONTAINING POLYPEPTIDE FROM BOVINE THYROGLOBULIN* (Received for publication, February 2, 1981)
Steven B. Chernoff and Allen B. Rawitch+ From the Department of Biochemistry, University of Kansas Medical Center, Kansas City, Kansas 66103
Upon reduction and alkylation, 19 S bovine thyroglobulin gave rise to a family of polypeptides ranging in molecular weight from 10,000 to 330,000. T h i s group of polypeptides has been fractionated by chromatography on agarose gel in 6 M urea. After observing significantly increased iodine and thyroxine contents in the lowest molecular weight fraction obtained from the agarose gel-6 M urea chromatography, subsequent fractionation led to theisolation of a single, iodine-rich polypeptide in homogeneous form. While several low molecular weight polypeptides were found to contain iodine, onepeptide, designated TG-F, was distinguished by the fact that its iodine content exceeded 2% and virtually all of the iodine found within its structure was in theform ofthyroxine. TG-F was purified from pooled normal bovine thyroid tissue with an average yield of 0.9 mol of polypeptide/mol of 19 S thyroglobulin. It contains 2% iodine, only traces of carbohydrates, and 0.4 molof thyroxine/mol of peptide. The molecular weight of the polypeptide was found to be 10,300 A 1,300 as determined from sodium dodecyl sulfate gel electrophoresis, thin layer gel filtration in 6 M guanidine hydrochloride, and gel filtration on a calibrated column in ammonium bicarbonate. TG-F contains no free NH2-terminalacid as judged from automated sequenator analysis as well as the dansyl-amino acid procedure. Amino acid analysis revealed 2 lysines, 7 arginines, and a single methionine/mol of peptide. Sodium dodecyl sulfate polyacrylamide gels following cyanogen bromide digestion contained two peptide fragments of similar size and near half the size of TG-F. The absence of significant amounts of iodinated tyrosine derivatives except thyroxine in TG-F suggests that this peptide contains a specific site at which thyroxine formation proceeds both immediately and to completion following iodination of this region by thyroid peroxidase.
Thyroglobulin, the iodinated glycoprotein of the thyroid gland, forms the matrix within which thyroid hormones are synthesized. While much is known about this protein (1-4), structure-function studies have been difficult due to its very large size as well as the physical and chemical microheterogeneity described by a number of workers (5-7). Newly synthesized thyroglobulin is a substrate for thyroid peroxidase, a membrane-associated glycoprotein (a), which catalyzes both * This work was supported by Grant AM18896 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom inquiries may be addressed.
iodination and subsequent coupling of iodinated tyrosines to form thyroid hormones (9, 10). The nature of the thyroid peroxidase reactions has been studied in several laboratories (9, lo), and while significant questions remain to be answered with regard to the mechanism of this enzyme, it has been reported that those tyrosine residues in thyroglobulin which are iodinated early are also those residues which participate in hormone formation (11) and that the efficient formation of hormone occurs over a relatively narrow range of iodine content (12). While the interesting recent report of Cahnman (13), describing small peptides capable of thyroid hormone synthesis, suggests that a specific juxtaposition of 2 tyrosines in a polypeptide structure is important in thyroxine biosynthesis, the nature of the amino acid sequence in the thyroglobulinmoleculewhich produces thyroid hormone has not been defined. Althoughan initial attempt by Dunn (14) to address this question has provided some data derived from thyroxine-containing small peptides isolated from a pronase digest of rabbit thyroglobulin, only the immediate neighbors to thyroxine within the amino sequence of thyroglobulin were suggested and the specific amino acid sequence surrounding the site of hormone formation remains to be defined. While there are in excess of 100 tyrosine residues/mol of thyroglobulin in those vertebrate species which have been studied (15, 16), less than a quarter of those tyrosines are normally iodinated in uiuo and a still smaller number of such iodinated tyrosines (typically between 4 and 8) are actually involved in hormone synthesis. That very specific tyrosines are involved in hormonogenesis is further suggested by the unique ability of thyroglobulins to form thyroid hormone at very low levels of iodination (17) and by the recent observation of Dunn (18) that specific peptide fragments derived from rabbit thyroglobulin are involved in thyroxine biosynthesis. While thyroid hormone synthesis has been reported in several other proteins at high levels of iodination (17), the thyroglobulins alone, among the many proteins studied, are capable of significant thyroid hormone synthesis at low levels of iodination. In this paper, we report the isolation and partial characterization of a polypeptide fragment, derived from normally iodinated bovine thyroglobulin upon reduction and alkylation. This peptide contains significant amounts of thyroxine and no significant amounts of other iodoamino acids. This polypeptide fragment contains an amino acid sequence which is clearly important inhormone formation since it contains onethird of the total thyroxine in the native thyroglobulin. Moreover, it is of sufficiently small size to permit amino acid sequence studies.
*
9425
EXPERIMENTAL PROCEDURES
Preparation of Thyroglobulin-Bovine thyroid glands were ob-
9426
Isolation Thyroxine-containing of a Polypeptide
tained at a local slaughter house from healthy cattle. The thyroids 50% glycerol.Five % polyacrylamide gels were employed. Sodium were removed and kept on ice until use (within 30 min) or frozen dodecyl sulfate-containing gels were run according to the method of within 10 min after the killing of the animals in the case of later use. Weber and Osborn (30). The gels were stained for protein with The bovine thyroids were sectioned into 1-to 2-mm wide slices with Coomassie brilliant blue and in some cases for carbohydrate by the a single-edged razor, and extracted overnight a t 4 'C in 400 ml of 0.15 procedure of Fairbanks et al. (31). Gels were made up at acrylamide M NaC1, pH 7.0/100 g of tissue. When frozen thyroids were used, they concentrations from 4% to 15%,depending on the molecular size of were partially thawed prior to slicing. AU procedures were carried out the sample (see figure legends). Densitometry of stained gels was at 4 "C, unless otherwise noted. The extract was then filtered through carried out on a Gilford spectrophotometer equipped with a gelseveral layers of cheesecloth, made 42% saturated with (NH&S04, scanning apparatus. and stirred for 12 h. The solution was then centrifuged (12,000 X g ) NH2-TerminalGroup Determination-Samples of purified TG-F for 20 min and the resulting pellet reprecipitated between ammonium peptide from the P-60 column were subjected to NHn-terminal analsulfate concentrations of 37% and 44%of saturation as previously ysis of the dansyl amino acid technique of Woods and Wang (32). described (5).The resulting final pellet a t 44% saturation was resus- The peptide was also subjected to automated analysis (33) in a pended in 0.1% NH4HC03, dialyzed a t 4 "C against multiple changes Beckman model 890C sequenator (using Beckman Program 102974). of the same buffer, and lyophilized. The resulting partially purified The fractions obtained from the sequenator were subjected to both thyroglobulin was stored in this form at -20 "C until its further high performance liquid chromatography analyses (34) and backpurification by gel fitration. Thepartially purified thyroglobulin was hydrolysis followed byamino acid analysis in order to identify released redissolved in 0.1%NH4HC03and applied to a column of Bio-Gel A- amino acids (35). 15M agarose (Bio-Rad) and elutedwith the same buffer. The fractions Pyroglutamic Acid Aminopeptidase Digestion-TG-F was treated corresponding to the 19 S form of thyroglobulin were pooled and with pyroglutamate aminopeptidase according to the method of Polyophilized. Purified thyroglobulin was stored in this form at -20 "C dell and Abraham (36). until its use in experiments. Molecular Weight Determinations-The molecular weights of the Reduction and Alkylation-The method of Crestfield et al. (19) reduced and alkylated peptides were determined from migration in was employed for reduction and alkylation of 19 S thyroglobulin.' SDS-gel electrophoresis (37),migration in the thinlayer gel filtration The alkylation phase of the procedure with iodoacetate was quenched system of Hung et al. (38) in 6 M guanidine HC1, and from elution with a 10-fold excessof 2-mercaptoethanol. A typical reduction mix- behavior on a calibrated column of Bio-Gel P-60 (Bio-Rad) (39). In each case, standards consisted of reduced and alkylated proteins or ture contained approximately 200 mg of thyroglobulin. Fractionation of Reduced and Alkylated Proteins-Samples of polypeptides of known molecular weight, as indicated in the figure reduced and alkylated thyroglobulin were applied directly to a Seph- legend. High Perfonnance Liquid Chromatography Analyses-High perarose CL-4B column (Pharmacia), equilibrated with 0.1 M sodium phosphate, pH 7.0, in 6 M urea, and eluted with the same buffer. The formance liquid chromatography of reduced and alkylated peptides effluent of the column was monitored for absorption a t 280 nm. Peak was carried out on a Beckman-Altex model 332 liquid chromatograph material were pooled and dialyzed briefly against distilled water to using a Waters 1-125 protein column in 0.1% ammonium bicarbonate. remove most of the urea andthen extensively against 0.1%NH4HC03 Column effluent was monitored at 230 nm. Tryptic Digestion and Peptide Mapping-Tryptic digests were before lyophilization. The various fractions (designated A through F in order of decreasing size) were subjected to gel electrophoresis and carried out as previously described (40) in 1%ammonium bicarbonate. iodine analysis as described below. The smallest fraction, fraction F, Aliquots of the tryptic digest were spotted on a sheet (20 X 20 cm) of was further purified on a column of Bio-Gel P-60 (Bio-Rad) in 0.1% thin layer cellulose (Eastman-Kodak (without fluorescent indicator)) N&HC03. The effluent of this column was also monitored at 280 nm. and chromatographed in an ascending chamber in butanolpyridine: The threepeaks detected in the elution pattern of this column were acetic acidwater (1510212). After drying, the plates were sprayed designated TG-E-1, TG-E-2, and TG-F, in order of decreasing size. with a dilute pyridine/acetic acid buffer at pH 3.5 and electrophoresed Fraction TG-F was rechromatographed in this system in those cases in the second dimension. Tryptic peptides were detected with a spray where gel electrophoresis indicated any contamination with fractions of0.1% ninhydrin in ethanol or with fluorescamine and ultraviolet light. Iodine-containing peptides were detected with the G e d i n and E-1 and E-2. Analytical Procedures-Amino acid analyses were carried out on Virtanen reagent (41)in the form of a fine spray. Iodine-containing a Beckman model 121 automated analyzer following hydrolysis in peptides were revealed initially as dark blue spots against a yellowredistilled 5.7 N HC1 for 22 h a t 110 "C. Tryptophan was determined green background. Substantial background deterioration occurred spectrophotometrically (20). Neutral sugars were determined accord- with time, necessitating observation and recording of the iodine ing to the procedure of Kim et al. (21), but using Dowex 1 resin to staining within 5 to 10 min. Cyanogen Bromide Cleavage-TG-F was subjected to CNBr remove H,S04 and a Supelco SP-2340column in a Hewlett-Packard model 5830A gas chromatograph for separation and quantitation of cleavage in 70%formic acid (42).After removal of the formic acid and the alditol acetate derivatives. Amino sugar analyses were carried out CNBr, the sample was concentrated by rotary evaporation and an on the shortcolumn of the amino acid analyzer as previously described aliquot applied to an SDS-polyacrylamide gel for analysis of cleavage (22).Sialic acid was determined by a modification of the thiobarbituric products. Estimation of Stoichiometry-The stoichiometry of TG-F per mol acid assay of Warren (23) after hydrolysis of the protein with 0.1 N of reduced and alkylated thyroglobulin was determined by densitomH&04 at80 "C for I h. Protein concentrations were estimated by the Hartreemodification etry of 4% SDS-polyacrylamide gels. Coomassie blue staining was of the Lowry method (24) using purified 19 S thyroglobulin as a assumed to be proportional to the amount of each polypeptide comstandard. The E% for the thyroglobulin was assumed to be 10.0 (25). ponent. The amount of TG-F recovered from the CL-4B column Iodine analyses were carried out by the method of Sandell and chromatography was determined by integration of the absorbance at Kolthoff (26) followinga wet-ashing procedure using perchloric acid. 280 nm. An E:& of 15.8 based on amino acid analyses and Lowry Thyroxine contents were determined in duplicate and at two sample determinations on pureTG-F was used in the latterdetermination. levels by specific radioimmunoassay of pronase digests according to RESULTS the procedure of Regard et al. (27). These analyses were carried aut in the laboratory of Dr. A. Taurog at theUniversity of Texas Health 19 S Thyroglobulin Starting Material-The 19 S thyroScience Center in Dallas. Qualitative iodoamino acid analyses were carried out by thin layer chromatography after pronase digestion globulin usedin these studies was isolated from pooled bovine thyroidsfollowingammoniumsulfatefractionationand 4% according to the method of Gleason (28). Gel Electrophoresis-Polyacrylamide gel electrophoresis under agarose gel chromatography.The fractions pooled fromsuch nondenatwing conditions was carried out as described by Ornstein a column yield a preparation which is over 95%in the 19 S (29), but without stacking or sample gels and the samples applied in form when subjected to direct analytical ultracentrifugation
before freeze-drying.The remaining components inthe prep-
' T h e abbreviations used are: 19 S thyroglobulin, thyroglobulin aration, the 12 S and 27 S forms of thyroglobulin, are present which sediments in the ultracentrifuge with a sedimentation coefficient near 19 S; SDS, sodium dodecyl sulfate; TG-F, the lowest in slightly increased amounts following lyophilization dueto molecular weight polypeptide derived from reduced and alkylated dissociation but do not exceed 15% of the total sedmenting of the material showedthe usual bovine thyroglobulin; dansyl, 5-dimethylaminonaphthalene-1-sul- material. Gel electrophoresis fonyl. three-banded pattern seen in thyroglobulins (15). The iodine
Isolation of a Thyroxine-containing Polypeptide content of the 19 S thyroglobulin was 1.0% with 2.5 mol of thyroxine found/mol of thyroglobulin by radioimmunoassay.
Fractionation of Reduced and Alkylated Thyroglobulin-
9427
that fraction F, the lowest molecular weight fraction, contained a substantial amount of thyroid hormone. Moreover, the per cent of the total iodine present in this form in this fraction was very high (>90%) compared to thatof the starting protein ( 19%). The material in fraction F of the CL-4B Sepharose column was next applied to a column of Bio-Gel P-60in an attemptto further purify the principal component in this fraction. The results of such an experiment are shown in Fig. 3.Three peaks were seen in the elution profile with the major peak representing material which corresponded to the principal component seen in gel electrophoresis of the impure fraction F from the urea-CL-4B column. The two lesser peaks seen in the elution profile corresponded with the two principal components seen in fraction E of the urea-CL-4B column. These components were designated TG-E-1, TG-E-2, and TG-F, in order of their elution.
Sodium dodecylsulfate electrophoresis of reduced and alkylated thyroglobulin revealed a family of polypeptides ranging in size from M, approximately 10,000 to near 330,000. No significant difference was observed between the SDS-gel electrophoresis patterns of the thyroglobulin prepared from fresh tissues and that prepared from frozen glands. Therefore, in mostcases, the thyroglobulin was prepared from frozen glands. This mixture of reduced and alkylated polypeptide chains was applied to a column of Sepharose CL-4B in 6 M urea, pH 7.0, 0.1 M sodium phosphate (Fig. 1).The elution profile revealed a large initial peak of high molecular weight material with several shoulders on the low molecular weight side followed by four additional peaks at lower molecular sue. These fractions were designated A through F in order of Characterization of the Purified Low Molecular Weight decreasing molecular size (Fig. 1). After dialysis against 0.1% NI&HC03, each pooled fraction was freeze-dried. The frac- Iodinated Peptides-Peptides TG-E-1, TG-E-2, and TG-F tions were each analyzed for iodinecontent (see Table I) and were shown to be enriched in both iodine and thyroxine (see were subjected to SDS-gel electrophoresis (Fig. 2). Due to Table 11). Most striking was TG-F, which not only showedan their iodine content and our earlier observations (45), we focused our attention on the lower molecular weight fractions. TG-A TG-B TGCTGDTGE TG-F Fractions E and F were found to contain increased amounts Cathode of iodine (1.6% and 2.1%, respectively) when compared to the starting protein (15).While fraction D wascomplex and contained a number of bands of intermediate molecular sue, fraction E contained two principal components with molecular weights near 20,000 and a small amount of a third component which correspondedto theprincipal constituent of fraction F. Fraction F contained reduced amounts of the two components seen in fraction E and a principal constituent of M , near 10,000. Analyses of fractions E and F for thyroxine indicated ~~
~w
El E2
F Anode
FIG. 2. Four 5% sodium dodecyl sulfate polyacrylamide slab gel electrophoresis of reduced and alkylated thyroglobulin fractions from the C U B column. The fust threeslots are 200 pg of TG-A, TG-B, and TG-C. The fourth slot is 100 pg of TG-D. The
last two slots are 40 pg each of TG-E and TG-F. Gels contained 4% bisacrylamide cross-linking. o
.
220 1 40 "
L 60 L
00 J
FRACTION NUMBER
FIG. 1. C W B gel chromatography. Sepharose CL4B equilibrated with 0.1 M sodium phosphate, pH 7.0, in 6 M urea or a column (100 X 5 cm). Samples of reduced and alkylated 19 S thyroglobulin (200 mg in 25 ml) were applied to the column and 15-ml fractions collected. The column profile wasbroken into six fractions, designated
1
F CI
TG-A,TG-B,TG-C,TG-D,TG-E,andTG-F. TABLE I Iodine contentof thyroglobulin fractions obtained from CL-4B column Iodine Tg fraction"
s 19 S TG
TG-A TG-B TG-C TG-D TG-E TG-F a Tg = thyroglobulin.
1.0 0.97 1.1 0.95 0.88 1.6 2.1
FRACTION NUMBER
FIG. 3. P-60gel chromatography. Bio-Gel P-60was equilibrated with 0.1% NRHCOJ in a column (110 X 2.5 cm) and 5-ml fractions were collected. This profile shows the purification of TG-F. Thefirst two peaks are components of TG-E, El,and Ez, contaminatingTG-F. Similarly, TG-E was freed of contamination by TG-F. Samples of 6 mg in 1.0 ml were used.
9428
Isolation of a Thyroxine-containing Poljpeptide
increased iodine content, but virtually all of its iodine was in the form of thyroxine as determined by specific radioimmunoassay. Because of its unique properties and composition, specific attention was directed to thecharacterization of TGF. The Properties of TG-F-SDS-polyacrylamide gel electrophoresis of TG-F revealed a single band migrating at a rate consistent with a molecular weight of near 10,OOO (Fig. 4). The data obtained using three different techniques for the determination of molecular weightare summarized in Fig. 5. TG-F showed an apparent molecular weight of 10,300 1,300 and was seen as a single component in all three procedures. The results of amino acid, iodoamino acid, and carbohydrate analyses of TG-F are summarized in Table 111. TG-F contains a single methionine residue, 2 lysine, and 7 arginine residues. The small amounts of neutral sugar and glucosamine found in the peptide do not correspond to any of the known
*
TABLE 11 Thyroxine content of the purified small molecular weight fractions of 19 S thyroglobulin Iodine
Tg fraction
x EI
I
0.95 1.7 0.2 2.1
E2
F
0.4
Amount T4/mol peptide d
0.1
b
oligosaccharide units in bovine thyroglobulin (43). Since the carbohydrate-staining procedure of Fairbanks (31) was negative for gels containing TG-F, definition of significance of the neutral sugars found in the analyses must await further characterization of the peptide. The amino acid and carbohydrate compositions shown in Table I11 are consistent with a molecular weight of approximately 9,800.This compares very well with the value obtained in physical studies, 10,300 f 1,300. Treatment of TG-F with CNBr followed bySDS-polyacrylamide gel electrophoresis resulted in two bands after Coomassie blue staining with similar molecular sizes near half that of TG-F, suggesting that thesingle methionine found in the peptide occurs near the middle of the polypeptide chain. Two-dimensional peptide maps of a tryptic digest of TG-F contained 11 spots when sprayed with ninhydrin or fluorescamine. Spraying of such a tryptic peptide map of TG-F with the iodine spray of Gmelin and Virtanen (41) revealed two rapidly developing positive spots. When TG-F was subjected to 10 cycles of automated Edman degradation in a sequenator, no free NHz-terminal sequence could be found either by chromatography of phenylthiohydantoin amino acid derivatives or back-hydrolysisfollowed by amino acid analyses. When this observation was c o n f i i e d in a repeat experiment at higher sample load, dansylation of TGF followed byhydrolysis and thin layer chromatography with standard dansyl amino acid markers was carried out and revealed no spot which corresponded to a known a-dansylated amino acid. Since Hayashi et al. (44)reported that pyroglutamic acid is a blocking group at the NHz terminus of hog thyroglobulin, TG-F was treated with pyroglutamic acid aminopeptidase (36) and then resubmitted to the automated Edman procedure. While negative results were obtained again, pyroglutamate TABLE I11 Amino acid and carbohydrate composition 19 S Tgb
TG-Fa mOl'/mOl peptide
Carboxymethylcysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan' N-Acetylglucosamine Fucose Mannose Galactose Monoiodotyrosine Diiodotyrosine Triiodothyronine Thyroxine
FIG. 4. Ten 5% sodium dodecyl sulfate polyacrylamide tube gel electrophoresis of TGF. The upper portion shows the gel; the lower portion shows the densitometer scan at 595 nm. Sample size was 40 pg. 5.0
? - 4.2i W
-
3.8 4.6:\-: 3.4
io
a0 rnl
60
0.4 0.2
0.6 0.8 0.2 0.4 0.8 0.8 Relative Migration
FIG. 5. Calibration curves for the three methods used to determine the molecular weight of TG-F. The following reduced and alkylated standards were used with numbers corresponding to those in the figure: 1) bovine serum albumin; 2) ovalbumin; 3) chymotrypsinogen;4 ) horse myoglobin; 5) a-lactalbumin; 6) ribonu(M, clease; 7) cytochrome c; 8) horsemyoglobinCNBrfragment 8,000); and 9) horsemyoglobinCNBrfragment (M,6,000).The arrows represent the position of TG-F in each case. Panel A shows the data from a calibrated P-60column in 0.1% NKHC03. Panel B shows the results of thin layer gel chromatography in G-75,in 6 M guanidine HC1. Panel C shows the data from sodium dodecylsulfate polyacrylamide gel electrophoresis in10% gels.
3.22 8.00 2.12 5.97 14.19 6.25 5.02 5.69 4.74 0.57 1.78 5.30 1.41 2.08 2.14 0.15 6.47 1.90 0.98 0.74 0.78 1.15
x
Wtd
3.75 10.18 2.40 5.86 20.61 6.83 3.23 4.55 5.30 0.85 2.28 6.75 2.59 3.46 3.10 0.23 11.38 3.53 0.86 0.59 0.69 1.01
x Wtd 3.65 7.02 4.02 6.70 14.60 6.53 3.92 4.52 4.93 0.86 2.74 8.97 3.53 6.91 2.96 1.34 8.92 2.5 0.4 2.7 1.6 0.237' 0.518/ 0.026/ 0.224
'
0.4 3.04 Average of five analyses. 'These data are taken from Gleason (28). e Determined by assuming 8.00 mol of aspartic acid/mol of TgF. Per cent of weight recovered. 'Determined spectrophotometrically. Per cent recovered from ion exchange column. a
'
Isolation of a Thyroxine-containing Polypeptide TABLEIV Recovery of TG-F at each step Procedure
Amount TGF recovered/ 100 mg reduced and alkylated thyroglobulin
Recovery"
mg
B
alkylation Reduction and 3.1b 100 CL-4B chromatography 2.9' 94 P-60 chromatography 1.4 45 a These are representative values obtained from over 20 individual samples of reduced and alkylated thyroglobulin. Based on densitometry of Coomassie Blue stained4%SDS-polyacrylamide gel electrophoresis experiments. 'Based on integration of the area under the TG-F peak and an E;% of 15.8, determined from purified TgF. can not be n,ded out completely since the pyroglutamic acid aminopeptidase may not act on such large polypeptides (36). Samples of TG-F were digested with pronase and subjected to radioimmunoassay for thyroxine (see Table 11).The thyroxine found by radioimmunoassay accounted for more than 95% ofthe totaliodine present in the peptide. This observation was qualitatively confirmed by thin layer chromatography of another aliquot of a pronase digest where the major iodinated species observed was thyroxine with only traces of other iodinated amino acids (in particular, monoiodotyrosine and diiodotyrosine were present in almost undetectable amounts). Densitometry of SDS-polyacrylamide gels of typical preparations of reduced and alkylated thyroglobulin after staining with Coomassie blueindicated that TG-F represented 3.1% of the total protein staining. While this value was based on the assumption of equal staining by Coomassie blue of the various polypeptides of thyroglobulin, it agrees well with the estimate obtained from the integration of 280-nm absorbance associated with the TG-Fpeak in the CL-4B columnelution profile. These observations suggest that very near 2 mol of TG-F occur/mol of thyroglobulin. TG-F is recovered in amounts of -0.9 mol/mol of thyroglobulin. Thus, the isolation procedure results in a typical net yield of pure peptide of approximately 45% (see Table IV). Since these thyroglobulin preparations are derived from pooled tissue of normal animals, this yield must be regarded as a representative yield of TG-F from normally iodinated thyroglobulin isolated under these conditions. DISCUSSION
In 1975,we reported that the smallest polypeptide fragments observed upon reduction and alkylation of bovine thyroglobulin were specifically enriched in both iodine and thyroid hormone (45). That observation has been recently confirmed and extended by Dunn et al. (18)in rabbit and human thyroglobulins.' In thispaper, we describe the isolation of one such small peptide (TG-F) derived from bovinethyroglobulin upon reduction and alkylation. This peptide is found in thyroglobulin prepared either from fresh or frozen thyroid glands. An average of 2 mol of TG-F are derived from 1 mol of 19 S thyroglobulin under these conditions, or 1 mol of TG-F/12 S subunit. The stoichiometry must be regarded as approximate since it is based on an assumption of equal Coomassie blue staining of the various polypeptide fragments derived from thyroglobulin and it may represent a minimum value since the larger molecular weight fractions may contain ad-
* Reported by J. T. Dunn,P. S. Kim, and A. D. Dunn at the annual meeting of The American Thyroid Association, San Diego, CA, November, 1980 (Abstr. T-1).
9429
ditional TG-F sequences covalently linked within their structures. TG-F contains less than 1 residue of thyroxine/mol of peptide and no significant amounts of other iodoamino acids, suggesting that some of the TG-Fis not iodinated. Nevertheless, the thyroxine present in TG-F represents at least 32% of the total thyroxine found in the whole bovine thyroglobulin (assuming 2 mol of TG-F/19 S thyroglobulin molecule). Furthermore, if the TG-F were fullyiodinated and coupled to the level of 1 mol of thyroxine/TG-F molecule, it could account for up to 2 mol of thyroxine/mol of thyroglobulin (80%of the amount of thyroxine found in the bovine thyroglobulin). The absence of significant amounts of monoiodotyrosine and/or diiodotyrosine in TG-F is in stark contrast to the iodoamino acid compositionof whole thyroglobulin and other iodinated peptides where substantial amounts of both of these iodoamino acids are found. This difference suggeststhat when the tyrosine residues in TG-Fare iodinated, subsequent coupling to form thyroxine proceeds immediately and completely. While these data may be interpreted in other ways, this suggestion is consistent with the fact that thyroid peroxidase is known to catalyze both the iodination and coupling reactions (8). TG-F was observed to be homogeneous (greater than 90% in the form of a single species) in SDS-gel electrophoresis, high performance liquid chromatography on a molecular sieve (1125) column, thin layer gel filtration in the presence of guanidine hydrochloride, and gel filtration on a column of Bio-Gel P-60. The amino acid composition of TG-F is not particularly unusual, although the presence of a single methionine near the middle of the TG-F sequence is notable in that theCNBr cleavage products should be useful in providing overlaps for amino acid sequence studies. The number of tryptic peptides observed in peptide maps (10-12) is consistent with the 2 lysine and 7 arginine residues found. The absence of histidine in TG-F should also be noted. It is of interest that thetyrosine content in TG-F is significantly lowerthan thatof thyroglobulin. Moreover, when one assumes that both of the tyrosines which form the thyroxine come from the TG-Fsequence, the remaining 1.4 residues of noniodinated tyrosine found in TGF would be totally consumed in hormone formation if the peptide werefully iodinated and coupled (to a level of 1 thyroxine/mol of TG-F). Even if only one of the tyrosines in T4 comes from the TG-F sequence, the fully iodinated and coupled peptide would contain only a single remaining tyrosine residue not used during hormone formation. This observation is quite consistent with the concept of very specific sites on bovine thyroglobulin for hormonogenesis since, of approximately 120 tyrosine residues found in thyroglobulin, only 25 to 30 are iodinated and less than 6 (-5%) participate in thyroxine formation. While small amounts of carbohydrate werefoundupon analysis of TG-F, the relative and absolute amounts of sugars do not correspond to any of the known oligosaccharide units reported in bovine thyroglobulin, and it is possible that these small amounts of carbohydrate reflect either a new oligosaccharide type in bovine thyroglobulin or possibly some minor contamination of the preparation with a glycosylated component(s). Thefact that staining of polyacrylamide electrophoresis gels containing TG-F with the Schiff-periodate stain according to the method of Fairbanks et al. (30) was negative suggests that the peptide may, in fact, be nonglycosylated. The absence of a free NH2 terminus in TG-F is significant in view of the fact that in multiple studies by several groups (15,46,47), only limited amounts of NHn-terminal amino acids have been reported (insufficientamounts to be consistent with the multiple polypeptide fragments observed in almost every
9430
Isolation of a
Thyroxine-containing
thyroglobulin preparation studied upon reduction). The origin of the TG-Fpolypeptide is an importantquestion which is currently being addressed in this laboratory. Since it is found in disflide linkage with the 19 S thyroglobulin complex, the simplest assumption is that it is derived from a parent polypeptide chain of thyroglobulin by either proteolysis or some nonclassicalpeptide bond cleavage. Whether this cleavage is part of the normal processing of thyroglobulin polypeptide chains and whether the cleavage proceeds or follows iodination and coupling are questions which remain to be answered. On the other hand, the possibility of independent synthesis of TG-F followed by disulfide linkage to thethyroglobulin complexcannot be ruled out at this time, particularly in view of the recent observations by Dunn and co-workers (48,49)that thyroglobulin from different thyroids within the same species and even from different parts of a single gland may have different amino acid compositions and presumably different amino acid sequences. If the observations of Dunn and co-workers are confirmed, clearly, the generally accepted scheme for thyroglobulin biosynthesis as unique chains of M, 330,000 (50) must be re-examined. Regardless of its origin, TG-F represents a fragment of the thyroglobulin complex whichcontains an amino acid sequence of key significancein thyroid hormone biosynthesis and thyroglobulin structure-function and therefore merits further study including the determination of the sequence and the location of the hormonogenic site within that sequence. It should be noted that recent reports from Dunn and coworkers (18)have described the isolation of a similar peptide derived from reduced and alkylated rabbit thyroglobulin following in vivo iodination of the protein with radioiodine. The polypeptide fragment described by Dunn et al. differs in several respects from TG-F. It has a reported molecular size of 20,000 (twice the size of TG-F), contains 10%carbohydrate and a free NH2-terminal aspartic acid. Moreover, the rabbit peptide contains significant amounts of other iodoamino acids in addition to thyroxine. It wil be of interest to compare amino acid sequence data on these two hormonogenicpeptides as they become available. Acknowledgments-We would like to thank Linda Fox for able technical assistance. The contribution of Dr. M. J. Gleason to the early stages of this work is also gratefully acknowledged. We would also like to thank Dr. A. Taurog at TheUniversity of Texas Center for the Health Sciences, Dallas, for carrying out the thyroxine radioimmunoassays and Dr. John Dunn at The University of Virginia School of Medicine, Charlottesville, for allowing us to see a copy of his manuscript on a similar peptide from rabbit thyroids prior to its publication. REFERENCES 1. Edelhoch, H. (1960) J. Biol. Chem. 235, 1326-1334
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