THE MOLECULAR WEIGHT AND ISOELECTRIC POINT OF THYROGLOBULIN

1. The sedimentation constant of hog thyroglobulin is 19.2ċ10^–13. That of human thyroglobulin is essentially the same. 2. The specific volume of hog thyroglobulin is 0.72. 3. The isoelectric point of native hog thyroglobulin is at pH 4.58, that of denatured thyroglobulin at pH 5.0. 4. The molecular weight of hog thyroglobulin is, in round numbers, 700,000, as calculated from the sedimentation and diffusion constants, or 650,000, as calculated from the sedimentation equilibrium data. 5. The thyroglobulin molecule deviates markedly from the spherical.     

Iodination and oxidation of thyroglobulin catalyzed by thyroid peroxidase

The kinetics of iodination and oxidation of hog thyroglobulin were studied with purified hog thyroid peroxidase and the results were compared with the reactions of free tyrosine. From Lineweaver-Burk plots and on the basis of a value of 0.83 for delta epsilon mM at 289 nm/iodine atom incorporated, the rate constant for transfer of an assumed enzyme-bound iodinium cation to thyroglobulin was estimated to be 6.7 X 10(7) and 2.3 X 10(7) M-1 s-1 in native (iodine content = 1.0%) and more iodinated (iodine content = 1.2%) thyroglobulins, respectively. This iodine-transferring reaction was stimulated by iodothyronines, similarly as observed in the reaction with free tyrosine. The iodination of thyroglobulin was inhibited by GSH, the inhibition being competitive with thyroglobulin. Thyroglobulin was oxidized in the presence of a thyroid peroxidase system without giving any appreciable change in absorbance around 300 nm. From stopped flow data, the oxidation was concluded to occur by way of two-electron transfer and the rate constant for the reaction of thyroid peroxidase Compound I with thyroglobulin was estimated to be 1.0 X 10(7) M-1 s-1. The stopped flow kinetic pattern was similar to that observed on the reaction with free tyrosine and monoiodotyrosine. About 6 mol of hydrogen peroxide were consumed per mol of thyroglobulin. Thyroid peroxidase catalyzed thyroglobulin-mediated oxidation of GSH, but lactoperoxidase did not.     

C-terminal extremity of ovine thyroglobulin shows strong interspecies homologies

We report on the 466 nucleotides long, extreme 3' end of the ovine thyroglobulin (Tg) mRNA. The nucleotide sequence and the deduced carboxyl terminal region of the protein have been compared with those reported in other species. Comparison with hog peptides known to contain all the triiodothyronine (T3) of the mature Tg suggests that the antepenultimate amino acid, a tyrosine residue could be involved in hormone formation. This also agree with the data reported for bovine and murine Tg.     

C cell (parafollicular cell) -- immunoreactive thyroglobulin: purification, identification and immunological characterization.

In relation to our earlier finding that the thyroglobulin-like material responsible for the cytochemical immunoreaction of C cells was obtained in the peak I fraction of Bio-Gel A-5 m, which included faster sedimenting components of thyroglobulin, the present study has identified the positive reacting component and clarified its immunochemical and immunohistochemical properties. 1. The peak I fraction of dog and hog thyroglobulin was chromatographed on a Bio-Gel A-50 m column. Antiserum to the faster eluted peak I'1 only immunoreacted with C cells. The peak I'1 was then refiltered on Bio-Gel A-150 m column. Antiserum to peak I'1 fraction of both species which was eluted in the first part had high immune specificity for C cells. 2. When 4-30% and 2-16% continuous gradient gels of polyacrylamide were employed, peak I'1 represented a single electrophoretic band corresponding to the component with the largest molecular weight in thyroglobulin. The protein was named C-thyroglobulin. The molecular weight was approximately 2,600,000, four times as large as 19 S, as calculated by relative mobility on the 2-16% gradient gel. 3. In double diffusion tests, anti-peak I'1 antiserum produced two immunoprecipitin lines with its own antigen. The reaction was different from that of anti-19 S antiserum which formed a single line. 4. On immunoperoxidase staining, anti-peak I'1 antiserum reacted to C cells in exactly the same way as anti-calcitonin antiserum. 5. When anti-peak I'1 antiserum was absorbed with calcitonin, the subsequent reaction of the C cells was greatly decreased. The absorption of anti-calcitonin antiserum with increased amounts of peak I'1 abolished the C cell reaction. On the basis of these observations, the possibility that C-thyroglobulin is a biosynthetic precursor of calcitonin exists.     

A procedure for the separation and quantitation of tryptophan and amino sugars on the amino acid analyzer

Tryptophan, 5-methyl tryptophan, glucosamine, and galactosamine can be separated from each other and hydrolysis products including lysinoalanine by chromatography on a 6 × 260-mm column of W-3H resin. The column is developed at 70°C for 20 min with pH 3.95 (0.4 Na⁺) buffer, followed by pH 6.4 (1 Na⁺) buffer for 55 min using a Beckman 119 CL amino acid analyzer. The recovery of the internal standards, 5-methyl tryptophan and galactosamine, can then be used to correct for tryptophan and glucosamine losses, respectively. The procedure uses the column and buffers normally employed for protein hydrolysate analysis and does not require additional resin columns, special buffers, or flow rate changes.     

Acid-induced conformational change in hog thyroglobulin

The molecular behavior of hog thyroglobulin in acid solutions has been examined by fluorescence, absorption, proton binding, and velocity centrifugation. The precipitation of thyroglobulin which normally occurs in the pH region near its isoelectric point was prevented by reducing the salt concentration to 0.01 m and the protein concentrations to the lowest levels needed for measurements. The rate of denaturation is very slow near pH 5.0 but increases rapidly with decreasing pH. The molecular properties of acid-denatured thyroglobulin, though still retaining some aspects of its native tertiary structure, are quite different from those of the native protein. In the molecular transition: (a) buried tryptophanyl residues become exposed, (b) the anomalous dissociation of certain groups becomes normalized, (c) subunits are formed, and (d) the molecular form of thyroglobulin and its subunits becomes partially unfolded.     

Subunit structure of 27 S thyroid iodoprotein.

The dissociation of thyroid 27 S iodoprotein by sodium dodecyl sulfate (SDS) and by succinic anhydride was investigated by means of ultracentrifugation and polyacrylamide gel electrophoresis. The iodoprotein obtained from either a human or hog was dissociated into three kinds of subunits (S-19, S-17 and S-12) by SDS treatment. At increased concentrations of SDS, the S-12 subunit was predominant among the dissociation products. The succinylation of 27 S iodoprotein showed essentially the same dissociation pattern as in the case of SDS treatment. This dissociation products of the protein preparations of different animals were qualitatively the same as those of thyroglobulin of the respective animals, confirming the hypothesis that 27 S iodoprotein was composed of two molecules of thyroglobulin. However, the extent of dissociation of 27 S iodoprotein measured by S-12 formation showed higher resistancy of the protein to the dissociating agents than that of thyroglobulin. The contents of sialic acid and hexose as well as iodoamino acids of 27 S iodoprotein were found to be the same as, or not far from, those of thyroglobulin; The dissociability and chemical composition of 27 S iodoprotein was discussed with reference to the subunit structure of the protein.     

The effect of iodide administration on hog thyroid gland and the composition of thyroglobulin and 27-S iodoprotein.

The effect of excess iodide on hog thyroid gland has been examined with regard to the change in the chemical composition of thyroglobulin and in the accumulation of 27-S iodoprotein by the in vivo treatment of hogs with iodide for various lengths of time. The iodine content of thyroglobulin was either unchanged by short term administration of excess iodide, or somewhat lowered. However, the iodine content as well as the total amount of thyroglobulin increased in the glands enlarged by prolonged treatment with iodide. The iodine highest reached 1.17% of the protein on an average. On the other hand, 27-S iodoprotein decreased and finally disappeared after the chronic treatment. Monoiodotyrosine and diiodotyrosine increased in parallel with the increase in the iodine content (0.15 to 1.17%) caused by the iodide treatment, while thyroxine increased but reached a plateau at the level of three residues per mole of thyroglobulin, and no change was observed even in the proteins with the higher iodine content than 0.75%. Proteolytic activity measured by amino acid release from the thyroid protein was depressed by the chronic treatment. On the other hand, the amount of iodocompound released by the autoproteolysis, which may reflect hormone secretion, increased, possibly because of the marked increase in the iodine content of thyroglobulin.     

The effect of scurvy on glycosaminoglycans of granulation tissue and costal cartilage

1. The effect of ascorbic acid deficiency on glycosaminoglycans of granulation tissue and cartilage of guinea pigs was investigated by determination of the changes in the glucosamine and galactosamine contents 12 days after tendonectomy. 2. In normal granulation tissue, the glucosamine and galactosamine contents rose to a peak at 5 and 10 days respectively, whereas the hydroxyproline and proline contents continued to rise throughout the 20 days after tendonectomy. 3. The galactosamine in scorbutic granulation tissue, but not in that of pair-fed controls, decreased significantly in absolute amount and relatively to glucosamine, which remained practically unchanged; the cartilage galactosamine did not decrease during the 22 days of deficiency owing to the presence of excess of preformed galactosaminoglycans, which masked the small amount of newly formed glycosaminoglycans. 4. The chemical results were confirmed by radioactivity studies in vivo of incorporation of [U-(14)C]glucose into galactosamine and glucosamine of scorbutic granulation tissue and cartilage. The incorporation of (14)C into galactosamine decreased significantly in scurvy in both tissues. 5. The results indicated in both tissues a decreased formation of galactosamine during scurvy, although an increased degradation of polymerized glycosaminoglycans could not be entirely ruled out. It is concluded that, if lack of ascorbic acid causes an impaired galactosamine formation, the most likely position for the block may be in the UDP-N-acetylglucosamine 4-epimerase reaction.     

Isolation and characterization of hog thyroid actin

When the thyroglobulin content is subtracted, actin represents approximately 4.6% of the total protein content in the hog thyroid gland. Actin has been isolated from acetone-dehydrated slices and purified to homogeneity by gel filtration, DEAE-cellulose chromatography and two polymerization-depolymerization cycles. Purified actin (Mr = 42000) contains the beta and gamma species with a 2 to 1 stoichiometry. In the presence of 0.1 M KCl and 2 mM MgCl2 thyroid actin polymerized into 6 nm diameter filaments; under these conditions the critical concentration was 30 micrograms/ml and the intrinsic viscosity 4.7 dl/g.     

Occurrence of lysinoalanine in calcified tissue collagen

Lysinoalanine was identified in hydrolysates of dentine and bone collagen. The compound was isolated and purified by ion exchange chromatography on P-cellulose and QAE-Sephadex columns. Identity with lysinoalanine was demonstrated by ¹H-nmr spectroscopy, amino acid analysis and paper chromatography. This is the first example of occurrence of lysinoalanine in native proteins.     

Solubilization, purification, and characterization of (H+, K+)ATPase from hog gastric microsomes

The (H+,K+)ATPase-enriched microsomal fraction prepared from hog gastric mucosa by sucrose density gradient centrifugation was effectively solubilized with Emulgen, with apparent preservation of the enzyme activity, and then the ATPase was highly purified by polyethylene glycol fractionation, and Blue Sepharose CL-6B and amino-hexyl Sepharose chromatographies. The purified enzyme showed a single band, with an apparent molecular mass of approximately 94 kDa, on SDS-PAGE, and exhibited both K+-ATPase and K+-stimulated-p-nitrophenyl phosphatase (pNPPase) activities. The optimum pH for the ATPase activity was 7.0. Amino acid analysis of the purified enzyme showed that it contains a large amount of hydrophobic amino acid (42%) and a small amount of glucosamine and galactosamine. The rabbit antibody monospecific for the ATPase, in the Ouchterlony double immunodiffusion and Western blotting tests, markedly inhibited both the K+-ATPase and K+-pNPPase activities.     

Enzymatic deglycosylation of human thyroglobulin: fluorescence studies

The interaction between the carbohydrate and the amino acid residues in human thyroglobulin has been studied. Previous reports showed that the removal of the two terminal carbohydrates of the complex chains leads to an increase in thyroglobulin binding to thyroid membranes. In our study, after enzymatic release with glycosidases of the sugar moieties from thyroglobulin, a time-dependent decrease in tryptophan fluorescence has been observed. This decrease was also associated with a shift in the emission peak from 335 to 340 nm. The strong quenching of tryptophan emission was also accompanied by a decrease in the exposure of tryptophan residues, as shown by a Stern-Volmer analysis with the neutral quencher acrylamide. These data, together with the increase in fluorescence of the dansylated deglycosylated thyroglobulin, strongly suggest that a significant conformational change of thyroglobulin follows the deglycosylation of the protein.     

Ion-exchange chromatographic analysis of iodothyronines.

An ion-exchange chromatographic procedure for the analysis of iodothyronine mixtures containing iodotyrosines is described. Eight different iodothyronines are separated into five peaks and one shoulder, the order of their elution being related to the number and position of iodine substitution in the compounds. The resolution of monoiodotyrosine and diiodotyrosine from each other and from iodothyronines is excellent. Quantitation of iodoamino acids is carried out by the automatic analysis of the effluent based on the ceric-arsenite reaction. This procedure has been applied to the determination of the iodoamino acid distribution in hog thyroglobulin and to the analysis of photodegradation products of thyroxine and triiodothyronine.     

The structure and properties of 27S and larger iodoproteins in the thyroid gland.

Iodoproteins larger than 19S were isolated from hog and calf thyroid glands using gel filtration on Sepharose 6B and one or two centrifugations in glycerol density gradients. Purified protein fractions were analysed in the analytical ultracentrifuge and characterized by the combined use of electrophoresis in continuous polyacrylamide gel gradients and electron microscopy. Three bands migrating more slowly than 19S could be identified in the polyacrylamide gels. Electron microscopy of the fastest of these species, having a sedimentation constant of 27S, showed pairs of 19S thyroglobulin molecules which had the same size and the same ovoid shape as normal, well-iodinated thyroglobulin molecules. The ovoids were randomly attached side to side, end to end. The more slowly migrating proteins were shown to consist of similar aggregates of three, four or more randomly attached molecules. Iodine and sialic acid determinations in 27S and 19S separated from the same pool of well iodinated protein showed no difference in iodine content but a larger amount of sialic acid in 27S than in 19S.     

A comparative review of the structure and biosynthesis of thyroglobulin

Thyroglobulin, the major iodoglycoprotein of the thyroid (M_r 669 kDa) has a sedimentation coefficient of 19 S and an isoelectric point (pI) of 4.4–4.7. The protein has been isolated and purified from saline extracts of the gland of several animal species, by methods such as ammonium sulfate fractionation, DEAE-cellulose chromatography and Sepharose 4B/6B gel-filtration. DEAE-cellulose chromatography of thyroglobulin from many species, by linear gradient, yielded a complex elution pattern, while camel thyroglobulin showed only a major and minor peak. As an iodoprotein, the protein has 0.1–2.0% iodine. The amino acid and iodoamino acid composition of thyroglobulins, in general, is similar. However, a high thyroxine content (15 mol/mol protein) has been noted for buffalo species. Asparagine or aspartic acid has been reported as the major N-terminal amino acid for thyroglobulins of several animal species whereas glutamic acid is the sole N-terminal amino acid for buffalo thyroglobulin. As a glycoprotein, thyroglobulin contains 8–10% total carbohydrate with galactose, mannose, fucose, N-acetyl glucosamine and sialic acid residues. The carbohydrate in the protein is distributed as two distinct units, A and B. In addition, human thyroglobulin has carbohydrate unit C. The occurrence of sulfate and phosphate as Gal-3-SO₄ and Man-6-PO₄, respectively, has been reported in few species. The quaternary structure of native thyroglobulin is comprised of two equal sized subunits of 330 kDa. However, the protein appears to contain 4–8 non-identical units in few species. The synthesis of thyroid hormones occurs in the matrix of the protein and is regulated by pituitary thyrotropin. The role of tyrosine residues 5 and 130 in thyroxine synthesis has been well documented.     

黑色菜豆(Phaseolus sp.)凝集素的纯化和性质

黑色菜豆(phaseolussp.)种子中含有对人A型血专一凝集的凝集素。用猪胃粘蛋白-Sepharose 4B作亲和吸附剂和Sephadex G-200凝胶过滤,可以纯化这种凝集素。纯化的凝集素在pH8.9,Tris-EDTANa_2-borate缓冲液的PAGE中,呈现单一蛋白带;酚-硫酸法测得总糖含量为3.22％。在SDS-PAGE中发现其分子由两种亚基所组成,亚基分子量分别为38,000和35,000。当凝集素浓度分别为0.98μg/ml和1.95μg/ml时能强烈地凝集人A型和AB型血细胞。在凝集素浓度高达500μg/ml时,B型血细胞能发生弱凝集反应,但对O型血和兔红细胞则完全不发生凝集反应。其凝集活性可被GalNAC、L-Fuc、猪甲状腺球蛋白和卵粘蛋白所抑制。该凝集素对人外周血中淋巴细胞的转化率达80％,细胞分裂比率高达37.1％;氨基组成分析表明,凝集素分子中Asp和Glu含量较高,而cys和Met含量很低。     

Purification and Characterization of Camel Thyroglobulin

Thyroglobulin (669 kDa), the major protein of the camel thyroid, has been isolated and purified from saline extract of the gland by ammonium sulfate fractionation and DEAE-cellulose chromatography. Ultracentrifugal analysis of the purified material, with an iodine content of 0.39%, showed a major and minor component with S_20,w values of 17 and 24, respectively. Separation of the protein from thyroid of individual animals by linear salt gradient on DEAE-cellulose showed a major and minor peak, indicating heterogeneity. Native gel electrophoresis of camel thyroglobulin showed a doublet, revealing microheterogeneity. A similar pattern was observed for the slower migrating components (24 S iodoprotein). N-terminal analysis of the purified protein revealed asparagine as the major N-terminal amino acid. Glycine and alanine were observed as the minor N-terminals. No differences in N-terminals between the major and minor peak were observed. Camel thyroglobulin, as thyroglobulin of other animal species, is a glycoprotein with a total carbohydrate content of 10.7%, comprising 6.0% neutral sugar, 3.67% glucosamine and 1.04% sialic acid. The iodoamino acid and amino acid composition of camel thyroglobulin is similar to that of other mammalian species.     

Separation and characterization of two carnosine-splitting cytosolic dipeptidases from hog kidney (carnosinase and non-specific dipeptidase)

High performance anion-exchange chromatography was used to separate two carnosine-hydrolysing dipeptidases from hog kidney. Both enzymes (peaks I and II) were cytosolic and were activated and stabilized by Mn2+ and dithiothreitol. Peak I had a narrow specificity when assayed without added metal ions, but a broad specificity in the presence of Mn2+ or Co2+. Peak II was inactive unless both Mn2+ and dithiothreitol were present. Bestatin and leucine inhibited peak II, but not peak I. Peak I had a Km of 0.4 mM carnosine, a pI of 5.5 and a Mr of 57,000. Peak II had a Km of 5 mM carnosine, a pI of 5.0 and a Mr of 70,000. Hog and rat brain and liver carnosinase activity was completely inhibited by bestatin, indicating that these organs contained peak II, with little or no peak I enzyme. Hog kidney peak I contained the classical carnosinase of Hanson and Smith, who first described this enzyme. It also contained activity against homocarnosine ("homocarnosinase") and showed "manganese-independent carnosinase" activity. These three activities could not be separated using 8 different chromatographic procedures; it was concluded that they are attributable to one enzyme. It is recommended that the name carnosinase be retained for this enzyme and the names "homocarnosinase" and "manganese-independent carnosinase" be withdrawn. The properties of hog kidney peak II closely resembled those of human tissue carnosinase (also known as prolinase, a non-specific dipeptidase), mouse "manganese-dependent carnosinase" and a rat brain enzyme termed "beta-Ala-Arg hydrolase". Since these terms appear to represent closely related enzymes with broad specificity, the recommended name for each is "non-specific cytosolic dipeptidase".     

Presence of beta-linked GalNAc residues on N-glycans of human thyroglobulin

Hepatic asialoglycoprotein receptor, which may mediate the clearance of circulating thyroglobulin, is known to have a high affinity for GalNAc. Recently, the receptor has been reported to be present also in the thyroid, implicating interaction with thyroglobulin. Here, mammalian thyroglobulins were analyzed for GalNAc termini by Western blotting with GalNAc-recognizing lectins labeled with peroxidase or (125)I. Wistaria floribunda lectin was found to bind human thyroglobulin and, to some extent, bovine, but not porcine thyroglobulin. After desialylation, the lectin bound all of the thyroglobulins tested. The binding was inhibited by competitive inhibitor GalNAc. Peptide N-glycanase treatment of human desialylated thyroglobulin resulted in the complete loss of reactivity with W. floribunda lectin, indicating that the binding sites are exclusively on N-glycans. The binding sites on human desialylated thyroglobulin were partly sensitive to beta-galactosidase, and the remainder was essentially sensitive to beta-N-acetylhexosaminidase. On the other hand, the binding sites of bovine and porcine desialylated thyroglobulins were totally sensitive to beta-galactosidase. Thus the lectin binds beta-Gal termini, as well as beta-GalNAc. GalNAc-specific Dolichos biflorus lectin also bound human thyroglobulin weakly. In contrast to W. floribunda lectin, desialylation diminished binding, suggesting that these two lectins recognize different GalNAc-terminated structures. Again, the binding was inhibited by GalNAc and by treatment with peptide N-glycanase. These results strongly indicate the presence of distinct GalNAc termini of N-glycans on human thyroglobulin.