Biomedical subjects

A Taurog

Publications and source records attributed to A Taurog.

At least 37 records · Page 2Linked to original sources

PubMed1990

Studies with purified human thyroid peroxidase and thyroid microsomal autoantibodies.

We have isolated highly purified thyroid peroxidase (TPO) from human thyroid tissue to study further the relationship between TPO and the thyroid microsomal antigen that elicits the production of microsomal autoantibodies in patients with autoimmune thyroid disease. Serum samples were obtained from 24 patients with suspected autoimmune thyroid disease, and from 7 normal subjects. Microsomal autoantibodies in the patient sera, as determined by the microsomal hemagglutination assay (MCHA), varied between 1:100 and 1:102,400. Antithyroglobulin antibodies, however, were very low (less than 1:100). Binding of serum autoantibodies to purified human TPO, as determined by enzyme-linked immunosorbent assay, correlated fairly well with MCHA titers (r = 0.72; P less than 0.001). An immunoblot procedure was developed to study the binding of serum antibodies to the major active fragment of TPO (93 kDa), after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Binding under both conditions correlated very well with MCHA titers (r = 0.80-0.84; P less than 0.001). Studies were performed to determine the inhibitory effect of patient serum on the enzymatic activity of purified human TPO. A marked inhibitory effect on guaiacol activity was observed when TPO was preincubated with as little as 10 microL high titer serum. There was a significant correlation (r = 0.47; P less than 0.01) between MCHA titer and inhibitory effect. The addition of 2 micrograms purified human TPO completely or almost completely inhibited the binding of serum antibodies to thyroid microsomes (enzyme-linked immunosorbent assay) in 10 of 11 patient sera with high MCHA titers (1:25,600 or greater).

Antibody Formation↗

PubMed1990

Lack of effect of propylthiouracil and methylmercaptoimidazole on thyroglobulin biosynthesis.

Experiments were performed both in vivo and in vitro to test a previous proposal that part of the antithyroid action of the thioureylene drugs, propylthiouracil (PTU) and methylmercaptoimidazole, can be attributed to inhibition of thyroglobulin (Tg) biosynthesis. Rat thyroid lobes were incubated in leucine-free Eagle's medium containing bovine thyroid-stimulating hormone and 0, 0.1-0.2, or 1 mM drug. After a 30-min preincubation, 5 mu Ci of [14C]leucine were added and the incubation was continued for 4 hr. The soluble fraction was analyzed by sucrose density gradient centrifugation, and the fractions corresponding to the 19S Tg peak were pooled and assayed for 14C. No inhibition of 14C incorporation into 19S Tg was observed, even in thyroid lobes incubated in the presence of 1 mM methylmercaptoimidazole or 2 mM PTU. At the same time, 14C incorporation into 19S Tg was completely inhibited when lobes were incubated in the presence of 0.1 mM puromycin. In vivo, rats received an injection of PTU (1 mumol/100 g body wt), followed 60 min later by an injection of 25 mu Ci of [14C]leucine. Blood samples and thyroids were taken 5 hr after the [14C]leucine injection. Serum thyroid-stimulating hormone was not significantly affected by the PTU injection. The thyroid-soluble fraction was analyzed by sucrose density gradient centrifugation. No significant differences between saline and PTU-injected groups were observed in [14C]leucine incorporation into 19S Tg. We conclude from both our in vitro and our in vivo studies that PTU and methylmercaptoimidazole have no inhibitory effect on thyroglobulin synthesis in rat thyroids and that such inhibition does not play a significant role in the antithyroid action of these drugs.

Animals↗

PubMed1989

Metabolism of 35S- and 14C-labeled 1-methyl-2-mercaptoimidazole in vitro and in vivo.

We previously described an in vitro incubation system for studying the mechanism of inhibition of thyroid peroxidase (TPO)-catalyzed iodination by the antithyroid drug 1-methyl-2-mercaptoimidazole (MMI). Inhibition of iodination in this system may be reversible or irreversible, depending on the relative concentrations of iodide and MMI and on the TPO concentration. Metabolism of the drug occurs under both conditions, and in the present investigation we used 35S- and 14C-labeled MMI together with reverse phase HPLC to examine the metabolic products associated with reversible and irreversible inhibition of iodination by MMI. Under conditions of reversible inhibition, MMI was rapidly metabolized and disappeared completely from the incubation mixture. With [35S]MMI, the earliest detectable 35S-labeled product was MMI disulfide, which reached a peak after a few minutes and then declined to undetectable levels. Coincident with the decrease in disulfide was the appearance of two 35S peaks, the major one corresponding to sulfate/sulfite, and the other to a component eluting at 7.5 min. Similar results were obtained for the disulfide and for the 7.5 min metabolite with [14C]MMI. The major 14C-labeled metabolite containing no S appeared to be 1-methylimidazole. Under conditions of irreversible inhibition, MMI disulfide was also the earliest detectable 35S-labeled metabolite. However, MMI decreased more slowly, and after reaching a nadir at about 6 min returned gradually to a level about halfway between the initial and the minimum value. The reformation of MMI appeared to involve the nonenzymatic disproportionation of MMI disulfide. Formation of the 7.5 min peak was also observed, but there was no formation of sulfate/sulfite. The difference in metabolic pattern between the reversible and irreversible conditions is primarily related to the rapid inactivation of TPO that occurs under irreversible conditions. The metabolism of [35S]MMI in thyroids of rats injected with the labeled drug resembles more closely conditions of reversible inhibition, since sulfate/sulfite is the only 35S-labeled metabolite. Neither [35S]MMI disulfide nor the 7.5 min component was detected in rat thyroids in vivo. However, it was demonstrated that these components do not survive homogenization with thyroid tissue, and failure to detect them in vivo does not exclude them as likely intermediates in intrathyroidal MMI metabolism. Based on the observations reported in this study, we present a revised scheme for the mechanism of inhibition of TPO-catalyzed iodination by MMI.

Animals↗

PubMed1989

Metabolism of 35S- and 14C-labeled propylthiouracil in a model in vitro system containing thyroid peroxidase.

In previous communications we described an in vitro model system containing highly purified thyroid peroxidase (TPO) for studying the mechanism of inhibition of thyroid hormone biosynthesis by the antithyroid drugs, 6-propylthiouracil (PTU) and 1-methyl-2-mercaptoimidazole (MMI). We showed that inhibition of iodination of thyroglobulin in this system may be reversible or irreversible depending on the relative concentrations of iodide and drug and the TPO concentration. Metabolism of the drugs occurred under both conditions, but was more limited under irreversible conditions of inhibition. It was of interest to examine the nature of the drug metabolites associated with reversible and irreversible conditions of inhibition. For this purpose we have employed the 35S- and 14C-labeled drugs and a recently developed reverse phase HPLC procedure. Results of a similar study with MMI were reported in an earlier communication. In the present study we report our findings with PTU. Under conditions of reversible inhibition, PTU was readily metabolized and by 15 min was reduced to a few percent of the starting value. The earliest detectable metabolite with both [35S]- and [14C]PTU was the disulfide, which reached a peak in about 15 min and then slowly declined. Coincident with the decline in the disulfide was the appearance of more polar metabolites. In the case of [35S]PTU, these corresponded to sulfate/sulfite, PTU sulfonate, and a product tentatively identified as PTU sulfinate. The latter two were also observed as 14C-labeled metabolites produced from [14C]PTU. Two nonpolar desulfurated 14C-labeled metabolites were also observed. Surprisingly, these did not correspond to either propyluracil or propyldeoxyuracil, the anticipated most likely products of PTU desulfuration. The identity of these desulfurated metabolites of PTU in the TPO model system remains to be determined. Under conditions of irreversible inhibition of iodination, a relatively small fraction of PTU was metabolized. PTU disulfide was, again, the earliest detectable metabolite, and it declined with time. However, only small amounts of other metabolites were observed, in contrast to the results obtained under conditions of reversible inhibition of iodination. As in the case of MMI, the difference in metabolic pattern between reversible and irreversible conditions is primarily related to the rapid inactivation of TPO that occurs under irreversible conditions. In general, the metabolism of PTU by the TPO model system resembled that previously observed with MMI. With both drugs, the disulfide was the earliest detectable metabolite, and under conditions of reversible inhibition of iodination, an appreciable fraction of the sulfur was oxidized as far as sulfate/sulfite.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

A reexamination of the proposed inactivation of thyroid peroxidase in the rat thyroid by propylthiouracil.

The antithyroid drug 6-propylthiouracil (PTU) was previously shown in our laboratory to have an unexpectedly prolonged inhibitory effect on iodination in the thyroid glands of rats. Eighteen hours after injection of a relatively small dose, iodination in the thyroid remained inhibited by more than 90%. We previously suggested that the prolonged inhibitory effect might be due to inactivation of thyroid peroxidase (TPO), a reaction previously shown to occur under certain conditions in an in vitro iodinating system containing highly purified TPO. However, the analytical procedure used in our earlier study did not exclude the possibility that sufficient PTU remained in the thyroid even after 18 h to inhibit TPO-catalyzed iodination by a reversible mechanism. Development of an improved analytical procedure, based on HPLC, led us to reexamine the mechanism of the prolonged inhibitory effect of PTU on iodination in rat thyroid glands. Rats were injected with [35S]PTU (1 mumol/100 g BW), and ultrafiltrates prepared from their homogenized thyroid glands were analyzed by HPLC. The major 35S-labeled metabolites were identified as sulfate/sulfite, PTU sulfinate, and PTU sulfonate. However, even after 18 h, a significant amount of unchanged [35S]PTU was also present. The calculated mean concentration of residual PTU was 20 microM, a sufficiently high level to explain the observed inhibition of iodination on the basis of a reversible mechanism. Experiments were also performed to examine the intrathyroidal distribution of 35S at intervals after the injection of [35S]PTU. All of the oxidation products of PTU showed marked increases between 2 and 16 h after injection. Based on our view that TPO is the major mediator of intrathyroidal metabolism of PTU, this observation is inconsistent with our previous proposal that TPO is inactivated after PTU injection. The results of the present study, therefore, lead us to withdraw our previous suggestion that TPO is inactivated after injection of PTU into rats. It is more likely that inhibition of iodination by PTU in the rat thyroid involves competition between PTU and tyrosyl residues of thyroglobulin for oxidized iodine, comparable to the reversible mechanism of inhibition observed in the TPO model system.

Animals↗

PubMed1989

Thyroid peroxidase and thyroid microsomal autoantibodies.

The antithyroid microsomal antibodies found in the serum of patients with autoimmune thyroid disease are directed largely, if not entirely, against thyroid peroxidase (TPO). In this study we used a highly purified, well characterized, large tryptic fragment of porcine TPO (hereafter referred to as purified porcine TPO) to examine possible differences among microsomal antibodies in patients with autoimmune thyroid disease. Antibodies against this TPO preparation and also against a synthetic peptide corresponding to residues 780-793 of the deduced sequence of the native enzyme were compared with microsomal antibodies from patients in immunoblot experiments. The antiporcine TPO and antisynthetic peptide antibodies reacted with crude preparations of human TPO. Binding of serum microsomal antibodies to purified porcine TPO was also found. Purified porcine TPO shows two fragments after gel electrophoresis under reducing conditions: a 59K fragment corresponding to the amino end of the molecule, and two approximately 30K fragments corresponding to the carboxyl end. Using an immunoblot procedure with purified porcine TPO as the antigen, we found that at least two epitopes were involved in microsomal antibody production: one associated with the 59K fragment and the other with the approximately 30K fragment(s). The distribution of serum antibodies against these epitopes differed among the patients, indicating that these antibodies comprise a heterogeneous group. Serum from patients with autoimmune thyroid disease significantly inhibited human TPO activity, raising the possibility that microsomal antibodies may contribute to the impaired thyroid function that occurs in some patients with autoimmune thyroid disease.

Adolescent↗

PubMed1988

Hormone-containing peptides from normal and goiter human thyroglobulins.

A series of low iodine human thyroglobulin samples derived from colloid-rich goiter tissue was examined by HPLC mapping of tryptic digests and compared to normal human thyroglobulin. These samples ranged in iodine content from 2 to 8 gram-atoms of iodine (g.a. I) per mole and were not further iodinated in vitro. Peptides containing the principal hormonogenic sequence were detected using the long wavelength absorbance of the iodotyrosine derivatives at 325 nm. Two such peptides were isolated and sequenced. Their thyroxine content was confirmed by radioimmunoassay. The number of 325-nm-absorbing peaks was significantly lower in the normally iodinated human thyroglobulin than that observed the thyroglobulins of cattle and dog. This suggests a more restricted iodination in the human protein. Sodium dodecyl sulfate gel patterns of the reduced and alkylated proteins showed significant molecular size heterogeneity in all of the samples. Polypeptide fragments ranged in molecular size from approximately 330 to 45 kDa in the goiter derived material and from approximately 330 to 15 kDa in the normal human material. This difference between the proteins is consistent with earlier observations that peptides less than 45 kDa appear concomitantly with hormone formation. These data confirm that the human thyroglobulin molecule is capable of forming at least limited amounts of thyroid hormone at iodine levels as low as 4 g.a. I per mole. The hormone detected in this study was located at residue 5 near the amino terminus of the thyroglobulin molecule.

Chromatography, High Pressure Liquid↗

PubMed1988

Spectral studies with lactoperoxidase and thyroid peroxidase: interconversions between native enzyme, compound II, and compound III.

Spectral scans in both the visible (650-450 nm) and the Soret (450-380 nm) regions were recorded for the native enzyme, Compound II, and Compound III of lactoperoxidase and thyroid peroxidase. Compound II for each enzyme (1.7 microM) was prepared by adding a slight excess of H2O2 (6 microM), whereas Compound III was prepared by adding a large excess of H2O2 (200 microM). After these compounds had been formed it was observed that they were slowly reconverted to the native enzyme in the absence of exogenous donors. The pathway of Compound III back to the native enzyme involved Compound II as an intermediate. Reconversion of Compound III to native enzyme was accompanied by the disappearance of H2O2 and generation of O2, with approximately 1 mol of O2 formed for each 2 mol of H2O2 that disappeared. A scheme is proposed to explain these observations, involving intermediate formation of the ferrous enzyme. According to the scheme, Compound III participates in a reaction cycle that effectively converts H2O2 to O2. Iodide markedly affected the interconversions between native enzyme, Compound II, and Compound III for lactoperoxidase and thyroid peroxidase. A low concentration of iodide (4 microM) completely blocked the formation of Compound II when lactoperoxidase or thyroid peroxidase was treated with 6 microM H2O2. When the enzymes were treated with 200 microM H2O2, the same low concentration of iodide completely blocked the formation of Compound III and largely prevented the enzyme degradation that otherwise occurred in the absence of iodide. These effects of iodide are readily explained by (i) the two-electron oxidation of iodide to hypoiodite by Compound I, which bypasses Compound II as an intermediate, and (ii) the rapid oxidation of H2O2 to O2 by the hypoiodite formed in the reaction between Compound I and iodide.

Hydrogen Peroxide↗

PubMed1988

Propylthiouracil and methimazole display contrasting pathways of peripheral metabolism in both rat and human.

We have developed HPLC procedures for analyzing the metabolites of [35S]methylmercaptoimidazole [( 35S] MMI) and [35S]propylthiouracil [( 35S]PTU) in bile, urine, serum, and liver of rats. We also studied urinary metabolites of [35S] MMI and [35S]PTU in one human subject. In bile collected from [35S]MMI-injected rats, two major metabolites accounted for 80-90% of the total 35S. Incubation of these metabolites either with or without beta-glucuronidase led to the appearance of a 35S-labeled compound less polar than MMI. In contrast, the major [35S]PTU metabolite in bile (greater than 50% of total 35S) was completely converted to [35S]PTU on incubation with beta-glucuronidase and showed no conversion in a control incubation. From these results we conclude that the major biliary metabolites of MMI in rats are not glucuronides. They appear to be labile conjugates of a metabolite of MMI. After [35S]MMI injection into rats, two major and at least four minor metabolites were observed in urine. In one human who received [35S]MMI orally, the HPLC profile of 35S in urine was similar to that of the rat. Incubation of human urine or of its isolated major component with beta-glucuronidase had no significant effect on the HPLC profile. On the other hand, the major urinary metabolite of [35S]PTU in human and rat urine was completely converted to [35S]PTU on incubation of whole urine with beta-glucuronidase. These results indicate that glucuronides comprise at most only a minor fraction of MMI metabolites in urine of rats or humans. Based on similarities in elution time, the metabolites of [35S]PTU in urine closely resembled those in bile of rats. In contrast, the metabolites of [35S]MMI in urine were strikingly different from those in bile. PTU displays noncovalent binding to serum protein to a much greater extent than does MMI. However, after injection of [35S]MMI into rats, a significant fraction of the 35S was firmly bound to protein in both serum and liver. This binding appeared to be covalent and involved metabolism of [35S]MMI. This type of binding was much less detectable after the injection of [35S]PTU into rats.

Animals↗

PubMed1988

Porcine thyroid peroxidase: relationship between the native enzyme and an active, highly purified tryptic fragment.

We previously described the preparation of highly purified porcine thyroid peroxidase by a procedure that involved initial solubilization of the enzyme with trypsin plus detergent. Recently, the complete amino acid sequence of porcine thyroid peroxidase (TPO) was determined by cDNA cloning, and it became of interest to compare the structure of the purified trypsin-solubilized enzyme with that of the native enzyme. For this purpose we employed antibodies to the purified enzyme and to two synthetic peptides representing defined regions of the protein. We also obtained N-terminal amino acid sequence data on TPO fragments separated by gel electrophoresis. Trypsin cleavage sites in the purified enzyme were observed after arg residues 109 and 561, and also at two undetermined sites close to the putative membrane spanning region at the carboxyl end. Major fragments of approximately 60, 32, and 29 kilodaltons were observed when the purified enzyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. This observation is explained by assuming that the cleavage site after arg residue 561 occurred within a disulfide loop. The Mr of the trypsin-solubilized enzyme is approximately 88,000 compared to approximately 106,000 for the native enzyme. The difference can be accounted for by the loss of approximately 90 residues from the amino terminus and of at least 80 residues from the carboxyl end. Despite the loss of these fragments totaling approximately 18 kilodaltons and cleavage of the peptide bond after arg residue 561, the purified trypsin-solubilized TPO appears to retain full enzyme activity.

Amino Acid Sequence↗

PubMed1987

Molecular cloning of the structural gene for porcine thyroid peroxidase.

We have isolated and determined the nucleotide sequence of overlapping cDNA clones, representing the entire structural gene for pig thyroid peroxidase. The protein coding region extends from an ATG residue at base 252 to a termination codon at base 3030, coding for a 100.4-kDa apoprotein of 926 amino acids. The derived amino acid composition agrees well with the experimentally determined amino acid composition of purified pig thyroid peroxidase. Five potential glycosylation sites are present in the protein. Potential membrane spanning regions are present at the amino-terminal end (1-23) and near the carboxyl-terminal end (845-870) of the protein. These data indicate that pig thyroid peroxidase is synthesized as a single polypeptide that is membrane-bound.

Amino Acid Sequence↗

PubMed1987

Digestion of thyroglobulin with purified thyroid lysosomes: preferential release of iodoamino acids.

[131I]Thyroglobulin [( 131I]Tg), prepared by either enzymatic iodination of human goiter Tg in vitro or isolation from the thyroids of rats previously injected with 131I, was digested with a solubilized enzyme mixture prepared from purified hog thyroid lysosomes. The digestion was performed at 37 C for 24 h under nitrogen at pH 5.0 in the presence of 4 mM dithiothreitol. Under these conditions the release of free [131I] iodoamino acids (MIT, DIT, T4, and T3) was quantitatively very similar to that observed with a standard pronase digestion procedure. To determine whether other amino acids in Tg were released as quantitatively as the iodoamino acids, free amino acids in the lysosomal digest were measured, and total free amino acid release was compared with a similar analysis performed after digestion of [131I]Tg with 6 N HCl. Total amino acid release was much less complete than iodoamino acid release, indicating preferential release of iodoamino acids from Tg by lysosomal digestion. Analysis of the lysosomal digest by HPLC on a size exclusion column indicated that Tg was degraded to peptides with a mol wt less than 4000. Assuming that the in vitro lysosomal digestion system represents a valid model for the physiological proteolytic system that degrades Tg, the results of the present study suggest that a substantial portion of the Tg in the thyroid is not degraded to free amino acids and that peptide fragments of Tg are normally present in the thyroid. In such a case, the fate and possible physiological activity of these fragments require further elucidation.

Amino Acids↗

PubMed1986

Isolation and characterization of a cDNA clone for porcine thyroid peroxidase.

We undertook the molecular cloning of porcine thyroid peroxidase (TPO). Four oligonucleotide probes were synthesized on the basis of amino acid sequences of 3 tryptic peptides from highly purified porcine TPO. These probes were used to screen a pig thyroid cDNA library. Seven of 16 selected clones (0.45-1.15 kb in size) reacted with all 4 probes. Nucleotide sequencing of the 1.15 kb at the 3'-end of the structural gene revealed the complementary sequence to all 4 probes as well as the nucleotides coding for the entire length of the 3 tryptic peptides. There is an open reading frame of 332 amino acid residues. On Northern blot analysis this gene codes for an mRNA species of 2.85 kb, corresponding to the anticipated size of the mRNA for the intact TPO molecule. We have therefore cloned and characterized a cDNA clone coding for approx. 36% of porcine thyroid peroxidase.

Animals↗

PubMed1986

A new class of propylthiouracil analogs: comparison of 5'-deiodinase inhibition and antithyroid activity.

After in vivo administration, propylthiouracil (PTU) inhibits not only thyroid iodide uptake and organification, but also T4 5'-deiodinase activity in most peripheral organs. The present report describes the effects of some previously untested 6-substituted 2-thiouracil derivatives on in vivo and in vitro iodide uptake and organification, and on T4 5'-deiodinase activity in liver and pituitary homogenates. When added to homogenates, many analogs were as potent or more potent than PTU in inhibiting hepatic T4 5'-deiodinase activity. Three derivatives, 6-anilino-2-thiouracil (A compound), 6-(p-ethylanilino)2-thiouracil (B compound), and 6-(p-n-butylanilino) 2-thiouracil (C compound), which were among the most potent inhibitors of hepatic T4 5'-deiodinase, when added in vitro inhibited T4 5'-deiodinase activity in liver homogenates after in vivo administration. When added to pituitary homogenates prepared from hypothyroid rats, these compounds also significantly inhibited pituitary T4 5'-deiodinase activity. In a concentration of 1 mM in the presence of 20 mM dithiothreitol, the percent inhibition of pituitary T4 5'-deiodinase activity was 19.7 +/- 7.4 (mean +/- SE), 34.0 +/- 3.2, 47.3 +/- 3.1, and 89.0 +/- 1.0 for PTU and the A, B, and C compounds, respectively (P less than 0.05 for all groups vs. one another and vehicle). Despite their ability to inhibit hepatic T4 5'-deiodinase activity, none of the 13 analogs tested altered thyroid iodide uptake or organification after administration of 0.1 mg/rat. PTU, in the same dose, inhibited thyroid iodide uptake by 78.2 +/- 2.4% (P less than 0.001) and thyroid iodide organification by 36.4 +/- 7.3% (P less than 0.01). Furthermore, the A, B, and C compounds did not inhibit thyroid iodide uptake or iodide organification when administered in higher doses of 5, 5, and 1 mg/rat, respectively. In contrast to these in vivo results, the A, B, and C compounds were more potent than PTU in inhibiting iodide organification in a purified thyroid peroxidase system and in porcine thyroid slices. The concentrations causing 50% inhibition of iodide organification in the purified thyroid peroxidase system were 30, 7, 8, and 14 microM for PTU and the A, B, and C compounds, respectively. However, PTU was far more potent in inhibiting iodide organification in intact incubated thyroid lobes compared to the A, B, and C compounds.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1986

Physiological role of thiol proteases in thyroid hormone secretion.

To determine the physiological role of the thiol proteases in T4 and T3 release from thyroglobulin, experiments were performed with 131I-prelabelled rat thyroid lobes incubated in vitro in the presence and absence of leupeptin, an inhibitor of thiol proteases. Basal secretion of [131I]T4 and [131I]T3 from rat thyroid lobes prelabelled in vivo was quite low, but in the presence of 10 mU/ml bovine TSH a marked stimulatory effect was observed. The stimulatory effect of TSH was completely abolished by leupeptin. This was associated with marked inhibition of lysosomal proteolytic activity, suggesting that the inhibitory effect of leupeptin on T4 and T3 secretion could be attributed to its inhibitory action on proteolysis of thyroglobulin. Further evidence for an inhibitory effect of leupeptin on intralysosomal hydrolysis of thyroglobulin was obtained when thyroid lobes were incubated with 131I- in the presence and absence of leupeptin and TSH. The crude lysosomal preparation was fractionated on a Percoll density gradient, which separates 131I-containing particles into a dense peak containing purified lysosomes and a buoyant peak containing pinocytotic vesicles. A marked increase in the 131I-content of the dense peak was observed in the presence of TSH + leupeptin. Analysis of the 131I in the dense fraction by sucrose density gradient centrifugation and by SDS-polyacrylamide gel electrophoresis demonstrated that leupeptin inhibited degradation of 19S thyroglobulin, especially the formation of [131I]peptides of MW less than 14K.

Acid Phosphatase↗

PubMed1985

Purification of thyroid lysosomes by colloidal silica density gradient centrifugation.

A procedure was devised for fractionating crude thyroid lysosomal particles (P750-15,000) by self-forming density gradient centrifugation with colloidal silica. Two discrete particle-containing peaks were observed, based on 131I-labeling and acid phosphatase activity: a heavy peak (density, 1.11-1.12) and a light peak (density, 1.05). Ultrastructural analysis revealed that the heavy peak consisted almost entirely of lysosomes, whereas the light peak represented a heterogeneous mixture of small vesicles and fragments of other intracellular organelles. In thyroids removed from rats 30 min after 131I injection, almost all of the 131I was present in the low density peak. This 131I appeared on sucrose density gradient centrifugation as a 19S peak, and it was almost completely insoluble in trichloroacetic acid. This was interpreted as indicating that the low density peak contained pinocytotic vesicles. In thyroids removed 4 days after 131I injection, the radioactivity appeared largely in the high density peak. Both the trichloroacetic acid solubility and the pattern on sucrose density gradient centrifugation indicated that the [131I] thyroglobulin had undergone extensive proteolysis. Thyroglobulin proteolytic activity was found primarily in the high density particles and to only a small extent in the low density particles. Studies performed at intervals after 131I injection combined with double labeling (131I and 125I) experiments provided evidence that radioactivity was transferred from the low density to the high density particles. Heterogeneity existed within the dense peak, related to the degree of thyroglobulin degradation, as it was observed that thyroid lysosomes become denser with increasing proteolysis of thyroglobulin. The acid phosphatase in the low density particles could be distinguished from that in the high density (lysosomal) particles by its elution pattern on Sephadex G-200 column chromatography, its response to freezing and thawing, and its reactivity with p-nitrophenylphosphate. It was concluded, therefore, that the acid phosphatase in the low density fraction was derived from prolysosomal structures such as vesiculated Golgi-endoplasmic reticulum-lysosomes. The prolysosomal acid phosphatase associated with the low density fraction appeared to be a large membrane-bound molecule which could be transformed into lysosomal acid phosphatase by incubation at pH 5.0.

Acid Phosphatase↗

PubMed1985

Lysosomal digestion of thyroglobulin: role of cathepsin D and thiol proteases.

Purified hog thyroid lysosomes, prepared by a procedure previously developed in this laboratory, were used to study lysosomal digestion of [131I]thyroglobulin [131I]Tg). The lysosomal proteases were solubilized with 0.1% Triton X-100. Rates of proteolytic digestion, measured by the release of ethanol-ammonium acetate-extractable 131I, were greatly stimulated by thiol reagents. The pH optimum was also affected by the presence of thiols. In the absence of a thiol reagent, a broad pH optimum was observed, ranging from 3.5-4.5. However, in the presence of 1 mM mercaptoethanol, the maximum rate of digestion occurred at pH 5.0, very close to reported values for the internal pH of lysosomes. Pepstatin, an inhibitor of cathepsin D, markedly inhibited lysosomal digestion of [131I]Tg at concentrations as low as 0.01 micrograms/ml. Its inhibitory effect was greater at pH 3.5 (pH optimum of cathepsin D) than at pH 5.0. Leupeptin, an inhibitor of thiol proteases, was not as potent as pepstatin, but it was significantly inhibitory at a concentration of 1 microgram/ml. In contrast to pepstatin, leupeptin displayed a greater inhibitory effect at pH 5.0 than at pH 3.5. The pH optimum of hog thiol proteases has been reported to range from 5.5-6.5. The effects of the two inhibitors were additive at pH 5.0. We conclude from these results that both cathepsin D and thiol proteases play a role in lysosomal digestion of Tg. Cathepsin D appears to be quantitatively more important than thiol protease in the initial phase of the digestion. The stimulatory effect of thiols on lysosomal digestion of [131I]Tg probably involves two separate effects: 1) stimulation of thiol proteases, and 2) reduction of S-S bonds in Tg, making the protein more susceptible to attack by proteolytic enzymes. Poorly iodinated [131I]Tg was more rapidly hydrolyzed than well iodinated [131I]Tg, based on the release of ethanol-ammonium acetate-extractable 131I. However, there was little or no difference in the rate of total peptide bond cleavage between poorly iodinated and well iodinated Tg. These results suggest that the first sites of iodination of Tg are preferentially attacked by lysosomal proteases. Long term (24-h) digestion of [131I]Tg with solubilized thyroid lysosomes at pH 5.0 in the presence of thiol compounds was just as effective as digestion with pronase at pH 8.0 in liberating free 131I-labeled iodothyronines and 131I-labeled iodotyrosines. Thus, thyroid lysosomes contain the full complement of proteases and peptidases required for cleaving free iodoamino acids from Tg.

Animals↗

PubMed1984

Mechanisms of thyroid peroxidase- and lactoperoxidase-catalyzed reactions involving iodide.

In a previous communication we proposed a reaction scheme to explain our observation that thyroid peroxidase and lactoperoxidase degrade H2O2 catalatically in the presence of low concentrations of iodide. An essential feature of the scheme was the proposal that enzyme-bound hypoiodite, designated [EOI]-, is a common intermediate in various peroxidase-catalyzed reactions involving iodide. In the present investigation, we tested the validity of this scheme by studying the predictions that it makes concerning the formation of OH-, O2, I2, and organically bound iodine. Stoichiometric and kinetic measurements were made to correlate formation of these various products. Three different peroxidase-catalyzed reactions were studied: 1) oxidation of I- to I2; 2) iodide-dependent catalytic degradation of H2O2 to O2; and 3) iodination of tyrosine or thyroglobulin. Reaction 2 was also studied nonenzymatically using I2, for comparison with the enzyme-catalyzed reaction. In all three reactions, both the stoichiometric and kinetic results with thyroid peroxidase agreed closely with the predictions made by the proposed scheme. This was largely the case with lactoperoxidase also. However, in the case of lactoperoxidase-catalyzed iodination of tyrosine or thyroglobulin, we observed a marked discrepancy between initial rates of OH- release and iodination, inconsistent with the mechanism originally proposed for the iodination reaction. As a possible explanation for this kinetic discrepancy, we postulate that lactoperoxidase generates hypoiodous acid and that the latter is the active intermediate in the various reactions involving iodide.

Hydrogen Peroxide↗