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PubMed1978

The characterization of n-butanol-pseudosolubilized and trypsin-solubilized porcine thyroid iodide peroxidase.

Porcine thyroid peroxidase (Iodide: hydrogen-peroxide oxidoreductase, EC 1.11.1.8) was solubilized by proteolytic and non-proteolytic procedures. A kinetic and physical study was undertaken to ascertain the catalytic properties of the peroxidase prepared by the two purported solubilization procedures. Where possible, the properties of the two enzyme preparations were compared with the original microsomal preparation. The n-butanol-solubilized thyroid iodide peroxidase is not truly soluble, but exists as a large molecular weight lipoprotein aggregate. The trypsin-solubilized thyroid iodide peroxidase is truly soluble, active, and contains lipids. The microsomes, butanol-pseudosolubilized enzyme, and trypsin-solubilized enzyme have similar kinetic properties such as pH optima, Km for iodide and H2O2, sigmoid character of the saturation curves, substrate inhibition, and inhibition by 3,5-diiodotyrosine. Since the proteolytic solubilization procedure produced a soluble peroxidase with catalytic properties similar to the microsomal preparation, trypsin-solubilized peroxidase can be studied with reasonable assurance that its properties are essentially unaltered and are not artifacts of the solubilization procedure.

Animals↗

PubMed1980

Inactivation of galactosyltransferase by lactoperoxidase and N-acetylimidazole.

Galactosyltransferase (UDPgalactose:D-glucose 4-beta-D-galactosyltransferase, EC 2.4.1.22) was totally inactivated by iodination with lactoperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). Substrates protected against inactivation. The presence of 10 mM Mn2+ and 1 mM UDPgalactose gave partial protection which was enhanced by the addition of 10 mM N-acetylglucosamine, but not by glucose. These results are consistent with a conformational change upon binding of UDPgalactose. Only monoiodotyrosine and diiodotyrosine were identified in the pronase digest of iodinated galactosyltransferase. Galactosyltransferase was also inactivated with N-acetylimidazole and partial activity was restored by treating acetylated galactosyltransferase with hydroxylamine. These results suggest that tyrosine(s) is essential for galactosyltransferase activity.

Acetylglucosamine↗

PubMed1980

Specificity of the acid protease from Monascus kaoliang towards the B-chain of oxidized insulin.

The proteolytic specificity of the acid protease from Monascus kaoliang has been investigated using the B-chain of performic acid-oxidized insulin as peptide substrate. Six splittings were detected after 1 h digestion and 12 splittings were found after 12 h incubation at 37 degrees C, pH 4.8. The bonds most susceptible to the acton of M. kaoliang acid protease were Phe(24)-Phe(25), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). Among the acid proteases compared, the specificity of M. kaoliang acid protease on the B-chain of oxidized insulin is more closely related to that of penicillopepsin with which it has ten cleavage sites in common. N-Acetyl-L-phenylalanyl-L-3,5-diiodotyrosine, a synthetic substrate for pepsin, was resistant to the hydrolysis of M. kaoliang acid protease.

Ascomycota↗

PubMed1981

Substrate requirement for inactivation of iodothyronine-5'-deiodinase activity by thiouracil.

Preincubation of rat liver microsomal fraction with 1 microM 2-thiouracil and 0.01-1 microM 3,3',5'-triiodothyronine or 3',5'-diiodothyronine, 0.1-10 microM thyroxine or 3,5-diiodothyronine led to a progressive, irreversible and concomitant decrease in subsequently assayed 3,3',5'-triiodothyronine- and 3',5'-diiodothyronine-5'-deiodinase activity. Preincubation with thiouracil alone, with iodothyronines alone or with thiouracil and 10 microM thyronine or 3,5-diiodotyrosine had no or virtually no effect. The results indicate that (1) a previously proposed ping-pong mechanism for thyroid hormone deiodination, involving the formation of an enzyme-sulphenyl iodide intermediate, is correct; (2) thyroxine, 3,3',5'-triiodothyronine and 3',5'-diiodothyronine are substrates for a common 5'-deiodinase; (3) this 5'-deiodinase is not fully specific as regards the position of the iodine substituents in the substrate, since it also appears to catalyse the 5-deiodination of 3,5-diiodothyronine.

Animals↗

PubMed1977

Preparation of monoiodotyrosine-13-glucagon.

Glucagon was iodinated with the lactoperoxidase method at pH 10.0 in the presence of propylene glycol using a substitution of 0.3 g-atom I/mol glucagon. Under these conditions the reactivity of the iodine to tyrosine at position 13 is found to be 4-fold that of the tyrosine at position 10. The amount of diiodotyrosine was less than one-twentieth that of the monoiodotyrosine at either tyrosine residue. Relatively pure monoiodo[125I]tyrosine-13-glucagon can be separated from other iodoglucagons by means of DEAE-chromatography. Such a homogeneous preparation with a known position of the iodine makes it possible to study a specific interaction between the monoiodoglucagon and the glucagon antisera or the glucagon receptor.

Amino Acids↗

PubMed1982

One enzyme for the 5'-deiodination of 3,3',5'-triiodothyronine and 3',5'-diiodothyronine in rat liver.

Many studies suggest that one enzyme is involved in the phenolic ring deiodination of iodothyronines in rat liver and kidney and another one in the tyrosyl ring deiodination. This study describes some characteristic of the phenolic ring (5'-) deiodination of rT3 and 3',5'-T2 by rat liver microsomes. At pH 7.2 the Km values of the 5'-deiodination of rT3 and 3',5'-T2 were 0.103 and 0.77 microM, respectively. 3',5'-T2 and rT3 inhibited the respective 5'-deiodination reactions competitively, the Ki values being 1.05 and 0.134 microM, respectively. Several radiographic contrast agents markedly inhibit the 5'-monodeiodination or rT3 and 3',5'-T2, the type of inhibition being competitive. Of these compounds iopanoic acid, ipodic acid and iophenoxic acid are the most potent inhibitors with Ki values of approximately 2 microM for both reactions. The non-iodine containing compound 8-anilino-1-naphthalene sulphonic acid (ANS) appeared to be a very strong competitive inhibitor of both 5'-deiodinations (Ki 4.3-4.7 microM), whereas salicylic acid, which as ANS inhibits the binding of iodothyronines to T4-binding globulin, inhibited these reactions to a much lesser extent (Ki 300-500 microM). On the other hand, diiodosalicylic acid was a very strong inhibitor. The beta-adrenergic blocker D,L-propranolol was a weak noncompetitive inhibitor of both 5'-deiodinations (Ki 0.4-0.7 mM). These reactions were also inhibited by various 2,6-diiodophenol derivatives, triiodophenol being the strongest and diiodotyrosine the weakest inhibitor tested. Comparing the Ki values of various inhibitors for the 5'-deiodination of rT3 and 3',5'-T2, a positive correlation between these values was found (r = 0.97). It was concluded that rT3 (to 3,3'-T2) and 3'-5'-T2 (to 3'-T1) monodeiodinating activities are very similar to each other and that there may just be one monodeiodinase catalyzing the 5'-deiodination of iodothyronines in rat liver.

Anilino Naphthalenesulfonates↗

PubMed1989

Isoniazid-mediated irreversible inhibition of the myeloperoxidase antimicrobial system of the human neutrophil and the effect of thyronines.

During aerobic myeloperoxidase-catalysed oxidation of isoniazid at pH 7.8, compound III was generated. Oxidation of isoniazid or hydrazine sulphate at pH values of 6.5 or 7.8 in a myeloperoxidase-H2O2 system caused considerable haem loss, which was associated with compound III formation. Haem loss and also compound III formation could be inhibited when 8 microM thyroxine was included in the reaction mixtures. During the reaction with isoniazid, an intense pink-coloured pigment with maximum absorbance at 500 nm was formed which could be bleached with ascorbate or hypochlorous acid. The pigment was more stable at pH 7.8 than at pH 6.5. A similar pink colour was generated when a mixture of isoniazid and thyroxine in alkaline solution was irradiated with light of wavelength greater than 300 nm. A possible product of thyroxine oxidation, 3,5-diiodotyrosine, could not protect the enzyme against isoniazid-mediated haem loss and no colour formation was observed. Haem loss was most extensive when isoniazid was oxidised in a myeloperoxidase system at pH 7.8 in the presence of 0.1 M NaCl. Thyroxine (8 microM), however, could still inhibit haem loss under these conditions. A good correlation was found between haem loss and irreversible loss of peroxidase activity.

Chlorides↗

PubMed1977

Induction of the thyroxine (T4) to triiodothyronine (T3) converting enzyme in rat liver by thyroid hormones and analogs.

In thyroidectomized, unsubstituted rats the T4 to T3 converting activity of liver homogenate is reduced to about 30% of that in unoperated control animals. The enzyme activity can be reinduced dose-dependently with T4. To achieve a normal activity, high, non-physiological plasma T4 concentrations are needed. Plasma T3 levels are much better correlated to the T4 to T3 converting activity. Pure T3 proved to be a more potent enzyme inducer than T4. No difference could be detected between L- and D-T3. Tyrosine, diiodotyrosine, 3,3'-diiodothyronine and 3,3',5'-T3 (reverse-T3) showed no inductive effect for the enzyme. These results demonstrate that the T4 to T3 converting enzyme is specific and shows regulatory properties.

Animals↗

PubMed1983

alpha-Neo-endorphin: receptor binding properties of the tritiated ligand.

Tritiated porcine alpha-neo-endorphin has been prepared from its corresponding iodinated analog. The iodinated analog (diiodotyrosine at position 1) was synthesized, along with its non-iodinated counterpart, by the solid-phase method. Catalytic exchange of this iodinated analog in the presence of tritium yielded tritiated porcine alpha-neo-endorphin having a specific activity of 45.5 Ci/mmole. Both the native, iodinated and tritiated alpha-neo-endorphin analogs were shown to be homogenous by chromatography on carboxymethylcellulose, paper chromatography, paper electrophoresis, high performance liquid chromatography and amino acid analysis. For the first time binding of alpha-neo-endorphin to rat membrane preparations is described using [3H2-Tyr1]alpha-neo-endorphin as the ligand. The binding is time-dependent and saturable with respect to alpha-neo-endorphin. Scatchard analysis was bi-phasic with KDs of 0.20 and 3.75 nM. Displacement binding studies indicate that the receptor for alpha-neo-endorphin has "kappa" and possibly "epsilon" binding characteristics.

Animals↗

PubMed1985

Peroxidatic degradation and ether link cleavage of thyroxine in a particulate fraction of human thyroid.

The present study was undertaken to investigate degradation of thyroxine (T4) mediated by thyroid peroxidase in man. A particulate fraction (1,000-100,000 x g) of normal human thyroid tissue was prepared and used as crude enzyme. 125I-T4 and unlabeled T4 were incubated with the particulate fraction in buffer containing glucose and glucose oxidase for generation of H2O2. After incubation, iodoamino acids were extracted with ethanol and the products of T4 degradation were analyzed by thin layer chromatography. In this system, T4 was degraded in time-, temperature- and pH-dependent manners, but not in the absence of the H2O2-generating system. The rate of degradation was related to concentration of the particulate fraction. The reaction was inhibited by methimazole, propylthiouracil and catalase. When [3',5'-125I] T4 was used as a tracer, major labeled products of T4 degradation were inorganic iodide and ethanol-unextracted fraction and no detectable labeled 3,5,3'-triiodothyronine (T3) or 3,3',5'-triiodothyronine (rT3) was generated. From a kinetic study by adding various doses of unlabeled T4, the apparent Km value for T4 was 30 microM and the Vmax value was 230 pmol/mg protein/min. When [3,5-125I] T4 was incubated with enzyme preparation, one third of degraded T4 was recovered as diiodotyrosine (DIT) and half of 125I-DIT was degraded in parallel incubation. No formation of radiolabeled DIT was observed in incubation with Na- 125I done in tandem. These findings suggest that thyroid hormones can be metabolized by peroxidase in human thyroid by pathways that include cleavage of ether linkage.

Autoradiography↗

PubMed1984

Isolation and characterization of camel pepsins.

Camel (Camelus dromedarius) pepsins were precipitated from the extract of the fundic gastric mucosa by ammonium sulfate between 35 and 80% saturation. DEAE--cellulose chromatography of this fraction produced two isoenzymes, I and II, which were further purified to homogeneity on sephadex G-100. Their Km with N-acetyl-L-phenylalanyl-diiodotyrosine were 0.10 and 0.90 mM, and their molecular weights which were determined on sodium dodecylsulfate gel electrophoresis exhibited 35,500 and 34,700, respectively. The two pepsins were essentially free of carbohydrates, but contained 0.3 and 1.0 mol of organic phosphate per 1 mol of protein, respectively. The apparent mobilities of the phospho- and dephosphoforms of each pepsin were indifferent in polyacrylamide gel electrophoresis at pH 8.9. The N-terminal residues of pepsins I and II were found to be alanine and leucine, respectively. Pepsin I was inactivated at a faster rate than that of pepsin II at pH 8 and 0 degrees C, and at pH 7.5 and 37 degrees C; but both were denatured under these conditions. The properties of these enzymes are compared with those of other mammalian and avian pepsins.

Animals↗

PubMed1984

Cellular processing of follicle-stimulating hormone by Sertoli cells in serum-free culture.

Specific binding of radioiodinated human follicle-stimulating hormone [( 125I]hFSH) to receptors on the cell surface of immature rat Sertoli cells in serum-free culture (4-7 days) was concentration-, time- and temperature-dependent with an apparent steady state of binding stained after 2 h at 4, 18 or 32 degrees C. Analysis of [125I]hFSH binding data indicated an apparent Ka of 3 X 10(9) M-1 with approximately 670 fmoles binding sites/mg protein. The radioiodinated hFSH (60-70 microCi/micrograms) bound to receptor and stimulated estradiol production, thus confirming its biological activity and Sertoli cell viability. Approximately 50% of the specifically bound radioactivity was lost from the cell surface after 0.25 h at 32 degrees C, 1 h at 18 degrees C or 4 h at 4 degrees C. Most (greater than 90%) of the receptor-bound hormone was removed from the cell surface and degraded to [125I]mono- and diiodotyrosine within 4 h at 32 degrees C. FSH degradation was inhibited at low temperatures (4 degrees C) and by the lysosomotropic agents chloroquine, NH4Cl or concanavalin A. Bacitracin and methylamine, inhibitors of internalization, were effective in blocking degradation but not loss of membrane-bound [125I]hFSH. Thus it appears that, as with small peptide and simple protein hormones, complex glycoprotein hormone-receptor complexes, such as FSH Sertoli cell receptors, are endocytosed by normal target cells and subsequently degraded, most probably within the lysosomes.

Animals↗

PubMed1975

[In vitro biosynthesis of iodothyronines from diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine (author's transl)].

A comparative study of two types of in vitro synthesis of iodothyronines has been done from 3,5-diiodotyrosine and from diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine (Tyr(I)2-Tyr(I)2) (equimolecular in tyrosyl rings). Incubations are made with rat thyroid gland minces in Eagle's medium or with thyroid microsomal fraction. Synthesis of thyroid hormones from Tyr(I)2-Tyr(I)2 is faster and more important than from diiodo-3,5-L-tyrosine (Tyr(I)2). A mechanism of iodothyronine formation via Tyr(I)2-Tyr(I)2 and different from the one occuring for Tyr(I)2 is suggested.

Animals↗

PubMed1979

Phenolic and nonphenolic ring deiodinations of iodothyronines in cultured hepatocarcinoma cell homogenate from monkey.

Iodothyronine monodeiodinase activities in homogenates of cultured monkey hepatocarcinoma cells were measured by the deiodination of [3.5-(125)I]-diiodo-L-thyronine or 3-[3',5'-(125)I]triiodo-L-thyronine (phenolic ring-labeled 'reverse' triiodothyronine). The assay system utilized a small ion-exchange column (AG50W-X4, O.9 X approximately 1 cm) to measure 125I-. Both deiodinases were destroyed by boiling for 1 min. Maximal nonphenolic ring deiodination was observed at pH 7.9 whereas maximal phenolic ring deiodination was at pH 6.3. Both reactions were enhanced strongly by dithiothreitol (0.1-5mM), and slightly by 5 mM beta-mercaptoethanol. Phenolic ring deiodination was strongly inhibited by 0.1 mM propylthiouracil. Nonphenolic ring deiodination was accelerated by EDTA (1.2 MM) and inhibited by Mg(2+) (5mM). Methylmercaptoimidazol and Mg(2+), Ca(2+) and Mn(2+) (0.1-1.0 mM) had little or no effect on either reaction, but Zn(2+) (0.1 mM) strongly inhibited both. Both reactions were inhibited by excess iodothyronine analogues at 10 mM to 10 micron M, and thyroxine was shown to be a competitive inhibitor in both cases. On the basis of relative affinities and inhibitory effects, it appears that the order of affinity for the phenolic ring deiodinase is 3,3',5'-triiodo-L-thyronine(rT3) greater than L-thyroxine(T4) greater than 3,5,3'-triiodo-L-thyronine(T3), whereas for the nonphenolic ring deiodinase the order is T3 greater than T4 greater than rT3. Diiodotyrosine did not affect their deiodination.

Animals↗

PubMed1979

Clearance of circulating desialylated thyroglobulins in the rat.

Canine and rat thyroglobulins were labeled with 125I either in vitro or in vivo, and were utilized for plasma clearance studies performed with rat. The half-life of physiologically radioiodinated asialothyroglobulins was about 6 min, while that of chemically radioiodinated asialothyroglobulin was about 12 min. No marked species difference was observed in this clearance. The label which had disappeared from the blood was recovered mainly in the liver, and this uptake was blocked by the simultaneous injection of desialylated orosomucoid but not by native orosomucoid. Radiolabeled monoiodotyrosine, diiodotyrosine, triiodothyronine and thyroxine were detected in the liver 17 min after intravenous injection of asialothyroglobulin labeled with 125I in vivo, suggesting the possible production of thyroid hormones in extrathyroidal tissues.

Animals↗

PubMed1980

Phenolic ring deiodination in cultured rat hepatoma cells, and subcellular localization of deiodinases in cultured rat hepatoma, monkey hepatocarcinoma cells and normal rat liver homogenates.

The cultured rat hepatoma cell (R117--21B) homogenates metabolized 3,[3',5'-125I]triiodothyronine by phenolic ring deiodination and produced radioactive iodide and 3,3'-diiodothyronine. Thyroxine (T4) was converted to 3,3',5-triiodothyronine (T3). The production of 125I- represented the deiodination was observed to be pH 6.0--7.0. This enzyme reaction was accelerated by dithiothreitoil. Propylthiouracil strongly inhibited the phenolic ring deiodination at 0.1 mM, whereas an effect of 20 mM methylmercaptoimidazol on the deiodination was very weak or absent. Excess unlabeled iodothyronines (T4, T3 and 3,5-diiodo-L-thyronine) inhibited the phenolic ring deiodination of labeled 3,3',5'-triiodothyronine, although their inhibitory effect was slightly different. Triiodothyroacetic acid was a better inhibitor than T3. Diiodotyrosine did not affect phenolic ring deiodination in cultured rat hepatoma cell homogenates. Phenolic and nonphenolic ring deiodinase activities of cultured monkey hepatocarcinoma cell and rat liver homogenates were also studied by the use of 3,[3',5'-125I]triiodothyronine and [3,5-125I]thyroxine, respectively. Both deiodinase activities were observed in particulate fractions (mitochondrial and microsomal) of cultured cell and rat liver homogenates.

Animals↗

PubMed1981

Identification of an intracellular pathway of thyroxine synthesis by dispersed thyroid cells.

This study deals with the identification of the biochemical events involved in the metabolic sequence leading from the synthesis to the release of thyroxine in the dispersed thyroid cell system. (1) Using an experimental model allowing the differentiation between intracellular and extracellular sites of iodination, it is shown that thyroxine is synthesized inside the cells by an iodinating system sensitive to thyrotropin stimulation. (2) The secretion of thyroxine synthesized inside the cells is not mediated by an exocytotic-endocytotic phenomenon. Colchicine, vinblastine, fluoride, propanolol and chlorpromazine, at concentrations equal to or 10--100-times higher than those required to inhibit hormone release in follicular-organized thyroid tissue have no effect on thyrotropin-stimulated thyroxine secretion. (3) The secretion involves the intracellular proteolysis of hormone-containing iodoprotein(s) which, in addition to free thyroxine, generates free mono- and diiodotyrosines. Free thyroxine is released into the incubation medium and iodotyrosines are deiodinated under normal conditions and accumulate in the presence of an inhibitor of iodotyrosine deiodinase: 3,5-dinitrotyrosine. This proteolysis is inhibited by 5 mM chlorpromazine. These data indicate that the complete metabolic sequence leading from the uptake of iodide to the release of free thyroxine into the incubation medium can be described as an 'intracellular metabolic sequence for thyroxine synthesis'.

Animals↗

PubMed1991

Determination of proteolytic hydrolysis of thyroglobulin.

A method for the determination of the free thyronine- and tyrosine-like amino acids in the thyroidal protein thyroglobulin is presented. The compounds of interest are monoiodotyrosine, diiodotyrosine, thyronine, diiodothyronine, triiodothyronine and tetraiodothyronine. The extent of proteolysis was followed by high-performance liquid chromatographic monitoring of both the remaining peptides and the formation of the free thyroidal amino acids. Total hydrolysis was achieved by a combination of proteolytic enzymes. A number of enzymes were tested, such as trypsin, chymotrypsin, pronase, aminopeptidase-M, carboxypeptidase-A, carboxypeptidase-P and carboxypeptidase-Y. The best combination turned out to be pronase followed by aminopeptidase-M. The relative amounts of the enzymes, with respect to the substrate thyroglobulin, and the time of incubation were optimized to achieve total proteolysis in 4 h. The method was applied successfully to samples from a toxicological experiment with sodium bromide.

Animals↗