Search PubMed⌕ Search

Biomedical subjects

J Nunez

Publications and source records attributed to J Nunez.

At least 109 records · Page 6Linked to original sources
PubMed1977

Spatial requirement for coupling of iodotyrosine residues to form thyroid hormones.

A linear random copolymer of tyrosine and lysine and two synthetic oligopeptides containing two tyrosine residues in addition to lysine residues give thyroid hormone (thyroxine and triodothyronine) residues in good yield upon enzymatic iodination with thyroid peroxidase. These synthetic peptides may serve as simple models for thyroglobulin, the protein in which biosynthesis of the thyroid hormone takes place. For the formation of significant amounts of hormone, such model compounds must contain at least two properly spaced tyrosine residues.

Iodide Peroxidase↗
PubMed1976

Kinetics of thyroglobulin iodination and of hormone synthesis catalysed by thyroid peroxidase. Role of iodide in the coupling reaction.

The kinetics of tyrosine iodination and of thyroxine synthesis in thyroglobulin, different reactions catalyzed by the same enzyme (thyroid peroxidase), have been compared. Thyroxine synthesis always began after a lag period of 3-5 min. This lag was constant whatever the rate of iodination; this rate of iodination was increased either by increasing the concentration of iodide or enzyme or by decreasing the concentration of thyroglobulin. Increasing the rate of iodination resulted in increasing the number of iodine atoms incorporated during the lag period. Thus the lag observed for thyroxine synthesis was constant and did not depend on the fact that free iodide or non-iodinated tyrosine residues of thyroglobulin were exhausted before thyroxine synthesis occurred. Finally, it appeared that, whatever the explanation of the lag, the enzyme catlyzes thyroid hormone synthesis at a slower rate than iodination. The existence of a lag also allowed us to prepare thyroglobulin samples with different iodine contents but without thyroid hormones. Thus iodination and thyroxine synthesis could be studied independently and the following results were obtained. 1. Iodotyrosine residues which can couple to form thytoxine are made considerably before coupling occurs. 2. H2O2 is required for coupling of these hormonogenic residues; thus the coupling reaction requires enzymic oxidation of the iodotyrosine residues. 3. In addition a strict requirement for iodide was needed for coupling; the requirement was dependent on the concentration of iodide. Thus iodide, a substrate of the iodination reaction, may also have other effects on the activity of thyroid peroxidase.

Goiter↗
PubMed1976

Maximal number of hormonogenic iodotyrosine residues in thyroglobulin iodinated by thyroid peroxidase.

Almost non-iodinated human goiter thyroglobulin has been iodinated in vitro by thyroid peroxidase to levels as high as 75 iodine atoms per mol of protein. The following results were obtained. 1. The iodine distribution obtained in vitro with human thyroglobulin strongly ressembles that obtained in vivo for rat thyroglobulin. Thus the distribution of iodine seems to depend essentially on the structure of thyroglobulin and on the reactivity of the different tyrosine residues. 2. Although the number of hormone residues increased with iodination the highest efficiency of hormone synthesis was obtained in a very narrow range of iodination: in vitro (40%) between 25 and 30 iodine atoms, and in vivo (48%) between 10 and 20 atoms. This result suggests that the tyrosines which are coupled with a high efficiency are iodinated sequentially. 3. Maximal thyroxine content was found to be lower than approximately 3 mol/mol of thyroglobulin. This result might mean that the two 12-S subunits of thyroglobulin are not identical and that one of them is able to produce 2 mol of hormone while the second only 1 mol.

Animals↗
PubMed1976

Role of cortisol on the glycogenolytic effect of glucagon and on the glycogenic response to insulin in fetal hepatocyte culture.

The effects of insulin and glucagon on glycogen metabolism were studied in cultured fetal hepatocytes transplanted from 15-day-old fetuses. The effects of these hormones were examined just after transplantation, when the cells contained only minute amounts of glycogen, and during the 3 to 4 day culture period, when the hepatocytes were exposed to 10 muM cortisol and actively accumulated glycogen. At all stages of the culture, glucagon addition (10 nM) was followed by a rapid depletion of labeled glycogen, previously synthesized during a pulse labeling with [14C]glucose: this effect was mimicked by N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) (0.3 to 1 nM). Such a glycogenolytic effect of glucagon was observed even 6 hours after transplantation, i.e. at a time when cortisol was not present. In addition, glucagon clearly induced cyclic adenosine 3':5'-monosphosphate (cyclic AMP) accumulation in cells grown for 18 hours in the absence of cortisol. With cells grown for 3 days in the presence of cortisol, glucagon-dependent glycogenolysis was also obtained when cortisol was removed from the medium 20 hours before hormone addition. Thus the presence of cortisol is not necessary either to maintain a response to glucagon or for the onset of the glycogenolytic effect of glucagon. Insulin addition (10 nM) stimulated [14C]glucose incorporation into glycogen at all stages of the culture when grown in the presence of cortisol; no glycogenic response to insulin was observed 6 hours after transplantation where cortisol was not previously introduced. In addition, if the hepatocytes were grown in the presence of insulin alone (i.e. in the absence of cortisol) no significant storage of glycogen occurred. Maximal storage (or labeling) of glycogen was observed when hepatocytes were grown in the presence of both cortisol and insulin. The presence of cortisol was therefore necessary for the expression of the glycogenic effect of insulin. These data show that marked difference exist between the onset of developmental responses towards glucagon and insulin. The glucagon-dependent regulatory pathway should be present very early in fetal development and should not depend on cortisol. On the contrary, the onset of the insulin-dependent regulatory pathway seems to be induced during culture, and it is likely that this is caused by cortisol.

Animals↗
PubMed1976

Phosphorylation of microtubule-associated proteins.

1. Tubulin is not an adenosine-3':5'-monophosphate-dependent (cyclic-AMP-dependent) protein kinase. Both entities have been clearly separated by sucrose gradient ultracentrifugation. With a tubulin preparation obtained by the polymerization-depolymerization technique protein kinase had a sedimentation coefficient of 8.7 S whereas tubulin sedimented with 6.4 S. After preincubation with both cyclic AMP and histone the kinase dissociated into its catalytic subunit with a sedimentation coefficient of 3.4 S. 2. Tubulin prepared by the polymerization-depolymerization technique was neither phosphorylated in vivo nor in vitro. On the contrary if this preparation was further purified by the Weisenberg's procedure (DEAE-Sephadex batch absorption) before incubation with [gamma-32 P]ATP, phosphorylation occurred. Thus, phosphorylation depended on the method used to purify tubulin i.e. was likely to an an artefact.

Brain↗
PubMed1976

Additive effects of norepinephrine and cyclic AMP on the activation of the protein kinase from adipose tissue.

In this work the kinetics of activation of the cyclic AMP-dependent protein kinase by several catecholamines and ACTH, have been studied in rat epididymal fat pads and isolated fat cells. The method of Soderling and co-workers which permits the measurement of the state of activation of the protein kinase after hormonal stimulation in adipose tissue, has been used. Kinetics experiments where norepinephrine was used showed that the results obtained with isolated cells conform to the models of Sutherland and Brostrom and co-workers. Wtih intact tissue, norepinephrine not only stimulates the protein kinase activity measured without exogenous cyclic AMP but also the total activity measured in the presence of cyclic AMP (5 X 10(-6) M); thus the effect of norepinephrine, obtained during incubation of the tissue, and that of cyclic AMP, added to the soluble fraction after incubation, were additive. This effect seems to be of the beta type because it is blocked completely by propranolol. A weak, additive but significant effect was also obtained with epinephrine and isoproterenol but not with ACTH. Neither cyclic GMP nor cyclic IMP seems implicated in this effect. It was shown that stroma vascular cells which are present in the fat pads are not involved. These results suggest that the effects of norepinephrine on the protein kinase of the fat pads cannot be completely explained by the model of Brostrom and colleagues.

Adipose Tissue↗
PubMed1976

Thyroid iodine organification defects: a case with lack of thyroglobulin iodination and a case without any peroxidase activity.

Two patients (G2, G3) with iodine organification defect were studied. The first patient (G2), a 25-year-old women with no clinical hypothyroidism, had had her goiter for 10 years; 62% of the thyroidal iodine was released by perchlorate indicating iodine organification defect. The thyroid tissue obtained at thyroidectomy contained a normal concentration of thyroid peroxidase (I2 formation from I-) when tested after solubilization of the enzyme by trypsin and digitonin treatment of the particulate material. 1. The enzymatic activity (G2-TPO) behaved on DEAE cellulose chromatography very differently from those of hog (P-TPO) or another human goiter peroxidase (G1-TPO) (Pommier, et al., J Clin Endocrinol Metab 39: 69, 1974): the molarity of elution was 2M NaCl instead of 0.15 mM. 2. Both P-TPO and G2-TPO catalyzed iodide peroxidation (I- leads to I2) but the Km (iodide) value for G2-TPO was much lower (2.3 x 10(-2) M) when compared with that of P-TPO (3.7 x 10(-3) M) or G1-TPO (3.5 x 10(-3) M). In addition, the optimum pH for this reaction differed markedly (pH 6.1 instead of 7.9). 3. G2-TPO was poorly efficient in catalyzing the oxidation of gaiacol to tetragaiacol. 4. G2-TPO was unable to perform the iodination of non-iodinated goiter thyroglobulin whatever the pH and the iodide concentration. 5. Thyroglobulin from this goiter (G2) was almost not iodinated (0.0014%), i.e., 0.07 atoms iodine/mole thyroglobulin), and its total content in the gland was very low (0.3-4 g/1000 g wet tissue instead of 25 g). A clear discrepancy was thus shown between the euthyroid state of this patient and the total lack of iodinating activity of the isolated peroxidase. The second patient (G3), a 17-year-old man with clinical hypothyroidism, had had his goiter for 5 years. 100% of the thyroidal iodine was released by perchlorate indicating a complete iodine organification defect. The thyroid tissue obtained at thyroidectomy contained no peroxidase activity when tested before and after treatment of the particulate material by trypsin and digitonin and even in the presence of hematin. Thyroglobulin from this goiter, which was almost non-iodinated (0.0014%), was present in normal amounts in the gland (congruent to 25 g/1000 g).

Adolescent↗
PubMed1975

9 S thyroid particulate iodoprotein.

Particulate iodoproteins have been studied in the rat thyroid gland using the isotopic 125I equilibration method. Pulse experiments were also performed with a second isotope, 131I. Labelled iodoproteins, both soluble and solubilized by digitonin from the thyroid particulate material, were analyzed by sucrose-gradient ultracentrifugation. 1. At isotopic equilibrium and irrespective of the iodide content of the diet two particulate iodoproteins, with sedimentation coefficients of 27 and 19 S, were solubilized by digitonin. In addition a 9 S iodoprotein was also present but its proportion varied markedly with the iodine content of the diet: it accounted for 50-60% of the label found at the particulate level when the dialy diet of iodine was high (75-500 mug/day) but was almost absent when the diet was only 2 mug/day. 2. Most of this 9 S protein was found in the 600-15 000 times g particulate pellet, i.e. a fraction enriched in lysosomes and phagolysosomes. 3. The iodoamino acid composition of the 9 S fraction was very similar to that of the 19 S particulate thyroglobulin: its hormone content was 19%. 4. The double precipitation technique showed that the 9 S fraction is immunochemically related to thyroglobulin. 5. Pulse experiments showed that the 9 S particulate iodoprotein was slowly labelled by 131I. 6. The amount of 9 S iodoprotein was increased by thyrotropin (30-40% increase versus control experiments 5 min after thyrotropin injection). These properties of the 9 S particulate iodoprotein are discussed in relation to the assumption that it might be a product of partial proteolysis of thyroglobulin after endocytosis and partial digestion by the phagolysosomes.

Amino Acids↗
PubMed1975

Glycogenolytic response to glucagon of cultured fetal hepatocytes. Refractoriness following prior exposure to glucagon.

The glycogenolytic effect of glucagon has been studied in fetal hepatocytes cultured for 3 to 4 days in the presence of cortisol (10 muM). The hepatocytes, when transplanted from young fetuses (15-day-old), contain only minute amounts of glycogen, whereas when cultured 3 to 4 days in the presence of cortisol, they contain high levels of stored glycogen. Glucagon induced a rapid but partial mobilization of glycogen, which was maximal after 2 hours. The half-maximal response was observed with about 0.1 nM glucagon. The glycogenolytic effect of glucagon in fetal hepatocytes is probably mediated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) as in adult liver. This effect was mimicked by cyclic AMP and N-6, O-2-dibutyryl cyclic AMP, (dibutyryl cyclic AMP), and potentiated by theophylline. Glucagon addition was followed by accumulation of cyclic AMP in the cells within 2 min. Glucagon produces a marked stimulation of the rate of glycogen breakdown and an inhibition of the rate of incorporation of [14-C] glucose into glycogen. The glycogeneolytic effect of a single addition of glucagon was reversed within 4 hours. A second addition of glucagon at this time was unable to induce a new glycogenolytic response. A resistance to glucagon stimulation appeared in the cells after a first exposure to the hormone. This refractoriness was also shown by the loss of glucagon-dependent cyclic AMP accumulation and was not linked to the release by the cells of a "hormone antagonist" into the medium. The hepatocytes resistant to the action of glucagon retained their response to cyclic AMP, dibutyryl cyclic AMP, and norepinephrine. Finally, glycogenolytic concentrations of cyclic AMP and of its dibutyryl derivative failed to induce a refractoriness to glucagon.

Animals↗
PubMed1975

Free diiodotyrosine effects on protein iodination and thyroid hormone synthesis catalyzed by thyroid peroxidase.

Free diiosotyrosine exerts two opposite effects on the reactions catalyzed by thyroid peroxidase, thyroglobulin iodination and thyroid hormone formation. 1. Inhibition of thyroglobulin iodination catalyzed by thyroid peroxidase was observed when free diiodotyrosine concentration was higher than 5 muM. This inhibition was competitive, suggesting that free diiodotyrosine interacts with the substrate site(s) of thyroid peroxidase. Free diiodotyrosine also competively inhibited iodide peroxidation to I2. 2. Free diiodotyrosine, when incubated with thyroid peroxidase in the absence of iodide was recovered unmodified; in the presence of iodide an exchange reaction was observed between the iodine atoms present in the diiodotyrosine molecule and iodide present in the medium. Using 14C-labelled diiodotyrosine, 14C-labelled non-iodinated products were also observed, showing that deiodination occurred as a minor degradation pathway. However, no monoiodo[14C]tyrosine or E114C]tyrosine were observed. Exchange reaction between free diiototyrosine and iodide is therefore direct and does not imply deiodination-iodination intermediary steps. Thyroglobulin inhibits diiodotyrosine-iodide exchange and vice versa, again suggesting competition for both reactions. These results support, by a different experimental approach, the two-site model for peroxidase previously described by us in this journal. 3. Free diiodotyrosine when present at a very low concentration, 0.05 muM, exerts a stimulatory effect on throid hormones synthesis. The relationship between diiodotyrosine concentration and thyroid hormone synthesis give an S-shaped curve, suggesting that free diiodotyrosine acts as a regulatory ligand for thyroid peroxidase. Evidence is also presented that free diiodotyrosine is not incorporated into thyroid hormones. Therefore, thyroid peroxidase catalyzes only intra-molecular coupling between iodotyrosine hormonogenic residues. 4. Finally, although no direct proof exists that these free diiodotyrosine effects upon thyroglobulin iodination and thyroid hormone synthesis are physiologically significant, such a possibility deserves further investigation.

Animals↗