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The Journal of Biological Chemistry Jun 1990Thyroid hormonogenesis in thyroglobulin results in the conversion of an "acceptor" iodotyrosine to a hormone residue and a "donor" iodotyrosine to a dehydroalanine...
Thyroid hormonogenesis in thyroglobulin results in the conversion of an "acceptor" iodotyrosine to a hormone residue and a "donor" iodotyrosine to a dehydroalanine residue. Altogether five acceptor sites have been located as hormone residues in thyroglobulin of different animal species. To search for donor sites, we treated bovine thyroglobulin with 4-aminothiophenol to specifically modify dehydroalanine residues to S-(4-aminophenyl)cysteine (APC) residues, according to the principle of dehydroalanine determination developed by us (Kondo, T., Kondo, Y., and Ui, N. (1988) Mol. Cell. Endocr. 57, 101-106). After digesting thyroglobulin with lysyl endopeptidase, APC-containing peptides were separated from other peptides by trapping them on immobilized naphthylethylenediamine and from each other by size-exclusion and reverse-phase high performance liquid chromatography (HPLC). The HPLC patterns showed about 10 APC-containing peptides. Among them, four different peptides were purified by repeated reverse-phase HPLC. The results of partial sequencing of the four peptides by manual Edman degradation disclosed that Tyr5, Tyr926, Tyr1375, and Tyr986 or Tyr1008 are available for hormonogenesis as donor sites. These results strongly suggest that only specific tyrosine residues behave as donors.
Topics: Alanine; Amino Acid Sequence; Animals; Binding Sites; Cattle; Chromatography, High Pressure Liquid; Molecular Sequence Data; Monoiodotyrosine; Peptide Fragments; Phenylthiohydantoin; Serine Endopeptidases; Thyroglobulin
PubMed: 2345166
DOI: No ID Found -
The Journal of Biological Chemistry May 1990In most highly structured native proteins, as well as in thyroglobulin, the reactivity in vitro of the various tyrosyl residues toward iodine is widely different. The...
In most highly structured native proteins, as well as in thyroglobulin, the reactivity in vitro of the various tyrosyl residues toward iodine is widely different. The present work demonstrates that of nearly 70 tyrosyl residues present in rat thyroglobulin, there is one, residue number 5 from the NH2-terminal end, which has in vivo the highest affinity toward iodine, being the first one to be iodinated. In fact, when 6-(n-propyl)-2-thiouracil (PTU)-treated, iodine-deficient animals were injected with 125I and killed shortly after, we isolated from thyroid glands poorly iodinated thyroglobulin (about 1 iodine atom/thyroglobulin molecule), nearly 90% of the radioactivity of which was found as monoiodotyrosine. Although CNBr cleavage of this protein gave several fragments after gel electrophoresis only one of these, with apparent mass 27,000 Da, contained 125I. This fragment was isolated and fully characterized. Twelve cycles of automated Edman degradation were performed; the sequence found, i.e. N-I-F-E-X-Q-V-X-A-Q-X-L, indicated that the 27,000-Da fragment is the NH2 terminus of thyroglobulin. This portion of the polypeptide chain contains several tyrosyl residues which may well all be potentially involved in the early iodination of the protein. The observation that the removal of seven amino acids from the NH2 terminus is accompanied (at the fifth step) by the total disappearance of radioactivity in the resulting shortened peptide suggested that the fifth residue was the only one iodinated under these conditions. A second, more quantitative experiment was performed on thyroglobulin obtained from 6-(n-propyl)-2-thiouracil-treated animals whose death was postponed 24 h after the injection of 125I. In this case the radioactivity was found not only in a single CNBr fragment (27,000 Da) but also in other discrete species of lower molecular mass. The mixture of these peptides was subjected to seven steps of manual Edman degradation. Fragments before and after partial degradation were run in parallel on a polyacrylamide gel and the distribution of 125I compared. Besides some change in the background, the two profiles were identical except for the absence of the 27,000-Da species. This proves that all the 125I present in the 27,000-Da species was localized at the fifth residue, the same site at which the hormone molecule is preferentially synthesized under normal conditions. This result is not unexpected and is in accord with the known properties of thyroglobulin which has a polypeptide chain designed for efficient synthesis of the hormone even at low levels of iodination.
Topics: Amino Acid Sequence; Animals; Cyanogen Bromide; Iodine; Iodine Radioisotopes; Male; Molecular Sequence Data; Molecular Weight; Peptide Mapping; Propylthiouracil; Protein Binding; Radioisotope Dilution Technique; Rats; Rats, Inbred Strains; Thyroglobulin
PubMed: 2187874
DOI: No ID Found -
The Journal of Biological Chemistry Mar 1983The specific binding of various concentrations of 125I-labeled nerve growth factor (NGF) to PC12 cells at 37 degrees C reached maxima after 90 min and then declined to...
The specific binding of various concentrations of 125I-labeled nerve growth factor (NGF) to PC12 cells at 37 degrees C reached maxima after 90 min and then declined to 25% of maximal binding after 10 h. Decreased binding was accompanied by degradation of 125I-NGF and the appearance of acid-soluble biologically inactive 125I (mainly 125I-monoiodotyrosine) in the medium as well as a decrease in the number of surface NGF receptors. The time-dependent decrease in binding and the degradation of 125I-NGF were inhibited by low temperature and the lysosomotropic agent chloroquine while degradation was inhibited by metabolic energy inhibitors in the absence of glucose. Chloroquine also produced an increase in the accumulation of 125I-NGF which was not readily removed from the cells. These data suggest that 125I-NGF bound to PC12 cells is efficiently internalized by receptor-mediated endocytosis and degraded by the lysosomes. It appears from other data that this process does not produce the intracellular signals regulating neurite outgrowth.
Topics: Adrenal Gland Neoplasms; Cell Line; Chloroquine; Cold Temperature; Humans; Kinetics; Nerve Growth Factors; Pheochromocytoma; Receptors, Cell Surface; Receptors, Nerve Growth Factor
PubMed: 6298216
DOI: No ID Found -
Journal of the American Society For... Aug 2007L-Tyrosine and iodinated L-tyrosines, i.e., 3-iodo-L-tyrosine and 3,5-diiodo-L-tyrosine, are successfully used as chiral references for the chiral discrimination of...
L-Tyrosine and iodinated L-tyrosines, i.e., 3-iodo-L-tyrosine and 3,5-diiodo-L-tyrosine, are successfully used as chiral references for the chiral discrimination of aliphatic, acidic, and aromatic amino acids. Chiral discrimination is achieved by investigating the collision-induced dissociation spectra of the trimeric complex [Cu(II)(ref)(2)(A) - H](+) ion generated by electro spraying the mixture of D- or L-analyte amino acid (A), chiral reference ligand (ref) and M(II)Cl(2) (M = Ni and Cu). The relative abundances of fragment ions resulted by the competitive loss of reference and analyte amino acids are considered for measuring the degree of chiral discrimination by applying the kinetic method. The chiral discrimination ability increases as the number of iodine atom increases on the aromatic ring of the reference and the discrimination is better with Cu when compared with Ni. A large chiral discrimination is obtained for aliphatic and aromatic amino acids using iodinated L-tyrosine as the reference. Computational studies on the different stabilities of the diastereomeric complexes also support the observed differences measured by the kinetic method. The suitability of the method in the measurement of enantiomeric excess over the range of 2% to 100% ee with relative error 0.28% to 1.6% is also demonstrated.
Topics: Amino Acids; Copper; Iodine; Molecular Conformation; Monoiodotyrosine; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism; Tyrosine
PubMed: 17588770
DOI: 10.1016/j.jasms.2007.05.006 -
The Journal of Biological Chemistry Dec 1989Charybdotoxin (ChTX), a peptidyl inhibitor of the high conductance Ca2+-activated K+ channel (PK,Ca), has been radiolabeled to high specific activity with 125I, and...
Characterization of high affinity binding sites for charybdotoxin in sarcolemmal membranes from bovine aortic smooth muscle. Evidence for a direct association with the high conductance calcium-activated potassium channel.
Charybdotoxin (ChTX), a peptidyl inhibitor of the high conductance Ca2+-activated K+ channel (PK,Ca), has been radiolabeled to high specific activity with 125I, and resulting derivatives have been well separated. The monoiodotyrosine adduct blocks PK,Ca in vascular smooth muscle with slightly reduced potency compared with the native peptide under defined experimental conditions. [125I]ChTX, representing this derivative, binds specifically and reversibly to a single class of sites in sarcolemmal membrane vesicles prepared from bovine aortic smooth muscle. These sites display a Kd of 100 pM for the iodinated toxin, as determined by either equilibrium or kinetic binding analyses. Binding site density is about 500 fmol/mg of protein in isolated membranes. The addition of low digitonin concentrations to disrupt the vesicle permeability barrier increases the maximum receptor concentration to 1.5 pmol/mg of protein, correlating with the observations that ChTX binds only at the external pore of PK,Ca and that the membrane preparation is of mixed polarity. Competition studies with ChTX yield a Ki of about 20 pM for native toxin. Binding of [125I]ChTX is modulated by ionic strength as well as by metal ions that are known to interact with PK,Ca. Moreover, tetraethylammonium ion, which blocks PK,Ca with moderately high affinity when applied at the external membrane surface, inhibits [125I]ChTX binding in an apparently competitive fashion with a Ki similar to that found for channel inhibition. In marked contrast, agents that do not inhibit PK,Ca in smooth muscle (e.g. tetrabutylammonium ion, other toxins homologous with ChTX, and pharmacological agents that modulate the activity of dissimilar ion channels) have no effect on [125I]ChTX binding in this tissue. Taken together, these results suggest that the binding sites for ChTX which are present in vascular smooth muscle are directly associated with PK,Ca, thus identifying [125I]ChTX as a useful probe for elucidating the biochemical properties of these channels.
Topics: Animals; Aorta; Calcium; Cattle; Cell Membrane; Charybdotoxin; In Vitro Techniques; Kinetics; Muscle, Smooth, Vascular; Potassium Channels; Receptors, Cholinergic; Sarcolemma; Scorpion Venoms; Sodium Channels
PubMed: 2480347
DOI: No ID Found -
Proceedings of the National Academy of... Oct 1971Tyrosine hydroxylase activity of synaptosomes isolated from rat brain was examined. A modified tritium-displacement assay was used, which allowed the measurement of...
Tyrosine hydroxylase activity of synaptosomes isolated from rat brain was examined. A modified tritium-displacement assay was used, which allowed the measurement of tyrosine hydroxylase activity without the addition of either inhibitors of the metabolism of the hydroxylated products or added exogenous cofactor. The enzyme activity was strongly inhibited by the addition of exogenous catecholamines and 3,4-dihydroxy-L-phenyl-alanine. Aromatic amines other than catechols did not markedly influence tyrosine hydroxylase activity. These in vitro findings support the hypothesis that synthesis of catecholamines is regulated by a mechanism of end-product inhibition at the tyrosine hydroxylase step.
Topics: Animals; Autoradiography; Brain; Catecholamines; Chromatography, Ion Exchange; Dihydroxyphenylalanine; Dopamine; Male; Methyldopa; Methyltyrosines; Mixed Function Oxygenases; Monoiodotyrosine; Norepinephrine; Normetanephrine; Octopamine; Phenethylamines; Rats; Serotonin; Synaptic Vesicles; Time Factors; Tritium; Tyramine; Tyrosine; Tyrosine 3-Monooxygenase
PubMed: 4400211
DOI: 10.1073/pnas.68.10.2370 -
The Journal of Biological Chemistry Jan 1994The high-conductance Ca(2+)-activated K+ (maxi-K) channel from bovine tracheal smooth muscle was purified to apparent homogeneity by a combination of conventional...
The high-conductance Ca(2+)-activated K+ (maxi-K) channel from bovine tracheal smooth muscle was purified to apparent homogeneity by a combination of conventional chromatographic techniques and sucrose density gradient centrifugation. Fractions with the highest specific activity for binding of monoiodotyrosine charybdotoxin, [125I]ChTX, were enriched approximately 2000-fold over the initial digitonin-solubilized material up to a specific activity of 1 nmol/mg protein. Silver staining after SDS-polyacrylamide gel electrophoresis of the fractions from the last step of the purification indicates that binding activity is correlated with a major component of the preparation that displays an apparent molecular weight of 62,000. Labeling the same preparation with 125I-Bolton-Hunter reagent reveals the existence of both 62 (alpha)- and 31 (beta)-kDa subunits, in an apparent stoichiometry of 1:1, comigrating with binding activity. The beta subunit is heavily glycosylated. Deglycosylation studies indicate that the beta subunit represents the protein to which [125I]ChTX is covalently incorporated in the presence of the bifunctional cross-linking reagent disuccinimidyl suberate. Binding of [125I]ChTX to the purified ChTX receptor displayed the same pharmacological profile that has been found previously for toxin binding to native membranes, including inhibition by iberiotoxin, limbatustoxin, tetraethylamonium, potassium, cesium, and barium. The purified preparation was reconstituted into liposomes which were then fused with artificial lipid bilayers. Single channels were readily observed with a conductance of 235 picosiemens in 150 mM KCl that displayed selectivity for potassium over chloride and that were blocked by ChTX. The open probability of these channels was increased by depolarizing membrane potentials and by raising the internal calcium concentration. These data suggest that the maxi-K channel purified from tracheal smooth muscle is composed of two subunits.
Topics: Animals; Binding Sites; Calcium; Cattle; Charybdotoxin; Electrophoresis, Polyacrylamide Gel; Ion Channel Gating; Membrane Potentials; Muscle, Smooth; Potassium Channels; Scorpion Venoms; Trachea
PubMed: 7506261
DOI: No ID Found -
The Journal of Experimental Medicine Nov 1967Mouse peritoneal macrophages take up I*-HSA from their medium during in vitro cultivation. Conditions which promote I*-HSA uptake are the same as those which stimulate...
Mouse peritoneal macrophages take up I*-HSA from their medium during in vitro cultivation. Conditions which promote I*-HSA uptake are the same as those which stimulate formation of pinocytic vesicles. Autoradiography of cells pulsed with (125)I-HSA showed that intracellular isotope is localized in perinuclear granules, or secondary lysosomes. Following a pulse of (125)I-HSA, intracellular radioactivity decreases and the amount of TCA-soluble isotope in the medium increases correspondingly. About 50% of the intracellular isotope is lost in 5 hr. The release of isotope from pulsed cells is not inhibited by parafluorophenylalanine, 2,4-dinitrophenol or by a reduction of the serum concentration of the medium. However, the processing of ingested (125)I-HSA is reversibly inhibited by reduced temperature. The TCA-soluble radioactive material excreted by pulsed macrophages was identified as monoiodotyrosine.
Topics: Animals; Autoradiography; Chromatography, Paper; Culture Techniques; Lysosomes; Macrophages; Mice; Nucleoproteins; Pinocytosis; Serum Albumin; Serum Albumin, Radio-Iodinated
PubMed: 6062005
DOI: 10.1084/jem.126.5.941 -
The Journal of Physiology Nov 1975Acetylcholine (ACh) receptors in rat diaphragm muscle were blocked by intrathoracic injection of alpha-bungarotoxin (alpha-BuTx) or [125I]alpha-bungarotoxin...
Acetylcholine (ACh) receptors in rat diaphragm muscle were blocked by intrathoracic injection of alpha-bungarotoxin (alpha-BuTx) or [125I]alpha-bungarotoxin ([125I]alpha-BuTx). The stability in vivo of the toxin-receptor complex formed by receptors in normal muscles and receptors in extrajunctional regions of denervated muscles was compared. Toxin was lost from junctional regions of normal muscles with a half-time of approximately 6 days. The loss of toxin was accompanied by a corresponding increase in the number of free toxin-binding sites. In contrast, 65% of the toxin bound to extrajunctional regions of denervated muscle was lost in 24 hr. 2. In a second series of experiments, animals were injected with [125I]alpha-BuTx and the muscle subsequently cultured for 24 hr. Loss of toxin again occurred more rapidly from extrajunctional receptors than from junctional receptors. The loss from extrajunctional receptors was described by a single first-order rate constant whose corresponding half-time was 8-11 hr. Loss was almost completely blocked by sodium cyanide and dinitrophenol and was inhibited by puromycin and cycloheximide. The radioactivity recovered in the medium was largely monoiodotyrosine. These results are consistent with the hypothesis that toxin loss reflects intracellular degradation of toxin-receptor complex. 3. Neonatal rats were injected with [125I]alpha-BuTx and the diaphragms cultured. Radioactive toxin was lost rapidly from extrajunctional regions of muscle and more slowly from regions containing end-plates. 4. These results could be explained by a difference in turnover rates for junctional and extrajunctional receptors.
Topics: Animals; Animals, Newborn; Bungarotoxins; Cyanides; Cycloheximide; Diaphragm; Dinitrophenols; Male; Muscle Denervation; Muscles; Neuromuscular Junction; Organ Culture Techniques; Puromycin; Rats; Receptors, Cholinergic
PubMed: 1206575
DOI: 10.1113/jphysiol.1975.sp011169 -
The Biochemical Journal Jun 1990The binding, internalization and degradation of 200 pM monoiodinated human atrial natriuretic factor-(99-126) (125I-hANF) by cultured bovine aortic endothelial cells...
The binding, internalization and degradation of 200 pM monoiodinated human atrial natriuretic factor-(99-126) (125I-hANF) by cultured bovine aortic endothelial cells (BAECs) were studied at 37 degrees C. 125I-hANF was rapidly cleared from the extracellular medium (t1/2 approximately 10 min), whereas preincubation of the cells in the presence of 20 mM-NH4Cl or 0.2 mM-chloroquine resulted in a significant inhibition of this process. The BAECs rapidly produce three major degradation products of 125I-hANF, namely [125I]iodotyrosine 126 (125I-Y), Arg125-[125I]iodotyrosine126 (125I-RY) and Phe124-Arg125-[125I]iodotyrosine126(125I-FRY), which were detected in the extracellular medium. NH4Cl and chloroquine acted to inhibit the generation of 125I-Y and 125I-RY, but not that of 125I-FRY. Furthermore, excess unlabelled hANF (300 nM) completely blocked the rapid production of 125I-Y and 125I-RY in the first 5 min, but only partially (49%) inhibited the generation of 125I-FRY. Thus, in contrast with our previous findings with cultured smooth-muscle cells [Johnson, Arik & Foster (1989) J. Biol. Chem. 264, 11637-11642], BAECs bind, internalize and rapidly degrade 125I-hANF, resulting in the release of 125I-Y and 125I-RY into the extracellular medium. Similarly to smooth-muscle cells, the BAECs generate 125I-FRY from 125I-hANF via an extracellular proteolytic event. The rapidity of the receptor-mediated process and its sensitivity to NH4Cl and chloroquine suggest that the 125I-hANF is proteolytically processed in the endosomes of BAECs and that its receptors cycle between the cell surface and intracellular stores.
Topics: Ammonium Chloride; Animals; Atrial Natriuretic Factor; Cattle; Cells, Cultured; Chloroquine; Dipeptides; Endothelium, Vascular; Iodine Radioisotopes; Monoiodotyrosine; Oligopeptides; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface
PubMed: 2163622
DOI: 10.1042/bj2680771