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The Journal of Neuroscience : the... Dec 1990This paper compares the localization of neurotensin receptors and of endopeptidase 24-16, a peptidase likely involved in the inactivation of neurotensin in primary...
This paper compares the localization of neurotensin receptors and of endopeptidase 24-16, a peptidase likely involved in the inactivation of neurotensin in primary cultures of neurons. Neurotensin binding sites were radiolabeled with 125I-Tyr3-neurotensin, whereas endopeptidase 24-16 was stained by immunohistochemical techniques using a monospecific polyclonal antibody. Endopeptidase 24-16 is present in 80-85% of the nondifferentiated neurons. The proportion of immunoreactive neurons decreased during maturation to reach 35-40% after 4-8 d of culture. By contrast, neurotensin receptors were not detectable in nondifferentiated cells and appear during maturation. Specific 125I-Tyr3-neurotensin labeling is maximal after 4 d of culture and is located on about 10% of differentiated neurons. Double-labeling experiments show that about 90% of cortical, hypothalamic, and mesencephalic neurons bearing the neurotensin receptor also contained endopeptidase 24-16, supporting the hypothesis that one of the functions of endopeptidase 24-16 is the physiological inactivation of neurotensin. However, the presence of endopeptidase 24-16 on numerous neurons that do not contain neurotensin receptors also suggests that the enzyme could be involved in the degradation and/or maturation of other neuropeptides.
Topics: Animals; Brain; Cell Differentiation; Cells, Cultured; Iodine Radioisotopes; Metalloendopeptidases; Mice; Monoiodotyrosine; Neurons; Neuropeptides; Neurotensin; Receptors, Neurotensin; Receptors, Neurotransmitter
PubMed: 2176676
DOI: 10.1523/JNEUROSCI.10-12-03916.1990 -
The Journal of Cell Biology Oct 1976125I-labeled human epidermal growth factor (hEGF) binds in a specific and saturable manner to human fibroblasts. At 37 degrees C, the cell-bound 125I-hEGF initially may...
125I-labeled human epidermal growth factor (hEGF) binds in a specific and saturable manner to human fibroblasts. At 37 degrees C, the cell-bound 125I-hEGF initially may be recovered in a native form by acid extraction; upon subsequent incubation, the cell-bound 125I-hEGF is degraded very rapidly, with the appearance in the medium of 125I-monoiodotyrosine. At 0 degrees C, cell-bound 125I-hEGF is not degraded but slowly dissociates from the cell. The data are consistent with a mechanism in which 125I-hEGF initially is bound to the cell surface and subsequently is internlized before degradation. The degradation is blocked by inhibitors of metabolic energy production (azide, cyanide, dinitrophenol), some protease inhibitors (Tos-Lys-CH2Cl, benzyl guanidobenzoate), a lysosomotropic agent (chloroquine) various local anesthetics (cocaine, lidocaine, procaine), and ammonium chloride. After the binding and degradation of 125I-hEGF the fibroblasts are no longer able to rebind fresh hormone. The binding capacity of these cells is restored by incubation in a serum-containing medium; this restoration is inhibited by cycloheximide or actinomycin D.
Topics: Ammonium Chloride; Anesthetics, Local; Azides; Binding, Competitive; Cell Membrane; Chloroquine; Cyanides; Cycloheximide; Dinitrophenols; Epidermal Growth Factor; Fibroblasts; Fluorides; Humans; Iodine Radioisotopes; Kinetics; Peptides; Temperature; Trypsin Inhibitors
PubMed: 977646
DOI: 10.1083/jcb.71.1.159 -
The Journal of Biological Chemistry May 1985When normal human fibroblasts are brought to a steady state with 125I-labeled epidermal growth factor (125I-EGF), greater than 90% of the radioactivity is intracellular....
When normal human fibroblasts are brought to a steady state with 125I-labeled epidermal growth factor (125I-EGF), greater than 90% of the radioactivity is intracellular. We investigated this material to determine whether the 125I-EGF is intact or degraded. Our results show that 125I-EGF is rapidly processed after internalization and can be resolved into four peaks by native gel electrophoresis. These different forms were isolated and tested for their ability to bind to cell-surface EGF receptors. The first processed form was fully capable of binding to EGF receptors, but the second processed form could not. The third form was a collection of small degradation products. We calculated that at steady state about 60% of internalized "125I-EGF" was in a form still able to bind to EGF receptors. We then investigated the ability of different reported inhibitors of EGF "degradation" to block the processing of EGF. Although inhibitors of cathepsin B (leupeptin, antipain, N alpha-p-tosyl-L-lysine chloromethyl ketone, and chymostatin) were able to inhibit the release of monoiodotyrosine from treated cells in a time- and concentration-dependent manner, they had little effect on the processing step that apparently inactivates 125I-EGF. In contrast, agents that raised intravesicular pH, such as methylamine and monensin, inhibited the initial steps in EGF processing as well as the later steps. Low temperatures inhibited the transfer of 125I-EGF to the lysosomes and inhibited the conversion of EGF to a nonbindable form, but had little effect on the initial processing. We conclude that the intracellular processing of EGF is a multistep process that is initiated prior to lysosomal fusion, involves cathepsin B activity, and requires an acidic pH. In addition, many of the protease inhibitors that have been utilized to investigate the role of EGF degradation in mitogenesis do not block the conversion of EGF to a form that is apparently unable to interact with its receptor.
Topics: Animals; Cathepsin B; Cathepsins; Cell Line; Cold Temperature; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Humans; Hydrogen-Ion Concentration; Leupeptins; Methylamines; Mice; Models, Biological; Monensin; Receptors, Cell Surface
PubMed: 2985587
DOI: No ID Found -
Journal of Nuclear Medicine : Official... Mar 1980A method is described that incorporates resin extraction and thin layer chromatography to isolate and separate radioiodinated thyroxine (T4), triiodothyronine (T3),...
A method is described that incorporates resin extraction and thin layer chromatography to isolate and separate radioiodinated thyroxine (T4), triiodothyronine (T3), iodoprotein, and iodide in samples of human plasma up to 3 ml. Tracer studies using this method showed that reverse T3 and 3',5' diiodothyronine (T2), as well as T4, were detected in the "T4 fraction," and that 3-3' T2 and 3' monoiodothyronine, as well as T3, were detected in the "T3 fraction." Monoiodotyrosine and diiodotyrosine (DIT) migrated more slowly than did T4 on the chromatogram, and a large amount of DIT was in the unextracted "iodoprotein fraction." Kinetic studies in 14 normal subjects given intravenous commercial [125I]T3 (T3*) and [131I]T4 (T4*), confirmed the quantitative importance of an iodoprotein in later samples after T3* administration, and its presence after T4*. T4* contamination of commercial T3* also became quantitatively important. On the other hand, despite confirmation of in vivo conversion of T4* to T3*, T3* contributed little quantitatively to the total concentration of radioactivity present even late after T4* injection, due to the more rapid turnover and greater distribution volume of T3*.
Topics: Chromatography, Thin Layer; Humans; Iodides; Iodoproteins; Kinetics; Resins, Plant; Serum Albumin, Radio-Iodinated; Thyroxine; Triiodothyronine
PubMed: 7365515
DOI: No ID Found -
The Journal of Cell Biology Nov 1972An enzymatic iodination procedure utilizing lactoperoxidase (LPO), radioactive iodide, and hydrogen peroxide generated by a glucose oxidase-glucose system has been...
An enzymatic iodination procedure utilizing lactoperoxidase (LPO), radioactive iodide, and hydrogen peroxide generated by a glucose oxidase-glucose system has been described and utilized for a study of the red cell membrane. 97% of the incorporated isotope is in the erythrocyte ghost and 3% is associated with hemoglobin. No significant labeling of the red cell membrane occurs in the absence of LPO or by the deletion of any of the other reagents. A 6 million-fold excess of chloride ions inhibits iodination by no more than 50%. Incorporation of up to 1 x 10(6) iodide atoms into a single erythrocyte membrane results in no significant cell lysis. The incorporated label is exclusively in tyrosine residues as monoiodotyrosine. 10-15% of the trichloroacetic acid-precipitable radioactivity can be extracted with lipid solvents but is present as either labeled protein or (125)I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins reveals only two labeled protein bands out of the 15 present, and the presence of 50-1 x 10(6) iodide atoms per ghost does not alter this pattern. Component a has a molecular weight of 110,000, is carbohydrate poor, and represents 40% of the total label. Component b has an apparent molecular weight of 74,000, contains all of the demonstrable sialic acid, and accounts for 60% of the total label. Trypsinization of iodinated, intact red cells results in the disappearance of only component b, the appearance of labeled glycopeptides in the medium, and the absence of smaller, labeled peptides remaining in the membrane. Pronase treatment hydrolyzes component b in a similar fashion, but also cleaves component a to a 72,000 mol wt peptide which is retained in the membrane. A combination of protease treatment and double labeling with (125)I and (131)I does not reveal the appearance of previously unexposed proteins.
Topics: Cell Membrane; Chromatography, Gel; Chromatography, Thin Layer; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Humans; Hydrogen Peroxide; Iodides; Iodine Isotopes; Methods; Molecular Weight; Neuraminidase; Peptide Hydrolases; Peroxidases; Pronase; Trypsin; Tyrosine
PubMed: 5076780
DOI: 10.1083/jcb.55.2.390 -
The Journal of Biological Chemistry Apr 1949
Topics: Humans; Monoiodotyrosine; Thyroid Gland; Tyrosine; Viscera
PubMed: 18117022
DOI: No ID Found -
Nucleic Acids Research Jun 2010Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase-tRNA pairs orthogonal to the host translation system. In the...
Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase-tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS-tRNA(Tyr) pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNA(Tyr). The endogenous TyrRS and tRNA(Tyr) genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS-tRNA(Tyr) pair. In this engineered strain, 3-iodo-L-tyrosine and 3-azido-L-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-L-tyrosine and was also found to recognize 3-azido-L-tyrosine. The structural basis for the 3-azido-L-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.
Topics: Azides; Escherichia coli; Gene Deletion; Genetic Code; Genetic Complementation Test; Methanococcales; Monoiodotyrosine; Mutation; Protein Biosynthesis; Protein Engineering; RNA, Transfer, Tyr; Saccharomyces cerevisiae; Suppression, Genetic; Tyrosine; Tyrosine-tRNA Ligase
PubMed: 20159998
DOI: 10.1093/nar/gkq080 -
The Journal of Cell Biology Jul 1980A method has been developed to deliver an idoinating system into the confines of the phagolysosome, allowing us to study the nature of the phagolysosomal membrane....
A method has been developed to deliver an idoinating system into the confines of the phagolysosome, allowing us to study the nature of the phagolysosomal membrane. Lactoperoxidase (LPO) is covalently coupled to carboxylated latex spheres (LPO-latex) in a stable, enzymatically active form. The addition of LPO-latex to cultured macrophages leads to their rapid attachment, ingestion, and enclosure in a plasma membrane-derived phagocytic vacuole. These organelles rapidly fuse with preexisting lysosomes and are converted to phagolysosomes (PL) that demonstrates both acid phosphatase and lactoperoxidase activities. The exposure of LPO-latex containing cells to 125I- and an extracellular peroxide-generating system, glucose oxidase-glucose, at 4 degrees C leads to incorporation of label into TCA-precipitable material. The incorporated cel-associated label was present as monoiodotyrosine, and negligible amounts were found in lipids. Cell viability remained > 99%. Autoradiography at both the light and EM level revealed that > 97% of the cells were labeled, and quantitative analysis demonstrated the localization of grains to LPO-latex containing PL. PL were separated on sucrose gradients, and their radiolabel was confined almost exclusively to the membrane rather than soluble contents. SDS-polyacrylamide gel electrophoretic analysis of the peptides iodinated from within PL demonstrated at least 24 species with molecular weights ranging from 12,000 to 250,000. A very similar group of proteins was identified on the plasma membrane (PM) after surface iodination, and on latex phagosomes derived from iodinated PM. No novel proteins were detected in PL, either immediately after phagosome-lysosome fusion or after 1 h of intracytoplasmic residence. We conclude that the membrane proteins accessible to LPO-catalyzed iodination on the luminal surface of the PL and on the external face of the PM are similar, if not identical.
Topics: Animals; Cell Fractionation; Female; Intracellular Membranes; Iodoproteins; Lactoperoxidase; Lysosomes; Macrophages; Membrane Lipids; Membrane Proteins; Mice; Organoids; Phagocytosis; Vacuoles
PubMed: 7419579
DOI: 10.1083/jcb.86.1.292 -
The Journal of General Physiology Nov 1959When I(131)-albumin is given intravenously to rabbits, the radioactive breakdown products that are released into the plasma and urine can be extracted into acetone....
When I(131)-albumin is given intravenously to rabbits, the radioactive breakdown products that are released into the plasma and urine can be extracted into acetone. Paper chromatography and paper electrophoresis show that about 80 per cent of these are I(131)-iodide and the remainder are organic I(131)-iodine compounds. When I(131)-iodide is given to rabbits taking iodide in their drinking water, the radioactivity is quantitatively excreted, without being accumulated in the tissues and without becoming attached to the plasma proteins. The rate of excretion can be defined by a first order rate process with a rate constant, a, ranging between 1 and 3day(-1). The organic I(131)-iodine compounds liberated during the metabolism of I(131)-albumin can be closely matched by a mixture of the organic I(131)-iodine compounds liberated during the metabolism of I(131)-monoiodotyrosine, I(131)-diiodotyrosine, and the amino acids released by digestion from I(131)-albumin. These organic I(131)-iodine compounds are not accumulated in the body and their radioactivity does not become attached to the plasma proteins. Their radioactivity is excreted as fast or faster than that of I(131)-iodide, and, to a satisfactory approximation, the same equations describing the excretion of I(131)-iodide with the same constants may be used for describing the excretion of the organic I(131)-iodine. These results permit improved estimates of the distribution and catabolism of I(131)-albumin.
Topics: Animals; Biological Transport; Iodine; Iodine Radioisotopes; Rabbits; Serum Albumin
PubMed: 13847673
DOI: 10.1085/jgp.43.2.397 -
Structure (London, England : 1993) Mar 2009We developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-L-tyrosine at desired sites, and we used this system for structure... (Comparative Study)
Comparative Study
We developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-L-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-L-tyrosine. The 1.7 A crystal structure of the engineered variant, iodoTyrRS-mj, bound with 3-iodo-L-tyrosine revealed the structural basis underlying the strict specificity for this nonnatural substrate; the iodine moiety makes van der Waals contacts with 5 residues at the binding pocket. E. coli cells expressing iodoTyrRS-mj and the suppressor tRNA were used to incorporate 3-iodo-L-tyrosine site specifically into the ribosomal protein N-acetyltransferase from Thermus thermophilus. The crystal structure of this enzyme with iodotyrosine was determined at 1.8 and 2.2 Angstroms resolutions by SAD phasing at CuK alpha and CrK alpha wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation.
Topics: Acetyltransferases; Arylamine N-Acetyltransferase; Bacterial Proteins; Crystallography, X-Ray; Escherichia coli; Escherichia coli Proteins; Methanococcaceae; Monoiodotyrosine; Protein Engineering; Structure-Activity Relationship; Thermus thermophilus
PubMed: 19278648
DOI: 10.1016/j.str.2009.01.008