Low Iodide Concentrations Research Articles

Purification and properties of glutamine synthetase from liver of Squalus acanthias

Ammonia assimilation for urea synthesis by liver mitochondria in marine elasmobranchs involves, initially, formation of glutamine which is subsequently utilized for mitochondrial carbamoyl phosphate synthesis [ P. M. Anderson and C. A. Casey (1984) J. Biol. Chem. 259, 456–462 ]. The purpose of this study was to determine if the glutamine synthetase catalyzing this first step in urea synthesis has properties uniquely related to this function. Glutamine synthetase has been highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme has a molecular weight of approximately 400,000 in the presence of Mg 2+, MgATP, and l-glutamate, but dissociates reversibly to a species with a molecular weight of approximately 200,000 in the absence of MgATP and l-glutamate. Association with the glutamine- and acetylglutamate-dependent carbamoyl phosphate synthetase, also located in the mitochondria, could not be demonstrated. The subunit molecular weight is approximately 46,000. The pH optimum of the biosynthesis reaction is 7.1–7.4. The purified enzyme is stabilized by MgATP and glutamate and by ethylene glycol, and is activated by 5–10% ethylene glycol. The apparent K m values for MgATP, l-glutamate, and ammonia (NH 4 +-NH 3) are 0.7, 11.0, and 0.015 m m, respectively. Mg 2+ in excess of that required to complex ATP as MgATP is required for maximal activity; Mn 2+ cannot replace Mg 2+. The enzyme is activated by low concentrations of chloride, bromide, or iodide; this effect appears to be related to decreases in the apparent K m for glutamate. The enzyme is inhibited by physiological concentrations of urea, but is not significantly affected by physiological concentrations of trimethylamine- N-oxide. Except for activation by halogen anions and the very low apparent K m for ammonia, this elasmobranch glutamine synthetase has properties similar to those reported for mammalian and avian glutamine synthetases. The very low apparent K m for ammonia may be specifically related to the unique role of this glutamine synthetase in mitochondrial assimilation of ammonia for urea synthesis.

Oxidant membrane injury by the neutrophil myeloperoxidase system. I. Characterization of a liposome model and injury by myeloperoxidase, hydrogen peroxide, and halides.

Neutrophils and other phagocytes can injure cells by means of oxygen-dependent mechanisms, particularly the myeloperoxidase (MPO)-H2O2-halide system. The extent of such damage depends in part on the antioxidant defenses of the target cell. To facilitate the study of this phenomenon, we developed a model system in which we employed liposomes as targets for the myeloperoxidase system. The most useful species of liposomes employed 51Cr as the aqueous space marker and phosphatidyl choline with or without dicetyl phosphate and cholesterol as the structural lipid. Marker entrapment was established on the basis of 1) resolution of free from lipid-associated 51Cr by gel exclusion chromatography, 2) latency of 51Cr on rechromatography of detergent-treated liposomes, and 3) a correlation between entrapment and surface charge density. Exposure of liposomes to the complete MPO system resulted in release of 50 to 75% of the entrapped 51Cr. Release was abrogated by omission of myeloperoxidase or H2O2, heating of MPO, or addition of azide, cyanide, or catalase. Reagent H2O2 could be replaced by glucose plus glucose oxidase. Kinetic studies indicated a rapid process, lysis reaching half-maximal levels in less than 2 min. The addition of cyanide at various times interrupted lysis at once, indicating a requirement for ongoing myeloperoxidase-dependent reactions. Liposome disruption by the MPO system was pH dependent, increasing dramatically as pH was decreased from neutrality to 6.0. In the absence of halides, no lysis was observed. Maximum lysis was found with chloride at 10 to 100 mM, although at 1 mM concentrations, iodide, bromide, and thiocyanate were more active than chloride. Fluoride was inactive. Antagonism between halide species was demonstrated in that low concentrations of iodide or bromide inhibited the effect of optimal concentrations of chloride. Using 125I, we found that exposure of liposomes to the MPO system resulted in an association between iodide and liposomes; moreover, there was a close correspondence between this phenomenon and 51Cr release, suggesting that halogenation may be one mechanism of injury. These studies establish the usefulness of the liposome as a model of oxidant injury by a physiologically relevant system. They bear a striking parallel to work being done on MPO-mediated injury to eukaryotic and prokaryotic cells. By using this simplified model system, it should be possible to explore a number of determinants of target cell injury at a biochemical and molecular level.

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.

Optical and optically detected magnetic resonance studies of AgBr:I−

Low-temperature emission and optically detected magnetic resonance (ODMR) spectra of AgBr containing various amounts of iodide have been obtained. The ODMR spectrum at low iodide concentrations (1-100 ppm) is assigned to transitions of the iodine-bound exciton. Time and wavelength evolution of the emission from high-iodide-concentration (\ensuremath{\sim}3 at.%) samples provides evidence for donor-acceptor recombination. ODMR spectra of high-iodide-concentration samples suggest an iodide-related hole trap.

Iodide-dependent catalatic activity of thyroid peroxidase and lactoperoxidase

Thyroid peroxidase (TPO) and lactoperoxidase (LPO) display significant catalatic activity at pH 7.0 in the presence of low concentrations of iodide, based both on measurements of H 2O 2 disappearance and O 2 evolution. In the absence of iodide only minor catalatic activity was detected. The stimulatory effect of iodide could not be explained by protection of the enzymes against inactivation by H 2O 2. A mechanism is suggested involving an enzyme-hypoiodite comolex as an intermediate.

Regulation of Enzymatic Iodothyronine Synthesis in Thyroglobulin by Low Concentrations of Iodide

Evidence is presented that free iodide in the reaction medium during iodination of thyroglobulin by lactoperoxidase or thyroidperoxidase in vitro and a limiting H2O2 supply actively regulates the efficiency of the simultaneously occurring synthesis of iodothyronines. Our arguments are as follows. 1 For any level of iodination achieved in vitro, the amount of iodine incorporated in newly formed iodothyronine residues (but not that of the overall iodination) was linearly and inversely correlated to the logarithm of free iodide remaining in the incubation medium. This finding holds true for iodide concentrations in the μM range, regardless of the native iodine content of a 19-S thyroglobulin sample, of the concentration of the enzymes and of the incubation time. 2 When thyroglobulins, iodinated either in the absence or in the presence of excess iodide, are compared the latter contained (for similar degrees of iodination) more newly synthesized diiodotyrosine residues but fewer iodothyronine residues, although over 50 % of these diiodotyrosine residues were hormonogenic. 3 An additional important increase in hormone synthesis was obtained by extending the reaction time beyond the point were all iodide was organified. The latter phenomenon is but another demonstration that coupling always lags behind iodination. 4 The lag between the onset of iodination and that of coupling is a function of the amount of newly organified iodine. Iodothyronine synthesis starts as soon as 8–10 atoms of iodine are incorporated per molecule of thyroglobulin. It is suggested that the inhibition of the coupling reaction by inorganic iodide in vitro may well have a physiological significance insofar as it could be one of several autoregulatory mechanisms in the thyroid gland preventing excessive intrafollicular storage of iodothyronines in response to an increased iodide supply.

InP and CdS Photoanodes in Concentrated Aqueous Iodide Electrolytes

Conditions for the efficient photo‐oxidation of iodide for both and are demonstrated by theoretical collection properties in rotating ring‐disk electrode configurations. A specific surface interaction promoting hole transfer kinetics is evidenced by open‐circuit potential shifts of illuminated disks with iodide concentration and nearly quantitative disk iodine production at low iodide concentrations. For , however, high concentrations of and control of hydrogen ion levels are both needed to maintain a thin, charge conducting semiconductor‐oxide film and adequate kinetics for solution iodide oxidation. Acidified concentrated iodide is the first electrolyte with an photoanode to handle solar current densities.

39] Iodination of rhodopsin and transmembrane topology

This chapter discusses the iodination of rhodopsin and transmembrane topology. In the presence of H 2 O 2 and NaI, lactoperoxidase catalyzes the iodination of accessible tyrosyl residues of proteins. The reaction at neutral pH and low iodide concentration proceeds through an enzyme-substrate complex that transfers the iodine atom directly onto the protein. As most intact cells or sealed membrane vesicles are impermeable to lactoperoxidase, the enzymatic iodination technique is ideally suited for probing the spatial arrangement of proteins in membranes. Several approaches have been developed to identify transmembrane proteins in membranes using this technique. These include iodination of both the exterior and interior surfaces of large sealed vesicles, proteolytic digestion and iodination of “inside-out” and “right-side-out” vesicles, and sequential proteolysis and iodination of reconstituted membrane vesicles containing symmetrically oriented proteins. Rhodopsin is asymmetrically oriented in the native rod outer segment disk membrane. Several sites on the portion of the rhodopsin polypeptide chain exposed to the cytoplasm are accessible to cleavage by papain. As the vectorial orientation of rhodopsin is not preserved after the reconstitution, the percent of rhodopsin molecules in the reconstituted membrane vesicles that is susceptible to papain proteolysis is expected to be different.

Intramolecular catalysis in the enolisation of β-piperidinopropiophenone and its methiodide derivative

Measurements have been made on the rates of iodination of β-piperidinopropiophenone and its methiodide derivative, and on the rates of bromination of β-piperidinopropiophenone. The rates are independent of halogen concentration, except at low pH and high iodide concentrations when the iodination reactions are reversible, and represent the rate of ionisation or enolisation of the substrates. The reactions are base-catalysed and catalytic constants for a number of bases have been determined. The rates of ionisation of the protonated and N-methylated aminoketones are closely similar, and are up to 4 000 times larger than those of corresponding reactions of the neutral aminoketone and acetophenone. The close similarity between the reactivities of the protonated and N-methylated aminoketone suggests that the effect is due to an intramolecular electrostatic stabilisation of the negative charge developing on the carbonyl oxygen in the transition state. The results are discussed in relation to those of earlier studies on related systems.

Isolation and surface labeling of murine polymorphonuclear neutrophils

Methods for the induction of an exudate of polymorphonuclear neutrophilic leukocytes (PMN) in the peritoneal cavity of C57BL, BALB/c, SJL and CBA mice were analysed. Peritoneal exudates in male mice were highly enriched for PMN (80-90%) three hours after a single injection of calcium caseinate whereas eosinophils comprised less than 1% of the exudate population. Female mice were a less satisfactory source of PMN because the proportion of eosinophilis in the exudate was variable. Purification of PMN from peritoneal exudate cells was performed on the basis of light scattering using a Becton-Dickinson cell sorter or by density gradient centrifugation with graded polyvinylpyrrolidone-coated silica particles (Percoll). Both techniques yielded approximately 97% pure PMN preparations. Electrophoretic analysis of the PMN proteins revealed an abundance of lactoferrin and actin, but several other proteins were also present in high concentrations. Proteolytic degradation of several high molecular weight proteins (greater than 90,000) was prevented by the addition of phenylmethylsulphonyl fluoride (PMSF) and ethylene diamine tetracetic acid (EDTA). Surface iodination, using diphenyl, tetrachloroglycouril (IODO-DEN), indicated that there were six tyrosine-containing proteins present on the external cell membrane. The apparent molecular weights of these surface proteins ranged from 185,000 to 90,000 and the major 125I-labeled protein had an apparent molecular weight of 90,000. Neither actin nor lactoferrin was labeled with 125I unless cell viability was lost during the iodination procedure. Standard conditions for labeling the cell surface only, required low iodide and IODO-GEN concentrations. Biosynthetic labeling of PMN using S-methionine increased the sensitivity of detection for most of the proteins, but some of the granule storage proteins (such as lactoferrin) were not effectively labeled within three hours.

Behaviour of iodide-selective electrodes at low concentrations of iodide

The influence of adsorption of iodide ions and redox processes on the response mechanism of solid-state iodide-selectives electrode is discussed. Such effects can cause non-Nernstian calibration curves for iodide concentrations below 10 -5 M. A semi-empirical equation is given which explains the electrode response under the conditions normally applied.

Tryptophan distribution in yeast hexokinase isoenzyme B

Titrations of the quenching of the tryptophan fluorescence of yeast hexokinase isoenzyme B by Cs +, I − and glucose as quenchers, singly and in various combinations, have been performed at pH 5.5, 8.3, and 10.1 at 20°C. The iodide and glucose titrations at pH 8.3 indicated that the four tryptophan residues of the monomer subunit can be classified as: (1) a highly accessible surface tryptophan (Trp-S 1); (2) a surface tryptophan (Trp-S 2) with restricted accessibility (possibly in a crevice); (3) a glucose-quenchable cleft tryptophan (Trp-G); and (4) a ‘buried’ tryptophan (Trp-B) in the hydrophobic interior. The Stern-Volmer constants, k 1, calculated for iodide quenching of the two surface residues at pH 8.3 are 22 and 3.3 M −1. The k 1 value of Trp-G at this pH is iodide dependent, increasing from 1.2 to 1.4 M −1 as the iodide concentration increases from 0.1 to 0.65 M, probably because of iodide-induced alteration of the cleft structure. An iodide concentration above 0.65 M quenches Trp-B detectably, demonstrating that a high iodide concentration produces a large structural change in the isoenzyme. The percentages of the total 350 nm emission attributable to the individual tryptophan residues at pH 8.3 are 9, 22, 28, and 41%, for Trp-S 1, Trp-S 2, Trp-G, and Trp-B, respectively. These results agree qualitatively with previous results deduced from the wavelength dependence of glucose quenching. I − and glucose bind non-competitively to produce a synergistic enhancement of the quenching of the cleft tryptophan fluorescence. At pH 5.5 even a low iodide concentration causes some quenching of Trp-B, especially in presence of saturating glucose. Glucose increases the iodide quenchability at this pH. At pH 10.1 glucose does not quench the tryptophan emission, and it does not influence the iodide-quenching action. However, at this high pH the k 1 of Trp-S 1 is reduced to the point where it is not resolvable from the k I values of Trp-S 2 and Trp-G, even though the sum of the fractional fluorescence contributions of these three residues is the same as at pH 8.3.

Chloride/bicarbonate exchange in human erythrocytes.

1. The exchange of chloride and bicarbonate across the human erythrocyte membrane has been followed by measuring the changes in extracellular pH which occur when chloride-rich erythrocytes are added to chloride-free media containing varying concentrations of bicarbonate and carbonic anhydrase. 2. The dependence of the rate of chloride/bicarbonate exchange on the extracellular concentration of bicarbonate was consistent with the existence of a saturable membrane anion transporter exhibiting Michaelis--Menten kinetics. In a medium containing sodium gluconate buffered to pH 7.0 with imidazole--malate the Km for bicarbonate activation of transport was 0.39 (+/- 0.03) mM and the Vmax was 2033 (+/- 80 m-mole anions exchanged/3 X 10(13) cells. min, at 10 degrees C. 3. Chloride/bicarbonate exchange was temperature-dependent with an Arrhenius activation energy of 19.4 kcal/mole in the temperature range 2--10 degrees C. 4. Exchange of intracellular chloride for extracellular bicarbonate was inhibited by the presence of extracellular halides. Inhibition by chloride, bromide and fluoride was competitive and the affinity of the transport system decreased in the order HCO-3 greater than Cl- greater than Br- greater than F-. The kinetics of inhibition by iodide were complex, but inhibitory effects of low concentrations of iodide were less than those of chloride and bromide.

The influence of Ca2+ ions on the difference absorption spectra of phosphorylase kinase.

A low iodide concentration causes a positive difference band in the 250-260 mm UV range. The effect of Ca2+ changes the direction of difference spectra. The concentration dependence is explained by multistage interaction. The addition of EDTA the solution restores the positive band.

A mechanism and an evaluation of surface specific iodination by the chloramine-T procedure

Both internal and external proteins in vesicular stomatitis virus were labeled when intact virions were iodinated with 50 μ m iodide; however, only the surface proteins were labeled when the same procedure was carried out at low iodide concentrations (below 0.5 μ m). This result together with similar observations reported earlier with another enveloped virus, Rous-associated virus-61 (RAV-61), suggest that viral envelopes provide a barrier to iodination by chloramine-T at low, but not at high, iodide concentrations. By monitoring the permeability of the RAV-61 envelope to successive iodinations and to iodination in the presence of chaotropic thiocyanate ions, it was shown that the permeability of the viral envelope was not altered at the higher concentrations of iodide. Further results support the hypothesis that iodination mediated by chloramine-T inolves two different iodinating species: (a) a membrane impermeable one, possibly “iodamine-T,” which predominates at low iodide concentrations, and (b) a membrane permeable species, possibly molecular iodine, which predominates at high concentrations of iodide. These results reinforce the proposal that the chloramine-T procedure is a useful method for specifically labeling surface proteins of lipid-enveloped structures.

On the Use of Chloramine-T to Iodinate Specifically the Surface Proteins of Intact Enveloped Viruses

Rous-associated virus-6I was used as a model for studying the labelling specificity of the chloramine-T iodination procedure when applied to intact enveloped viruses. The specificity of labelling depended markedly on the concentration of iodide in the reaction mixture. At low concentrations of iodide (below 0-5 muM) only the surface proteins and lipid of intact virions were iodinated; there was no detectable labelling of internal proteins. At 10 muM-iodide, however, both internal and external proteins were iodinated; moreover there was a marked change in the reactivity of the surface proteins. It appears that the lipid envelope provides an effective barrier to the iodinating complex generated at low, but not at high, concentrations of iodide. These and other observations suggest that the chloramine-T procedure has a previously unrecognized potential for specifically labelling the surface proteins of lipid-enveloped structures.

The anodic dissolution of iron—VI. The behaviour of variously pre-treated iron electrodes and of cold-worked cobalt electrodes in moderately acid solutions containing iodide or carbon monoxide

In strongly acid, iodide containing solutions the anodic Tafel diagram of iron is characterized by a restricted. Tafel region at low anodic potentials followed by a very steep increase in current at more anodic potentials. It is shown that there is another Tafel region subsequent to the steep increase in current, and that the current level at the beginning of the steep increase is not related to the rate of diffusion of iodide ions to the metal surface. In a higher range of pH (2 to 3·5) it is demonstrated that a family of variously shaped Tafel diagrams (including the one obtained at low pH) may be obtained by successive changes in pH and iodide concentrations. These features are shown to be in agreement with a model of coupled, parallel reactions. Annealing or cold-working of the electrodes gives rise to different kinetic parameters for the reaction observed at low potentials, and minor changes in the parameters of the transition between the coupled, parallel reactions are also observed. The reaction order with respect to iodide is negative for the reaction at low potentials (I 1), but positive for the reaction observed at higher potentials (I 2). At low iodide concentrations the Tafel slope of the I 1 reaction is low (30 to 60 mV), and high (100 mV) for the I 2 reaction, but with increasingly high iodide concentrations this situation becomes reversed. For the I 1 reaction the reaction order with respect to pH is +2 for cold-worked iron and ca. + 1 for annealed iron, while for the I 2 reaction it is ca + 0·2 in both cases. A phenomenon similar to passivation is occasionally apparent at very high potentials. The restricted amount of data is consistent with further oxidation of a reaction intermediate (Fe(II) to Fe(III)), further uptake of hydroxyl ions and of iodide ions, but also, instead of real passivation, the possible operation of yet another dissolution process (I 3) at even higher potentials. Some less detailed experiments involving iron in solutions saturated with carbon monoxide (and cobalt in solutions containing iodide or carbon monoxide) gave results in general agreement with the above description for the case of iron in iodide solutions. It is concluded that the transition between the I 1 and the I 2 reaction follows a pattern, which has been shown to be a common feature for iron dissolution in the presence of several other electrolytes. The two individual processes follow much the same general behaviour revealed in other electrolytes, although extreme cases of behaviour are observed at high iodide concentrations. On the basis of these extreme behaviours a tentative mechanism for the I 2 reaction is suggested.

Rate of the Dushman Reaction in Iodic Acid at Low Iodide Concentration. Complexity of Iodic Acid

Abstract Three rate laws have been postulated for the Dushman reaction, 6H++5I−+IO3−=3H2O+3I2, with total reaction orders of four, five and six, at iodide ion concentrations below 10−8 M. New direct measurements of the rate and an examination of thermodynamic data for iodic acid solutions have eliminated some of the confusion. The complexity of iodic acid solutions, with and without added salt, makes it impossible to establish a single reaction order from the radioactive iodine exchange measurements. The new results are best expressed by a quasiempirical rate equation, −d[IO3−]⁄dt=8.59×104[H+]2(I−)[IO3−] mol l−1 min−1, which is closely approximated by −d[IO3−]⁄dt=γ±p4(H+)2(I−)(IO3−){k′+k″(HIO3)} mol l−1 min−1, with k′=2.83×105 mol−3 l3 min−1 and k″=6.60×106 mol−4 l4 min−1 for 25 °C. The second rate law is interpreted in terms of a reasonable mechanism that takes into account the thermodynamically established polymerization in iodic acid solutions.

Effect of iodide concentration on thyroxine synthesis catalysed by thyroid peroxidase.

Thyroglobulin with a low content of iodine atoms per molecule of protein was incubated with thyroid peroxidase and increasing iodine concentrations (0.01 to 4 mM). When iodide was in great excess (4 mM), I2 was the only product, protein iodination being markedly or completely inhibited. At low iodide concentrations (0.2 mM and below) no I2 was formed. A clear competition between these two possible reactions catalysed by thyroid peroxidase was therefore demonstrated. When iodide concentration was less than 0.5 mM, thyroid peroxidase catalysed only the iodination of thyroglobulin. Thyroid hormones began to accumulate when the iodination reaction leveled off. Different observations suggest that thyroxine was synthesised mainly by the coupling of two diiodotyrosine residues rather than by the iodination of 3,5,3′‐triiodothyronine residues. With very low iodide concentration, the synthesis of hormones began soon after the formation of hormonogenic residues. However the iodination reaction being faster than the coupling reaction, highly iodinated thyroglobulin could be obtained which did not contain thyroid hormones. From these results it appears that thyroid peroxidase is able to enter into at least three reactions: iodide peroxidation, tyrosine residues iodination and coupling of iodotyrosine residues to thyroid hormones. Surprisingly, this relative unspecificity of thyroid peroxidase may have some regulatory physiological significance.

Effect of Dietary Iodine on Thyroxine Secretion Rate of Lactating Cows

Supplemental iodine, 100mg daily, was added to low iodine diets of seven Holstein cows which were compared to paired controls. Treatment was from 6 to 8 weeks prepartum through 28 weeks of lactation. Plasma thyroxine (T4) and T4 disappearance rates after injection of 20mg T4 were determined four times at 2-month intervals. Iodine supplemented cows averaged higher than controls in plasma total iodine, protein-bound iodine, and T4. Average differences in T4 turnover rates were not significant, but overall T4 secretion or disappearance rates were higher for the supplemented cows in the last two periods. Plasma thyroxine secretion rates of both controls and iodine supplemented cows increased slightly with advancing lactation. Incipient iodine deficiency was indicated in the control cows at the end of the trial by low milk iodine and blood iodide concentrations. Milk yields of control cows averaged 733kg higher than yields of supplemented cows in 28 weeks. There was no indication that the iodine deficient diet caused hypothyroidism in high producing cows.