ESR signals of NADH and NADPH under illumination

ESR method was applied to investigate the formation of NADH and NADPH free radicals. It was shown that under the action of light (340 nm) in water and other solutions of these compounds a reaction occurred resulting in the formation of free radicals having the typical ESR spectrum. The analysis of the temperature dependence showed the light-induced ESR signals to be registered at −30°C to −120°C, the most intensive ones being observed at −50°C. It was concluded that the observed ESR signals belonged to the products of one-electron oxidation of the coenzymes-free radicals NAD^. and NADP^..     

Ion transport and permeability in the mouse egg

Sodium, potassium, and chloride unidirectional fluxes have been studied in the mature mouse egg. Their relationship to cell membrane potential and conductance has been investigated. Unidirectional Na efflux is composed of a ouabain sensitive component, presumably representing an active Na efflux, an external Na-dependent component and a diffusional component. The data indicate that the external Na-dependent component represents a Na:Na exchange mechanism. There also exists an ouabain-sensitive component of K influx. The stoichiometry of the ouabain-sensitive fluxes is approx. 2.7:1 (Na to K). From the diffusional components of Na and K flux, the membrane permeability to these cations has been estimated. P_Na and P_K are 1.2 × 10⁻⁷ cm sec⁻¹ and 0.8 × 10⁻⁷ cm sec⁻¹ respectively. These permeabilities, in conjunction with the internal exchangeable fractions of Na and K and the external concentrations, predict an egg membrane potential of −11 mV (inside negative). Microelectrode measurements yield an egg membrane potential of −14 ± 0.4 mV, indicating that the cell membrane potential is predominantly a result of the Na and K permeabilities and distributions. Internal exchangeable Cl is 67 ± 3 mM in standard medium, as determined from ³⁶Cl distribution. The chloride equilibrium potential is therefore −15 mV, which is not significantly different from the egg membrane potential. This suggests that Cl distributes passively across the egg membrane, reflecting the egg membrane potential. Hyperpolarization of the egg membrane potential to −27 ± 1.5 mV by reduction of external Na results in an exchangeable internal Cl of 49 ± 8 mM. This yields a Cl equilibrium potential of −24 mV, indicating that the Cl distribution shifts in the predicted manner upon a change in cell membrane potential. Tracer flux data indicate that Cl conductance comprises the bulk of the total membrane conductance with Na and K sharing the remainder in approximately equal amounts.     

Genetic and physiological evidence concerning the development of chemically sensitive voltage-dependent ionophores in L6 cells

The electrophysiological properties of a tissue culture muscle line, L₆, and a K⁺ resistant mutant (MK₁) derived from L₆ were determined to elucidate certain aspects of membrane differentiation and function. MK₁ was selected as a clone of myoblasts resistant to the toxic effects of 55 mM K⁺. The resting potentials of L₆ and MK₁ myoblasts and myotubes were K⁺ dependent and equal. The amplitudes of the action potentials were equal in normal medium, but 27.7 mM K⁺ interfered with or eliminated the ability of L₆ myotubes to produce action potentials. MK₁ myotubes produced nearly normal action potentials under these conditions. Thus, the K⁺ resistant myoblasts differentiate into myotubes which have an action potential generating mechanism much less sensitive to K⁺ than the normal mechanism. Also, both d-tubocurarine and α-bungarotoxin enhance the amplitude of the action potentials produced by L₆ myotubes in the presence of 27.7 mM K⁺; these compounds do not enhance the amplitude of the action potentials produced by MK₁ myotubes under the same conditions. It is proposed that as a consequence of differentiation a type of ionophore present in myoblasts becomes a voltage-dependent ionophore in myotubes. Furthermore, these voltage-dependent ionophores can be chemically sensitive.     

Dopamine-stimulated cyclic AMP and binding of [H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10⁻⁸ M and was half-maximal at 7.9±3.4·10⁻⁷M. The increase at 1·10⁻⁵ M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10⁻⁹ M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10⁻⁵ M dopamine was 2.3±0.9·10⁻⁶M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The K_d value for high affinity binding sites was 0.43±0.1·10⁻⁷M and 4.7±1.6·10⁻⁷M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10^/t-7M noradrenaline, 2·10^/t-7 M epinine, 4.1±1.8·10^/t-6M fluphenazine, 8.0±1.6·10^/t-6M haloperidol, 4.2±1.2·10⁻⁶Mcis-flupenthixol, 2.7±0.4·10⁻⁵Mtrans-flupenthixol, >1·10⁻⁵M apomorphine, sulpiride, naloxone and isoproterenol.     

Permeability of bilayer lipid membranes for superoxide (O2) radicals

Egg yolk phosphatidylcholine monolamellar liposomes (1000 Å in diameter) loaded with cytochrome c were placed into an external solution, in which superoxide radicals, O₂⁻, were generated by a xanthine-xanthine oxidase system. The penetration of the superoxide radicals across the liposomal membrane was detected by cytochrome c reduction in the inner liposome compartment. The effects of modifiers and temperature on this process were studied. The permeability of liposomal membrane for O₂⁻(P′_O2⁻ = (7.6 ± 0.3) · 10^-8 cm/s), or HO₂^• (P′_HO2⁻ = 4.9 · 10^-4 cm/s) were determined. The effect of the transmembrane electric potential (K⁺ concentration gradient, valinomycin) on the permeability of liposomal membranes for O₂⁻ were investigated. It was found that O₂⁻ can penetrate across liposomal membrane in an uncharged form. The feasibility of penetration of superoxide radicals through liposomal membrane, predominantly via anionic channels, was demonstrated by the use of an intramolecular cholesterol-amphotericin B complex.     

Reactivity of hypotaurine and cysteine sulfinic acid toward carbonate radical anion and nitrogen dioxide as explored by the peroxidase activity of Cu,Zn superoxide dismutase and by pulse radiolysis

Abstract

Hypotaurine and cysteine sulfinic acid are known to be readily oxidized to the respective sulfonates, taurine and cysteic acid, by several oxidative agents that may be present in biological systems. In this work, the relevance of both the carbonate anion and nitrogen dioxide radicals in the oxidation of hypotaurine and cysteine sulfinic acid has been explored by the peroxidase activity of Cu,Zn superoxide dismutase (SOD) and by pulse radiolysis. The extent of sulfinate oxidation induced by the system SOD/H₂O₂ in the presence of bicarbonate (CO₃^?– generation), or nitrite (^?NO₂ generation) has been evaluated. Hypotaurine is efficiently oxidized by the carbonate radical anion generated by the peroxidase activity of Cu,Zn SOD. Pulse radiolysis studies have shown that the carbonate radical anion reacts with hypotaurine more rapidly (k = 1.1 × 10⁹ M^?1s^?1) than nitrogen dioxide (k = 1.6 × 10⁷ M^?1s^?1). Regarding cysteine sulfinic acid, it is less reactive with the carbonate radical anion (k = 5.5 × 10⁷ M^?1s^?1) than hypotaurine. It has also been observed that the one-electron transfer oxidation of both sulfinates by the radicals is accompanied by the generation of transient sulfonyl radicals (RSO₂^?). Considering that the carbonate radical anion could be formed in vivo at high level from bicarbonate, this radical can be included in the oxidants capable of performing the last metabolic step of taurine biosynthesis. Moreover, the protective effect exerted by hypotaurine and cysteine sulfinate on the carbonate radical anion-mediated tyrosine dimerization indicates that both sulfinates have scavenging activity towards the carbonate radical anion. However, the formation of transient reactive intermediates during sulfinate oxidation by carbonate anion and nitrogen dioxide radical may at the same time promote oxidative reactions.     

Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity

The free radical scavenging properties and possible antioxidant activity of folic acid are reported. Pulse radiolysis technique is employed to study the one-electron oxidation of folic acid in homogeneous aqueous solution. The radicals used for this study are CCl₃O₂^•, N₃^•, SO₄^•−, Br₂^•−, √OH, and O^•−. All these radicals react with folic acid under ambient condition at an almost diffusion-controlled rate producing two types of transients. The first transient absorption maximum is around 430 nm, which decays, and a simultaneous growth at around 390 nm is observed. Considering the chemical structure of folic acid, the absorption maximum at 430 nm has been assigned to a phenoxyl radical. The latter one is proposed to be a delocalized molecular radical. A permanent product has been observed in the oxidation of folic acid with CCl₃O₂^• and N₃^• radicals, with a broad absorption band around 370–400 nm. The bimolecular rate constants for all the radical-induced oxidation reactions of folic acid have been measured. Folic acid is seen to scavenge these radicals very efficiently. In the reaction of thiyl radicals with folic acid, it has been observed that folic acid can not only scavenge thiyl radicals but can also repair these thiols at physiological pH. While carrying out the lipid peroxidation study, in spite of the fact that folic acid is considerably soluble in water, we observed a significant inhibition property in microsomal lipid peroxidation. A suitable mechanism for oxidation of folic acid and repair of thiyl radicals by folic acid has been proposed.     

Cytochrome c2 and reaction center of Rhodospeudomonas spheroides Ga. membranes. Extinction coefficients, content, half-reduction potentials, kinetics and electric field alterations

1. The reduced minus oxidized extinction coefficients (Δ^red-ox) of reaction center P605 when in the chromatophore is about 20% smaller than in the detergent-isolated state. Presumably the coupling of the reaction center protein to the antenna bacteriochlorophylls and carotenoids causes this hypochromism. The chromatophore values for P605 are 19.5 mM⁻¹ · cm⁻¹ with the spectrophotometer on single beam mode at 605 nm, and 29.8 mM⁻¹ · cm⁻¹ on dual wavelength mode set at 605 – 540 nm. Cytochrome c₂, which is not affected by detergent, has a Δ^red-ox value at 550-540 nm of 19.0 mM⁻¹ · cm⁻¹.2. The total bacteriochlorophyll to reaction center bacteriochlorophyll protein (P) ratio is about 100 : 1. The cytochrome c₂: reaction center protein ratio approaches 2. In current French press chromatophore preparations, about 70% of the reaction centers are each associated on a rapid kinetic basis with two cytochrome c₂ molecules (intact P-c₂ units). The remaining reaction center proteins are not associated with cytochrome c₂ on a kinetically viable basis and may be the result of damage incurred during mechanical rupture of the cells.3. The half-reduction potential of cytochrome c₂ in the isolated state is 345 mV. In the chromatophore, two electrochemical species of cytochrome c₂ are recognized. The majority has a value of approx. 295 mV and is identifiable with cytochrome c₂ in a reaction center protein-associated state (kinetically active, intact P-c₂ units); the remainder has an approx. 350 mV half-reduction potential and is probably cytochrome c₂ in the “free” or reaction center-dissociated state (possibly from damaged P-c₂ units). It appears that there is no exchange of cytochrome c₂ between the reaction center-associated and the reaction center-dissociated state.4. The half-reduction potential of cytochrome c₂ is pH independent (from pH 5 to 9) whether measured in the free state or when associated with the chromatophore membrane. This shows that a proton is not involved in the oxidation and reduction of cytochrome c₂ in the physiological pH range.5. The kinetics of the intact reaction center, P, and cytochrome c₂ units in chromatophores and whole cells of Rhodopseudomonas spheroides are described. The two cytochrome c₂ molecules which are associated with one P exhibit similar oxidation kinetics; both are biphasic. The fast phase is estimated to be 20–40 μs in half time. The second slower phase is variable depending on the ionic strength of the medium used for the preparation of the chromatophores; it varies from 0.3 to 8 ms.6. An equilibrium for cytochrome c₂ and the reaction center and/or the membrane is suggested. The two states of the equilibrium are described by a population of cytochrome c₂ functionally “close” to the P⁺, and a population functionally distant from the P⁺, which might be physically off the binding site, or orientated unfavorably to the P⁺. The former population is identified by the 20–40 μs oxidation rate; the latter variable and somewhat slower oxidation (0.3–8 ms) is that whose rate is governed by the diffusional processes of the equilibrium which brings the cytochrome to the close position.7. Carotenoid bandshifts are kinetically compatible (a) with the P oxidation which is too fast to measure, and (b) with the two phases of cytochrome c₂ oxidation. These are interpreted as arising from local electric field alterations occurring during the electron transfer events in the reaction center and cytochrome c₂.     

A sensitive and precise isotopic assay of ATPase activity

A manual ATPase assay which measures the release of ³²P_i from [γ-³²P]ATP is described. Sodium dodecyl sulfate is used to terminate the enzyme reaction and extraction of the phophomolybdate complex into xylene: isobutanol is used to separate ³²P_i from [γ-³²P]ATP for quantitation by scintillation counting. The three-step assay is rapid (75–90 samples/h) and minimizes hydrolysis of ATP due to exposure to acidie conditions. The extraction procedure separates 10⁻¹⁵ to 10⁻⁷ mol of ³²P_i from aqueous solution with an efficiency of 100,7 ± 0.62%. Less than 1% of unhydrolyzed [γ-³²P]ATP is extracted. Extraction efficiency is not affected by protein or salts commonly present in enzyme incubation mixtures. Results obtained with this assay are precise, with an intraassay coefficient of variation of 0.6% and an interassay coefficient of variation of 1.8%. The results are comparable to results obtained with a spectrophotometric assay, with a correlation coefficient of 0,996, though assay performance and sensitivity are greatly improved with the isotopic assay.     

A chemical and spectral characterization of the extrapallial fluid of Mytilus edulis

The extrapallial fluid of Mytilus edulis is analyzed for inorganic and organic constituents. Disc gel electrophoresis reveals the presence of at least five protein components. The insoluble fraction is sulfated carbohydrate material while the ultrafiltrable portion contains free amino acids. The concentration of calclium is 9.8 ± 0.4 m , 84.7% of which is complexed; 74.3% of the calcium is bound to small molecules, 9.2% to insoluble carbohydrate, 0.88% to soluble macromolecular components, and 15.3% is free. The data suggest that CaCO₃ deposition may be controlled by complexation of Ca²⁺ by small chelates produced by the animal. EPR measurements indicate that the majority of manganese present in the fluid is also bound to small molecules. Titrations of the native fluid with Mn²⁺ and H⁺ establish that the chelating capacity of the fluid for Mn²⁺ is nominally 10⁻⁴ . Mn²⁺ forms 1:1 complexes in the fluid with ligands having an average pK_a′ value of 5.2     

Quantification of hydrogen peroxide and glucose using 3-methyl-2-benzothiazolinonehydrazone hydrochloride with 10,11-dihydro-5H-benz(b,f)azepine as chromogenic probe

A sensitive, selective, and rapid enzymatic method is proposed for the quantification of hydrogen peroxide (H₂O₂) using 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) and 10,11-dihydro-5H-benz(b,f)azepine (DBZ) as chromogenic cosubstrates catalyzed by horseradish peroxidase (HRP) enzyme. MBTH traps free radical released during oxidation of H₂O₂ by HRP and gets oxidized to electrophilic cation, which couples with DBZ to give an intense blue-colored product with maximum absorbance at 620 nm. The linear response for H₂O₂ is found between 5 × 10⁻⁶ and 45 × 10⁻⁶ mol L⁻¹ at pH 4.0 and a temperature of 25 °C. Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 0.415 × 10⁶ M⁻¹ min⁻¹ and 9.81 × 10⁻⁴ min⁻¹, respectively. The catalytic constant (k_cat) and specificity constant (k_cat/K_m) at saturated concentration of the cosubstrates were 163.2 min⁻¹ and 4.156 × 10⁶ L mol⁻¹ min⁻¹, respectively. This method can be incorporated into biochemical analysis where H₂O₂ undergoes catalytic oxidation by oxidase. Its applicability in the biological samples was tested for glucose quantification in human serum.     

Rate of RNA synthesis as a function of temperature in Chinese hamster lung fibroblasts (V-79)

The rates of intracellular RNA synthesis at various temperatures between 33 and 41 °C were determined in Chinese hamster lung fibroblasts by measuring average amounts of [³H]uridine incorporated per cell per unit of time. The energy of activation and Q₂₀ for intracellular RNA synthesis were calculated from the slopes of the relative rates of RNA synthesis in hamster fibroblasts vs time, plotted on Arrhenius coordinates. The incorporation of uridine into RNA is characterized by an energy of activation of 19 200 calories/mole and a Q₁₀ of 2.71. The absolute rates of RNA synthesis were determined at various temperatures, with values ranging from 1.55 to 0.60 × 10⁻¹⁵ g RNA/min/cell at 41 to 33 °C, respectively.     

Direct oxidation of boronates by peroxynitrite: Mechanism and implications in fluorescence imaging of peroxynitrite

In this study, we show that boronates, a class of synthetic organic compounds, react rapidly and stoichiometrically with peroxynitrite (ONOO⁻) to form stable hydroxy derivatives as major products. Using a stopped-flow kinetic technique, we measured the second-order rate constants for the reaction with ONOO⁻, hypochlorous acid (HOCl), and hydrogen peroxide (H₂O₂) and found that ONOO⁻ reacts with 4-acetylphenylboronic acid nearly a million times (k = 1.6 × 10⁶ M^− 1 s^− 1) faster than does H₂O₂ (k = 2.2 M^− 1 s^− 1) and over 200 times faster than does HOCl (k = 6.2 × 10³ M^− 1 s^− 1). Nitric oxide and superoxide together, but not alone, oxidized boronates to the same phenolic products. Similar reaction profiles were obtained with other boronates. Results from this study may be helpful in developing a novel class of fluorescent probes for the detection and imaging of ONOO⁻ formed in cellular and cell-free systems.     

Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking

These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C₁-BODIPY-C₁₂ in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis–Menten kinetics; the apparent efficiency (k_cat/K_T) of this process increases over 2-fold (2.1 × 10⁶–4.5 × 10⁶ s⁻¹ M⁻¹) upon adipocyte differentiation. The V_max values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 × 10⁶ s⁻¹ M⁻¹ versus 1.5 × 10⁶ s⁻¹ M⁻¹). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V_max values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The same cells had reduced efficiency for fatty acid transport (ranging from 0.82 × 10⁶ s⁻¹ M⁻¹ to 1.35 × 10⁶ s⁻¹ M⁻¹).     

One-electron reduction of selenomethionine oxide

Experimental evidence is provided that selenomethionine oxide (MetSeO) is more readily reducible than its sulfur analogue, methionine sulfoxide (MetSO). Pulse radiolysis experiments reveal an efficient reaction of MetSeO with one-electron reductants, such as e^-_aq (k = 1.2 × 10¹⁰M^-1s^-1), CO^·-₂ (k = 5.9 × 10⁸ M^-1s^-1) and (CH₃)₂) C^·OH (k = 3.5 × 10⁷M^-1s^-1), forming an intermediate selenium-nitrogen coupled zwitterionic radical with the positive charge at an intramolecularly formed Se_∴ N 2σ/1σ* three-electron bond, which is characterized by an optical absorption with λ_max at 375 nm, and a half-life of about 70 μs. The same transient is generated upon HO^· radical-induced one-electron oxidation of selenomethionine (MetSe). This radical thus constitutes the redox intermediate between the two oxidation states, MetSeO and MetSe. Time-resolved optical data further indicate sulfur-selenium interactions between the Se_∴ N transient and GSH. The Se_∴ N transient appears to play a key role in the reduction of selenomethionine oxide by glutathione.     

Determination of free radical reaction products and metabolites of salicylic acid using capillary electrophoresis and micellar electrokinetic chromatography

Hydroxylated radical products of salicylic acid are often used as a relative measurement in free radical research. Several analytical methods exist to determine the amount of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid. In this study we use capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) in order to determine these free radical products. The CZE experiment was optimized with a CZE simulation program in order to achieve an optimal pH. Calibration curves were recorded in the range 10⁻⁶–10⁻⁴ M and the detection limit was determined. For both CZE and MECC it was 2·10⁻⁷ M. Both methods resulted in a reproducible analysis of salicylate and its hydroxylated free radical products in 6 min.     

Photolysis of d-fructose in aqueous solution

In the 254-nm photolysis of aqueous solutions of d-fructose, only the openchain form, which is present to an extent of 0.8% in equilibrium with the cyclic forms, absorbs the light. A study of the products and their quantum yields reveals that the main, primary process is C--- bond cleavage α to the carbonyl group. In the absence of oxygen, the subsequent reactions of the resulting radicals are (a) loss of CO from the hydroxyalkylacyl radicals (estimated rate constant a 3 × 10⁶ s⁻¹); (b) consecutive elimination of two molecules of water from the tetritol radicals; and (c) disproportionation and combination reactions. A peculiar products is trans-4-hydroxy-2-butenal, whose precursor is formed from the tetritol radical through elimination of two molecules of water. This compound is a good radical-scavenger and during photolysis quickly attains a low steady-state concentration. One of the products derived from it is a 2,3-dideoxy-2,3-di-C-hydroxymethyltetrose. In the presence of oxygen, the CO elimination process is partly, and the water elimination reactions are fully, suppressed by the fast addition of oxygen to the acylalkyl and hydroxyalkyl radicals. The peroxyl radicals react through unimolecular elimination of HO₂ from α-hydroxyalkylperoxyl radicals and bimolecular dismutation with loss of O₂, accompanied by loss of CO₂ when hydroxyalkylacylperoxyl radicals are involved.     

Kinetics of oxidation of tyrosine by a model alkoxyl radical

Abstract

Oxidation of tyrosine moieties by radicals involved in lipid peroxidation is of current interest; while a rate constant has been reported for reaction of lipid peroxyl radicals with a tyrosine model, little is known about the reaction between tyrosine and alkoxyl radicals (also intermediates in the lipid peroxidation chain reaction). In this study, the reaction between a model alkoxyl radical, the tert-butoxyl radical and tyrosine was followed using steady-state and pulse radiolysis. Acetone, a product of the β-fragmentation of the tert-butoxyl radical, was measured; the yield was reduced by the presence of tyrosine in a concentration- and pH-dependent manner. From these data, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 6?±?1 × 10⁷ M^?1 s^?1 at pH 10. Tyrosine phenoxyl radicals were also monitored directly by kinetic spectrophotometry following generation of tert-butoxyl radicals by pulse radiolysis of solutions containing tyrosine. From the yield of tyrosyl radicals (measured before they decayed) as a function of tyrosine concentration, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 7?±?3 × 10⁷ M^?1 s^?1 at pH 10 (the reaction was not observable at pH 7). We conclude that reaction involves oxidation of tyrosine phenolate rather than undissociated phenol; since the pK_a of phenolic hydroxyl dissociation in tyrosine is ～ 10.3, this infers a much lower rate constant, about 3 × 10⁵ M^?1 s^?1, for the reaction between this alkoxyl radical and tyrosine at pH 7.4.     

The cytoprotective properties of prostaglandin E2 against the toxic effects of actinomycin C on embryonic neural retina cells

Prostaglandins (PGs) have been shown to cytoprotect various tissue types against the toxic effects of many chemicals. The mechanism of this protection is poorly understood, but the involvement of cAMP is often implied. Only one previous study examined nervous tissue and PG protection. The present study was designed to determine if PGE₂ affords cytoprotection to a more specific nervous tissue (embryonic neural retina) from the toxicity of actinomycin C (AMC) using a trypan blue exclusion assay. The lowest concentration of PGE₂ (2 × 10⁻⁵M) had no effect, but as the concentration increased (3 × 10⁻⁵M and 5 × 10⁻⁵M), PGE₂ did afford protection against AMC in a dose dependent fashion. Theophylline treated cells were not protected, suggesting that cAMP may not be the primary mechanism of protection.