Diazenedicarboxylic Acid Bis Research Articles

ATP synthase complex from beef heart mitochondria. Role of the thiol group of the 25-kDa subunit of Fo in the coupling mechanism between Fo and F1.

In order to assess the role of thiol groups in the Fo part of the ATP synthase in the coupling mechanism of ATP synthase, we have treated isolated Fo, extracted from beef heart Complex V with urea, with thiol reagents, primarily with diazenedicarboxylic acid bis-(dimethylamide) (diamide) but also with Cd2+ and N-ethylmaleimide. FoF1 ATP synthase was reconstituted by adding isolated F1 and the oligomycin-sensitivity-conferring-protein (OSCP) to Fo. The efficiency of reconstitution was assessed by determining the sensitivity to oligomycin of the ATP hydrolytic activity of the reconstituted enzyme. Contrary to Cd2+, incubation of diamide with Fo, before the addition of F1 and OSCP, induced a severe loss of oligomycin sensitivity, due to an inhibited binding of F1 to Fo. This effect was reversed by dithiothreitol. Conversely, if F1 and OSCP were added to Fo before diamide, no effect could be detected. These results show that F1 (and/or OSCP) protects Fo thiols from diamide and are substantiated by the finding that the oligomycin sensitivity of ATP hydrolysis activity of isolated Complex V was also unaltered by diamide. Gel electrophoresis of FoF1 ATP synthase, reconstituted with diamide-treated Fo, revealed that the loss of oligomycin sensitivity was directly correlated with diminution of band Fo 1 (or subunit b). Concomitantly a band appeared of approximately twice the molecular weight of subunit Fo 1. As this protein contains only 1 cysteine residue (Walker, J. E., Runswick, M. J., and Poulter, L. (1987) J. Mol. Biol. 197, 89-100), the effect of diamide is attributed to the formation of a disulfide bridge between two of these subunits. These results offer further evidence for the proposal, based on aminoacid sequence and structural analysis, that subunit Fo 1 of mammalian Fo is involved in the binding with F1 (Walker et al. (1987]. N-Ethylmaleimide affects oligomycin sensitivity to a lesser extent than diamide, suggesting that the mode of action of these reagents (and the structural changes induced in Fo) is different.

Radiosensitization, Thiol Oxidation, and Inhibition of DNA Repair by SR 4077

The mechanism of radiosensitization by diazenedicarboxylic acid bis(N),N-piperidide (SR 4077), a less toxic analog of diamide, was studied using Chinese hamster ovary cells. SR 4077 gave an average SER of 1.58 for postirradiation incubations of 0.5, 1.0, or 2.0 h. Intracellular GSH and protein thiols decreased rapidly following drug addition and GSSG increased. The GSH/GSSG ratio shifted to 1/1.6 after SR 4077 addition but returned to greater than 10/1 between 0.5 and 1.0 h. After 4 h, total intracellular GSH was only 58% of pretreatment level and extracellular GSSG increased. Protein thiols decreased to 18% of pretreatment values, recovered most rapidly between 0.5 and 1.0 h, and reached 87% of pretreatment level after 4 h. A decrease in DNA single-strand break repair as measured by alkaline filter elution rate over 0.5 h was seen, and the initial rate of repair was slower than in cells not treated with SR 4077. DNA double-strand break repair as measured by neutral filter elution rate was delayed during the first hour after irradiation when cells were treated with SR 4077. The times for maximum radiosensitization, GSH and protein thiol oxidation and recovery, and DNA strand break repair kinetics were closely linked. We propose that a protein thiol(s) required in repair processes was reversibly oxidized during SR 4077 treatment.

Formation of disulfides with diamide

Publisher Summary This chapter discusses the formation of disulfides with diamide. Several diazenecarbonyl derivatives were found to be active for the conversion of thiols to disulfides. The most convenient and chemically simple agent was diazenedicarboxylic acid bis(N,N-di-methylamide), now known by the trivial name, “diamide.” Diamide is a yellow, nonhygroscopic solid, easily soluble in both water and organic solvents, and rather stable toward hydrolysis. Diamide penetrates cell membranes within seconds and also reacts within the cell at a high rate (seconds to minutes) at physiological pH. Diamide is effective in the absence of oxygen, conditions under which metabolic activity is minimal. There is a simple stoichiometric relationship between the amount of agent added and the quantity of thiol reacted. Because the reaction of diamide with thiols has a low activation energy, thiol oxidation is fast at low temperatures. Reaction can be terminated through removal of diamide by washing of the cells or stopped instantaneously by the addition of acid. In most cases, diamide treatment does not cause any irreversible damage, and, after incubation of the cells with appropriate substrates at a suitable temperature, the original thiol status is recovered. Diamide treatment thus allows the study of cell functions altered by a perturbation in thiol status.

Radiosensitization by diamide analogs and arsenicals

Several analogs of the glutathione (GSH) oxidizing reagent diamide (diazenedicarboxylic acid bis[(N,N′-diethylamide)] were tested as radiosensitizers of aerobic cells. In general, radiosensitization correlates with the rate of reaction with cellular reducing agents and occurs only when the reductive capacity of the cell is exceeded. SR-4077, [diazenedicarboxylic acid bis(N,N-piperidide], is particularly suitable for mechanistic studies, because it is less cytotoxic than diamide, but is equally reactive toward cellular GSH. SR-4077 sensitizes CHO cells to X rays under aerobic conditions, even when the drug is added after irradiation. Radiosensitization is expressed both as a change in the exponential slope of the radiation cell survival curve and as a decrease in the shoulder of the survival curve. Phenylarsine oxide, a dithiol-binding reagent, sensitizes aerobic CHO cells to X rays by modification of the shoulder of the survival curve. The results are consistent with the hypothesis that the shoulder-modifying effect of GSH oxidants is caused by the loss of protein thiols, which might be involved in the repair of X ray-induced DNA damage.

Effect of thioloxidant diazene dicarboxylic acid bis-(N′-methylpiperazide) (DIP) on 45Ca 2+ net uptake into rat pancreatic islets

The effect of DIP (an oxidant of glutathione) on 45Ca 2+ net uptake induced by a variety of stimulators of insulin secretion was studied in rat pancreatic islets. In addition the effect of exogenous glutathione (GSH) on 45Ca 2+ net uptake in response to glucose was tested. DIP (0.1 mM) inhibited the increase of 45Ca 2+ net uptake in the presence of glucose (16.7 mM) and glyceraldehyde (10 mM). A similar inhibitory effect could be demonstrated, when 45Ca 2+ net uptake was enhanced by tolbutamide (100 μg/ml), glibenclamide (0.5 μ/ml), b-BCH (20 mM), 2-ketoisocaproate (20 mM), arginine (20 mM) in the presence of 3 mM glucose or by high extracellular potassium (20 mM). The increase of 45Ca 2+ net uptake stimulated by leucine (20 mM) plus glucose (3 mM) was further augmented by DIP. Exogenous GSH did not affect 45Ca 2+ net uptake in the presence of (5.6 – 16.7 mM) glucose. It is suggested that 45Ca 2+ net uptake of pancreatic islets depends on the redox state of islet thiols regardless of whether uptake is promoted via inhibition of potassium efflux (nutrients, sulfonylureas) or by high potassium and arginine. The voltage sensitive calcium-channel is the site of action of critical thiols. It is possible that these thiols are localized at the inner side of the plasma membrane.

Thiols in oxidative phosphorylation: inhibition and energy-potentiated uncoupling by monothiol and dithiol modifiers

Three apparently different modifications of submitochondrial particles (SMP) or ATP synthase preparations (complex V) inhibit oxidative phosphorylation and ATP-32Pi exchange activities, all of which are reversible by addition of mono- or dithiols. (a) Triphenyltin chloride inhibits ATP synthesis and hydrolysis without uncoupling. The inhibition by triphenyltin chloride is reversible by addition of beta-mercaptoethanol, dithiothreitol, or dihydrolipoamide. (b) Factor B is a water-soluble protein of Mr (11-12) X 10(3), contains a vicinal dithiol, and is required for energy transfer to and from F1-ATPase when tested with SMP-rendered factor B deficient by extraction with ammonia-ethylenediaminetetraacetic acid (EDTA) (AE-SMP). Treatment of factor B with mono- and dithiol modifiers, such as p-(chloromercuri)benzenesulfonate (PCMPS), Cd2+, or diazenedicarboxylic acid bis(dimethylamide) (diamide), inhibits factor B. This inhibition is reversed by addition to modified factor B of appropriate mono- and dithiol compounds. Preparations of AE-SMP are partially F1 deficient and partially uncoupled. The uncoupling can be repaired completely by addition of factor B or low levels of oligomycin, or to a large extent by addition of F1-ATPase + oligomycin sensitivity conferring protein. (c) SMP, AE-SMP, and complex V can be completely uncoupled by treatment at 30 degrees C with phenylarsine oxide, Cd2+, diamide, PCMPS, monobromobimane, and mono- and bifunctional maleimides. The uncoupling by these reagents is potentiated by membrane energization. Uncoupling by diamide is greater than or equal to 80% reversed by dihydrolipoamide or beta-mercaptoethanol, the former being much more potent. Dithiothreitol and dithioerythritol are poorly effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of rheological properties of human erythrocytes by crosslinking of membrane proteins

The crosslinking of membrane proteins of human erythrocytes by diamide (diazene dicarboxylic acid bis(N,N- dimethylamide )) was quantified by 4% polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. The relation between the crosslinking of membrane proteins and erythrocyte functions (rheological and oxygen transporting) was quantitatively examined. (i) The crosslinking of membrane protein was induced by diamide, without changing the shape and the contents of intracellular organic phosphates (adenylates and 2,3-diphosphoglycerate). The intensity of spectrin 2 in SDS-polyacrylamide gel electrophoresis decreased proportionally to diamide concentration. The percentage decrease in spectrin 2 (using band 3 as an internal standard) was the most appropriate indicator for crosslinking (‘% crosslinking’). (ii) The suspension viscosity of erythrocytes increased in proportion to the percentage of crosslinking, in the range of applied shear rates of 3.76–752 s −1. (iii) Erythrocyte deformability (measured by a high-shear rheoscope) was reduced by the crosslinking. The change was detectable even at 5% crosslinking. (iv) Rouleaux formation (measured by a television image analyzer combined with a low-shear rheoscope) was inhibited by the crosslinking. The inhibition was also sensitively detected at more than 5% crosslinking. (v) Hemoglobin in erythrocytes was chemically modified by higher dose of diamide (probably by the binding of diamide with sulfhydryl groups). Also the oxygen affinity of hemoglobin increased and the heme-heme interaction decreased. (vi) The reduction of the crosslinking of membrane proteins by dithiothreitol apparently reversed the intensity of spectrin bands in SDS-polyacrylamide gel electrophoresis and the erythrocyte functions (the suspension viscosity and the deformability), though not completely.

A possible role of intracellular and membrane thiols of rat pancreatic islets in calcium uptake and insulin release.

To distinguish between the significance of membrane thiols and intracellular thiols of rat pancreatic islets in calcium uptake and insulin secretion, lanthanum-nondisplaceable uptake of 45Ca and secretion of insulin were studied in the presence of the membrane-penetrating thiol oxidant diazene dicarboxylic acid bis-(N'-methylpiperazide) (DIP), on the one hand, and the corresponding nonpenetrating thiol oxidant salt of DIP (bis-N'-methyl iodide, i.e. DIP + 2), on the other hand. In contrast to DIP + 2, DIP inhibited glucose-induced (20 mM) uptake of 45Ca and insulin secretion. Furthermore, DIP + 2 stimulated 45Ca uptake and insulin secretion in the presence of low glucose (3 mM). DIP inhibited these effects of DIP + 2. 45Ca uptake and insulin release evoked by the ionophore A-23187 were not inhibited by DIP, however. The present data collected from isolated pancreatic islets have several implications. Firstly, the reduced state of the intracellular glutathione to GSSG ratio, which causes the superficial thiol to remain in the reduced state, constitutes a prerequisite for glucose-stimulated 45Ca uptake and insulin release. Secondly, direct oxidation of superficial thiols leads to 45Ca uptake and subsequent secretion of insulin. Finally, the redox state of the intracellular glutathione system does not appear to be of importance in the stimulus-secretion coupling steps following the uptake of calcium.

Parallel efflux of Ca2+ and Pi in energized rat liver mitochondria.

Addition of Ruthenium Red to energized rat liver mitochondria that have previously accumulated Ca2+ and phosphate from the external medium induces a parallel efflux of both these ions. Mersalyl or dithioerythritol, which decrease Ruthenium Red-insensitive Ca2+ efflux, also decrease phosphate efflux to the same extent. Conversely diazenedicarboxylic acid bis(NN-dimethylamide) (DDBA), which increases the Ruthenium Red-induced Ca2+ efflux concurrently increases phosphate release. Dithioerythritol and DDBA, reducing and oxidizing agents of thiol groups respectively, modify Ca2+ and Pi efflux without penetrating the mitochondrial inner membrane. Under all the adopted conditions the membrane potential is preserved. The release of resting respiration and the parallel efflux of Mg2+ and adenine nucleotides, events closely correlated to Ca2+ cycling, are equally prevented either by mersalyl, which inhibits phosphate transport, or dithioerythritol; DDBA has the opposite effect. These findings and the observation that suggest that Ca2+ and phosphate transport in energized liver mitochondria are closely related and dependent on the redox state of membrane-bound thiol groups.

Aggregation of intramembrane particles in erythrocyte membranes treated with diamide

Treatment of erythrocytes with diamide (diazene dicarboxylic acid bis-( N, N-dimethylamide) results in oxidation of sulfhydryl groups of the membrane, and cross-linking of membrane proteins into high molecular weight complexes. Concomitant freeze-etching studies show aggregation of intramembrane particles on the protoplasmic fracture face of erythrocyte ghost membranes treated with the oxidant. Furthermore, after a 3 h incubation of erythrocytes with 10 mM diamide at 37°C, cellular energy levels declined to about 70% of control values. The data suggest that disulfide cross-linking of the major membrane proteins releases the apparent physical occlusion of the band 3 proteins within the interstices of the cytoskeletal shell. This results in the translational mobility of band 3 proteins which is reflected ultra-structurally in the freeze-etch images.

Inhibition of D(--)-beta-hydroxybutyrate dehydrogenase by modifiers of disulfides, thiols, and vicinal dithiols.

D(--)-beta-hydroxybutyrate dehydrogenase of beef heart mitochondria catalyzes the reversible oxidation of D-(--)-beta-hydroxybutyrate to acetoacetate in the presence of NAD. Both the membrane-bound and the soluble forms of the enzyme are inhibited by modifiers of thiols [N-ethylmaleimide (NEM) and p-(chloromercuri)phenylsulfonate (pCMS)], vicinal dithiols [phenylarsine oxide and diazenedicarboxylic acid bis(dimethylamide) (diamide)], and disulfides (sulfite, sulfide, and cyanide). NAD and NADH, but not beta-hydroxybutyrate and acetoacetate, protect the enzyme against inhibition by NEM, pCMS, phenylarsine oxide, and diamide. As tested with NEM and diamide, the inhibitions caused by mono- and dithiol modifiers were pseudo first order, and the reaction order with respect to the concentration of either inhibitor was unity, thus indicating the modification of a single essential thiol and/or dithiol. Sulfite and sulfide inhibitions appeared to be competitive with respect to beta-hydroxybutyrate, with Ki values of 10-15 and about 240 microM, respectively. Sulfite inhibition was uncompetitive with respect to NAD, NADH, and acetoacetate. The above results have suggested the presence in D(--)-beta-hydroxybutyrate dehydrogenase of an essential thiol and/or a vicinal dithiol associated with the binding site(s) of NAD and NADH. The inhibition by sulfite, sulfide, and cyanide might be indicative of the presence of an essential disulfide or due to a ternary complex formation involving the enzyme, NAD, and the above nucleophiles.

Glutathione and the mitochondrial reduction of some diazenes

Mitochondria reduce diazenedicarboxylic acid bis(dimethylamide) (diamide) and the corresponding bis( N'-methylpiperazide) (DIP) but not the bis( N'-methyl iodide) salt (DIP 2+). Without added substrates, DIP, but not diamide or DIP 2+, depletes mitochondrial glutathione. The depletion is less in the presence of some Krebs acids, especially succinate, and these acids proportionately increase the rate of DIP reduction. The reduction is inhibited by antimycin, thiol reagents and uncouplers. The results suggest that DIP 2+ is impermeable, but that DIP enters mitochondria chiefly as a cation and at a more rapid rate than diamide.

The effect of diamide on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes

The effect of diamide (diazene dicarboxylic acid bis[ N, N′-dimethylamide) on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes was examined. In the absence of mitogenic lectins, 5 · 10 −3−1 · 10 −4 M diamide markedly increased intracellular cyclic AMP with variable effects at higher levels. In the presence of phytohemagglutinin or concanavalin A, 5 · 10 · M or higher diamide concentrations consistently decreased cyclic AMP levels, usually to control levels or below, while 1 · 10 −4−1 · 10 −5 M diamide augmented the lectin-induced rise in cyclic AMP. When intact lymphocytes were incubated with diamide, phosphodiesterase activity against both cyclic AMP and cyclic GMP, assayed in homogenates of these cells, was inhibited at concentrations as low as 1 · 10 −6 M. In contrast, when diamide was incubated with phosphodiesterase extracted from lymphocytes there was a dual effect. At low substrate concentrations and high diamide concentrations diamide was a non-competitive inhibitor of phosphodiesterase with a K i of 1.3–2.5 mM for cyclic AMP and 3.3–10 mM for cyclic GMP. In contrast, at high substrate concentrations diamide was an ‘uncompetitive’ activator of phosphodiesterase activity for both cyclic AMP and cyclic GMP. The effects of diamide could be largely or completely blocked by glutathione or dithiothreitol, indicating that sulfhydryl reactivity was involved in diamide's action on lymphocyte phosphodiesterase activity and intracellular cyclic AMP levels. These data demonstrate that diamide is a phosphodiesterase inhibitor both on phosphodiesterase extracted from lymphocytes and when incubated with intact lymphocytes and that diamide may increase or decrease intracellular cyclic AMP levels depending on the concentration of diamide used.

Mammalian cell studies with diamide

Diamide, diazenedicarboxylic acid bis (N,N′-dimethylamide), is a glutathione oxidant that has several advantages over classical sulfhydryl reagents, including reversibility. It is used widely to study glutathione's role in cellular function and radioresistance. It is also an effective radiosensitizer of hypoxic cells and acts by a unique mechanismm increasing the slope of the survival curve of irradiated hypoxic mammalian cells at high concentrations and decreasing the shoulder at low concentrations. Increased yields of radiation-induced chromosome aberrations and DNA strand breaks suggest that diamide sensitizes by mechanisms involving DNA. These mechanisms may include direct interactions with DNA and removal of cellular reducing species that normally repair radiation damage. Diamide sensitizes hypoxic cells synergistically with nitrofuran or nitroimidazole sensitizers, is effective against ascites tumors in vitro, and is useful for elucidating the mechanisms of aminothiol radioprotection. If used carefully, and with due regard for its limitations, it provides a useful tool with which to expand our knowledge of the mechanisms that govern cellular radiosensitivity and to hasten development of effective radiosensitizers for clinical use.

Action of diamide on hormone stimulated lipolysis in rat adipose tissue

Diamide [diazenedicarboxylic acid bis (N,N-dimethylamide)], a thiol oxidizing agent, was able to inhibit noradrenaline and theophylline-stimulated lipolysis in rat adipose tissue. This inhibition was not paralleled by any modification of cAMP level and accumulation. Besides diamide showed a non competitive inhibitory effect (50 per cent) on lipolytic response of adipose tissue to dibutyryl-cAMP. The results provide an indirect but consistent evidence that the observed inhibitory effects could be due to an interaction of diamide at a level subsequent cAMP formation, probably to a modification of protein kinase activity.

The reversible inhibition of gluconeogenesis in kidney cortex by diazenedicarboxylic acid bis (N,N-dimethylamide).

Gluconeogenesis in rat kidney cortex slices was reversibly inhibited by diazenedicarboxylic acid bis(N,N‐dimethylamide). The rate of formation of glucose from pyruvate, malate, succinate and D‐fructose decreased at least 3‐fold in the presence of this inhibitor, commonly called diamide. The extent of inhibition of gluconeogenesis from pyruvate was dependent on the concentration of diamide and closely correlated with the degree of inhibition of protein kinase. The inhibition of gluconeogenesis and protein kinase were completely reversed after removal of the inhibitor. The effects of diamide on particulate enzymes which may be involved at rate‐limiting steps in transport processes which support gluconeogenesis in renal tissue was examined. Several enzymes present in kidney brush border and microsomal membrane preparations were inhibited by diamide. Na+, K+‐dependent ATPase and glucose‐6‐phosphatase were inhibited, but Mg2+‐dependent ATPase, alkaline phos‐phatase and y‐glutamyl transpeptidase, which are also present in these same subcellular fractions, were not inhibited by diamide. Based on the results obtained in these studies it is proposed that diamide may inhibit gluconeogenesis at rate‐limiting steps catalyzed by enzymes which are regulated by protein kinase. These enzymes, in turn, appear to play a role in the transfer of substrates across the plasma membrane.The mechanism of inhibition of protein kinase by diamide was also investigated. Protein kinase present in a wide variety of rat tissues as well as phosphorylase kinase and pyruvate dehydrogenase kinase was inhibited by diamide. Kinetic studies showed that the inhibition of protein kinase by diamide was non‐competitive with respect to Mg2+, ATP, protein substrates and cyclic AMP. Sedimentation studies in sucrose‐density gradients containing histone and cyclic AMP showed that diamide inhibited both the intact and dissociated forms of protein kinase. These results support earlier observations which showed that diamide did not bind at the active site of protein kinase, but instead formed inactive complexes by binding to hydrophobic areas on the surface of the enzyme.

Radiosensitization of Hypoxic Mammalian Cells by Diamide: II. Studies of Mechanism

Diamide (diazenedicarboxylic acid bis(N,N'-dimethylamide)) radiosensitizes hypoxic Chinese hamster cells by decreasing the shoulder of the survival curve (at low concentrations) and increasing the slope (at high concentrations). We studied the mechanisms responsible for these two phenomena by irradiating cells under different conditions of temperature and substrate availability and measuring either their survival or DNA damage (chromosome aberrations and strand breaks). The results confirm that different mechanisms are responsible for the two effects. The effect on the shoulder appeared to be due to oxidation of endogenous nonprotein sulfhydryls and reduced pyridine nucleotides, compounds that would normally effect rapid chemical repair of certain (single-hit type) lesions. The slope effect, on the other hand, may have been due to reactions with DNA similar to those that have been described for the electron-affinic compounds. These results, together with the finding that hypoxic cells remained sensitized after the removal of diamide when they were irradiated in the cold, provide a useful model and a new experimental approach to the question of chemical radiosensitization.

Distinctive perturbations of amino acid and sugar influx by sulfhydryl reagents in the avian small intestine

1. Influx of leucine, β-alanine and 3-O-methyl glucose, substrates transported by separate systems, has been studied in chicken small intestine. 2. Influx of leucine (J mc L eu ) was much more rapidly inactivated during a 15 min exposure to p-chloromercuriphenyl sulfonate (PCMBS) or 3-chloromercuri-2-methoxy-propylurea (chlormerodrin) than β-alanine or 3-O-methyl glucose influx. Diazenedicarboxylic acid bis (N,N-dimethylamide) (diamide) in short-term (min) exposures inhibited β-alanine influx to a greater degree than the other substrates. 3. Neither the organic mercurials nor diamide in 30 min exposures changed intracellular GSH levels, implying their effects were confined to the cell membrane. 4. Dithiothreitol (DTT) completely and rapidly reversed inhibition of J mc L eu caused by the mercurials, suggesting these reagents acted on memebrane sulfhydryls. DTT only slowly reversed diamide-induced inhibition, whereas added GSH was without effect, indicating an intra-membrane site of action. 5. The temporal effects of the reagents on the different substrates suggest that separate and distinct classes of spacially-organized sulfhydryl groups were present among the different systems.

Diamide acts intracellularly to enhance transmitter release: The differential permeation of diamide, DIP, DIP + 1 and DIP + 2 across the nerve terminal membrane

The actions of the new potent thiol oxidizing agents, diazene dicarboxylic acid bis (N′-methyl piperazide) (DIP) and the N′-methyl iodide (DIP + 1) and the bis-N′-methyl iodide (DIP + 2) salts of DIP, were tested at the frog neuromuscular junction. At 20°C, DIP was as fast as the thiol oxidizing agent, diamide, in evoking transmitter release but was appreciably less effective at 6°C. DIP + 1 and DIP + 2 did not increase transmitter release. Since the three agents are potent oxidizers of glutathione and since the effectiveness of the compounds appears to depend on their a ability to exist, at least in part, in a neutral form at physiological pH, it is concluded that their action as promoters of transmitter release depends on their ability to permeate nerve terminal membranes. Thus, both diamide and DIP act to increase transmitter release by the intracellular oxidation of glutathione. The two charged agents, DIP + 1 and DIP + 2, are potent muscular depolarizing agents. It is probable that the quaternary nitrogen groups of these compounds render them cholinomimetics.

Glutathione XI. Species variation of membrane diffusion coefficients derived from intracellular thiol oxidation with DIP homologs

The membrane diffusion coefficients of DIP homologs (diazenedicarboxylic acid bis (N′-alkylpiperazides, e.g., N′-alkyl = ethyl) (I) vary substantially in red cells of different species. The coefficients range from 2.0 to 23.0 cm 2 /sec × 10 9 at 1°C (sheep r.b.c., chimpanzee r.b.c.) and the increase is correlated with increasing fluidity of the membrane, as indicated by a “fluidity factor” based on the double bond content of the fatty acids of the membrane. High variability of the coefficient in rabbits might be related to cholesterol content.