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Pretreatment of renal iodothyronine 5'-deiodinase with sulfhydryl reagents, iodoacetate, iodoacetamide and N-alkylmaleimides, results in irreversible loss of catalytic activity. Iodoacetate and iodoacetamide were the most potent inhibitors, being 100- to 1000-times more potent than N-alkylmaleimides. Iodoacetate and iodoacetamide inactivation followed pseudo-first-order kinetics with maximum inactivation rate constants of 1.56 min-1 and 0.87 min-1, respectively. Thyroxine and 3,3',5'-triiodothyronine and the competitive inhibitor iopanoate, protected the enzyme against iodoacetate inhibition. Protection by 3,3',5'-triiodothyronine was competitive with iodoacetate with a dissociation constant (Kd) of 113 nM; in close agreement with the Km for rT3 of 190 nM determined under similar reaction conditions. [3H]Carboxymethylation of renal membranes in the absence and presence of 3,3',5'-triiodothyronine showed specific incorporation of iodo[3H]acetate into substrate-protected sites of 35-40% of total when non-essential residues were first blocked with excess unlabeled iodoacetate. ' Protectable ' [3H]acetate incorporation followed pseudo-first-order kinetics and the rate constant for incorporation was identical to the rate constant for inactivation. These results indicate that iodoacetate fulfills the minimum criteria for an active-site-directed reagent for renal 5'-deiodinase and that a sulfhydryl group is in close proximity to the iodothyronine-binding site.
Studies were conducted to determine whether the sulfhydryl inhibitor, sodium iodoacetate, administered to preleukemic AKR mice and to mature C3H mice altered the immunocompetence of their bone marrow. Parameters investigated included the splenic plaque-forming capacity directed to sheep erythrocytes of bone marrow transferred from iodoacetate-treated aanimals to irradiated syngeneic recipients and the mitogenic responsiveness of bone marrow cells from untreated and iodoacetate-treated preleukemic AKR mice to phytohemagglutinin and concanavalin A. The administration of two 0.5-ml doses of 10 mM iodoacetate to preleukemic AKR mice and to C3H mice resulted in a significant increase in bone marrow immunocompetence. Irradiated mice given marrow transplants from iodoacetate-treated syngeneic donors exhibited greater numbers of plaque-forming cells directed against sheep erythrocytes than did recipients of marrow from control animals. This effect was abrogated when the donor marrow was previously treated in vitro with rabbit antimouse brain serum and the complement to remove thymus-derived lymphocytes. The mitogenic responsiveness of marrow cultures from iodoacetate-treated AKR mice to phytohemagglutinin was similar to that observed for control mice, while the response to concanavalin A was decreased. These findings suggest that the administration of iodoacetate potentiated the immunocompetence of bone marrow by affecting thymus-derived cells.
When grown in monolayer culture, OK cells display endocytic uptake of soluble fluid-phase markers such as lucifer yellow (LY) and horseradish peroxidase (HRP). The response of this process to metabolic inhibitors was characterized in the present study. Inhibition of cell metabolism by cyanide produced a decrease in cell ATP content which was accompanied by a decrease in uptake of both LY and HRP, confirming the energy-dependence of fluid-phase endocytosis in OK cells. Use of iodoacetate also decreased cell ATP content but its action on endocytosis was unexpected. Cell uptake of HRP was decreased by iodoacetate, similar to the effect of cyanide, but there was a marked increase in LY uptake. Additional studies showed that cyanide did not change intracellular Na+ or intracellular K+ and did not interfere with the Na(+)-dependency of Pi uptake. In contrast, iodoacetate produced a marked increase in Na+, a decrease in K+, and abolished the Na(+)-dependency of Pi transport. The latter was due primarily to a 10-fold increase in Na(+)-independent uptake of Pi. These findings suggest, indirectly, that plasma membrane permeability to Na+, K+, Pi, and small molecules such as LY, may be increased by iodoacetate, possibly through its action as an alkylating agent. This mechanism may allow increased cell uptake of LY through a non-endocytic pathway, and may mask the inhibitory action of iodoacetate on endocytic uptake of LY. These additional effects complicate the use of iodoacetate to interrupt endocytosis.
OBJECTIVE: To determine the effect of matrix metalloproteinase (MMP) inhibitors in mono-iodoacetate-induced arthritis in rats. DESIGN: The ability of compounds to inhibit MMPs in vitro was assessed kinetically using a quenched fluorescent substrate. Rats were injected with iodoacetate intraarticularly in one knee joint and damage to the tibial plateau was evaluated from digitized images captured using an image analyser and by histology. Collagenase and gelatinase activity in cartilage from iodoacetate injected knees were evaluated using(3)H-rat type I collagen and gelatin zymography, respectively. RESULTS: Collagenase and gelatinase activity significantly increased in the knee cartilage of rats injected with iodoacetate with peak activity by day 7. Three MMP inhibitors were examined for their efficacy in the rat iodoacetate-induced arthritis model. Significant (P< 0.05) inhibition of cartilage damage was observed in animals treated orally with 35 mg/kg b.i.d. of the three different MMP inhibitors. Inhibition of cartilage damage by the MMP inhibitors ranged from 36-42%. CONCLUSION: MMP inhibitors are partially protective against cartilage and subchondral bone damage induced by iodoacetate. These results support an important role for MMPs in mediating the joint damage in this model of arthritis.
The toxic effects of two metabolic inhibitors, dinitrophenol and iodoacetic acid, were compared. Mouse neuroblastoma cell cultures (Neuro-2a) were exposed to different concentrations of the toxic compounds for 24, 48 and 72 h to study basal toxicity effects (cell proliferation by quantification of total protein content (PR) and relative neutral red uptake (RNRU) by lysosomes). The following biochemical indicators assessed in the in vitro test system were: cytosolic phosphofructokinase (PFK) and enolase (ENL) activities in glycolysis; mitochondrial succinate dehydrogenase (SDH) activity in the citric acid cycle; lysosomal beta-galactosidase (GAL) activity; and neuronal acetylcholinesterase (AChE) activity. The effects of the two metabolic inhibitors on the various indicators differed. Iodoacetic acid was found to be far more toxic than dinitrophenol to neuroblastoma cell proliferation at 24 h exposure. Though 2,4-dinitrophenol and iodoacetic acid both inhibited cell proliferation of the neuroblastoma cells, their effects on the other endpoints were opposite. Dinitrophenol was a general activator of the metabolism, particularly affecting lysosomal function. Iodoacetic acid did not significantly alter general metabolism, but considerably modified lysosomal function and AChE activity. The modification of lysosomal function of Neuro-2a cells by the two compounds was quite different: dinitrophenol increased RNRU and GAL activity, and iodoacetic acid decreased both parameters.
Iodoacetate inhibits glyceraldehyde-3-phosphate dehydrogenase activity in pancreatic islets and causes a time- and dose-related inhibition of glucose oxidation and lactate output by the islets. High concentrations of the drug (0.3 mM or more) fail to affect Ba2+-induced insulin secretion but inhibit glucose-stimulated proinsulin biosynthesis, 45Ca net uptake and insulin release. A mixture of fumarate, glutamate, and pyruvate, the oxidation of which is only partially reduced by iodoacetate, fails to protect the B-cell against the inhibitory effect of the drug. These findings are compatible with the view that glycolysis plays an essential role in the process of glucose-induced insulin release. At low concentrations of iodoacetate (up to 0.2 mM), the reduction in glucose metabolism coincides with a partial inhibition of proinsulin biosynthesis. However, the expected reduction in 45Ca net uptake and subsequent insulin release is masked by a concomitant facilitating action of iodoacetate, possibly due to interference with native ionophoretic processes. It is concluded that iodoacetate is not an adequate tool to dissociate, if they are dissociable, the fuel and secretory functions of glucose.
The influence of sulphydryl inhibitor iodoacetate on properties characteristically associated with T-lymphocytes was examined in young AKR mice. Thymocytes from mice receiving 100 microgram of iodoacetate showed a decrease in cortisone sensitivity, and responded more vigorously to phytohemagglutinin (PHA). Spleen cells from treated mice also exhibited greater reactivity to both PHA and concanavalin A (Con A). The uptake of 14C-iodoacetate by the thymus, spleen, lymph nodes, bone marrow, liver and kidneys also was investigated. One hour, 2 h and 18 h after the intraperiotoneal injection of 10-3 M iodoacetate, the thymus demonstrated a higher incorporation of the labelled compound than did the spleen, lymph nodes or other organs examined. These findings provide further evidence to indicate that the immuno-enhancement observed following iodoacetate treatment is related to its effect on T lymphocytes.
Three groups of dystrophic and non-dystrophic mice 129/Re were used for studying the effect of the myotoxic agent iodoacetate on dystrophic muscle. The mice of the first group were given intramuscular injections of iodoacetate. The mice of the second group were injected with normal saline and the third group was maintained as untreated controls. The most severe histopathological changes were found in the dystrophic mice treated with iodoacetate. The non-dystrophic mice of the same group showed a significant increase in the number of internal nuclei. Moderate changes were observed in saline-treated dystrophic controls. There was no significant decrease in the life expectancy in any of the groups. The body weight of dystrophic mice was reduced throughout the experiment. On the contrary the non-dystrophic group showed an increased in weight, regardless of the treatment. The aggravation of the histopathological changes of dystrophic mice by iodoacetate would probably give support to the cyclical necrosis/abnormal regeneration theory of pathogenesis of muscular dystrophy.
Both iodoacetic acid and (R,S)-2-bromo-3-(5-imidazolyl)propionic acid (BrImPpOH) react with liver alcohol dehydrogenase in an affinity labelling mechanism between pH 6.1 and 10.5. The buffer-independent dissociation constants and the first-order rate constants have been determined as a function of pH. With BrImPpOH a pKa close to 9 for the free enzyme is assigned to the zinc-water ionization. The buffers used exerted a protective effect upon the inactivation of the enzyme by iodoacetic acid and BrImPpOH. Phosphate buffer showed a high degree of protection especially at lower pH, while zwitterionic buffers like Mes (4-morpholineethanesulfonic acid), Pipes (1,4-piperazinediethanesulfonic acid), Epps [4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid] and Bicine [N,N-bis(2-hydroxyethyl)glycine] gave less protection to various degrees. An exception was Ches (cyclohexylaminoethanesulfonic acid) which had an anomalously high affinity for the iodoacetate binding site. The dissociation constants of the buffers were calculated for the case of inactivation by both iodoacetic acid and BrImPpOH.
In this comparative study, rat hearts were perfused at 37 degrees C with three clearly defined protocols: the Ca2+ paradox, the O2 paradox, and with 20 mM caffeine. Each protocol involved an initial priming (Ca2+o depletion or anoxia; stage 1) and subsequent full activation (Ca2+o repletion, caffeine or reoxygenation; stage 2) of the damage system of the sarcolemma. Iodoacetate (1 mM) provided complete protection in the O2 paradox and over 80% protection in the Ca2+ paradox and caffeine protocols against creatine kinase release when present throughout the experiment (P < 0.001). Almost identical protection was found when iodoacetate was present only in stage 2 (P < 0.001). However, it was concluded that iodoacetate had limited protective effects when present only in stage 1 in any of the three protocols and that its action is to inhibit the activity of the transsarcolemma damage system in stage 2 when it has been activated in stage 1. It is suggested that iodoacetate interacts with thiol groups on the damage system of the sarcolemma.
Neuronal damage associated with cerebral ischemia and hypoglycemia might be the consequence of the extracellular accumulation of excitatory amino acids. In previous studies we showed that elevation of glutamate and aspartate extracellular levels by inhibition of its uptake in vivo is not sufficient to induce neuronal damage unless mitochondrial energy metabolism is compromised. In the present study we show that chronic systemic administration of the glycolysis inhibitor iodoacetate (25 mg/kg) induces no damage to the brain per se but enhances neuronal vulnerability to glutamate-mediated neurotoxicity in the hippocampus. Tissue injury is well protected either by antagonizing NMDA glutamate receptors with MK-801 or by administration of pyruvate, a substrate of the tricarboxylic acid cycle. In contrast to systemic treatment, local infusions through a dialysis probe of 5 mM iodoacetate into the hippocampus induced acute lesions not sensitive to MK-801. Iodoacetate intrahippocampal perfusion induced substantial increases in the extracellular levels of glutamate (3.5-fold), taurine (8.8-fold), and particularly aspartate (35-fold). Neuronal damage under this conditions occurs very rapidly as revealed by the histological analysis of animals transcardially perfused immediately after iodoacetate perfusion. Aspartate might contribute to neuronal damage since intrahippocampal administration of this amino acid (600 nmol/microl) induces extensive lesions. The present study might suggest that impairment of glucose oxidation through the glycolytic pathway in vivo facilitates glutamate neurotoxicity. Additionally, the results indicate that pyruvate might prevent as efficiently as glutamate receptor antagonists glutamate-mediated neuronal damage associated with ischemia/hypoglycemia.
A highly purifed preparation of rat intestinal phosphodiesterase II (oligonucleate 3'-nucleotidohydrolase, EC 3.1.4.18) has been studied using a synthetic substrate, thymidine 3'(2,4-dinitrophenyl) phosphate. The enzyme was most active between pH 6.1 and pH 6.7 and was inhibited by Cu2+ and Zn2+ but unaffected by EDTA, Mg2+, Co2+, and Ni2+. The reaction rate decreased at high levels of enzyme because of competitive inhibition by deoxythymidine 3'-phosphate, a reaction product, which showed a Ki of 2-10(-5) M. The molecular weight of the enzyme by gel-filtration was 150 000-170 000. In electrofocusing experiments multiple peaks of activity were found at pH 3.4, 4.2-4.5and 7.2. Polyacrylamide gel electrophoresis of freshly purified phosphodiesterase II showed up to 10 protein bands in the gels. If the preparations were stored at 4 degrees C for some time only one or two bands appeared. Investigation of the reaction of rat intestinal phosphodiesterase II with a number of possible phosphodiesterase substrates indicated that the enzyme required a nucleoside 3'-phosphoryl residue for the initiation of hydrolysis. Thus compounds such as NAD, ATP, bis-(p-nitrophenyl)phosphate, thymidine 5'-(p-nitrophenyl)phosphate, glycerylphosphorylcholine, guanylyl-(2' leads to 5')-adenosine and 3',5'-cyclic AMP which contain phosphodiester bonds, nevertheless were not substrates for the enzyme. The enzyme was inhibited reverisbly by p-chloromercuribenzoate and p-chloromercuriphenylsulfonate and inactivated irreversibly by iodoacetic acid. Activity of the phosphodiesterase II was reduced to 50% by incubation with 2.0-10(-3)--5.0-10(-3) M iodoacetate for 20--30 min at 24 degrees C at pH 5.0--6.1. Iodoacetamide had no effect. The degree of inactivation by iodoacetate was reduced by the presence of a substrate for the enzyme or, more effectively by deoxythymidine 3'-phosphate, a competitive inhibitor. It is concluded that iodoacetic acid alkylates an essential residue at the active centre of the enzyme.
A two-step method for labeling the glutamine active site of formyglycinamide ribonucleotide (FGAR) amidotransferase from chicken liver has been developed in which reaction of all other reactive groups with unlabeled iodoacetate is followed by specific labeling of the glutamine site with radioactive reagent. A study of the reaction as a function of duration, temperature, and pH of the incubation as well as concentration of iodoacetate has revealed that two nonessential groups of the enzyme react in the presence of glutamine and that this modified enzyme is relatively resistant to further carboxymethylation. When this modified enzyme was incubated with radioactive iodoacetate in the presence of FGAR, ATP, and Mg2+ after removal of glutamine by dialysis, about 1 mol of radioactive iodoacetate was incorporated per mol of enzyme with inactivation. This method permits labeling of the active site for glutamine without the use of glutamine analogues.
Fluoride and iodoacetate inhibited the oxidation of glucose by islets of Langerhans isolated from the rat pancreas. Fifty % inhibition occurred with either 17 mM fluoride or 0.5 mM iodoacetate. The rate of insulin biosynthesis was more strongly inhibited by these inhibitors, especially fluoride. Fifty % inhibition occurred with approximately 1.5 mM fluoride. At high concentrations of iodoacetate and fluoride, the inhibitory effect on insulin synthesis was not reversed to a significant degree by the addition of pyruvate in the incubation medium. In addition to inhibiting the glycolysis and depriving islets of energy essential for the biosynthesis of insulin, fluoride probably exerts a direct inhibitory influence on the biosynthetic mechanism. A separate experiment with [6-14C]glucose indicated that 0.2 mM iodoacetate does not inhibit glycolysis completely.
Iodoacetate and iodoacetamide were compared as to their capacity to block islet glycolysis and interfere with glucose inhibition of glucagon release and glucose stimulation of insulin release. Glycolysis was measured in isolated rat islet by the rate of lactate formation from 27 mM glucose. Hormone release was investigated by perfusing isolated rat pancreas with a 10 mM mixture of 19 amino acids, with and without 5 mM glucose. In perfusion experiments, lactate (2.5 mM) and pyruvate (0.5 mM) were present to provide alternate source of energy independent of glycolysis. Iodoacetate was about twice as potent as iodoacetamide in blocking glycolysis in islets, 0.2 and 0.5 mM, respectively being needed for complete inhibition of lactate production. Levels of either agent lower than 0.05 mM did not affect lactate accumulation. Iodoacetate, at the level which completely inhibited glycolysis did not interfere with the permissive action of glucose for insulin release. In contrast, iodoacetamide at a level (0.05 mM) which had no effect on lactate production, changed the response of the beta-cell dramatically: amino acids now released insulin even in the absence of glucose and insulin release by 5 mM glucose alone was greatly augmented. Both thiol reagents at 0.025 mM concentration completely prevented glucose from suppressing amino acid stimulated glucagon release, iodoacetamide being more potent than iodoacetate. These data indicate that the opposite physiological actions of glucose in alpha and beta-cells are in each case dissociable from the fuel function of the sugar molecule, and the results best support the concept that glucose and thiol reagents effect insulin and glucagon secretion by acting on sulfhydryl groups related to receptor sites in the alpha-and beta-cell membrane.
The present study was undertaken to examine the effects of iodoacetic acid, a non-phorbol tumor promoter, on metabolic cooperation between mutant human fibroblasts as measured by [14C]citrulline incorporation. Other thiol-reactive polyphenolic compounds such as hydroquinone and 2-hydroxyestrone were also examined. 12-O-Tetradecanoyl phorbol-13-acetate (TPA), a potent skin tumor promoter, inhibited the cell-cell communication by more than 60% at 20 ng/ml. However, iodoacetic acid, hydroquinone, and 2-hydroxyestrone, had no effect on the process even at cytotoxic concentrations. Induction of intercellular contact (agglutination) among lymphocytes during the course of phytohemagglutinin (PHA)-induced blastogenesis was monitored turbidometrically at 620 nm. Hydroquinone and 2-hydroxyestrone suppressed the PHA-induced lymphocyte agglutination at 1-2 microM in vitro concentrations while iodoacetic acid was devoid of any effects at concentrations up to 100 microM. Hydroquinone and 2-hydroxyestrone concomitantly suppressed PHA-induced lymphocyte blastogenesis at 1-2 microM in vitro concentrations while the suppression by iodoacetic acid was significant at 10 microM. All 3 compounds failed to disrupt microtubule assembly, a sulfhydryl-dependent process, in a rat brain crude extract. However, p-benzoquinone, an oxidation product of hydroquinone, did inhibit the process at 1 mM. In summary, these studies suggest that, unlike TPA, thiol-reactive non-phorbol tumor promoters and polyphenolic compounds do not inhibit cell-cell communication between mutant human fibroblasts. Although the compounds demonstrate diverse molecular mechanisms of action, they all inhibit in vitro immune functions suggesting that immunosuppression may play a role in tumor promotion.