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PubMed1977

Glutamine active site of formylglycinamide ribonucleotide amidotransferase. 1. Labeling of the enzyme with iodoacetate.

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.

Animals↗
PubMed1976

Effects of iodoacetate and fluoride on islate respiration and insulin biosynthesis.

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.

Animals↗
PubMed1975

Iodoacetate and iodoacetamide-induced alterations of pancreatic alpha- and beta-cell responses.

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.

Amino Acids↗
PubMed1987

Iodoacetic acid and related sulfhydryl reagents fail to inhibit cell-cell communication: mechanisms of immunotoxicity in vitro.

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.

Agglutination↗
PubMed1994

Site-specific cartilage changes in murine degenerative knee joint disease induced by iodoacetate and collagenase.

Degenerative joint disease was induced in the knee joints of mice by intraarticular injection of two different stimuli: iodoacetate and highly purified collagenase. Proteoglycan synthesis was measured in vivo at different time points in four topographical areas of the knee joint (central and peripheral parts of the patella and central parts of the medial and lateral tibial plateaus) and was compared with histological observations of localized damage to the joint. In vitro incubation with iodoacetate had a direct effect on proteoglycan metabolism. Intra-articular injection of iodoacetate in vivo inhibited the proteoglycan synthesis in cartilage from the central part of the patella. In the peripheral part of the patella, inhibition on day 1 was followed by stimulation of synthesis on days 3-30. Proteoglycan synthesis also was inhibited in the central parts of the medial and lateral tibial plateaus. The areas with inhibited synthesis had loss of safranin O staining on histology. In vitro incubation with collagenase did not have a direct effect on the proteoglycan metabolism of intact cartilage; this led to the assumption that osteoarthritis after injection of collagenase is caused by ligamentous injury, which leads to an unstable joint. Injection of collagenase in vivo stimulated the proteoglycan synthesis in cartilage from the central and peripheral parts of the patella. In an early stage of the process, the cartilage from the tibial plateaus also was slightly stimulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

The effect of iodoacetic acid on the electroretinogram and oscillatory potentials in rabbits.

It has been shown that a single injection of iodoacetic acid selectively (but temporarily) abolishes the b-wave of the electroretinogram. We examined whether such use of this chemical further substantiate our claim that the b-wave of the electroretinogram is a composite potential resulting from the summation (or integration) of faster retinal potentials, usually referred to as the oscillatory potentials. Full-field electroretinograms were recorded from adult New Zealand rabbits before and after a single, bolus injection of 15 mg/kg of buffered iodoacetic acid. Both the 1-1000 Hz electroretinogram and the 100-1000 Hz oscillatory potentials were recorded simultaneously. The oscillatory potentials considered in this study were those normally seen on the rising phase of the b-wave. Following the intravenous injection of iodoacetic acid, there was a progressive decrease in the amplitude and peak time of the b-wave. This observation also was reflected in the oscillatory potential recordings, in which the long-latency oscillatory potentials (3 and 4) progressively disappeared while oscillatory potential 2 remained. We believe that these findings further support our contention that the oscillatory potentials are major components of the b-wave.

Animals↗
PubMed1994

Bronchial smooth muscle energetics: effect of iodoacetate and hypoxia.

The active pressure (AP) and the oxygen consumption (VO2) of segments of bovine bronchi were measured during a 10 microM carbachol stimulation. VO2 did not increase during the carbachol-induced contraction whereas there was a twofold increase in the lactate production. Addition of the glycolytic blocker, iodoacetate (83 microM), decreased the AP to 68.9 +/- 6.4% of control value (n = 10, P < 0.05) whereas VO2 remained constant. The lactate concentration in the physiological solution decreased significantly (P < 0.05). When the solution was supplemented with pyruvate (10 mM), the effect of iodoacetate was antagonized. Under hypoxic condition, i.e. when the solution was bubbled with 5% CO2 in N2, VO2 decreased sharply to 7.7 +/- 3.1% of control (n = 8, P < 0.05) whereas AP did not change. The combined effect of iodoacetate and hypoxia led to a fall in both AP (12.4 +/- 3.0% of control, n = 7, P < 0.05) and VO2 (21.2 +/- 5.1%, P < 0.05). These results suggest that the energy required by bronchial smooth muscle to generate contraction could be supplied by either the aerobic or the anaerobic pathway.

Animals↗
PubMed1982

The specificity of active-site alkylation by iodoacetic acid in the enzyme thiosulfate sulfurtransferase.

The active-site sulfhydryl group in the enzyme thiosulfate sulfurtransferase (rhodanese; thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) is alkylated rapidly by iodoacetic acid in the free enzyme form, E, with complete loss of sulfurtransferase activity. Iodoacetic acid is completely ineffective with the sulfur-substituted form of the enzyme, ES. Iodoacetamide, on the other hand, has no effect on either enzyme form. The competitive enzyme inhibitor, toluenesulfonic acid, protects against inactivation in a strictly competitive way and analysis gives an apparent binding constant for toluenesulfonic acid of 12.5 mM, which is in agreement with studies of its effect on the catalyzed reaction. These results are taken to indicate that iodoacetic acid is an affinity analog for the substrate, thiosulfate, and inactivates because it can use the specific thiosulfate binding interactions, correctly orient its reactive center and displace intraprotein interactions which appear to protect the active-site sulfhydryl group in the E form.

Alkylation↗
PubMed1983

The effects of cyanide and iodoacetate intoxication and ischaemia on enzyme release from the perfused rat heart.

Isolated rat hearts perfused in the presence of iodoacetate show inhibition of glycolysis and release enzymes into the perfusate. Hearts perfused with cyanide, a mitochondrial inhibitor, show acceleration of glycolysis and no enzyme release. The adenine nucleotide content of the iodoacetate, but not the cyanide-perfused hearts was reduced. These results indicate that the membranes were permeable in the former treatment group. The adenylate energy charge and the ATP content of both the cyanide and iodoacetate treatment groups were similar but, as the extent of enzyme release was quite different, it appears that the energy state of the cell was not the prime factor controlling membrane integrity. Isolated perfused hearts were rendered ischaemic by placing a one-way ball valve in the aortic outflow tract. ATP concentration declined, as did ADP after an initial rise of short duration. AMP concentrations rose as the time of ischaemia increased. At the time at which enzyme release was first determined, the intracellular total adenine nucleotide content began to decline, suggesting that the membrane had become permeable to both small and large molecules. Glycolysis was stimulated by the hypoxia induced in the preparation and then this increase became inhibited. The point at which this inhibition was observed was also the point at which membrane permeability was evident. Taken together, the data from these experiments suggest that the energy derived from the activity of the glycolytic pathway may be important to the heart for maintenance of membrane function, particularly in ischaemia.

Adenine Nucleotides↗
PubMed1991

Inactivation of the RTEM-1 cysteine beta-lactamase by iodoacetate. The nature of active-site functional groups and comparisons with the native enzyme.

The pH-rate profile for inactivation of the RTEM-1 cysteine beta-lactamase by iodoacetate supports previous evidence [Knap & Pratt (1989) Proteins Struct. Funct. Genet. 6, 316-323] for the activation of the active-site thiol group by adjacent functional groups. The enhanced reactivity of iodoacetate, with respect to that of iodoacetamide, suggests the influence of a positive charge in the active site. The reactivity of iodoacetate is not affected by dissociation of an active-site functional group of pKa 6.7, which increases the reactivity of neutral reagents, probably because of a compensation phenomenon; it is, however, lost on dissociation of an acid of pKa 8.1. It is concluded that the active cysteine beta-lactamase has four functional groups at the active site, one nucleophilic thiolate of Cys-70, one neutral acid (most probably the carboxy group of Glu-166, from the crystal structures) and two cationic residues (most probably Lys-73 and Lys-234). A comparison of these results with the pH-dependence of reactivity of the native RTEM-2 beta-lactamase suggests that the active form of the latter enzyme is also monocationic, although the nucleophile (Ser-70) is likely to be neutral in this case and the carboxylic acid dissociated. A mechanism of class A beta-lactamase catalysis is discussed where the Glu-166 carboxylate acts as a general base/acid catalyst and Lys-73 is principally required for electrostatic stabilization of the anionic tetrahedral intermediate.

Binding Sites↗
PubMed1988

Restoration of enzymic activity and cytotoxicity of mutant, E553C, Pseudomonas aeruginosa exotoxin A by reaction with iodoacetic acid.

Pseudomonas aeruginosa exotoxin A (ETA) is inactivated greater than 1,000-fold when an active site glutamic acid, E553, is mutated to aspartic acid (Douglas, C.M., and Collier, R. J. (1987) J. Bacteriol. 169, 4967-4971). To test the effect of creating a carboxyl-containing side chain at position 553 longer than that of glutamic acid, we first replaced Glu-553 with cysteine by site-directed mutagenesis of cloned ETA and then carboxymethylated the cysteine side chain with iodoacetic acid. The E553C mutation reduced ADP-ribosyltransferase and cytotoxic activities greater than 10,000-fold. Reaction of the mutant with iodoacetic acid enhanced enzymic activity 2,500-fold, to a level approximately one-sixth that of wild type toxin, and restored cytotoxicity to a slightly lesser extent. Iodoacetamide did not activate the mutant, and neither iodoacetic acid nor iodoacetamide affected the activity of wild type toxin. These results show that the carboxyl group of Glu-553 is important for ADP-ribosylation activity and imply flexibility in the enzyme-substrate complex in accommodating the slightly longer S-carboxymethylcysteine side chain. This general approach may have applications in protein engineering as well as in studying carboxyl side chain functions in enzymes.

ADP Ribose Transferases↗
PubMed1982

Effect of cytochalasin-B, low Ca++ concentration, iodoacetic acid, and quinacrine-HCl on the attachment of Giardia trophozoites in vitro.

Giardia sp. trophozoites were isolated directly from the small intestines of rats and permitted to attach to polystyrene Petri dishes incubated at 37 C. Attached trophozoites were treated in vitro with various agents which inhibit cell motility (cytochalasin-B, low Ca++, colchicine) and metabolism (iodoacetic acid, 2,4-dinitrophenol) and chemotherapeutic agents (quinacrine-HCl, metronidazole). Trophozoite attachment was sensitive to cytochalasin-B, low Ca++, iodoacetate, and quinacrine-HCl. Contractile proteins (actin, myosin) have been demonstrated previously in the periphery of the ventral disc. The effect of cytochalasin-B and low Ca++ concentrations on trophozoite attachment is consistent with the suggested role of contractile proteins in the mechanism of attachment. The effect of iodoacetate suggests that energy for attachment is derived from glycolysis. The effect of quinacrine-HCl on attachment was rapid (less than 10 min with 10.0 micrograms/ml). Its mode of action on attachment is not understood.

Animals↗
PubMed1993

Effects of intravenous iodoacetate and iodate on pH outside rod photoreceptors in the cat retina.

PURPOSE: Effects of intravenous iodoacetate (a glycolysis inhibitor) and iodate (a metabolism inhibitor selective to retinal pigment epithelium) on light-evoked alkalinizations and hypoxia-induced acidifications were studied in the dark-adapted cat retina, in vivo, to learn about pH regulation. METHODS: pH was recorded in the extracellular space surrounding rod photoreceptors with double-barrelled H(+)-selective microelectrodes. RESULTS: Intravenous infusion of 5 mg/kg iodoacetate-induced alkalinizations in the outer nuclear layer and suppressed both light-evoked and hypoxia-induced pH responses immediately. Iodate injection (30 mg/kg) produced acidifications in the subretinal space and affected light-evoked alkalinizations gradually but not hypoxia-induced acidifications. CONCLUSIONS: These results suggest that rods glycolysis plays an important role in both light-evoked and hypoxia-induced pH responses. And the retinal pigment epithelium may have little concern with light-evoked alkalinizations except that it plays an important role in regenerating the rhodopsin to be needed for the light responses of photoreceptors. Furthermore, the finding of the intravenous-iodoacetate-induced alkalinization in the outer nuclear layer supports that acid production by rods in the dark is originated from glycolysis to support the dark current. The iodate-induced acidification in the subretinal space indicators that the retinal pigment epithelium might actively transport acids from the subretinal space to the choroid.

Animals↗
PubMed1976

Alkylation of cysteinyl residues of pig heart NAD-specific isocitrate dehydrogenase by iodoacetate.

Pig heart NAD-specific isocitrate dehydrogenase is inactivated by reaction with iodoacetate at pH 6.0. Loss of activity can be attributed to the formation of 1-2 mol of carboxymethyl-cysteine per peptide chain. The rate of inactivation is markedly decreased by the combined addition of Mn2+ and isocitrate, but not by alpha-ketoglutarate, the coenzyme NAD or the allosteric activator ADP. The substrate concentration dependence of the decreased rate of inactivation yields a dissociation constant of 1.6 mM for the enzyme-manganous-dibasic isocitrate complex, a value that is 50 times higher than the Km for this substrate. This result suggests that in protecting the enzyme against iodoacetate, isocitrate may bind to a region distinct from the catalytic site. Isocitrate and Mn2+ also prevent thermal denaturation, with an affinity for the enzyme close to that observed for the iodoacetate-sensitive site. The alkylatable cysteine residues may contribute to a manganous-isocitrate binding site which is responsible for stabilizing an active conformation of the enzyme.

Animals↗
PubMed1980

Iodoacetate inhibition of glyceraldehyde-3-phosphate dehydrogenase as a model of human myophosphorylase deficiency (McArdle's disease) and phosphofructokinase deficiency (Tarui's disease).

A model of the human neuromuscular disorders myophosphorylase deficiency and phosphofructokinase deficiency has been developed using intra-aortic injection of sodium iodoacetate in adult male rats. Iodoacetate selectively inhibits in vivo the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. The iodoacetate-injected rats develop electrically silent cramps in leg musculature during ischemic (or vigorous non-ischemic) exercise. Post-exercise rhabdomyolysis is evidenced by a 10-fold serum CPK elevation, excessive uptake of 99mTc-diphosphonate by cramped muscle, and type IIB fiber damage (histochemically-demonstrated) in cramped muscle. Further analysis of this model will allow a greater understanding of the clinical syndrome associated with the human disorders and permit development of successful treatment programs.

Animals↗
PubMed1977

Iodoacetate-induced inhibition and enhancement of spontaneous leukemia in AKR mice.

Either inhibition or enhancement of the spontaneous lymphoma exhibited by AKR mice was observed after treatment with different dosages of the sulfhydryl inhibitor, sodium iodoacetate. Treatment of the mice at 3 or 6 months of age with five ip injections of 0.10 mg of iodoacetate at 5-day intervals significnatly extended the survival of the animals. A single administration of this dosage elevated the responses of splenic lymphocytes to the T-cell mitogens phytohemagglutinin (PHA) and concanavalin A (Con A), and resulted in higher PHA and Con A response ratios than were noted for age-matched controls. Conversely, groups of 3- or 6-month-old AKR mice, subjected to the same regimen but with 0.01-mg dosages, exhibited an apparent accelerated development of the leukemia and survived for significantly shorter periods. In general, splenic lymphocytes harvested from mice given a single 0.01-mg treatment of iodoacetate were not as reactive as were control cell cultures when exposed to the T-cell mitogens.

Animals↗
PubMed1977

Effects of iodoacetate, mannoheptulose and 3-O-methyl glucose on the secretory function and metabolism of isolated pancreatic islets.

The ability of iodoacetate, mannoheptulose, and 3-O-methyl glucose to alter islet cell metabolism and glucose-stimulated insulin secretion was examined. A method for the sequential analysis of the releasing and fuel function of glucose in isolated islets was applied. Insulin release was measured by radioimmunoassay and the metabolism of glucose by determining the rate of tritiated water production from [5-3H]glucose and lactate accumulation. It was found that iodoacetate, in the range of 0.2-1.0 mM, inhibited the metabolism of glucose linearly while release was not blocked until metabolism was reduced by 30-40%. The KI for both processes, release and metabolism, was the same. Pyruvate did not protect against or reverse the effects of iodoacetate. Mannoheptulose inhibited both release and metabolism half-maximally at about 5 mM when 27.5 mM glucose was used as the stimulatory agent. A mannoheptulose-resistant component of glucose metabolism, amounting to 30% of the maximal rate was observed. 3-O-Methyl glucose had no effect on insulin release but reduced glucose utilization and lactate production from low glucose. The results are discussed in light of the two prevailing hypotheses explaining glucose induced insulin release, i.e., the receptor and the metabolism hypotheses.

Animals↗
PubMed1975

Sodium iodoacetate as an antiglycolytic agent in blood samples.

We evaluated the effect of sodium iodoacetate on glycolysis in a series of randomly selected blood samples from patients. Glucose values for serum and for serum with added sodium fluoride (2.5 g/liter) or sodium iodoacetate (2 g and 0.5 g/liter) were compared at room temperature. Respective declines in glucose values averaged 170, 40, 30, and 30 mg/liter after 24 h. Iodoacetate-preserved (0.5 g/liter) samples showed no visible hemolysis. Results of determinations of urea with urease and of other tests on SMA 12/60 (Technicon) panels were unaffected.

Autoanalysis↗