Investigation of the mechanism of the action of dimethyllaurylbenzyl-ammonium chloride (ajatin) on rough and smooth forms of yeast cells

Ajatin эффект от потреблен ия кислорода в присутствии некото рых промежуточных и продукция была Гликолиз расследование вмест е с эффект iodoacetic кислоты, для того что бы пролить свет еще выше чувствительность грубый формы дрожжей для Ajatin и объяснить синергический дейст вия Ajatin и iodoacetic кислоты в присутстви и глюкозы. Было установлено, что окисление этанола и ацетата в грубой форме distiller дрожжи гораздо боле е чувствительны для действий Ajatin чем окисление глюкозы, в то время как в виде гладких разница в чувствительности ме жду этими двух процессов меньш е. Потребление кислорода в присутст вии этанола или ацетата в качестве субстрата значитель но сдерживается путем iodoacetic кислоты в виде гладких, но не зависит от этого ингибитора в грубой форме. На основе результато в и данных в литературе метаболизма гладких и грубые формы дрожже й обсуждается и Причины синергическ ий действия Ajatin и iodoacetic кислоты на потребление кислоро да в присутствии глюк озы принимаются вверх. Она представляет интерес для обнаружи ть, что окислительные проце ссы не мешает arsenite, относительно устойчив к Ajatin с тем, что в присутстви и arsenite разница в чувствительности дв ух формах дрожжей в действие по чти Ajatin исчезает.     

Investigations of the effect of dimethyl-lauryl-benzyl ammonium chloride (ajatin) on the resistance of cell membranes in rough and smooth forms of yeast cells

Было установлено, чт о при анаэробных усл овиях Ajatin значительно повышает decolorization курсу ме тиленового голубог о в дрожжи клеток, это может быть объяснен о ущерб в клеточные м ембраны. Этот эффект Ajatin зависит от концент рации клеток, но не то лько зависит от веса Соотношение этого а гента и дрожжевой кл етки, как предлагало сь некоторыми автор ами. При более низких концентрациях дрож жей клеток рост конц ентрации Ajatin из причин резкого падения дея тельности, которая н е встречается на бол ее высоких клеток ко нцентраций. Эффект Ajatin по курсу метиленово го синий decolorization примерно равны в обоих видов к леток дрожжей и выше Чувствительность г рубой форме смертон осное эффект Ajatin таким образом не может быт ь объяснено в нижней сопротивление клет очных мембран грубы х формах к этому аген ту. Сравнение поведе ния грубый и гладкой формы клеток дрожже й применительно к Ajatin б ыл использован в кач естве основы для обс уждаем механизм сме ртельным исходом вл ияние этого веществ а.     

Different efficacy of iodoacetic acid and N-ethylmaleimide in high-performance liquid chromatographic measurement of liver glutathione

The widely used high-performance liquid chromatography (HPLC) procedure to determine glutathione in biological samples utilizing iodoacetic acid as thiol quenching agent and 1-fluoro-2,4-dinitrobenzene for derivatization has been modified regarding tissue sample processing and storage of the working solutions. The modified procedure compared with the original method reduces artifactual oxidation in rat liver glutathione measurement (1.47±0.8% vs. 2.84±0.69%, respectively). In both HPLC procedures, an increase in artifactual oxidation was found in both standard glutathione solutions and hepatic samples when N-ethylmaleimide instead of iodoacetic acid was used for thiol trapping.     

Development and function of B cells in monolayer islet cells of 3-week-old rat

Monolayer cultures of pancreatic B cells of 3-week-old rats were kept for 7 days in medium with 5.5 mM glucose plus 1 mM 2-deoxy-2-fluoroglucose or for 4 days in medium with 5.5 mM glucose alone, following exposure for 3 days to a medium with 5.5 mM glucose plus 5 microM iodoacetic acid. Addition of the deoxyglucose or iodoacetic acid caused a selective deletion of fibroblasts, yielding large clusters that consisted mostly of islet cells. At the early stage of culture in medium with 16.7 mM glucose (day 4), the response of B cells to 16.7 mM glucose included only a small rise in insulin secreted during the first and second phases, and that to 10 mM of leucine and 2-ketoisocaproate was monophasic. After culturing for 7 days, these three secretagogues markedly stimulated insulin secretion by B cells cultured in both media, with a significant rise in secondary phase secretion. However, quantitative relationships differed. Thus, the response (total insulin secreted during a 30-min stimulation) of B cells in 2-deoxy-2-fluoroglucose to glucose was 155%, to leucine 185% and 2-ketoisocaproate 126% of that of cells exposed to iodoacetic acid. In conclusion, the present results suggest that B cells of 3-week-old rat may be immature, and that medium containing 2-deoxy-2-fluoroglucose is beneficial to continued maturation of the response in vitro.     

On the active site of liver acetyl-CoA. Arylamine N-acetyltransferase from rapid acetylator rabbits (III/J)

A covalent, catalytic intermediate of cytosolic liver acetyl coenzyme A: arylamine N-acetyltransferase (EC 2.3.1.5) from rapid acetylator rabbits (III/J) was isolated and chemically characterized. The active site was further studied using two covalent inhibitors, [2-3H]iodoacetic acid and bromoacetanilide. Inhibition experiments with [2-3H]iodoacetic acid at pH 6.9 showed that the incorporation of 0.7 mol of [2-3H]iodoacetic acid/mol of N-acetyltransferase led to rapid, irreversible loss of enzyme activity. Preincubation of the enzyme with acetyl coenzyme A (acetyl-CoA) completely protected against inactivation by [2-3H]iodoacetic acid. After incubating the N-acetyltransferase with [2-3H]acetyl-CoA in the absence of an acceptor amine, an acetyl-cysteinyl-enzyme intermediate was isolated and characterized. Preincubation of N-acetyltransferase with iodoacetic acid prevented the incorporation of the [2-3H]acetyl group into the enzyme. The product analog, bromoacetanilide, caused a rapid irreversible loss of N-acetyltransferase activity. The reaction was pseudo first-order and saturated at high bromoacetanilide concentrations (KI = 0.67 mM; k3 = 1 min-1). Preincubation of the enzyme with acetyl-CoA prevented inactivation by the inhibitor. The acceptor amine 4-ethylaniline did not prevent inhibition. Incorporation of the inhibitor was directly proportional to the loss of activity showing a 1:1 stoichiometry of enzyme to inhibitor. The target amino acid was identified as cysteine by amino acid analysis of inhibitor-treated enzyme.     

The use of 3-bromopropionic acid for the determination of protein thiol groups

S-Carboxymethylcysteine, formed by the reaction of iodoacetic acid with cysteine, was found to undergo intramolecular cyclization to yield 3-oxo-(2H,3H,5H,6H-1,4-thiazine)-5-carboxylic acid. The cyclization was studied under various conditions and the product was isolated and characterized. S-Carboxyethylcysteine, formed by the reaction of 3-bromopropionic acid with cysteine, did not undergo the cyclization reaction. The use of 3-bromopropionic acid was examined as an alternative to iodoacetic acid for the protection and determination of protein thiol groups.     

Cloning and characterization of an intracellular isoamylase gene from Pectobacterium chrysanthemi PY35

The melibiose transport carrier of Escherichia coli (coded by melB gene) is a cotransport system which couples the transport of a-galactosides to protons, sodium, or lithium ions. The charged amino acid residues in membrane-spanning helices are of considerable interest because many of them have important function in substrate recognition. In most cases changing these charged residue to an uncharged residue (cysteine) results in total loss of activity. In this communication we describe experiments in which the cysteine substitution for a charged residue was chemically changed by sulfhydryl reagents (MTSEA and MTSET to restore a positive charge and MTSES a negative charge) or by iodoacetic acid or through oxidation by hydrogen peroxide so as to regain the original negative charge. In two cases (D55C and D124C) the reconstructed negative charges via the oxidation of the thiol to the sulfinic and/or sulfonic acid resulted in partial recovery of transport: D55C up to 27% of the normal and D124C up to 4% of the normal in melibiose accumulation; D55C up to 36% of the normal and D124 up to 4.5% of the normal in downhill transport. Sulfhydryl reagents and iodoacetic acid failed to recover transport in all cases. We infer that the configurations of the charges as well as the structure of the side chains that carry them are critical in the maintenance of the transport.     

Metabolism of leukotriene E4 to 5-hydroxy-6-mercapto7,9-trans-11,14-cis-eicosatetraenoic acid by microfloral cysteine-conjugate beta-lyase and rat cecum contents

Leukotriene E4 was incubated with cysteine-conjugate beta-lyase isolated from the intestinal bacterium Eubacterium limosum. The reaction was terminated by addition of iodoacetic acid or dimethyl sulfate, and the products formed were isolated by reverse-phase high-performance liquid chromatography. The structures of two adducts of a metabolite were determined by uv spectroscopy, by gas-liquid radiochromatography, and by comparisons with chemically synthesized reference compounds. They were 5-hydroxy-6-S-carboxymethylthio-7,9-trans-11,14-cis-eicosatetraeno ic acid (iodoacetic acid adduct) and 5-hydroxy-6-S-methylthio-7,9-trans-11,14-cis-eicosatetraenoic acid (dimethyl sulfate adduct) indicating that the structure of the underivatized metabolite was 5-hydroxy-6-mercapto-7,9,11,14-eicosatetraenoic acid (5,6-HMETE). The latter product is formed by beta-lyase-catalyzed cleavage of the cysteine C-S bond in leukotriene E4. Leukotriene E4 was also metabolized to 5,6-HMETE by rat cecal contents. A product formed was trapped as the iodoacetic acid derivative and identified as 5-hydroxy-6-S-carboxy-methylthio-7,9,11,14-eicosatetraenoic acid. It is concluded that intestinal leukotriene E4, originating from biliary excretion of systemic cysteinyl leukotrienes or produced in the intestine, is converted by microfloral cysteine-conjugate beta-lyase to 5,6-HMETE.     

Partial reduction of disulfide bonds in the hormone-specific subunits of TSH and LH.

Partial reduction at pH 7.0 of the hormone specific (β) subunit of either bovine thyrotropin or luteinizing hormone with dithioerythritol results primarily in the opening of a single disulfide bridge. The partially reduced subunits were alkylated with [1-¹⁴C] iodoacetic acid, followed by complete reduction and alkylation with non-radioactive iodoacetic acid. Isolation and degradation of the radioactive tryptic peptides shows that the bond primarily reduced in each β subunit links analogous half-cystine residues in the two sequences (88–95 in TSH-β and 93–100 in LH-β). These results are the first direct evidence of similar disulfide structures in hormone specific subunits of glycoprotein hormones.     

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.     

Effect of N,N′-bis (alkyldimethyl)-α,ω-alkanediammonium dibromides on bacteria of the genusclostridium

Antibacterial effect of 17 ammonium compounds of the type of N,N′-bis(alkyldimethyl)-α,ω-alkanediammonium dibromides was tested on anaerobically sporulating bacteria of the genusClostridium. A sizable antibacterial activity was displayed by five N,N′-bis(alkyldimethyl)-1,6-hexanediammonium dibromides and by four N,N′-bis(decyldimethyl)-α,ω-alkanediammonium dibromides. These compounds exhibited activity higher than, or comparable with, that of the reference standards Ajatin and Septonex. The maximum antibacterial activity was found in compounds whose alkyl chain contained 9–12 carbon atoms. Compounds with a lower number of carbon atoms in the chain (less than 8) exhibited a low activity.     

Survey of Chemical Compounds Tested In Vitro against Rumen Protozoa for Possible Control of Bloat

Over 170 chemical agents were screened for antiprotozoal action in bovine ruminal fluid. Compounds were tested at 0.1 and 0.05% concentrations. Tested compounds included inorganic compounds, antibiotics, biocides, neuromuscular agents, arsenicals, plant and animal hormones, antimalarials, surface-active agents, anthelmintics, and many others. The most active compounds were cupric sulfate, nickel sulfate, nitrofurazone, hydrogen peroxide, dodecyl sodium sulfate, pelargonic acid, iodoacetic acid, 1-diethylaminoethylamino-4-methylthiaxanthrone, sodium arsanilate, sodium arsenate, bismuth glycolyl arsanilate, 1-β-hydroxyethyl-2-methyl-5-nitroimidazole, and p-nitroaniline. Copper ion was not particularly effective against entodinia; nickel ion had no effect on holotrichs. Hydrogen peroxide and iodoacetic acid were effective at a concentration of 0.005%. Anionic surface-active agents were very effective, especially long-chain sulfates and phosphates. These antiprotozoal agents warrant further in vivo studies for possible use in treating or curing bloat in ruminants.     

Modification of alpha-crystallin subunit chains and formation of alpha-neoprotein molecules: role of SH groups

Immunoadsorbents with bound antibodies restricted to determinants dependent on alpha-crystallin's quaternary structure permitted the fractionation of the population of 125I-labeled alpha-crystallin molecules, treated by iodoacetic acid, into molecules in which the native structure was still preserved and molecules with a completely different quaternary structure than the native protein. Parallel experiments with [14C]iodoacetic acid yielded information on the percentage of blocked SH groups in each of the above two fractions. The presence of molecules formed by A with B-chain association was established by sequential binding first to an immunoadsorbent with antibodies restricted to determinants located on alpha-crystallin's A-subunit chains as ligand and second, after desorption, to an immunoadsorbent with antibodies to B chains as ligand. With the aid of these techniques, it was established that (i) The modified alpha-crystallin molecules with quaternary determinants of the native protein contained a maximum of 23% blocked SH groups, indicating that the carboxymethylation involved only the fast-reacting surface SH groups. (ii) The modified alpha-crystallin molecules without the native protein's quaternary structure were built by a different association between A and B subunits than in alpha-crystallin, indicating formation of alpha-neoprotein molecules. (iii) Monomeric A chains with all SH groups carboxymethylated, and monomeric B chains in a ratio of 1A:5B, 2A:1B, and 5A:1B in urea solution, associate on dialysis, forming alpha-neoprotein molecules.     

Characteristics of a highly thermostable neutral protease produced fromBacillus stearothermophilus

A highly thermostable neutral protease was found in culture filtrates ofBacillus stearothermophilus. The optimum reaction pH and temperature of this protease were 6.0 and 60°C, respectively, and 90% activity remained even after heat treatment at 90°C for 30 min. The protease was markedly inactivated by diisopropyl fluorophosphate, but EDTA and iodoacetic acid hardly affected it. The neutral protease therefore could be defined as a highly thermostable, neutral(-serine) protease.     

Exploitation of hormone-induced conformational changes to label selectively a component of rat liver plasma membranes.

The kinetics for inactivation of rat liver plasma membrane adenylate cyclase by iodoacetic acid and iodoacetamide has been measured in the presence and absence of glucagon. Glucagon stimulated the rate of iodoacetic acid inhibition by a factor 9f 2.3-fold and iodoacetamide inhibition by 10-fold. These results suggest that interaction of glucagon with its receptor in the membrane resulted in conformational changes which increased either the exposure or nucleophilicity of one or more sulfhydryl groups crucial for adenylate cyclase activity. Membranes were treated with radioactively labeled iodoacetamide or iodoacetic acid in the presence or absence of glucagon and run on 5 and 7.5% sodium dodecylsulfate polyacrylamide gels. These labeling experiments revealed that two membrane components were more extensively labeled in the presence of glucagon. The first component had an apparent molecular weight of 240,000 on sodium dodecyl sulfate gels and stained positive with Coomassie blue and periodate Schiff reagent. This polypeptide accounted for approximately 1.3% of the total membrane protein. The second component had an apparent molecular weight less than 10,000 and could not be correlated directly with a well defined protein band on sodium dodecyl sulfate polyacrylamide gels. The enhancement in labeling of the 240,000 molecular weight component seen in the presence of glucagon agreed very well with that predicted from the kinetics for inhibition of adenylate cyclase activity in the presence and absence of glucagon. This correlation suggests that the component selectively labeled by this technique may be an integral component of the adenylate cyclase system and that hormone-induced conformational changes may be used to selectively label components of the adenylate cyclase system in mammalian membranes.     

CARBOXYMETHYLATION OF SULPHYDRYL GROUPS IN PROTEOLIPIDS1

(1) The sulphydryl groups of brain white matter proteolipids were studied by alkylation with iodoacetic acid and iodoacetamide in an organic solvent medium. To make sterically hindered sulphydryl groups available, the reaction was also carried out in the presence of sodium dodecyl sulphate. (2) In all cases, iodoacetamide was a better alkylating agent than was iodoacetic acid. (3) Only minimal alkylation of crude white matter proteolipids was obtained in the absence of detergent; addition of sodium dodecyl sulphate increased the availablity of SH groups. (4) Purified proteolipids prepared by column chromatography were alkylated to a lesser degree than were crude proteolipids. (5) Prior reduction with mercaptoethanol resulted in the quantitative conversion of cysteine to S-carboxymethylcysteine with either alkylating agent and in both preparations. (6) The possibility of a conformational difference between the protein in the crude and purified preparations is discussed.     

Effects of lodine in Various Formulations on the Growth of Barley and Pea Plants in Nutrient Solution Culture

The forms of iodine added to cultures of barley were potassiumiodide, potassium iodate, potassium periodate, and iodoaceticacid at iodine concentrations of 1.0 ppm and 10.0 ppm. Withpea, only iodide and iodate at 1.0 ppm iodine concentrationwere used. For both species, comparisons were made with culturesto which no iodine was added. In barley, growth was increased by 1.0 ppm iodine, the relativeeffectiveness of the different formulations being in the order:iodoacetic acid > iodide > iodate > periodate. With10.0 ppm, iodide and iodoacetic acid treatments gave reducedgrowth, iodate was without effect, and periodate enhanced growth. In pea, 1.0 ppm iodine was inhibitory, iodide being more toxicthan iodate. Analysis of dry matter showed iodine content according to treatmentto be in the order: iodide > iodoacetic acid > iodate> periodate     

Alteration of Oxygen Consumption and Energy Metabolism During Repetitive Exposure of Mice to Hypoxia

Changes in oxygen consumption, body temperature and energy metabolism were studied while mice were repeatedly exposed to a sealed environment. The average tolerance limits of environmental oxygen level (vol%) and the average oxygen consumption rates (ml/g.min) were exponentially decreased and the average body rectal temperatures (°C) were linearly declined while the average tolerable times (min) to hypoxia were linearly increased as animals were repeatedly exposed to hypoxia for 5 runs. The average survival times (min) in sealed environments after administration of normal saline, iodoacetic acid, malonic acid, potassium cyanide, and potassium cyanide plus iodoacetic acid in group exposed repeatedly to hypoxia for three runs were, respectively, 3.1, 3.9, 1.4, 2.6, and 2.8 times those of the control groups that had corresponding administration of the different chemicals, but no exposure to hypoxia. The results indicate that progressive increase in hypoxia tolerance is related to progressively lower rate of oxygen consumption and heat production, and the lowered energy requirement during repetitive exposure to hypoxia is achieved mainly via pathways of the respiratory chain and glycolysis.     

Production of hybrid calluses via donor-recipient fusion between Microcitrus papuana and Citrus sinensis

X-ray irradiated embryogenic protoplasts of Microcitrus papuana Swing. were electrically fused with iodoacetic acid-treated embryogenic protoplasts of Newhall navel orange [Citrus sinensis (L.) Osb.]. Seven cell lines were established by low-melting agarose embedding culture of fusion-treated protoplasts. Cytological examination of 4 cell lines showed that each cell line consisted of many aneuploid (45.10%, 38.98%, 32.69% and 34.85%, respectively) and diploid cells (52.94%, 59.33%, 63.46% and 62.12%. respectively), whereas only a few tetraploid cells (1.96%, 1.69%, 3.85% and 3.03%, respectively) were detected. Analyses of random amplified polymorphic DNA with four 10-mer primers confirmed the hybrid characteristics of the cell lines, which in combination with chromosome counting proved that the cell lines were asymmetric hybrids. This revised version was published online in June 2006 with corrections to the Cover Date.     

Domain-level stability of an antibody monitored by reduction, differential alkylation, and mass spectrometry analysis

Human immunoglobulin G1 (IgG1) contains 12 domains, and each has an intrachain disulfide bond that connects the two layers of antiparallel β-sheets. These intrachain disulfide bonds are shielded from solvents under native conditions. Therefore, accessibility of the disulfide bonds to reduction under conditions that unfold antibody has the potential to be a good indicator of the thermodynamic stability of each domain. The stability of a recombinant monoclonal antibody at the domain level was investigated using a novel method involving reduction of the disulfide bonds in the presence of increasing amounts of guanidine hydrochloride and alkylation with [¹²C]iodoacetic acid, which was followed by reduction of the remaining disulfide bonds and alkylation with [¹³C]iodoacetic acid. The percentage of modification by [¹²C]iodoacetic acid of each cysteine residue was calculated using mass spectra of the cysteine-containing tryptic peptides and used to follow the unfolding of each domain. It demonstrated that the CH2 domain was the least stable domain of the antibody, whereas the CH3 domain was the most stable domain of the antibody. Other domains showed intermediate resistance to the denaturant concentration, similar to the overall unfolding transition monitored by the intrinsic tryptophan fluorescence wavelength shift.