SH Compounds Research Articles

Purification and characterization of thiol aminopeptidase from the cytosolic fraction of human placenta.

A thiol-dependent aminopeptidase was purified from the cytosolic fraction of human placenta. The purified enzyme consisted of a single polypeptide chain with a mol wt of 95,000. The enzyme was most active in the neutral region with Ala-pNA as substrate, and the activity was increased about 20-fold in the presence of some -SH compounds. The results of substrate specificity studies indicated that the enzyme hydrolyzes bonds involving the amino groups of neutral and basic amino acid residues. However, higher thiol-dependent activity was only detected with neutral ones. The enzyme was strongly inhibited by microbial aminopeptidase inhibitors, puromycin, o-phenanthroline, and sulfhydryl reactive-reagents. As to several naturally occurring peptides tested, the enzyme showed N-terminal Tyr-releasing activity toward enkephalins and kinin-converting activity.

False-positive results for ketone with the drug mesna and other free-sulfhydryl compounds.

All free-sulfhydryl compounds tested produced false-positive reactions in the Legal test for ketones. The color developed in the ketone pad of urine dipsticks [N-Multistix SG, Multistix 10 SG (Ames), and Chemstrip 9 (Boehringer-Mannheim)] was misinterpreted for ketone bodies, both by visual and automated reading. In contrast to the reaction with true ketones, a drop of glacial acetic acid added onto the ketone pad of dipsticks discharged the false-positive red color. A red-violet also developed instantly with free -SH compounds in the Acetest tablet assay (Ames), but quickly faded. In general, the presence of acidic groups such as -COOH and -SO3H in the structure appeared to increase the nitroprusside reactivity of free -SH compounds, whereas the presence of a -NH2 group appeared to decrease it. Currently, false-positive ketone reactions ascribable to a free -SH group are most likely to be seen for urine containing mesna. The false-positive test for ketones caused by free -SH compounds can be recognized and ruled out by proper procedures. On the other hand, this chromogenic reaction with free thiols might be used for monitoring urinary excretion of mesna.

Analysis and Frequency of Detection of Dichlofluanid in Foodstuffs

Dichlofluanid was determined at ppb levels in foodstuffs by ECD-GC using 4 kinds of liquid phases with no interference. Addition of silver nitrate to samples when they were homogenized with acetone was indispensable to protect dichlofluanid from decomposition caused by SH compounds which are present in foods, and in wheat an interfering substance showing a similar retention time to dichlofluanid was also eliminated by addition of silver nitrate. After evaporation of acetone, the residue was dissolved in n-hexane and the hexane solution was extracted with acetonitrile. After evaporation of acetonitrile, the residue was column chromatographed on Florisil. Investigation of commercially available vegetables, fruits and wheat showed a high frequency of detection of dichlofluanid in greenhouse products, especially tomatoes, though at low levels.

Purification and Characterization of Rice Lipoxygenase Component 3 from Embryos

Lipoxygenase-3, the major component of the enzyme in rice grain, was purified 2980-fold with a yield of 7% from embryos. The purified enzyme had a specific activity of 280 μmol O2 formed/min per mg protein. This enzyme was inactivated by SH compounds, such as cysteine and glutathione. The inactivation was prevented by the addition of catalase or replacement of the air by N2 gas. These two treatments were also effective for the stable storage of the purified enzyme. The molecular weights measured by sodium dodecyl sulfate gel and gradient gel electrophoresis were 93,000 and 89,000, respectively, indicating that the enzyme is a single polypeptide chain. The purified enzyme contained 0.73 Fe atom per molecule. The absorption spectrum suggested that the enzyme is a non-heme iron protein. Some similarities in amino-acid composition were observed between rice, soybean, and pea lipoxygenases. The purified enzyme specifically produced 9-d-hydroperoxy-10,12(E,Z)-octadecadienoic acid when linoleic acid was used as ...

Suppression of 7,12-dimethylbenz[ a]anthracene-induced chromosome aberrations in rat bone marrow cells by vegetable juices

A study was made of the in vivo effects of various vegetable juices on 7,12-dimethylbenz[ a]anthracene (DMBA)-induced chromosome aberrations (CA) in rat bone marrow cells. DMBA-induced CA consisted mainly of gaps and breaks. Exchanges were observed infrequently. Depending on the progressive severity of their chromosome damage, cells were classified into 4 catagories: cells with gaps only, cells with breaks, cells with exchanges, and cells with multiple CA (more than 10 aberrations). Multiple CA and the number of aberrations per cell, reflecting the severity of damage within a cell, were significantly suppressed by most vegetable juices investigated. The effect of fresh or boiled juices from 10 vegetables on the incidence of DMBA-induced aberrant metaphase cells (not including cells with gaps) revealed significant suppression of the incidence by fresh or boiled juice from onion, burdock, egg plant, cabbage and welsh onion. There was no difference between the effect of fresh juice or boiled juice except in the pumpkin. Fresh pumpkin juice, conversely, enhanced the incidence of aberrant cells, while boiled pumpkin juice significantly suppressed it. The present results may suggest that some vegetables, such as onion, suppress chemically induced cancer. Glutathione also suppressed DMBA-induced CA; it is, of course, well known that SH compounds analogous to GSH are plentiful in onion and welsh onion.

Angiotensin-converting enzyme inhibitors: new orally active 1,4-thiazepine-2,5-diones, 1,4-thiazine-2,5-diones, and 1,4-benzothiazepine-2,5-diones possessing antihypertensive activity.

The preparation of a series of 1,4-thiazepine-2,5-diones, 1,4-thiazine-2,5-diones, and 1,4-benzothiazepine-2,5-diones and their ability in inhibiting the activity of angiotensin-converting enzyme (ACE) in vitro and in vivo were examined. These compounds are assumed to act as prodrugs since they undergo rapid ring-opening reactions to give the corresponding biologically active free SH compounds when incubated with rat plasma or when treated with aqueous 0.1 N HCl or phosphate buffer (pH 7.4). The thiazepines 23-25 and 30 are potent inhibitors of ACE when administered po to rats and are comparable in potency to captopril (1). The most active thiazines in rats, po, were 42 and 45. Of the benzothiazepines studied, 22a was the most active in inhibiting ACE in the conscious normotensive rat, ID50 = 0.15 mg/kg, po. The acute antihypertensive effects of oral administration of a number of these compounds on mean arterial pressure and heart rate were studied in spontaneously hypertensive rats (SHR) maintained on a sodium-deficient diet.

Effects of various thiols on paraquat toxicity

1. 1. It was demonstrated that cysteine and d-penicillamine are able to replace reduced glutathione to some extent in the glutathione peroxidase reaction. 2. 2. An in vivo study was made of the role played by -SH compounds in the antioxidant enzyme system involved in the detoxication of the LD 50 of paraquat (PQ), and hence of their role in the detoxication of PQ. 3. 3. The effectiveness was d-PA > GSH > Cys in the liver and GSH > Cys > d-PA in the lung. Part XXVII of “Properties of enzymes”, serial publication.

Studies on antineoplastic activity of naphthomycin, a naphthalenic ansamycin, and its mode of action.

An antibiotic, identical with naphthomycin, was isolated from a soil Streptomyces. The antibiotic displayed significant therapeutic activity by ip administration against murine tumors: Ehrlich carcinoma and IMC carcinoma implanted ip. The maximum increase of life-span was more than 169% in Ehrlich carcinoma, and 128% in IMC carcinoma. The antibiotic exhibited a potent cytotoxicity against murine leukemic cells: P388, L1210, and L5178Y. IC50 was 0.4-1.3 microgram/ml in culture. The activity of naphthomycin was reversed by SH compounds: 2-mercaptoethanol, dithiothreitol, and glutathione. DNA and RNA syntheses were more markedly inhibited by naphthomycin than protein synthesis in L5178Y cells. Approximately 50% inhibition of nucleic acid syntheses was observed at an antibiotic concentration of 2 micrograms/ml. Naphthomycin blocked alkaline phosphodiesterase obtained from L5178Y cells: IC50 was ca. 7.6 micrograms/ml. The antibiotic neither caused metaphase arrest nor prevented tubulin polymerization. The results suggest that the mechanism of cytotoxicity of naphthomycin is the inhibition of various SH enzymes, particularly those involved in nucleic acid biosynthesis. The mode of action is unique in the ansamycin group of antibiotics.

Angiotensin-converting enzyme inhibitors. New orally active antihypertensive (mercaptoalkanoyl)- and [(acylthio)alkanoyl]glycine derivatives

A variety of N-substituted (mercaptoalkanoyl)- and [(acylthio)alkanoyl]glycine derivatives was synthesized and their ability in inhibiting the activity of angiotensin-converting enzyme (ACE) was examined in vitro and in vivo. The acylthio derivatives prepared are assumed to act as prodrugs since they are much less active than the corresponding free SH compounds in vitro and can be expected to act in vivo only after conversion to the free sulfhydryl compounds. A number of these compounds are potent ACE inhibitors that lowered blood pressure in Na-deficient, conscious spontaneously hypertensive rats (SHR), a high renin model. One of the most active members of the series was (S)-N-cyclopentyl-N-[3-[(2,2-dimethyl-1-oxopropyl)thio]-2-methyl-1 -oxopropyl]glycine (REV 3659-(S), pivopril). Structure-activity relationships are discussed.

Radio- and thermosensitivity of E. coli K1060 after thiol depletion by diethylmaleate.

The Escherichia coli auxotroph K1060 has been grown in a medium supplemented with either oleic acid (18:1) or linolenic acid (18:3) and its radiosensitivity and thermosensitivity established using bacterial cell survival as the assay system. No difference in radiosensitivity was observed when oleic and linolenic grown cells were exposed to gamma-radiation at room temperature. When heated at 49 degrees C linolenic grown cells were more sensitive than oleic grown cells. To investigate whether soluble -SH compounds, e.g., glutathione (GSH), were critical in protecting cells against radiation or heat, studies were performed using cells depleted of -SH by incubation with diethylmaleate (DEM). After reduction of water-soluble non-protein thiol compounds to 25% (10 mM DEM treatment) of control value, no major changes in radiosensitivity under oxic conditions were found. Radioresistance increased slightly when irradiation was performed under hypoxic conditions. Thermoresistance was clearly stimulated after DEM treatments between 1 and 10 mM DEM. The main conclusion of these experiments is that lowering the cellular level of reduced glutathione may not generally be correlated with a higher radio- and thermosensitivity.

Pulse radiolysis studies of WR-1065

WR-1065, the dephosphorylated sulfhydryl form of WR-2721 has been suggested as the metabolite responsible for the radioprotective abilities of the latter and as being active during the radiation chemical stage of damage production. Hence this study was performed to determine some of the radiation chemical parameters of the compound. Pulse radiolysis techniques, developed for use with SH compounds, were employed in the current work: Three systems were used to attempt to measure the rate constant for reaction of OH' radicals with WR-1065; (a) Competition with phenylalanine, in which the decrease in the amount of RSSR- caused by the addition of phenylalanine is monitored; this failed because of the absence of an absorbance attributable to RSSR-. (b) Competition with 2,2′-Azinobis-(3-ethylbenzthiazo6ne-6-sulfonate) (ABTS~ monitoring the decrease in OH' induced ABTS absorbance caused by WR-1065 addition. This was unsuccessful due to reaction of RS with ABTS. (c) Competition with CNS −, observing the decrease in (CNS) 2 −caused by WR-1065 addition indicates that the second order rate constant for reaction of OH' with WR-1065 is 9.2 ± 03 X 109 M −1 s −1 To investigate the ability of WR-1065 to react with DNA radicals the yield of ABTS radicals was monitored in a situation where DNA scavenges 50% of the OH' radicals in the presence of ABTS (20°6) and WR-1065 (3096). DNA radicals were shown not to react with ABTS but WR-1065 radicals do, the absence of additional ABTS absorbing species indicates that DNA radicals do not react with WR-1065 under the experimental conditions used. An attempt was made to investigate the possibility that WR-1065 is concentrated close to DNA macromolecules in solution: The rate constant for reaction Br 2 staggered− with WR-1065 was measured in the presence and in the absence of excess DNA (with which Br 2 staggered− was not observed to react). The observed rates of reaction were independent of the presence of DNA indicating that the latter does not affect the availability of WR-1065 for reaction with Br 2 staggered− radical. Reaction of WR-1065 with Br 2 − was used as a probe for determining the pK of the SH group. The rate of reaction increases from 1.8 × 10 8 M −1 s −1 at pH 6.3 to 1.6 × 109 M −1 s −1 at pH 8.4, the mid-point of the increase indicates a pK of 7.3.

A target-radical hypothesis of radiation damage fixation and modification in oxygenated and hypoxic cells

Radicals in radiation target molecules (presumed to be DNA), are produced by ionizing radiation in at least two ways: Solute and solvent radicals R’ react with target molecules, or ionizations in target molecules become radicals. T’. An intracellular radiation-chemical reaction scheme is therefore proposed in which solute and solvent radicals react with non-target molecules S. (scavengers) or with target molecules (DNA) to produce target radicals. The probability of target radical production is affected by scavenger molecules, and the probability of target radical decomposition to become “uncommitted damage” that the cell may repair is affected by the concentrations of 02, thiols and electronaff,nic sensitizers. which compete with one another to form target-radical products TO:, T&, and TL. This uncommitted damage is then subject to biochemical modification by the cell. The modification results in either “fixation” into F,T02 or F,TL (molecular lesions that are permanent) or enzymatic “repair” through T T’+L-TL:andT+&-T.$, which produce uncommitted chemical damage. Experimental data on strand-break induction in glutathioneproficient and -deficient cells. the inactivation of cells and the computer simulations generally agree that RSH molecules act as scavengers of solvent and solute radicals (R’) in an Ol-independent manner and react with target radicals to reverse T in competition with O2 and/or electronaffinic sensitizers It is postulated that the two categories of uncommitted target lesions TO2 and TL are preferentially repaired by different pathways. Evidence favoring this notion is available in existing literature. Reported values of dose-modification factors when DNA strandbreakage (uncommitted damage) and ceil survival (fixed, unrepaired damage) are the end-points and cells are treated with 02, SH compounds, SH depletion. or electronafTinic sensitizers are compared with calculated values to evaluate the plausibility of the proposed reaction scheme.

Enhancement of neutrophil response by SH-containing compounds: Modulation of superoxide and hydrogen peroxide production

Anti-inflammatory effects of SH compounds in vivo and their effects on lymphocytes and macrophages in vitro have been described, but little is known about the mechanism of action or their effects on the neutrophil. In the present study the activity of seven low molecular weight non-protein SH compounds was compared. At a concentration of 3 × 10 −4M all the compounds enhanced the activity of the HMP shunt of zymosan-stimulated neutrophils by 26–48% and that of PMA-stimulated cells by 6–44% above the control value (14.2 nmol CO 2/2.5 × 10 6neutrophilsJ30 min). Pretreatment of neutrophils with SH compounds for 15 min resulted in enhanced release of O 2 − by stimulated neutrophils in all cases, with the exception of GSH, by up to 87% above that of control. These effects were largely related to the ability of the compounds to modulate the release of O 2 − and H 2 by stimulated neutrophils when present in the reaction mixture. Only the compounds α-MPG and cysteine had a mild preserving effect on the intracellular GSH concentration of stimulated neutrophils. None of the compounds tested had any adverse effect on phagocytosis or killing of opsonized bacteria by the neutrophils. SH compounds may protect sensitive SH groups of functional proteins by providing an easily accessible source of oxidizable SH groups in times of high oxidative stress, and their ability to interact with oxygen products could in part explain their anti-inflammatory properties.

Purification and properties of indole 2,3-dioxygenase from maize leaves

An indole 2,3-dioxygenase was purified ca 38-fold from maize leaves. The enzyme had an MW of about 98000, an optimum pH of 5.0 and the energy of activation was 9.1 kcal/mol. The K max for indole was 1.4 × 10 −4 M. The enzyme was inhibited by diethyldithiocarbamate, salicylaldoxime and sodium dithionite. The inhibition by diethyldithiocarbamate was specifically reversed by Cu 2+. The dialysed enzyme was stimulated by Cu 2+. Four atoms of oxygen were utilized in the disappearance of 1 mole of indole. Inhibition of the enzyme by -SH compounds and -SH group inhibitors, and their partial removal by Cu 2+ only, suggested the involvement of -SH groups in binding of Cu 2+ at the catalytic site.

Experimental methyl mercury neurotoxicity: Similar in vivo and in vitro perturbation of brain cell-free protein synthesis

Perturbation of brain protein synthesis by methyl mercury chloride (MeHg) was compared in vivo and in vitro. MeHg-stimulated and/or inhibited brain cell-free protein synthesis following in vivo or in vitro administration. Although pretreatment with GSH protected the postmitochondrial supernatant (PMS) from the in vitro inhibition, direct addition of -SH compounds did not reverse the in vivo or in vitro perturbations in synthesis induced by MeHg. Inhibition of synthesis induced by both in vivo and in vitro methyl mercury administration resulted in inactivation of component(s) in brain pH 5 enzymes. Stimulation of amino acid incorporation following in vivo administration of MeHg was apparently associated with the ribosome fraction, but in vitro preincubation of PMS with MeHg produced stimulation associated with the pH 5 enzyme fraction. A model of MeHg neurotoxicity was proposed providing a common molecular locus of interaction in vivo and in vitro.

Ultraviolet-Induced Sister Chromatid Exchanges in V-79 Cells with Normal and BrdUrd-Substituted DNA and the Influence of Intercalating Substances and Cysteine

The influence of intercalating substances (proflavine, ethidium bromide) and of an SH compound (L-cysteine) on uv-induced sister chromatid exchanges (SCEs) was investigated in V-79 cells with normal and BrdUrd-substituted DNA. The results are discussed in relation to the primary damages leading to SCE induction produced by uv irradiation. The data indicate that neither the pyrimidine dimers nor DNA single-strand breaks are the primary cause of SCE induction, and that the damages leading to SCEs by uv irradiation differ from those which cause chromosome aberrations.

The effect of sulfhydryl compounds on sister-chromatid exchanges: II. The question of cell specificity and the role of H 2O 2

In contrast with earlier report on the induction of sister-chromatid exchanges (SCEs) by SH compounds in cell lines of the Chinese hamster, cysteine, cysteamine and cystamine did not cause an increase of the SCE frequency in human lymphocyte cultures. Differences in the treatment protocols or variations of the Brd Urd concentration had no effect on the induction of SCEs by these substances. The inclusion of H 2O 2 and comparative investigations with V79 cells of the Chinese hamster showed that the probable reason for the SCE induction by SH compounds is the inability of the cells to degrade H 2O 2. Furthermore, for cystamine it became clear that additional effects must exist besides the induction of SCEs through H 2O 2. The present study underlines the fact that the examination of a substance within one cell system does not necessarily permit a reliable statement about the DNA-damaging property of this substance.

Characterization of organomercury-decomposing activity in cell extract of mercury-resistant Clostridium cochlearium T-2P

Organomercury-decomposing activity in Clostridium cochlearium T-2P harboring a plasmid which mediates mercury resistance was studied. The results obtained indicated that the splitting of the CHg bond was catalyzed by an enzyme present in the cell extract which requires glucose, but not SH compounds, for its activity. The ability to degrade organomercurials was induced specifically by mercury compounds, particularly by organic mercury, but not by other metal ions. Alkylmercury was a better substrate for the decomposing enzyme than arylmercury. The properties of the decomposing enzyme were very different from those of the other splitting enzymes reported so far.

Induction of auto-immune syndromes by penicillamine therapy in rheumatoid arthritis and other diseases.

Penicillamine and some structurally related compounds have been shown to be inducers of autoimmune phenomena when administered to patients, regardless of the underlying disease for which they are being given. Most of these reactions are serious and often mandate discontinuance of treatment, but the development of a particular adverse reaction to one SH compound does not necessarily mean that others will elicit a similar or indeed any adverse effect. Of great theoretical interest is the mechanism by which penicillamine, as the prototype of this class, results in apparent breakdown of tolerance to self-antigens. No clear answer to this extremely important question is forthcoming, probably due to limitations in contemporary methodology. It is believed that if the mechanism of these drug-induced events were understood, new insights into the immunologic aberrations in RA might be achieved.

A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase

A new and convenient method for the determination of P i was developed. Phosphomolybdate is measured colorimetrically, without reduction to molybdenum blue, by dissolving the whole assay mixture in acetone, where phosphomolybdate is bright yellow. The hydrolysis of acid-labile phosphates (e.g., creatine phosphate) causes no problems, because extra molybdate is complexed with citrate immediately after the color has been developed. Strong reductants and SH compounds which interfere, if present in high concentrations, are eliminated by adding H 2O 2. Detergents, organic bases, protein, and sucrose do not interfere. The assay is as sensitive as most modifications of the Fiske-SubbaRow method. In the routine procedure the useful range is 50–1500 nmol of P i. The application of the method to the assay of inorganic pyrophosphatase in the cells of Escherichia coli is described.