Sulphydryl Inhibitors Research Articles

Platelet Aggregation by Thimerosal

Thimerosal, a sulphydryl inhibitor, induces aggregation of normal platelet rich plasma over a wide range of concentrations. Low doses induce a monophasic response preceded by a lag phase, high doses produce an immediate biphasic response. Thimerosal induces platelet aggregation through its binding by sulphydryl groups. Thimerosal induced aggregation is not mediated by ADP, it is not influenced by fibrinogen, von Willebrand factor, calcium, and magnesium ions of the medium. Thimerosal induced platelet aggregation is normal in patients affected by thrombocytopathia (defect of ADP release) but not in patients affected by Glanzmann's thrombasthenia. Mercaptopropionglycine, a substance which tends to preserve SH groups, inhibits platelet aggregation induced by thimerosal, thrombin, collagen, and ADP. A mechanism is proposed for thimerosal induced aggregation and the role of SH groups also in ADP, thrombin and collagen induced aggregation is indicated.

Microcalorimetric studies of tissue cells and bacteria

AbstractAnalytical microcalorimetric studies of cultures of Enterobacter aerogenes and Clostridium pasteurianum revealed a complex pattern of heat production. While both organisms gave thermograms which followed the exponential growth curves in the early stages, the later events in the heat effect appeared to reflect deficiencies in the growth medium and induction of “secondary” nutrients. Synchronously dividing Schizosaccharomyces pombe is known to give maxima in oxygen uptake during the cell cycle. These peaks are enhanced by the uncoupling agent, 2‐oxomalononitrile m‐chlorophenylhydrazone (CCCP). The rate of heat production in control cultures pumped through the LKB microcalorimeter increased at a linear rate, doubling over each complete cell cycle. Introduction of CCCP to cultures gave an altered thermogram in which the maxima of heat evolution which occurred corresponded in time to the peaks in oxygen uptake.When suspensions of muscle fibroblasts from 9 day old chick embryos were pumped through the aeration vessel, they produced heat at a rate which was proportional to cell concentration. Inhibitors of respiration decreased heat evolution by similar percentages to their effect on oxygen uptake. Cells of the same type in monolayer gave slightly lower levels of heat production. The thermogram was characterised by maxima over 4 h, which may be a reflection of temporal changes in intracellular ATP levels. When muscle fibroblasts were mixed with excess (zero order kinetics) ATP and pumped through the gold vessel of the microcalorimeter, the rate of heat production was proportional to cell concentration, the latter representing the hydrolysing enzyme. 4,4′‐Dithiodi(nicotinic acid), a sulphydryl inhibitor which does not enter cells, depressed the calorimetric response vs cell concentration curve by 78%, indicating that a sulphydryl enzyme was present at the cell surface. Batch calorimetric experiments to determine the heat of interaction of antibodies with cells gave thermograms which differed in form and value, the latter changing in sign and magnitude. The results illustrate the need for careful model experiments to disect this complex system. The present results underline the value of microcalorimetry in giving analytical data on growth and respiration in cells and organisms and illustrates the potential of the technique in studies of surface‐localised reactions.

Effects of sulphydryl reagents on an insect nerve-muscle preparation

The sulphydryl inhibitors N-ethyl maleimide (NEM), iodoacetic acid (IAA), p-chloromercuribenzoic acid (PCMB), and mersalyl at concentrations in the millimolar range blocked the neurally evoked mechanical response of an isolated nerve-muscle preparation from the grasshopper, Romalea microptera, and also blocked the contractions evoked by external application of l-glutamic acid, the suspected excitatory neurotransmitter. Blockade caused by PCMB could be reversed completely by post-perfusion with dithiothreitol (DTT); however, that caused by mersalyl was irreversible. Protection could be achieved by prior mixture of inhibitors with equimolar concentrations of the thiols, l-cysteine or reduced glutathione. The alkylating agents NEM and IAA also reduced the retractor unguis contracture caused by depolarization with high K + saline, whereas the organomercurials PCMB and mersalyl did not. The primary site of action appears to be the post-synaptic membrane. These findings indicate significant differences from the response of the eel electroplax preparation and suggest a functional rôle for free sulphydryl groups rather than disulphide linkages in this insect excitatory neuromuscular junction.

Glycolate oxidoreductase in Escherichia coli

Escherichia coli cells growing on glycolate as a sole source of carbon synthesise an enzyme which catalyses the oxidation of this compound to glyoxylate. The formation of glyoxylate from glycolate by extracts of such cells is dependent upon the presence of the artificial electron acceptors phenazine methosulphate and 2,6-dichlorophenolindophenol. The enzyme catalysing this conversion has been partially purified from cell extracts. The enzyme readily oxidised glycolate ( K m 4·10 −5M) and d(—)-lactate ( K m 7·10 −4M, but l(+)-lactate was oxidised only slowly. Maximum rates of glycolate oxidation were observed in the pH range 8.0–8.8. The enzyme was relatively insensitive to metal complexing agents but was sensitive to sulphydryl inhibitors. Addition of glycolate to the enzyme produced spectral changes characteristic of the reduction of flavin and cytochrome components, but no cofactors have been positively identified.

Microsomal NADPH-cytochrome c reductase from the housefly, Musca domestica: Properties of the purified enzyme

The characteristics of purified NADPH-cytochrome c reductase (E.C.1.2.6.3) from housefly microsomes have been studied. Like the mammalian reductase, the housefly reductase is specific for NADPH, utilizes 2,6-dichlorophenol-indophenol and ferricyanide as electron acceptor substrates, has a ping-pong reaction mechanism, and is competitively inhibited by NADP. Unlike the mammalian enzyme, the housefly reductase is much less sensitive to changes in ionic strength, has a lower affinity for substrates, and is more sensitive to sulphydryl inhibitors such as p-chloromercuribenzoate and N-ethyl maleimide.

Uptake of tritiated folates by human bone marrow cells in vitro.

Summary. Using incubation periods up to 4 hr, it was demonstrated that uptake of tritiated pteroylglutamic acid ([3H]PteGlu) by human bone marrow cells in vitro was in the range of six‐fold greater than uptake by reticulocytes. Uptake was temperature‐dependent, increasing during 4 hr at 37°C but not at 4°C; a similar temperature dependence was found for the uptake of [3H]methyltetrahydrofolate ([3H‐CH3]H4PteGlu). The pH optimum for [3H]PteGlu uptake was in the range of 7.4. Percentage uptake decreased as concentration of [3H]PteGlu increased. Preincubation with unlabelled PteGlu reduced uptake of subsequently added [3H]PteGlu by twice as much as did preincubation with methotrexate, suggesting that methotrexate may only partly share the uptake mechanism for [3H]PteGlu. [3H]PteGlu uptake was not affected by preincubation with diphenylhydantoin or a sulphydryl inhibitor. Uptake of [3H‐CH3]H4PteGlu by human bone marrow cells appeared to be approximately twice the uptake of [3H]PteGlu. The findings support the concept of two mechanisms for folate uptake by human reticulocytes and bone marrow cells: an energy‐dependent, active carrier mechanism probably of primary physiologic significance, and a seemingly passive diffusion‐like mechanism, probably primarily of pharmacologic significance.

Studies of the effect of sulphydryl and other inhibitors on reticulocyte uptake of doubly-labelled transferrin.

Summary. Inhibition of iron uptake from transferrin by reticulocytes has been further studied using doubly‐labelled 59Fe‐125I‐transferrin. The inhibition by p‐hydroxy mercuribenzoate, previously reported, is shown to occur also with the sulphydryl inhibitors p‐hydroxymercuriphenylsulphonate and N‐ethylmaleimide. The pattern of ‐SH inhibition is compared with and found to differ from that for two other inhibitors, trypsin, which alters membrane binding, and sodium fluoride, a metabolic inhibitor. When assessed by three variables, namely the effect of inhibitor concentration, the time of inhibitor exposure, and the effect on the rate of uptake, sulphydryl inhibitors are shown to act differentially on transferrin binding and on iron uptake. The latter is far more sensitive and is affected at lower concentrations. It is concluded that ‐SH groups are active in the dissociation of iron from its transferrin bond after surface binding has occurred. The effect of‐SH inhibitors on transferrin binding may be secondary to changes in membrane function induced by higher concentrations of these inhibitors.

Metabolic control mechanisms in mammalian systems. Regulation of pyruvate kinase in the rat cerebral cortex.

Abstract—The distribution of pyruvate kinase (ATP: pyruvate phosphotransferase; EC 2.7.1.40) in several areas of the central nervous system, the ontogenic changes in the cerebro‐cortical enzyme, and its modulation by certain metabolites were investigated in the rat. No significant differences in activity of pyruvate kinase in different regions of the nervous system were detected when activity was expressed per g of tissue. Studies on the ontogeny of pyruvate kinase in cerebral cortex revealed extremely low levels of enzymic activity at birth. A two‐fold increase occurred between 1 and 20 days of postnatal age, with a further two‐fold increase between 20 and 60 days of age. l‐Phenylalanine, when added directly into the reaction mixture, produced a dose‐dependent inhibition of cerebro‐cortical pyruvate kinase; a similar inhibition of the enzyme activity was observed with copper. The inhibition by l‐phenylalanine and copper was prevented and reversed by l‐alanine. Although the cerebro‐cortical enzyme was resistant to thermal inactivation at 37°C, incubation of the enzyme preparation at 55°C for 60 min resulted in approximately 60 per cent inhibition of its activity. Preincubation with l‐alanine provided partial protection against thermal inactivation of the enzyme. Although l‐alanine exerted no effect on the non‐competitive inhibition of pyruvate kinase produced by calcium, such inhibition was prevented and reversed by EDTA. The sulphydryl inhibitor, P‐chloromercuribenzoate, produced a dose‐dependent inhibition of cerebro‐cortical pyruvate kinase, whereas addition of penicillamine resulted in a slight activation of the enzyme. Although inhibition of pyruvate kinase by P‐chloromercuribenzoate was unaffected by l‐alanine, penicillamine effectively prevented and reversed the inhibition produced by P‐chloromercuribenzoate.

Effects of sulphydryl inhibitors on frog sartorius muscle: N-ethylmaleimide.

1. The characteristic action of the -SH inhibitor, N-ethylmaleimide (NEM), is muscle rigour. The dose-response curve indicates a biphasic effect with maximum rigour tension produced by 1.0 mM NEM; beyond 1.0 mM there was an inverse relationship between dose and response.2. NEM produces a membrane depolarization unrelated to rigour development.3. NEM causes a sustained increase in (45)Ca efflux from whole muscle. Pretreatment of a muscle with ethylenediamine tetra-acetic acid (EDTA, 5 mM) to remove membrane calcium does not alter the NEM induced (45)Ca efflux.4. It is suggested that the primary site of NEM action is inhibition of calcium uptake by the sarcoplasmic reticulum thereby producing rigour. At concentrations above 1.0 mM, NEM may affect the myofilaments.

Mechanism of enhanced streptokinase-induced clot lysis following in-vitro factor-XIII inactivation.

Summary. Factor‐XIII (fibrin stabilizing factor, FSF) activity in human plasma may be inhibited in vitro by a variety of sulphydryl inhibitors and by several derivatives of glycine. Incorporation of small amounts (40 units) of streptokinase (SK) into a clot formed from plasma after in vitro inactivation of Factor XIII, resulted in marked shortening of clot lysis times compared to controls without Factor‐XIII inactivation.Enhancement of SK‐induced clot lysis by the sulphydryl inhibitors is in part related to Factor‐XIII inactivation. Further acceleration of lysis is noted with concentrations greater than the minimal amount necessary for complete Factor‐XIII inhibition. At higher concentrations, clotting is delayed and ultimately completely inhibited. These reagents also produce marked changes on the thromboelastogram consistent with a weakened clot structure. No evidence to suggest increased plasminogen‐plasmin conversion, enhanced plasmin activity or inhibition of anti‐plasmin by sulphydryl inhibitors could be found.Glycine ethyl ester (GEE) incorporation results in marked enhancement of SK‐induced plasma clot lysis in vitro and pronounced changes on the thromboelastogram. The enhanced clot lysis is related to Factor‐XIII inhibition. No further acceleration of clot lysis was obtained once Factor‐XIII activity was completely inhibited. GEE alone shows an additional effect of inhibiting anti‐plasmin activity.Thus enhanced SK‐induced clot lysis following in vitro inhibition of Factor‐XIII activity with sulphydryl inhibitors and derivatives of glycine is considered to be due to the greater ease with which plasmin may act on a weakened clot. GEE, in addition to promoting the formation of a weaker clot, inhibits anti‐plasmin activity, and so further increases the relative effectiveness of streptokinase.

Effect of sulphydryl inhibition on the uptake of transferrin-bound iron by reticulocytes.

Iron transferrin, the highly stable carrier of plasma iron, may be dissociated, under physiological conditions, by sodium thioglycollate, a reaction which involves the formation of iron thioglycollate complexes. This suggested that sulphydryl radicals, which are the active groups in thioglycollate and related compounds, may play a part in the uptake of iron by immature red cells from iron transferrin. If this were so, it could be predicted that the sulphydryl inhibitor, p-hydroxymercuribenzoate, would depress the uptake of iron by reticulocytes. It is demonstrated here that inhibition of uptake of transferrin-bound iron by reticulocytes, which have been exposed to micromolar concentration of p-hydroxymercuribenzoate, is virtually complete. It is suggested that sulphydryl-containing compounds are intimately involved in the process of iron uptake by immature red cells.

Ion movements in human red cells independent of the sodium pump.

1. A study was made of the dependence on external Na of the movements of Na and K in human red cells. Special attention was given to ouabain-insensitive movements. The effect of internal Na on Na influx, and the influence of some sulphydryl inhibitors on ion movements and metabolism was also investigated.2. External Na stimulated ouabain-insensitive Na efflux and K influx. There was also a ouabain-insensitive component of Na influx that was raised on increasing the internal Na concentration. Exchange diffusion of Na appears to occur in the presence of ouabain and external K.3. Net transport of Na and K in the presence of ouabain was independent of external Na, as was also lactate production.4. Ethacrynic acid partially inhibited the Na pump; the Na-dependent components of Na efflux and K influx in the presence of ouabain were completely inhibited by ethacrynic acid. Both ouabain-sensitive and ouabain-insensitive adenosinetriphosphatase activities were inhibited by ethacrynic acid indicating a non-specific effect of this compound. Iodoacetamide decreased only the ouabain-insensitive ATPase activity.5. The results suggest that when the Na pump is blocked by ouabain, part of the residual ion movements can be attributed to exchange diffusion.

SODIUM‐INDUCED EFFLUX OF CALCIUM FROM BRAIN MICROSOMES

Abstract— A microsomal preparation from rat brain accumulated 45Ca in the presence of ATP. The uptake of calcium was associated with a corresponding uptake of 32P from [γ‐32P]ATP, with a 32P:45Ca molar ratio of about 0·65. Microsomes that were first loaded with 46Ca lost radioactivity during a subsequent incubation in the absence of ATP; efflux of previously accumulated 32P (from ATP) corresponded with the calcium efflux. By contrast, the uptake of 14C from [8‐14C]ATP was far less than of 32P, and the efflux did not follow calcium efflux. Thus the movement of 32P probably represented the phosphate anion in association with calcium. Calcium efflux diminished with increasing pH up to 8·6. Sodium specifically increased the rate of efflux of previously accumulated calcium. This sodium‐induced efflux was associated with a corresponding efflux of 32P (from ATP) and had a pH optimum near 7·8. It was not accompanied by a change in the amount of retained adenine nucleotides nor in the pattern of ATP metabolites, and was unaffected by ouabain or oligomycin. Low concentrations of certain sulphydryl inhibitors blocked the sodium‐stimulated efflux with little effect either on efflux in the absence of sodium or on 45Ca accumulation. Higher concentrations of these inhibitors, associated with an increase binding of the reagent, caused a generalized efflux of 45Ca and an inhibition of accumulation. The lipid solubility of a series of mercurial reagents corresponded with efficacy in promoting efflux; this suggested that this second class of reactive sites lay within a lipoidal permeability barrier.

Oestrogen binding sites in the chicken oviduct similar to those of the mouse uterus.

ABSTRACT Surviving oviducts and slices of the gizzard of 7–10 day old chickens were incubated at 37° C in a saline medium which contained tritium-labelled 17β-oestradiol. The oviducts took up more 17β-oestradiol than the slices of the gizzard. After washing the oviducts retained 10–20 times more 17β-oestradiol than the gizzard on a wet weight basis. The preferential uptake of 17β-oestradiol in the oviduct was reduced by dilution with unlabelled 17β-oestradiol and by several non-steroidal oestrogens. The optical isomers of the free phenol corresponding to Fenocyclin® and the stereoisomers of hexoestrol produced a quantitatively different inhibition of the uptake of 17β-oestradiol in the oviduct: the more oviductotrophic the isomer, the more potent was the inhibition of the uptake. Progesterone or testosterone did not inhibit the uptake in the oviduct. No hormone tested affected the residual concentration of 17β-oestradiol in the gizzard. A sulphydryl inhibitor, N-ethylmaleimide, inhibited the uptake and retention of 17β-oestradiol in the oviduct but not in the gizzard. These findings show that in vitro the oviduct takes up oestrogens in a very specific way. This was previously found to be the case with the mammalian uterus. A comparison between the present data on the chicken oviduct with those of the mammalian uterus reveals many similarities.

Selective effects of metabolic inhibitors on retinal cultures

Tissue cultures of retinas from young rats, rabbits, mice and chick embryos were treated with metabolic inhibitors in order to determine the sensitivity of the various retinal cell types to these agents. Sulphydryl blocking chemicals, i.e. mercurie compounds, arsenicals, arylating agents, alkylating agents and oxidizing agents, and sodium azide caused selective degeneration of all the rod cells without any apparent damage to the other cell types. A tenfold or more increase of the concentrations of these chemicals resulted in nonspecific damage to all the cell types in the explants. Sodium cyanide, sodium fluoride and other metabolic inhibitors did not reveal any such selective cytotoxic properties. N -Ethyl maleimide was the only sulphydryl inhibitor that did not cause selective rod cell degeneration. Nerve cells, neuroglial cells, cones and pigment epithelial cells in retinal cultures showed about the same sensitivity to the chemicals. The mesenchymal cells were the most resistant to the toxic agents. There was a large increase in the number of phagocytes and mast cells concomitant with exportmentally induced rod cell destruction. Nerve cells, neuroglial cells, and mesenchymal cells from the cerebellum and other tissues, however, did not show any such sensitivity to the metabolic inhibitors. Sulphydryl donors and the antioxidant dl -a-tocopherol protected the rod cells against the selective degeneration induced by the sulphydryl inhibitors. The sensitivity of the rod cells in culture increased during the first 8–10 days and did not change thereafter. Plasma clots and high serum concentrations both interfered with the inhibitors. Histochemical studies revealed changes of enzyme activities shortly after exposure to the inhibitors. At relatively low concentrations, the thiol inhibitors caused selective degeneration of all the rod cells in the retinal cultures; however, the same concentrations of inhibitors produced no effects in living animals. The reasons for this difference, as well as the similarities between experimentally induced rod cell degeneration and the degeneration of rod cells in tisstie cultures of dystrophie retinas, are discussed in the present publication. The most important difference between the latter two types of degeneration was the lack of cellular reaction accompanying rod cell degeneration in explants of dystrophic retinas.

Enzymic conversion of 3-hydroxyanthranilic acid to cinnabarinic acid by the leaves of Tecoma stans

An enzyme cinnabarinic acid synthase which catalyzes the oxidative dimerization of 2 moles of 3-hydroxyanthranilic acid to 1 mole of cinnabarinic acid has been partially purified from the leaves of Tecoma stans. The enzyme showed an absolute requirement for Mn++ ions. The optimum pH was around 8.0 and optimum temperature was 30°. The Km value is 3.6 × 10−4 M. Several divalent metal ions as well as metal chelating agents inhibited the reaction. The enzyme activity was highly susceptible to a variety of reducing agents. Sulphydryl inhibitors did not inhibit the reaction. Electron acceptors had no effect on enzyme activity, though cytochrome c slightly activated the reaction. Cytochrome c reduced by 3-hydroxyanthranilic acid was reoxidized by the enzyme preparation. The system was specific for 3-hydroxyanthranilic acid and cytochrome c reduced by o-aminophenol or ascorbic acid was not reoxidized.

Muscle Contraction produced by Sulphydryl Inhibitors

IT was recently reported1 that the sulphydryl (—SH) agent, parachloromercuribenzoate (PCMB), causes superprecipitation of actomyosin gel. Masking of an —SH group appears to prevent interaction between ATP and some inhibitory site on the protein, thus enabling ATP to induce superprecipitation through reaction with another site. If this phenomenon is related to changes occurring in the actomyosin system during muscle activity, then —SH agents should be expected to produce contraction. Accordingly, several such agents were tested on skeletal muscle, and they were found to cause various types of contractile phenomena.

The Effect of Sulphydryl and Enzyme Inhibitors on Platelet Aggregation in Vitro

SummaryThe glycolytic enzyme poisons potassium cyanide, sodium fluoride, and monofluoroacetate do not inhibit platelet clumping. The sulphydryl inhibitors, n-ethyl maleimide and p-hydroxymercuribenzoate, inhibit platelet aggregation produced by ADP, ATP, nor-adrenaline, 5HT, thrombin and connective tissue extract. This inhibitory activity is counteracted by 1-cysteine. These results suggest that platelet thiol groups are involved in the platelet adhesion occurring during aggregation.

Investigation of the mechanism of resistance to sodium arsenite in the blue tick, Boophilus decoloratus Koch

The glutathione and free sulphydryl levels of eggs and larvae of arsenic-resistant and arsenic-sensitive blue ticks were determined at intervals from 5 days after commencement of oviposition to approximately 18 days after hatching. From 15 to 20 days after commencement of oviposition the glutathione and free sulphydryl level was greater in the arsenic-resistant strain than in the sensitive strain. The combination of several known sulphydryl inhibiting materials with sodium arsenite resulted in a potentiation of arsenic toxicity in respect of adult female ticks. Arsenic-resistant adult female ticks were cross-resistant to some sulphydryl inhibitors.

Limitations in the histochemical demonstration of pyridine nucleotide-linked dehydrogenases (nothing dehydrogenase).

1. The ability of SH groups in well-dialyzed protein fractions to reduce exogenous pyridine nucleotides has been described in the biochemical literature. 2. This effect has been shown to be the cause of the positive histochemical result obtained when fresh tissue sections are incubated at alkaline pH levels with oxidized pyridine nucleotides (DPN or TPN) and tetrazolium salts, in the absence of any substrate. 3. The effect is pH-dependent, commencing at about pH 7.0 and rising to a maximum between pH 8.0 and 9.0. It is abolished by low concentrations of sulphydryl inhibitors or by heating the tissues. 4. The specific diaphorases are unaffected by sulphydryl inhibitors and these are presumed to transfer hydrogen and electrons from the reduced pyridine nucleotides (DPNH, TPNH) to the tetrazolium salt. 5. The development of histochemical methods for specific pyridine nucleotide-linked dehydrogenases is hindered by the activity described, to which Racker's convenient term "nothing dehydrogenase" has been applied.