Effect of scorpion venom, tityustoxin, on the release of acetylcholine from incubated slices of rat brain

Abstract— Purified tityustoxin (TsTX) from the venom of the scorpion, Tityus serrulatus, when incubated in vitro with slices of rat cerebral cortex, increased the amount of free ace-tylcholine (ACh) in the incubation medium and, simultaneously, reduced the amount of bound ACh in the slices. The effect was optimal at pH 7.4 and was dependent upon time of incubation, an energy source and the concentration of toxin. Tityustoxin increased the synthesis of ACh, but this effect seemed to be related to an increase in the release of ACh. The effect of the TsTX was independent of the concentration of K⁺ ion but was dependent on the presence of Na⁺ and Ca²⁺ in the incubation medium. Hexamethonium and hemicholinium reduced the effect of tityustoxin, but cocaine had no effect on the release of ACh stimulated by the TsTX. Tetrodotoxin blocked completely the stimulation caused by the tityustoxin. We suggest that probably both tityustoxin and tetrodotoxin exert different and antagonistic effects competing in the Na⁺ channels.     

Destruction of photosystem I iron-sulfur centers of spinach andAnacystis nidulans by mercurials

Several mercurials destroyed Photosystem I (PSI) Fe−S centers in thylakoids and PSI particles from spinach and fromAnacystis nidulans as revealed by EPR measurement and acid-labile sulfide determination. Of the mercurials tested, HgCl₂ was the most effective, followed by phenylmercuric acetate (PMA), Mersalyl and pCMB in the order of decreasing effectiveness. Fe−S centers in thylakoids were much more labile than those in PSI particles. InA. nidulans thylakoids, Center B was more susceptible than Center A and X to PMA. P700 was less susceptible to PMA than these centers. For 50% inactivation of Fe−S centers inA. nidulans thylakoids, about 0.4 mM PMA was required for Center B, and about 1 mM was required for Center A and X. These differential susceptibilities of Fe−S centers were more pronounced with HgCl₂ than with the other three mercurials.     

Induction of Metallic Mercury-releasing Enzyme in Mercury-resistant Pseudomonas

The induction of metallic mercury-releasing enzyme (MMR-Enz) which catalyzes the reduction of mercurials to metallic mercury was studied. The formation of MMR-Enz was induced when the organism was grown with mercurials such as phenyl mercuric acetate (PMA), p-chloromercury benzoate (pCMB), sodium ethyl mercuric thiosalicylate (merzonin), mercuric chloride (MC) and metallic mercury, but it was not induced when grown with HgS or other metals such as Ag⁺, Cd²⁺, Cu²⁺, Fe³⁺, Co²⁺, Sn²⁺, etc. Cd²⁺ and Cu²⁺ were inhibitory for the formation of MMR-Enz. d-glucose: NAD oxidoreductase (EC 1.1.1.47 GDH) or l-arabinose: NADP oxidoreductase (EC 1.1.1.46 ADH) and cytochrome c-I (cyt. c-I) which were all involved in the decomposition of mercurials together with MMR-Enz were found in the crude extract regardless of mercurial addition. These results suggest that MMR-Enz plays a conclusive role on the decomposition reaction of mercurials.     

Water exchange through erythrocyte membranes: Biochemical and nuclear magnetic resonance studies re-evaluating the effects of sulfhydryl reagents and of proteolytic enzymes on human membranes

Summary The water permeability of human red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on intact cells and resealed ghosts following exposure to various sulfydryl-reacting (SH) reagents and proteolytic enzymes. The main conclusions are the following: (i) When appropriate conditions for exposure of erythrocytes or ghosts to mercury-containing SH reagents (concentration, temperature and duration of incubation) were found, the maximal inhibition of water diffusion could be obtained with all mercurials (including HgCl₂ and mersalyl that failed to show their inhibitory action on RBC water permeability in some investigations). While previous studies claimed that long incubation times are required for the development of maximal inhibition of water diffusion by mercurials, the present results show that it can be induced in a much shorter time (5–15 min at 37°C) if relatively high concentrations of PCMBS (2–4mm) are used and no washings of the inhibitor are performed after incubation. Higher than optimal concentrations of mercurials and/or longer incubation times result in lower values of inhibition, sometimes a loss of inhibition, or can even lead to higher values of permeability compared to control RBCs. (ii) The conditions for inhibition by mercurials are drastically changed by preincubation of erythrocytes with noninhibitory SH reagents (such as NEM or IAM) or by exposure to proteolytic enzymes. If the cells are digested with papain, the duration of incubation with PCMBS should be decreased in order for inhibition to occur. This explains the lack of inhibition reported previously, when a relatively long duration of incubation with PCMBS was used subsequent to papain digestion. (iii) The degree of inhibition of water diffusion induced by mercurials appeared to be dependent upon the temperature of which the water permeability was measured. The values of maximal inhibition ranged from 45–50% at 37°C, increased 10–15% at 20°C and further increased at lower temperatures, reaching values above 75% below 10°C; these results clarify the conflicting reports of various authors. (iv) The inhibition of water diffusion, either reversible, or irreversible, was not accompanied by significant changes in the pattern of RBC membrane polypeptides fractionated by polyacrylamide gel electrophoresis. (v) The mean value of the activation energy of water diffusion (E _a,d) obtained on 42 donors was 25.6 kJ/mol. The values ofE _a,d increased in parallel with the values of the inhibition of water diffusion induced by PCMBS until the maximal inhibition was reached (whenE _a,d=41 kJ/mol) and then both sets of values decreased in parallel.     

The Effects of Scorpion Venom Toxin on the Release of Amino Acid Neurotransmitters from Cerebral Cortex In Vivo and In Vitro

Tityustoxin, the active component of the venom of the Brazilian yellow scorpion Tityus serrulatus, caused specific release of the neurotransmitter amino acids glutamate, aspartate and GABA in vivo from the superfused sensori-motor cortex of conscious unanesthetised rats and in vitro from rat cortical synaptosomes. The effects on synaptosomes appear to be due to a depolarising action. Synaptosomal potassium levels were depleted by the toxin. The action was also blocked both in vivo and in vitro by tetrodotoxin and was Ca2+-dependent. The uptake of [U-14C]GABA was inhibited by tityustoxin but this action was prevented by tetrodotoxin (1 microM). Since the release of [U-14C]GABA from synaptosomes due to the tityustoxin was also prevented by tetrodotoxin under identical circumstances, it is concluded that the tityustoxin has a primary action on release of neurotransmitters rather than on uptake.     

Action of mercurials on the active and passive transport properties of sarcoplasmic reticulum

The effect of Hg²⁺ and Ch₃-Hg⁺ on the passive and active transport properties of the Ca²⁺-Mg²⁺-ATPase-rich fraction of skeletal sarcoplasmic reticulum (SR) is reported. The agents abolish active transport, at 10^–5 and 10^–4 M concentrations, respectively. Addition of the mercurials was also shown to release actively accumulated Ca²⁺. The mercurials increase the passive Ca²⁺ and Mg²⁺ permeability in the absence of ATP at the same concentrations at which they inhibit transport. It is proposed that both effects are the result of direct binding of the mercurials to the SH groups of the Ca²⁺-Mg²⁺-ATPase pump, altering the conformational equilibria of the pump. The agents were also shown to increase the passive KCl permeability. The SR preparation consists of two vesicle populations with respect to K⁺ permeability, one with rapid KCl equilibration faciliated by a monovalent cation channel function and one with slow KCl equilibration. The mercurials increase the rates of KCl equilibration in both fractions, but produce higher rates in the fraction containing the channel function. The results are discussed in terms of pump and channel function and are compared with results for the electrical behavior of the Ca²⁺-Mg²⁺-ATPase and other SR proteins in black lipid membranes, as presented by others.     

Mobilization of the Readily Releasable Pool of Acetylcholine from a Sympathetic Ganglion by Tityustoxin in the Presence of Vesamicol

The present experiments tested whether preganglionic stimulation and direct depolarization of nerve terminals by tityustoxin could mobilize similar or different pools of acetylcholine (ACh) from the cat superior cervical ganglia in the presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol, AH5183), an inhibitor of ACh uptake into synaptic vesicles. In the absence of vesamicol, both nerve stimulation and tityustoxin increased ACh release. In the presence of vesamicol, the release of ACh induced by tityustoxin was inhibited, and just 16% of the initial tissue content could be released, a result similar to that obtained with electrical stimulation under the same condition. When the impulse-releasable pool of ACh had been depleted, tityustoxin still could release transmitter, amounting to some 10% of the ganglion's initial content. This pool of transmitter seemed to be preformed in the synaptic vesicles, rather than synthesized in response to stimuli, as tityustoxin could not release newly synthesized [3H]ACh formed in the presence of vesamicol, and hemicholinium-3 did not prevent the toxin-induced release. In contrast to the results with tityustoxin, preganglionic stimulation could not release transmitter when impulse-releasable or toxin-releasable compartments had been depleted. Our results confirm that vesamicol inhibits the mobilization of transmitter from a reserve to a more readily releasable pool, and they also suggest that, under these experimental conditions, there might be some futile transmitter mobilization, apparently to sites other than nerve terminal active zones.     

The kinetics of electron entry in cytochromec oxidase

Summary The kinetics of electron entry in beef heart cytochromec oxidase have been studied by stopped-flow spectroscopy following chemical modification of the Cu_A site with mercurials. In this derivative Cu_A is no longer reducible by cytochrome c while cytochromea may accept electrons from the latter with rates comparable to the native enzyme. The results indicate that Cu_A is not the exclusive electron entry site in cytochromec oxidase.     

Control Mechanisms at the Level of FDP-Aldolases in Algae

Abstract

The FDP-aldolase I from Euglena gracilis is a four-chain enzyme, as shown by the amino acid analysis of the C and N terminals. The protein is dissociated by acid pH to a monomer. The reassociation-dissociation process goes through a dimer stage. The enzyme, inhibited either by mercurials or by ATP, is dissociated to a dimer. This process is readly reversible either by mercaptoethanol and by AMP.     

The action of organic mercurials on the erythrocyte membrane

The solubilisation of proteins from erythrocyte membranes by treatment with organic mercurials has been studied with different species. The marked solubilisation previously reported for human membranes does not seem to be a general phenomenon. All of the other species examined showed less than 50% of the solubilisation shown by human membranes. The protein-solubilising effect seems to be dependent on hydrophobic mercury derivatives carrying a net negative charge. Uncharged compounds like phenylmercuric acetate blocked the effect, although $\text{N-}\text{ethylmaleimide}$ and iodoacetamide did not. With the aid of radioactively labelled compounds, and of atomic absorption spectrophotometry, the proteins reactive towards the mercurials were identified. The major integral protein, band 3, was the major protein capable of binding the mercurial. Reaction with the mercurial appears to disrupt interaction of band 3 with bands 2.1 and 4.2, allowing dissociation of the cytoskeleton from the membrane. In addition, band 4.9 was also found to react with the mercurials, possibly resulting in disruption of the cytoskeleton.     

A new class of chromophoric organomercurials and their reactions with d-glyceraldehyde 3-phosphate dehydrogenase

THE SYNTHESES OF THE FOLLOWING ORGANOMERCURIALS ARE DESCRIBED: 2-chloromercuri-4-nitrophenol, 2-chloromercuri-4,6-dinitrophenol, 4-chloromercuri-2-nitrophenol and 2,6-dichloromercuri-4-nitrophenol. All four organomercurials show large spectral changes in the visible spectrum when thiols displace a more weakly bound ligand such as EDTA from the mercury atom. These spectral changes are primarily associated with pK perturbation of the nitrophenols. The mercurials are therefore chromophoric probes for thiol groups in proteins and other thiols of biological interest. The enzyme d-glyceraldehyde 3-phosphate dehydrogenase from lobster muscle is used as a model system in which the properties of the organomercurials may be illustrated. In particular it is shown how d-glyceraldehyde 3-phosphate dehydrogenase carboxymethylated at the active site may be mercurated at a specific site. This mercurial derivative may be crystallized and shown to be isomorphous with the parent enzyme. The mercurials also act as ;reporter groups' by monitoring phosphate or pyrophosphate binding to the enzyme. The mercurials may also be used to estimate cations by an EDTA displacement method.     

Antifungal action of alkoxy ethyl mercuric acetates

The fungitoxic action of alkoxy ethyl mercuric acetates and phenyl mercuric acetate was studied withAspergillus niger, Penicillium italicum andUstilago maydis as test moulds. Concentrations of mercurials from 10^–3 m to 10^–5 m were used in each experiment. The inhibitory action was about the same for each of the compounds tested.     

Characterization of urease from the phototrophic bacteriumRhodobacter capsulatus E1F1

Rhodobacter capsulatus E1F1 showed high cytosolic urease activity when growing on urea, purines, and purine metabolites as nitrogen source. Molecular mass ofR. capsulatus enzyme is similar to that of other bacteria and greatly differs from that of jack bean. Kinetic parameters of partially purifiedR. capsulatus enzyme resemble those described in other bacterial ureases. The activity was inhibited by metal-chelating agents and by mercurials. Urease fromR. capsulatus E1F1 was negligible in nitrogen-starved cells or in cells cultured with nitrate, ammonium, or amino acids. Moreover, ammonium inhibited both the urea uptake and the urease activity expression inR. capsulatus cells.     

Some differences in the conjugation of o-aminophenol and p-nitrophenol by the uridine diphosphate transglucuronylase of mouse-liver homogenates

1. A study of the catalysis of the formation of the glucuronides of o-aminophenol and p-nitrophenol by the uridine diphosphate transglucuronylase of homogenates of female mouse liver has been made, with reference to the effect of reagents reacting with thiol groups. 2. The synthesis of both glucuronides was completely inhibited by organic mercurials and N-ethylmaleimide. The inhibition was only partial with arsenite and the arsenoxides, iodoacetamide and o-iodosobenzoate. 3. The o-aminophenol system was much more sensitive than that for p-nitrophenol to all the thiol reagents, except N-ethylmaleimide, which was equally active in both systems. 4. At very low concentrations of the organic mercurials, the o-aminophenol system was activated. 5. With o-aminophenyl glucuronide formation, complete protection was given by glutathione and cysteine against the organic mercurials, N-ethylmaleimide and iodoacetamide, and partial protection against the arsenicals. Reversal was complete against the mercurials, and very limited against the arsenicals and iodoacetamide. The effects of N-ethylmaleimide and o-iodosobenzoate were irreversible. Results with p-nitrophenol were very similar. 6. Uridine diphosphate transglucuronylase was partially protected against p-chloromercuribenzoate and lewisite oxide by uridine diphosphate glucuronate, but not by o-aminophenol. 7. Glutathione did not prevent the decline in the rate of conjugation of o-aminophenol when homogenates were aged by incubation at 30°. Cysteine was unable to prevent or reverse inactivation by ultrasonic radiation.     

The inhibition of the uridine diphosphate-transglucuronylase activity of mouse-liver homogenates by thiol reagents

1. A study of the catalysis of the formation of the glucuronides of o-aminophenol and p-nitrophenol by the uridine diphosphate transglucuronylase of homogenates of female mouse liver has been made, with reference to the effect of reagents reacting with thiol groups. 2. The synthesis of both glucuronides was completely inhibited by organic mercurials and N-ethylmaleimide. The inhibition was only partial with arsenite and the arsenoxides, iodoacetamide and o-iodosobenzoate. 3. The o-aminophenol system was much more sensitive than that for p-nitrophenol to all the thiol reagents, except N-ethylmaleimide, which was equally active in both systems. 4. At very low concentrations of the organic mercurials, the o-aminophenol system was activated. 5. With o-aminophenyl glucuronide formation, complete protection was given by glutathione and cysteine against the organic mercurials, N-ethylmaleimide and iodoacetamide, and partial protection against the arsenicals. Reversal was complete against the mercurials, and very limited against the arsenicals and iodoacetamide. The effects of N-ethylmaleimide and o-iodosobenzoate were irreversible. Results with p-nitrophenol were very similar. 6. Uridine diphosphate transglucuronylase was partially protected against p-chloromercuribenzoate and lewisite oxide by uridine diphosphate glucuronate, but not by o-aminophenol. 7. Glutathione did not prevent the decline in the rate of conjugation of o-aminophenol when homogenates were aged by incubation at 30°. Cysteine was unable to prevent or reverse inactivation by ultrasonic radiation.     

Interactions of fluorescent mercurial compounds and acidic fluorochromes with isolated living cells and nuclei

Summary Two fluorescent mercurials (fluorescein mercuric acetate and merbromin) and two acidic fluorochromes (brilliant sulfoflavine and primuline) were tested as supravital fluorochromes and compared with the fluorescent probe for hydrophobic groups, 8-anilino-1-naphthalene-sulfonic acid (ANS). Neither the mercurials nor the acidic fluorochromes appeared to penetrate intact cells, but all of the dyes fluorochromed damaged cells in a characteristic fashion. Expriments were then undertaken on nuclei isolated in 0.25 M sucrose. The fluorescent mercurials produced fluorescence of the nuclear envelope and nucleoli. More generalized fluorescence was induced if nuclei remained for prolonged periods in saline solutions balanced for intact cells or in nuclei exposed to 0.2 N hydrochloric acid. Acidic fluorochromes produced a more generalized distributional pattern of fluorescence. Primuline produced substantially more intense nuclear fluorescence than brilliant sulfoflavine at equimolar concentrations. Considered as a whole, these results indicate that an examination of the interaction of fluorescent dyes with unfixed cellular components could prove to be a useful tool in cell biology, particularly in the investigation of nuclear function.Supported in part by GRS-FR-5394 to the Albany Medical College.     

Properties of the inner membrane anion channel in intact mitochondria

The mitochondrial inner membrane possesses an anion channel (IMAC) which mediates the electrophoretic transport of a wide variety of anions and is believed to be an important component of the volume homeostatic mechanism. IMAC is regulated by matrix Mg²⁺ (IC₅₀=38 µM at pH 7.4) and by matrix H⁺ (pIC₅₀=7.7). Moreover, inhibition by Mg²⁺ is pH-dependent. IMAC is also reversibly inhibited by many cationic amphiphilic drugs, including propranolol, and irreversibly inhibited byN,N-dicyclohexylcarbodiimide. Mercurials have two effects on its activity: (1) they increase the IC₅₀ values for Mg²⁺, H⁺, and propranolol, and (2) they inhibit transport. The most potent inhibitor of IMAC is tributyltin, which blocks anion uniport in liver mitochondria at about 1 nmol/mg. The inhibitory dose is increased by mercurials; however, this effect appears to be unrelated to the other mercurial effects. IMAC also appears to be present in plant mitochondria; however, it is insensitive to inhibition by Mg²⁺, mercurials, andN,N-dicyclohexylcarbodiimide. Some inhibitors of the adenine nucleotide translocase also inhibit IMAC, including Cibacron Blue, agaric acid, and palmitoyl CoA; however, atractyloside has no effect.     

Release of Formaldehyde from Dimethylol Dimethylhydantoin,a Possible Antiseptic Agent

Dimethylol dimethylhydantoin (abbreviated as DMDMH), a water-soluble, stable, colorless and odorless hydantoin derivative, was investigated with various microorganisms for its potential applicability as an antiseptic agent. The possible mechanism of its action was elucidated. DMDMH inhibits bacterial and fungal growth at 15.6 to 250 µg/ml, and HeLa S3 cells at 13.7 µg/ml by its continuous presence. Its removal after 1-hr incubation at 5,000 µg/ml reversed the bacterial growth, thus indicating bacteriostatic action. The mechanism of action, in a study using a proton nuclear magnetic resonance, revealed a release of formaldehyde from DMDMH when pH was increased. Effect of DMDMH on antigen-antibody reaction in agar gel showed no adverse effect at moderate concentration. Since its toxicity to animal was much less than that of mercurials or phenol, it can be useful as an antiseptic agent.     

Role of hydrogen sulfide in mercury resistance determined by plasmid of Clostridium cochlearium T-2

Mercury resistance of Clostridium cochlearium T-2P was found to be controlled by a different mechanism from those reported so far since no mercury-reducing activity was detected in this strain. The H₂S generating ability as well as the demethylating activity of this bacterium was eliminated by the treatment with acridine dye and recovered by the conjugation of the cured strain with the parent strain. In addition, the strain which lost their abilities to generate H₂S and to decompose methylmercury, showed higher sensitivity to mercurials than the parent strain. From these results, the genes conferring both the activities seemed to reside on the plasmid and the mechanism of mercury resistance was probably based on a detoxification mechanism involving methylmercury decomposition and inactivation of the inorganic mercury with H₂S.     

THE STRUCTURAL SPECIFICITY OF THE HIGH AFFINITY UPTAKE OF l-GLUTAMATE AND l-ASPARTATE BY RAT BRAIN SLICES

Abstract— The high affinity uptake system for l -glutamate and l -aspartate in rat cerebral cortex may not be specific for these likely excitatory synaptic transmitters, as threo-3-hydroxy- dl -aspartate, l -cysteinesulphinate, l -cysteate and d -aspartate strongly inhibit the observed high affinity uptake of l -[³H]glutamate by rat brain slices in a manner consistent with linear competitive inhibition. These substances should therefore be considered as possible substrates for the transport system. Each of these four acidic amino acids excites central neurones in a manner similar to excitation induced by l -glutamate, and as each might occur in brain tissue, their possible synaptic role should be investigated.
l -Glutamate high affinity uptake was shown to be sodium-dependent, but under certain conditions appeared to be less sensitive than GABA uptake to changes in the external sodium ion concentration, and to drugs which modify sodium ion movements. This may be relevant to the efficiency of the glutamate uptake process during synaptic depolarization induced by glutamate.
l -Glutamate high affinity uptake was inhibited in a relatively nonspecific manner by a variety of drugs including mercurials and some electron transport inhibitors.