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Mercury redox transformation reactions in nature may be biological (enzymic) or nonbiological (nonenzymic). Biological mercury transformation has usually been explained in terms of the action of a number of specialized enzymes. While nonbiological transformation has been explained in terms of the action of certain biological products such as methylcobalamin (methyl vitamin B/sub 12/), methionine, ascorbate, humic acid and some proteins. It has been shown that mercuric salts (Hg/sup + +/) can be reduced and alkylated by the action of ethylene and acetylene (two known reducing agents in air), and it has been established that mercuric salts are reduced by propene and other higher molecular weight unsaturated hydrocarbons (olefins).

Methylphenylmercury reacts with two normally inaccessible cysteine residues in crystals of carbonmonoxyhaemoglobin, but not with the third, normally reactive one. It may, therefore, be useful in the preparation of new heavy atom derivatives for protein crystallography.

The disruption of erythyrocyte membrane cytoskeletons brought about by treatment with p- mercuribenzene sulphonate (PMBS) has been followed by measurements of turbidity and the binding of 203Hg-labelled PMBS. After pretreatment with N- ethylmaleimide to block readily reactive sulphydryl groups, incubation with [203Hg]PMBS showed incorporation of approximately 4 moles radiolabel per mole of spectrin and one per mole of actin. The incorporation of radiolabel paralleled the decrease in turbidity, and the labelling of spectrin paralleled that of actin. The kinetics were pseudo first order, and the pH dependence of the observed rate constant indicated a normal p K a value for the sulphydryl group involved. The calculated second-order rate constant for the reaction of the sulphydryl anion with PMBS, however, was several orders of magnitude less than expected from model compound studies. The results suggest that association between spectrin and actin may result in the steric hindrance of reactivity of a limited number of sulphydryl groups in each protein. Disruption of the spectrin-actin association may then be linked to the modification of the sulphydryl groups.

SummaryThe effects of the sulfhydryl reactive agent p-chloromercuribenzene sulfonic acid (pCMBS) were measured on isotopic Na and Cl fluxes across isolated fundic and antral mucosa. pCMBS inhibits active Na and Cl transport by both fundic and antral mucosa. Addition of p-chloromercuri-benzoate bound to aminoethyl-dextran (with a mol wt of approximately 10,000) (pCMB-AED) decreases H secretion as well as net Na and Cl transport. Since the effects of a presumably impermeable chloromercuribenzene compound (pCMB-AED) are qualitatively similar to those of a free chloromercuribenzene compound (pCMBS), the active transport effects of pCMBS appear to be due primarily to the oxidation of superficially located thiol groups.

A 7.9-kilobase (kb) chromosomal fragment was cloned from a mercury-resistant Bacillus sp. In Escherichia coli, in the presence of a second plasmid carrying functional transport genes, resistance to HgCl2 and to phenylmercury acetate (PMA) was expressed. Shortening the cloned fragment to 3.8 kb abolished resistance to PMA but not to HgCl2. In Bacillus subtilis, the 3.8-kb fragment produced mercuric reductase constitutively but did not produce resistance to HgCl2 or to PMA.

The effect of phenylmercury and methylmercury on rat liver glutathione peroxidase (GSH Px) is investigated and compared with that of Hg(II) and with some previously reported results for Cd(II). Analysis of the kinetics of metal binding to the enzyme gives apparent inhibition rate constants: kc = 9.7 mM−1 min−1 for all three mercury compounds and 75 mM−1 min−1 for CdCl2. Glutathione (0.2 mM) protects the enzyme from metal inhibition, decreasing the apparent inhibition rate constants (kc) by 3.6 times for mercury compounds and 4.4 times for CdCl2. K1 for the three mercury compounds is found to be 53 μM. It is unexpected that the same value of K1 exists for all three forms of mercury studied and that inhibition of the enzyme by the metals is a relatively slow process. For Cd(II) the value of K1 is 8.5 μM. It is suggested that inhibition of GSH·Px enzyme activity by cadmium, mercury, and organic mercury salts may not be due to simple complexation of the active site selenium moiety but may be due to a slower process, e.g., an alteration of the enzyme tertiary or quaternary structure.

The organomercurial reagent p -chloromercuribenzene sulfonate (PCMBS) is an inhibitor of osmotic water permeability in the human red cell membrane. We have found that thiourea, when added along with PCMBS to a red cell suspension, interferes with this inhibition and at high enough concentrations prevents the inhibition from developing altogether. For a 2 mM PCMBS concentration K i = ; 3 ± 1 mM. When thiourea is added at a later time, the PCMBS inhibition, which normally takes about 20 min to develop fully, is halted and remains fixed at the value attained by that time. Thiourea also inhibits the reversal of PCMBS inhibition by a 10 mM concentration of cysteine, the half-time for reversal increasing by more than an order of magnitude when [thiourea] = ; 50 mM. Possible implications for the nature of the water and urea transport pathways across the red cell membrane are discussed.

Incubation of Novikoff rat hepatoma cells; mouse L929, P388 and L1210 cells; and Chinese hamster ovary cells with sulfhydryl reagents, such as p -hydroxymercuribenzoate or p -hydroxymercuribenzenesulfonate, reduced the zero- trans influx of uridine in a concentration-dependent manner. The sensitivity of uridine transport to inhibition varied somewhat for the cell lines, Chinese hamster ovary cells being the most sensitive. Maximum inhibition by p -hydroxymercuribenzoate occurred in 10–20 min of incubation at 37 °C, and was associated with a decrease in maximum transport velocity without significant change in substrate affinity of the carrier. The development of inhibition of uridine influx correlated with binding of [ 14 C] p -hydroxymercuribenzoate to the cells. Inhibition of transport also roughly correlated with a decreased binding of 6-nitrobenzylthioinosine to high-affinity binding sites on the cells (presumably representing the nucleoside transporter) without affecting binding affinity. Treatment of cells with p -hydroxymercuribenzenesulfonate reduced uridine influx and efflux to a similar extent. Inhibition of uridine transport and binding of [ 14 C] p -hydroxymercuribenzoate were readily reversed by incubation of the cells with dithiothreitol. The results indicate that sulfhydryl groups are essential for the functioning of the nucleoside transporter, perhaps for the binding of substrate. Blockage of the sulfhydryl groups results in a reversible inactivation of the carrier. Treatment of the cells with the sulfhydryl reagents also caused a concentration-dependent increase in cell volume, which was readily reversed by incubation of the cells with dithiothreitol but seemed unrelated to the inhibition of nucleoside transport.

A histoenzymic study of cerebral phosphatases and esterases was performed on rats subjected to experimental intoxication with mercury phenylacetate. Following intragastric application of mercury phenylacetate to experimental animals, decreased activities of cerebral ATPase, acP and AChE were observed. The intoxicated animals displayed enhanced cerebral TPPase and partially also NsChE activities. Apart from changes in the histoenzymic pattern of the experimental brains, the ingestion of mercury phenylacetate brought about evident morphological changes in form of neuronal vacuolisation and spongious degeneration of the white matter. The extent of morphological as well as histoenzymic alterations was dependent on the duration of the experimental poisoning.

The reaction of phenylmercury hydroxide with chelating ligands such as salicylaldehyde, 8-hydroxyquinoline, benzoylphenyl hydroxylamine, 2-hydroxybenzophenones and some Schiff bases has been found to give chelated phenylmercury compounds. Stable phenylmercury titanoxanes containing the HgOTi group have been prepared by the reaction of chelated titanium diisopropoxides with phenylmercury hydroxide. Isopropyl titanate under similar conditions gave tetra-phenylmercury titanate.

Removal of the green colour of butyryl-CoA dehydrogenase by reagents specific for nucleophilic sulphur is shown to involve chemical modification of the tightly bound CoA persulphide. 5,5′-Dithiobis-(2-nitrobenzoic acid) (Ellman's reagent) de-greens the enzyme essentially irreversibly, with a stoicheiometry of approx. 1 mol/mol of FAD. A compound separated by subsequent gel filtration is eluted at the same position as a CoA-thionitrobenzoate standard. The 35S-labelled distal sulphur atom of CoA persulphide is separated from this material. The enzyme remains fully active. Phenylmercuric acetate also de-greens the enzyme. The extent to which thiols restore the green colour declines with time. Gel filtration of mercurial-treated enzyme separates low-Mr material containing a CoA moiety, an extra S atom and a phenylmercury moiety. This material, added to yellow butyryl-CoA dehydrogenase with an excess of thiol, re-forms green enzyme, but it loses this ability on storage. The results are explicable if it is assumed that the thionitrobenzoate derivative of CoA persulphide loses the extra sulphur atom much more readily than does the phenylmercury derivative.

Freshly prepared human red blood cells incubated with 5 mM ferricyanide, 0.2 mM iodoacetate and 2 mM adenosine in the presence of 5 mM EGTA demonstrate comparable increases in Na+ and K+ permeability (ferricyanide effect). This effect is unrelated to the Ca2+-activated K+ channel (Gardos effect) since influx of Ca2+ from outside the cell is excluded. Also this effect is different from the non-specific Na+ and K+ permeability change elicited by PCMBS. These differences become obvious by using various reagents. For example, A23187 and quinidine exert opposite effects in Gardos and ferricyanide experiments, where A23187 and atebrin react oppositely in the latter and in PCMBS experiments. The ferricyanide effect described here does not involve formation of nonspecific channels. The change in Na+ permeability separately from K+ permeability under certain circumstances suggests a more specific effect.

By combining radio-gas chromatography with liquid scintillation counting and with thin-layer chromatography-autoradiography, the conversion of symmetrical phenylmercury(II) compounds into phenylmercury(II) chloride and mercury(II) chloride in gas chromatography in the nanogram range has been demonstrated. The chlorine atoms necessary for this reaction are supplied by the Si-Cl-groups of cholorosilanized materials. The composition of the eluates depends on the concentration of the Si-Cl-groups.

The effect of sodium selenite on the activity of the selected enzymes in blood serum and on mercury concentration in some tissues of guinea pigs exposed to ethyl- (EtHg) or phenylmercuric chloride (PhHg) was investigated. Every second day for a 3-month period animals were given intragastrically a solution of mercuric compounds (2.5 mg Hg/kg) with or without sodium selenite (1 mg Se/kg). The activity of malate dehydrogenase (MDH, EC 1.1.1.37), phosphohexoizomerase (PHI, EC 5.3.1.9), and gamma-glutamyltranspeptidase (GGTP, EC 2.3.2.2) in blood serum of control animals was ca. 3.8, 325, and 48 IU. After 10 weeks of exposure to EtHg and PhHg, the activities (IU) of the above enzymes were, respectively, 5.9 and 6.5 (MDH), 585 and 600 (PHI) and 211 and 86.5 (GGTP). Sodium selenite administered with mercuric compounds did not prevent in increases in enzyme activity. During the experiment the level of inorganic as well as organic mercury accumulated in kidneys and liver was estimated. After a 12-week exposure, sodium selenite decreased the level of total mercury in the liver (in the case of both EtHg and PhHg: from 47.0 to 31.8 and from 41.3 to 25.4 micrograms Hg/g tissue, respectively). It also slightly decreased the mercury level in the kidneys of animals exposed to PhHg (from 889 to 73.3 micrograms Hg/g tissue) but did not change the mercury concentration in the kidneys of guinea pigs exposed to ethylmercuric chloride.

We have studied the influence of certain chemical groups, located on the surface of lymphocytes or sheep red blood cells, on the ability of these cells to form spontaneous rosettes. We found that fluorescamine, which reacts with amino groups, inhibits rosette formation, while p-chloromercuriphenylsulfonic acid, which binds to sulfhydryl groups, increases the percentage of the rosette-forming lymphocytes. The possible mechanism of action of the above reagents is discussed.

The purpose of these experiments was to determine if membrane-mediated glucocorticoid uptake by the AtT-20 cell was sensitive to the sulfhydryl group inhibitor, p -chloromercuri-phenylsulfonate (PCMPS). This agent was chosen because of its reported limited entry into the cell interior. Our experiments showed that cells could be incubated with 1 mM PCMPS for 1 h before intracellular concentrations of the inhibitor were sufficient to affect the intracellular cytosol receptor. In contrast less than 15 min of treatment inhibited intact cell steroid uptake. The inhibition was completely reversed by dithiothreitol. These studies show inhibition of glucocorticoid uptake under conditions which do not affect the intracellular receptor and infer that the AtT-20 cell membrane-mediated uptake mechanism for glucocorticoids contains sensitive sulfhydryl groups.

A T = 1 empty aggregate of alfalfa mosaic virus coat protein had been crystallized in a hexagonal unit cell and its orientation was determined with the rotation function. A single heavy-atom derivative has now been prepared and the position of the two Hg atoms per protein subunit were determined using a systematic Patterson search procedure, given the particle orientation. Phases, initially determined by single isomorphous replacement, were refined by six cycles of electron density averaging and solvent leveling to produce a 4.5 A resolution electron density map. The protein coat is confined between 95 and 58 A radius. The subunit boundary could be delineated easily. It has a central cavity reminiscent of the beta-barrel in other spherical plant viruses, but its topology could not be determined unambiguously. The spherical particle has large holes at the 5-fold axes, consistent with previous observations. The subunits have substantial interactions at the 2 and 3-fold axes. The structure of the elongated particles is discussed in relation to these results.

The bifunctional mercurial meso-1,4-bis(acetatomercuri)-2,3-diethoxybutane and mercuric chloride are capable of dimerizing papain, by the attachment of the thiol group of two molecules of papain to each molecule of reagent. This is evident from the titration data, gel filtration and sedimentation equilibrium. The conformational change of papain necessary for this reaction is discussed.

A new method for continuous in-cage monitoring of the activity of rats using passive sensors to detect the animals' infrared emissions, and its usefulness for toxicological screening tests is described. In order to obtain baseline values for toxicological tests, the differences between day and night activity, adaptation to changing day/night rhythm, the effects of handling the animals (measurement of body weight, food and water consumption) and the effect on activity patterns of the female estrus cycle were investigated. In two separate experiments, single doses of acetanilide and phenylmercuric acetate were administered. Passive infrared monitoring of motor activity proved to be a sensitive and discriminating method suitable for quantitative and qualitative assessment of acute toxic effects.

Lymphocytes from whole blood cultures from 28 subjects occupationally exposed to different mercury compounds and from seven control subjects were studied cytogenetically. Differences between aneuploidy of exposed and nonexposed subjects were found statistically significant for all analysed mercury compounds. No statistically significant difference however (except for ethylmercury exposure) was observed for the frequency of cells with structural chromosome aberrations although all of the observed frequencies in exposed subjects were higher than those of the controls. A significant correlation was found between the blood and urine mercury levels on the one hand and the total amount of cells with chromosomal aberrations and frequencies of cells with other than chromatid type aberrations on the other.

When pretreated with the organomercurial compound that reversibly blocks the adhesion of pyocin R1 to sensitive cells, this bacteriocin became susceptible to trypsin. Trypsinization resulted in the exclusive fragmentation of subunit protein no. 2 among 20 different subunit components and the disappearance of distal knobs from the fibers as well as in irreversible inactivation.

Seventy isolates of Providence were obtained from urinary tract infections in patients from five hospitals. They were all identified as Providence B (Providencia stuartii). A typing system was devised for these organisms based on their production of and sensitivity to bacteriophage and bacteriocins. Using this system, 27 distinct strains were recognized, and it was clear that many patients had been infected with the same strain. The minimum inhibitory concentrations of six antiseptics and disinfectants for these strains were determined. Many of the strains were up to 300-fold more resistant to the cationic antiseptics chlorhexidine, cetrimide, and Resiguard than the control strain Escherichia coli NCTC 10418. The sensitivities of the Providence strains to Hycolin, glutaraldehyde, and phenyl mercuric nitrate, however, were similar to those of the control organism. There were significant correlations between the sensitivities of the strains to the three cationic antiseptics. The loss of chlorhexidine resistance from a Providence strain after exposure of the cells to N-methyl-N' nitro N nitrosoguanidine resulted in a concomitant loss of resistance to cetrimide and Resiguard, but there was no significant increase in the sensitivity of the mutants to the other three antibacterials.

1. In guinea-pig papillary muscle PCMBS (0.03–0.6 mmol/l) produced, after a transient negative inotropic phase, a pronounced positive inotropic effect. Finally a contracture developed, while the force of contraction declined continuously. 2. Endogenously released noradrenaline contributed to the time course but not to the maximum of the positive inotropic effect of PCMBS. The latter was equal to the maximal effect obtained by noradrenaline. For further evaluation of PCMBS effects, only muscles from animals pretreated with reserpine were used. 3. The increase of the concentration of PCMBS (from 0.03–0.6 mmol/l) did not significantly alter the magnitude of its negative or positive inotropic effects, but distinctly accelerated their development. When contraction force was plotted versus timex[PCMBS], the time curves of the development of the inotropic effects became superimposable. The similar dependence of negative as well as positive inotropic effects on the concentration of PCMBS suggested that the same type of receptor, probably sulfhydryl groups, is involved in the mediation of both effects. 4. A mathematical analysis made evident that either the rate of permeation of PCMBS across some barrier far from equilibrium or the rate of binding of supramaximal concentrations of PCMBS to a receptor may be rate-limiting. Permeation is most likely the rate-limiting step, since the rapid inactivation of isolated sarcolemmal ATPase indicates a fast binding of PCMBS. 5. The negative inotropic effects of PCMBS correlated with a shortening of both contraction time and plateau duration of the action potential. This made it probable that the negative inotropic effect and the shortening of action-potential duration were causally related. 6. A reduction of [Na+]0 (from 140 to 70 mmol/l) or of stimulation frequency (from 1 to 0.004 Hz) or an increase of [K+]0 (from 2.4 to 9.6 mmol/l) delayed the appearance of the positive inotropic effect of PCMBS (t50% of maximal effects was 4 min at 2.4 mmol/l K+ and 27 min at 9.6 mmol/l). 7. Mg2++Na++K+- and Mg2+-dependent ATPases of sarcolemmal particles were inhibited by PCMBS (1–30 μmol/l). 8. The similarities in the influence of variation of contraction frequency and of [Na+]0 or [K+]0 on the positive inotropic effects of PCMBS and of cardioactive glycosides and their common inhibitory action on membrane ATPase suggest that inhibition of this enzyme by the Hg-compound is the cause of its positive inotropic effect.

Organomercurial pollution occurring in the Rhine river in 1986 led us to study the possibility of depollution by mercury-resistant environmental aquatic strains. Four species of Pseudomonas were investigated for their ability to biotransform phenylmercuric acetate (PMA). Such biological depollution was demonstrated to be due to an enzymatic activity in whole cells and in cell-free extracts from Pseudomonas fluorescens and other Pseudomonas species. PMA biotransformation was followed by high-performance liquid chromatography. Some of those bacteria growing between 4 and 41°C probably represent a natural means of organomercurial depollution, which acts slowly in interaction with other organisms and non-organic porous surfaces.

Human kidney prolinase, assayed with Pro-Ala, and non-specific dipeptidase, assayed with Gly-Leu, were purified by using DEAE-cellulose, gel-filtration, metal-ion-chelate, hydrophobic and adsorption chromatography and chromatofocusing. Both enzymes gave single peaks of activity that were congruent and the ratio of their activities was constant throughout the purification. Gel filtration indicated an Mr of 100 000 and chromatofocusing a pI of 5.4. Ni2+-chelate chromatography demonstrated the presence of exposed histidine residues on the enzyme and was an effective separative procedure. Polyacrylamide-gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. Both enzyme activities decayed at the same rate at 53 degrees C and were inhibited to the same extent by p-hydroxymercuribenzoate. Of six non-specific dipeptidase substrates tested Gly-Leu gave the highest activity, and of six prolinase substrates Pro-Leu had the highest activity. Gly-Leu was hydrolysed at double the rate of Pro-Leu. Pro-Ala was a competitive inhibitor of activity towards Gly-Leu, and Gly-Leu was a competitive inhibitor of activity towards Pro-Ala. Mixed-substrate studies strongly suggested that Gly-Leu and Pro-Ala were hydrolysed at a common active site. The data are consistent with prolinase and non-specific dipeptidase activity in human kidney being due to a single enzyme.

Xenopus transcription factor IIIA (TFIIIA) contains two tightly bound intrinsic Zn2+ ions that are released through treatment with either p-(hydroxymercuri)benzenesulfonate (PMPS) or diethyl pyrocarbonate (DEP) as monitored by the metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR). The inactivation of TFIIIA by DEP as detected by an in vitro 5S RNA gene transcription assay was correlated with the extent of modification of histidine residues and Zn2+ release. Following reaction with PMPS, the 7S particle was dissociated into free TFIIIA and 5S RNA. This dissociation could be correlated with the extent of modification of cysteine residues as well as the Zn2+ release. The dissociation of the 7S particle was reversed by the addition of excess thiol reagent. However, the reversibility could be inhibited by EDTA, suggesting that Zn2+ was required for the binding of TFIIIA to 5S RNA. In the presence of PMPS- or DEP-modified TFIIIA or Zn2+-depleted TFIIIA, the fluorescence emission maximum of the hydrophobic probe, 8-anilinonaphthalenesulfonate, was blue-shifted by 30 nm, while only less than a 10-nm blue shift was observed in the presence of either the 7S particle or TFIIIA. These results indicate that the two Zn2+ ions in TFIIIA are coordinated with the cysteine and histidine residues and are required for maintenance of the proper conformation of TFIIIA.

The sensitivity of nucleoside transport by rat erythrocytes to inhibition by nitrobenzylthioinosine (NBMPR) and the slowly permeating organomercurial, p-chloromercuriphenyl sulfonate (pCMBS), was investigated. The dose response curve for the inhibition of uridine transport (100 microM) by NBMPR was biphasic--35% of the transport activity was inhibited with an IC50 value of 0.25 nM, but 65% of the activity remained insensitive to concentrations as high as 1 microM. These two components of uridine transport are defined as NBMPR-sensitive and NBMPR-insensitive, respectively. Uridine influx by both components was saturable and conformed to simple Michaelis-Menten kinetics, and was inhibited by other nucleosides. The uridine affinity of the NBMPR-sensitive transport component was threefold higher than for the NBMPR-insensitive transport mechanism (apparent Km for uridine 50 +/- 18 and 163 +/- 28 microM, respectively). The two transport systems also differed in their sensitivity to pCMBS. NBMPR-insensitive uridine transport was inhibited by pCMBS with an IC50 of approximately 25 microM, while 1 mM pCMBS had little effect on NBMPR-sensitive transport by intact cells. pCMBS inhibition was reduced in the presence of uridine and adenosine and reversed by the addition by beta-mercaptoethanol, suggesting that the pCMBS-sensitive thiol group is located on the exterior surface of the erythrocyte membrane within the nucleoside binding site of the transport system. Inhibition of uridine transport by NBMPR was associated with high-affinity [3H]NBMPR binding to the cell membrane (apparent Kd 46 +/- 25 pM). Binding of inhibitor to these sites was competitively blocked by uridine and inhibited by adenosine, thymidine, dipyridamole, dilazep and nitrobenzylthioguanosine. Assuming that each NBMPR-sensitive transport site binds a single molecule of NBMPR, the calculated translocation capacity of each site is 25 +/- 6 molecules/site per sec at 22 degrees C. pCMBS had no effect on [3H]NBMPR binding to intact cells but markedly inhibited binding to disrupted membranes indicating that the NBMPR-sensitive nucleoside transporter probably has a thiol group located on the inner surface of the membrane. Exposure of rat erythrocyte membranes to UV light in the presence of [3H]NBMPR resulted in covalent radiolabeling of a membrane protein(s) (apparent Mr on SDS gel electropherograms of 62,000). Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine. We conclude that nucleoside transport by rat erythrocytes occurs by two facilitated-diffusion systems which differ in their sensitivity to inhibition by both NBMPR and pCMBS.

The cytotoxicity of two kinds of mercurial preservatives, thimerosal and phenylmercuric acetate, was studied using Chang's human conjunctival epithelia in cell culture. The cultured cells were exposed for 5 s, 2 min and 24 h to each of the two preservatives at various concentrations, obtained by serial dilution. The cytopathic effect and cell desquamation from the wall of culture flask were observed with an inverted microscope and LD50 was calculated by Van der Waerden's method. The LD50 values of thimerosal were 291.6, 47.4 and 2.2 micrograms/ml at exposure times of 5 s, 2 min and 24 h, respectively. Those of phenylmercuric acetate were 1,120.2, 227.5 and 2.6 microgram/ml at exposure times of 5 s, 2 min and 24 h, respectively. Cytotoxicity can be expressed quantitatively and precisely by LD50. LD50 is lower than the concentrations that are necessary to induce observable morphological changes. The results may help to choose the least toxic concentration of mercurial preservatives for addition to ophthalmic solutions.

Intraperitoneal injection of diphenylmercury into rats results in a much higher initial concentration of mercury in the brain than that following injection of mercuric chloride or phenylmercuric acetate. The concentration in the brain then decreases, and after several days it is similar to that resulting from mercuric chloride and phenylmercuric acetate injection.

The efficiency of the sodium salt of 2,3-dimercaptopropanesulfonic acid (DMPS) and meso-dimercaptosuccinic acid (DMSA) to mobilize mercury from tissues has been assessed in rats pretreated with different doses of HgCl2, phenylmercury acetate or exposed to different concentrations of mercury vapors. These pretreatments increase the mercury concentration in the kidney and to a lower extent in the liver. Only exposure to metallic mercury vapor leads to mercury accumulation in the brain. Both chelators mobilize mercury stored in the kidney and the amount of metal excreted in urine following a single administration of DMSA is a good indicator of the renal burden of mercury. The rate of removal is greater after DMPS administration than after DMSA but repeated administration of either agents eventually leads to the same total amount of mercury mobilized from the kidney. The loss of mercury from the liver can be slightly accelerated by repeated administration of the chelators. However, the chelators are inefficient in removing mercury from the brain.

Mercurial reagents inhibit the water permeability of erythrocytes and proximal renal tubule. We examined the effect of two such agents on vasopressin-induced water transport across toad urinary bladder. Water flows were measured in unfixed tissues and in tissues fixed either with N-ethylmaleimide (NEM) or with glutaraldehyde. When added concurrently with 20 mU/ml vasopressin, 1 mM mucosal p-chloromercuribenzene-sulfonic acid (p-CMBS) inhibited water flow within 1 h. p-CMBS also inhibited flow in tissues that had been fixed with mucosal NEM after stimulation with vasopressin. However, p-CMBS did not affect flow in glutaraldehyde-fixed tissues. In contrast, HgCl2 inhibited water flow and urea permeability even in tissues that had been fixed with glutaraldehyde after stimulation with vasopressin. Inhibition was more pronounced when HgCl2 was added to the mucosal rather than the serosal bathing medium and was not reversed by dithiothreitol. HgCl2 did not diminish the frequency or area of luminal membrane aggregates observed by freeze-fracture electron microscopy. HgCl2 also did not affect amphotericin-induced water permeability in glutaraldehyde-treated tissues, suggesting that it did not diminish the permeability of cellular barriers to flow. Our results parallel closely those reported by other investigators for water flow across erythrocytes and proximal renal tubule and suggest that mercurial reagents can directly block the vasopressin-induced water channel. The water channel at the apical membrane of the toad bladder may prove to share structural similarity with that constantly present in erythrocytes and proximal renal tubule.

The sulfhydryl reagent p- chloromercuribenzene sulfonate increased the I sc across substrate-replete toad urinary bladder when applied to the mucosal (apical) surface. This increase was accounted for by an increased mucosal to serosal net flux of Na + . In the absence of substrate, the rise in I sc was accompanied by an irreversible increase in tissue conductance which was not apparent in the replete preparation. These findings suggest that p- chloromercuribenzene sulfonate may be useful in marking mucosal functions associated with the Na + transport apparatus.

The human red cell anion transport protein, band 3, contains six pCMBS (p-chloromercuribenzene sulfonate) reactive SH groups, five of which react with N-ethylmaleimide. We have carried out equilibrium binding experiments using N-ethylmaleimide-treated red cell ghosts and found that the sulfhydryl reactive water transport inhibitor, pCMBS, inhibits the binding to band 3 of the specific anion exchange inhibitor DBDS (4,4'-dibenzoamido-2,2'-disulfonic stilbene) in a non-competitive manner. Stopped-flow kinetic studies, in which DBDS is mixed with ghosts in the presence of pCMBS, show that pCMBS slows the DBDS induced conformational change in band 3. A non-competitive reaction scheme has been developed which incorporates the quantitative results of equilibrium and kinetic studies. The pCMBS effect on DBDS binding and kinetics is reversed with 5 mM cysteine suggesting a sulfhydryl bond is involved in pCMBS binding to band 3. These data suggest that pCMBS has a specific binding site on band 3, consistent with the hypothesis that band 3 mediates red cell water transport.

Nucleosides cross the human erythrocyte membrane by a facilitated-diffusion process which is selectively inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR). The chemical asymmetry of the transporter was investigated by studying the effects of p- chloromercuriphenyl sulphonate (PCMBS) on uridine transport and high-affinity NBMPR binding in inside-out and right-side-out membrane vesicles, unsealed erythrocyte ghosts and intact cells. PCMBS was an effective inhibitor of the transporter (50% inhibition at 30 μM), but only when the organomercurial had access to the cytoplasmic membrane surface. PCMBS inhibition of NBMPR binding to ghosts was reversed by incubation with dithiothreitol. Both uridine and NBMPR were able to protect the transporter against PCMBS inhibition.

The interactions of 4-thiouridine and 5-((methylamino)methyl)-2-thiouridine in the tRNA and of phosphorothioate esters in nucleic acids with an organomercurial have been investigated. For this purpose, an affinity electrophoretic system has been developed in which the mercury derivative has been covalently immobilized in a standard polyacrylamide gel. The retardation of thiolated macromolecules was found to be sensitive to the chemical environment of the sulfur atom, giving characteristic interaction constants dependent on the nature of the modification and its accessibility to binding. The interaction could, in the case of tRNA, be abolished by conventional specific chemical modification of the thiolated bases, as well as by irradiation with /sup 32/P-derived ..beta..-emission. Not only has the fractionation of sulfur-modified from unmodified species been attained but a quantitative application of the technique has made it possible to study the binding of mercury and, by competition, that of magnesium in terms of the conformation of tRNA.

l-Octanol/water partition coefficients, mercury concentration in octanol divided by mercury concentration in water, provide a simple but limited model system for aspects of the biological behavior of methylmercury and commonly used organomercury medicinal compounds. In an octanol/water system some widely studied antidotes for mercury poisoning at least partly displace the biological thiols L-cysteine and glutathione from binding to methylmercury at pH 6.9. Addition of the antidote meso-dimercaptosuccinic acid to methylmercury in the presence of glutathione results in formation of metallic mercury. For organomercury (RHg) derivatives of L-cysteine and glutathione, octanol/water partition coefficients fol1ow the order R = Ph > Et > Me. An exceptional1y high value for diphenylmercury, compared with PhHg derivatives of L-cysteine and glutathione, is consistent with reported results of the distribution of mercury compounds in rats. Ethylmercury(II) is partly displaced from thimerosal by L-cysteine and glutathione in the octanol/water system, indicating that the active form of thimerosal in vivo may involve binding of ethylmercury to biological ligands.

Increase in urinary excretion of mercury and increase in blood concentration following the use of mercury based drugs has been checked by a limited volunteer study.

The toxicity of elements depends strongly on their chemical form. The separation of individual species must usually precede the determination by activation analysis or by other methods. The simple and rapid methods for the separation of inorganic mercury(II), methylmercury and phenylmercury compounds, arsenic(III) and arsenic(V) species including the separation of methylated forms, thallium(I) and thallium(III), chromium(III) and chromium(VI) using liquidliquid distribution reactions are presented.

Collagenase-isolated pancreatic islets of non-inbred ob/ob mice, containing more than 90% β-cells, were labelled with radioactive orthophosphate ( 32 P or 33 P) and then subjected to non-recirculating perifusion. The basal D -glucose concentration in the perifusion medium was 2.8 mM. When the concentration was suddenly raised to 5.6, 8.3 or 16.7 mM, D -glucose promptly elicited a transient and dose-dependent release of radiophosphate. In the presence ot 2.8 mM D -glucose, 0.1 mM of the poorly permeating sulphydryl blocker, chloromercuribenzene- p -sulphonic acid, also evoked a phosphate flush resembling the one induced by d -glucose. The basal radiophosphate release was partially inhibited by 1 mM 4-acetamido-4′-isothiocyanostilbene-2,-2′-disulphonic acid. However, the phosphate flush induced by 16.7 mM d -glucose was not noticeably inhibited by 4-acetamido-4′-isothiocyanostilbene-2,-2′-disulphonic acid. It is concluded that the phosphate flush emanates from β-cells and that membrane sulphydryl groups may participate in its regulation. Although at least the basal phosphate release may in part represent transmembrane transport through 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid-sensitive anion channels, other mechanisms are also likely to participate in the glucose-induced phosphate flush.

On the basis of evidence derived mostly from human erythrocytes, it has been suggested that water traverses cell membranes through membrane-spanning proteins such as the anion channel or the glucose transporter acting as water pores. However, specific inhibitors of such permeation processes have not been found to block water transport, and hence a precise identification of the water route has not been possible so far. We have investigated this issue by characterizing the osmotic flows across a fluid-transporting epithelium, the rabbit corneal endothelium. The rate of such flows was monitored continuously as a function of time. We confirmed prior findings of (a) an inhibition by PCMBS on osmotic water flow, and (b) lack of inhibition by DTNB and DIDS. On the other hand, we have found for the first time that several blockers of glucose facilitated diffusion, namely, phloretin (2 mM), phloridzin (2 mM), diallyldiethylstilbestrol (0.1 mM), cytochalasin B (20 μg/ml), and ethylidene- d -glucose (200 mM), all clearly inhibit osmotic flow. Our evidence is consistent with the hypothesis that both water and glucose may traverse these cell membranes through the same channel-like pathway contained in the glucose transporter membrane-spanning protein.

Ca2+-sensitive electrodes and the photoproteins obelin and aequorin were used with the oocytes of the anuran Xenopus laevis and the urodeles Ambystoma mexicanum and Pleurodeles waltlii in order to detect any changes in internal free Ca2+ which might result from progesterone or agonist stimulation. A dramatic Ca2+ surge was recorded: from 0.7 × 10−6 M in the unstimulated oocyte to 7 × 10−6 M after stimulation but before germinal vesicle breakdown (GVBD). Ca2+ efflux was also measured, but it accounted for less than 0.2% of the internal Ca2+ transient; this efflux did not take place in the absence of external calcium. The Ca2+ surge and maturation in response to progesterone, p-hydroxymethylenesulfonate (PHMPS), or Mn2+ occurred normally even when divalent cations were absent from the external medium. In contrast, external divalent cations were necessary for the induction of meiosis and a Ca2+ transient by the K+ ionophore valinomycin. HCO3− also triggers meiosis and causes Ca2+ release, but the release occurs with quite different kinetics. Incompletely grown or seasonally dormant oocytes as well as 10 mM theophilline- or procaine-treated oocytes neither release Ca2+ nor respond to the hormone. We conclude that intracellular released Ca2+ is likely to be the major “second messenger” following hormone stimulation in amphibian oocytes as in starfish.

It has been suggested that during the oxytocin-induced hydrosmotic response, water crosses the luminal membrane of urinary bladder epithelium cells through membrane-spanning proteins. Although specific inhibitors of osmotic water transport have not been found, certain sulfhydryl reagents such as mercurial compounds may help to identify the proteins involved in this permeation process. We tested the effects of p-chloromercuribenzene sulfonate (PCMBS) and of fluorescein-mercuric acetate (FMA) on the net water flux, the microtubule and microfilament structures of the frog urinary bladder, and the distribution of intramembrane particle aggregates in the luminal membrane. We observed that: (i) 5 mM PCMBS at pH 5 and 0.5 mM FMA at pH 8 added to the mucosal bath at the maximum of the response to oxytocin partially inhibited the net water flux. Inhibition then increased progressively when the preparation was repeatedly or continuously stimulated, until it reached a maximal inhibition at 120 min. This inhibition was not reversed even when cystein was added in the mucosal bath. PCMBS and FMA effects were also observed when cyclic AMP (3',5' cyclic adenosine monophosphate) was used to increase water permeability, (ii) PCMBS mucosal pretreatment did not modify the basal water flux but potentiated the inhibitory effect of PCMBS or FMA on the hydrosmotic response to oxytocin. (iii) Microtubule and microfilament network, visualized in target cells by immunofluorescence, was not affected by PCMBS. (iv) The maximal PCMBS or FMA inhibition was not associated with a reduction of aggregate surface area in the apical membrane. The persistence of the intramembrane particle aggregates associated with the oxytocin-induced hydrosmotic response during the net water flux inhibition by PCMBS, suggests that the PCMBS effect occurs possibly at the level of sulfhydryl groups of the water channel itself.