Covalent inhibition of P. falciparum ferredoxin-NADP+ reductase: Exploring alternative strategies for the development of new antimalarial drugs.

The protozoan Plasmodium falciparum is the main aetiological agent of tropical malaria. Characteristic of the phylum is the presence of a plastid-like organelle which hosts several homologs of plant proteins, including a ferredoxin (PfFd) and its NADPH-dependent reductase (PfFNR). The PfFNR/PfFd redox system is essential for the parasite, while mammals share no homologous proteins, making the enzyme an attractive target for novel and much needed antimalarial drugs. Based on previous findings, three chemically reactive residues important for PfFNR activity were identified: namely, the active-site Cys99, responsible for hydride transfer; Cys284, whose oxidation leads to an inactive dimeric form of the protein; and His286, which is involved in NADPH binding. These amino acid residues were probed by several residue-specific reagents and the two cysteines were shown to be promising targets for covalent inhibition. The quantitative and qualitative description of the reactivity of few compounds, including a repurposed drug, set the bases for the development of more potent and specific antimalarial leads.

Inhibition of Zn(II) binding type IA topoisomerases by organomercury compounds and Hg(II).

Type IA topoisomerase activities are essential for resolving DNA topological barriers via an enzyme-mediated transient single strand DNA break. Accumulation of topoisomerase DNA cleavage product can lead to cell death or genomic rearrangement. Many antibacterial and anticancer drugs act as topoisomerase poison inhibitors that form stabilized ternary complexes with the topoisomerase covalent intermediate, so it is desirable to identify such inhibitors for type IA topoisomerases. Here we report that organomercury compounds were identified during a fluorescence based screening of the NIH diversity set of small molecules for topoisomerase inhibitors that can increase the DNA cleavage product of Yersinia pestis topoisomerase I. Inhibition of relaxation activity and accumulation of DNA cleavage product were confirmed for these organomercury compounds in gel based assays of Escherichia coli topoisomerase I. Hg(II), but not As(III), could also target the cysteines that form the multiple Zn(II) binding tetra-cysteine motifs found in the C-terminal domains of these bacterial topoisomerase I for relaxation activity inhibition. Mycobacterium tuberculosis topoisomerase I activity is not sensitive to Hg(II) or the organomercury compounds due to the absence of the Zn(II) binding cysteines. It is significant that the type IA topoisomerases with Zn(II) binding domains can still cleave DNA when interfered by Hg(II) or organomercury compounds. The Zn(II) binding domains found in human Top3α and Top3ß may be potential targets of toxic metals and organometallic complexes, with potential consequence on genomic stability and development.

Ruthenium dihydroxybipyridine complexes are tumor activated prodrugs due to low pH and blue light induced ligand release.

Ruthenium drugs are potent anti-cancer agents, but inducing drug selectivity and enhancing their modest activity remain challenging. Slow Ru ligand loss limits the formation of free sites and subsequent binding to DNA base pairs. Herein, we designed a ligand that rapidly dissociates upon irradiation at low pH. Activation at low pH can lead to cancer selectivity, since many cancer cells have higher metabolism (and thus lower pH) than non-cancerous cells. We have used the pH sensitive ligand, 6,6'-dihydroxy-2,2'-bipyridine (66'bpy(OH)2), to generate [Ru(bpy)2(66'(bpy(OH)2)](2+), which contains two acidic hydroxyl groups with pKa1=5.26 and pKa2=7.27. Irradiation when protonated leads to photo-dissociation of the 66'bpy(OH)2 ligand. An in-depth study of the structural and electronic properties of the complex was carried out using X-ray crystallography, electrochemistry, UV/visible spectroscopy, and computational techniques. Notably, RuN bond lengths in the 66'bpy(OH)2 complex are longer (by ~0.3Å) than in polypyridyl complexes that lack 6 and 6' substitution. Thus, the longer bond length predisposes the complex for photo-dissociation and leads to the anti-cancer activity. When the complex is deprotonated, the 66'bpy(O(-))2 ligand molecular orbitals mix heavily with the ruthenium orbitals, making new mixed metal-ligand orbitals that lead to a higher bond order. We investigated the anti-cancer activities of [Ru(bpy)2(66'(bpy(OH)2)](2+), [Ru(bpy)2(44'(bpy(OH)2)](2+), and [Ru(bpy)3](2+) (44'(bpy(OH)2=4,4'-dihydroxy-2,2'-bipyridine) in HeLa cells, which have a relatively low pH. It is found that [Ru(bpy)2(66'(bpy(OH)2)](2+) is more cytotoxic than the other ruthenium complexes studied. Thus, we have identified a pH sensitive ruthenium scaffold that can be exploited for photo-induced anti-cancer activity.

N-containing Ag(I) and Hg(II) complexes: a new class of antibiotics.

Several classes of antimicrobial compounds are presently available; microorganism's resistance to these drugs constantly emerges. In order to prevent this serious medical problem, the elaboration of new types of antibacterial agents or the expansion of bioactivity of the naturally known biosensitive compounds is a very interesting research problem. The synthesis and characterization of metal complexes with organic bioactive ligands is one of the promising fields for the search. The biological activities of the metal complexes differ from those of either the ligand or the metal ion. The results obtained thus far have led to the conclusion that structural factors, which govern antimicrobial activities, are strongly dependent on the central metal ion. A review of papers dealing with the Ag(I) and Hg(II) complexes of N donor ligands is presented. These metal complexes of N-chelating ligands have attracted considerable attention because of their interesting physicochemical properties and pronounced biological activities. This review will mainly focus on the preparation procedures and antibacterial properties of free organic ligands and the corresponding complexes. Finally, a research about antimicrobial properties of new Hg(II) complexes with 5-methyl-5-(4-pyridyl)-2,4-imidazolidenedione (L) and various halogen ions, HgL2X2 (X = Cl¯ (49), Br¯ (50), and I¯ (51)), is reported. Noteworthy antimicrobial activities, evaluated by minimum inhibitory concentration, for these complexes were observed.

In vivo effects of traditional Ayurvedic formulations in Drosophila melanogaster model relate with therapeutic applications.

BACKGROUND: Ayurveda represents the traditional medicine system of India. Since mechanistic details of therapy in terms of current biology are not available in Ayurvedic literature, modern scientific studies are necessary to understand its major concepts and procedures. It is necessary to examine effects of the whole Ayurvedic formulations rather than their "active" components as is done in most current studies. METHODS: We tested two different categories of formulations, a Rasayana (Amalaki Rasayana or AR, an herbal derivative) and a Bhasma (Rasa-Sindoor or RS, an organo-metallic derivative of mercury), for effects on longevity, development, fecundity, stress-tolerance, and heterogeneous nuclear ribonucleoprotein (hnRNP) levels of Drosophila melanogaster using at least 200 larvae or flies for each assay. RESULTS: A 0.5% (weight/volume) supplement of AR or RS affected life-history and other physiological traits in distinct ways. While the size of salivary glands, hnRNP levels in larval tissues, and thermotolerance of larvae/adult flies improved significantly following feeding either of the two formulations, the median life span and starvation resistance improved only with AR. Feeding on AR or RS supplemented food improved fecundity differently. Feeding of larvae and adults with AR increased the fecundity while the same with RS had opposite effect. On the contrary, feeding larvae on normal food and adults on AR supplement had no effect on fecundity but a comparable regime of feeding on RS-supplemented food improved fecundity. RS feeding did not cause heavy metal toxicity. CONCLUSIONS: The present study with two Ayurvedic formulations reveals formulation-specific effects on several parameters of the fly's life, which seem to generally agree with their recommended human usages in Ayurvedic practices. Thus, Drosophila, with its very rich genetic tools and well-worked-out developmental pathways promises to be a very good model for examining the cellular and molecular bases of the effects of different Ayurvedic formulations.

Effect of mercury and organomercurials on cellular glucose utilization: a study using resting mercury-resistant yeast cells.

AIMS: Mercury compounds are highly toxic to all types of living cells. Isolated yeast strains of Rhodotorula rubra showed high and low resistance pattern towards mercury and organomercurial compounds. To investigate the basis of differential sensitivity of these two types of strains, glucose utilization was measured in the presence of mercury compounds. METHODS AND RESULTS: Glucose utilization process remained unaffected in resting cells of highly Hg(2+)-resistant strain in the presence of HgCl(2) but not in the presence of phenylmercuric acetate and thimerosal. However, HgCl(2) significantly affected glucose utilization in the case of low-resistant cells. The Hg-retaining ability of the cell wall of highly Hg(2+)-resistant yeast strain was greater than that of the weakly Hg(2+)-resistant strain. The spheroplast-bound Hg(2+) was also significantly less in the highly Hg(2+)-resistant strain than in the weakly Hg(2+)-resistant strain. CONCLUSIONS: Glucose uptake machinery was not affected in the presence of toxic metal ions in the case of high-resistant strains. But in the case of low Hg(2+)-resistant strain, glucose transport system may be affected either by inactivation of sensor proteins containing -SH group associated with glucose uptake. SIGNIFICANCE AND IMPACT OF THE STUDY: Cell wall of mercury-resistant yeast cells may play an important role in heavy metal bioremediation process.

Synthesis, characterization and antimicrobial activity of Fe(II), Zn(II), Cd(II) and Hg(II) complexes with 2,6-bis(benzimidazol-2-yl) pyridine ligand.

2,6-Bis(benzimidazol-2-yl)pyridine (L) ligand and complexes [M(L)Cl(2)] and [Fe(L)(2)](ClO(4))(2) (M=Zn, Cd, Hg) have been synthesized. The geometries of the [M(L)Cl(2)] complexes were derived from theoretical calculation in DGauss/DFT level (DZVP basis set) on CACHE. The central M(II) ion is penta-coordinated and surrounded by N(3)Cl(2) environment, adopting a distorted trigonal bipyramidal geometry. The ligand is tridentate, via three nitrogen atoms to metal centre and two chloride ions lie on each side of the distorted benzimidazole ring. In the [Fe(L)(2)](ClO(4))(2) complex, the central Fe(II) ion is surrounded by two (3N) units, adopting a octahedral geometry. The elemental analysis, molecular conductivity, FT-Raman, FT-IR (mid-, far-IR), (1)H, and (13)C NMR were reported. The antimicrobial activities of the free ligand, its hydrochloride salt, and the complexes were evaluated using the disk diffusion method in dimethyl sulfoxide (DMSO) as well as the minimal inhibitory concentration (MIC) dilution method, against 10 bacteria and the results compared with that for gentamycin. Antifungal activities were reported for Candida albicans, Kluyveromyces fragilis, Rhodotorula rubra, Debaryomyces hansenii, Hanseniaspora guilliermondii, and the results were referenced against nystatin, ketaconazole, and clotrimazole antifungal agents. In most cases, the compounds tested showed broad-spectrum (Gram positive and Gram negative bacteria) activities that were either more effective than or as potent as the references. The binding of two most biologically effective compounds of zinc and mercury to calf thymus DNA has also been investigated by absorption spectra.

Cleavage of double-strand DNA by linear and triangular trinuclear copper complexes.

Two trinuclear copper(II) complexes with linear or triangular metal-binding pendants, [Cu(3)(L(1))](6+) (1) and [Cu(3)(L(2))](6+) (2), have been synthesized and characterized, where L(1)=N,N,N',N'-tetra[(2-pyridyl)methyl]-5,5'-bis(aminomethyl)-2,2'-dipyridyl, L(2)=N,N,N',N',N'',N''-hexa[(2-pyridyl)methyl]-1,3,5-tris(aminomethyl)-benzene. Interactions of them with calf thymus DNA have been investigated by measuring the changes in the melting temperature. The obtained DeltaT(m) values indicated that both 1 and 2 exhibited very high affinities towards DNA and strong destabilization of DNA, and were far better than their mononuclear analogues. In the absence of any reducing agent, 1 showed markedly higher nuclease activity than 2, and its hydrolytic process was further clarified in the presence of a few of radical scavengers. The pseudo Michaelis-Menten kinetic parameters (k(cat)) were determined through DNA cleavage versus the concentration of the complexes, to be 6.05 and 0.19 h(-1) for 1 and 2 respectively. Much higher nuclease activity of 1 is probably attributed to its linear multiple metal sites that fit well to the phosphodiester backbone of nucleic acid. DNA cleavage versus the concentration of DNA shows that rate constants rapidly increase at beginning and then markedly decrease with increasing DNA concentration. It is likely that excess substrates would promote intermolecular interaction between a complex and more plasmid DNA(s) and weaken its intramolecular cooperative effect that is propitious to DNA cleavage.

The role of radical reactions in organomercurials impact on lipid peroxidation.

The organomercury compounds RHgX and R(2)Hg are broad-spectrum biocidal agents acting via diverse mechanisms in biological systems. Despite the enormous amount of studies carried out in last decades to elucidate the detailed mechanisms of organomercurials toxicity their biomolecular mode of action is still under debate. Among various toxicity mechanisms the action of RHgX and R(2)Hg at the membrane level due to the lipophilic properties of their molecules is discussed. Organomercurials are supposed to induce membrane associated oxidative stress in living organism through different mechanisms including the enhancement of the lipid peroxidation and intracellular generation of reactive oxygen species (ROS), H(2)O(2), O(2)(-), HO(). The perturbation of antioxidative defense system and the peroxidation of unsaturated fatty acids in membrane lipid bilayer are consequences of this impact. On the other hand, the involvement of organomercurials in radical and redox biochemical processes is manifested in carbon to metal bond cleavage that leads to the generation of reactive organic radicals R(). This pathway is discussed as one of the multiple mechanisms of organomercurials toxicity. The goal of this review is to present recent results in the studies oriented towards the role of organomercurials in the xenobiotic-mediated enhancement of radical production and hence in the promotion of lipids peroxidation. The application of natural and synthetic antioxidants as detoxification agents is presented.

Inhibition of interferon-gamma signaling by a mercurio-substituted dihydropsoralen in murine keratinocytes.

Psoralens and ultraviolet light A (PUVA) are used in the treatment of a variety of epidermal proliferative and inflammatory disorders. These compounds are known to intercalate and photo crosslink DNA. Specific receptor proteins for psoralens have also been identified. We describe a novel activity of a thiol reactive derivative, iodomercurio-4',5'-dihydrotrimethylpsoralen (iodomercurio-H2TMP) in keratinocytes. Without UVA, this psoralen was found to be an effective inhibitor of interferon-gamma (IFN-gamma)-signaling as measured by induction of nitric oxide biosynthesis (IC50 = 0.8 microM). This activity was increased (IC50 = 0.1 microM) when the cells were depleted of intracellular glutathione (GSH) with buthionine sulfoximine. In keratinocytes, IFN-gamma stimulates expression of inducible nitric oxide synthase (NOS2). Although iodomercurio-H2TMP did not alter NOS2 enzymatic activity, it blocked IFN-gamma-induced expression of NOS2 mRNA and protein, an effect that was enhanced in GSH-depleted cells. Iodomercurio-H2TMP was found to readily inhibit IFN-gamma signaling in transient transfection assays using NOS2 promoter/luciferase reporter constructs. NOS2 gene expression is known to require a variety of transcription factors including STAT-1, NF-kappaB and AP-1. Using mobility shift assays the psoralen, at concentrations that inhibit nitric oxide biosynthesis, had no effect on the DNA binding activity of STAT-1 or NF-kappaB. However, iodomercurio-H2TMP was found to suppress AP-1. These data indicate that iodomercurio-H2TMP acts at sulfhydryl-sensitive sites to inhibit NOS2. Moreover, this is dependent on early events in the IFN-gamma signal transduction pathway. Inhibition of AP-1 suggests that the psoralen functions by interfering with an important transcription factor that regulates expression of NOS2 in keratinocytes.

Existence of an iron-oxidizing bacterium Acidithiobacillus ferrooxidans resistant to organomercurial compounds.

Acidithiobacillus ferrooxidans MON-1 which is highly resistant to Hg2+ could grow in a ferrous sulfate medium (pH 2.5) with 0.1 microM p-chloromercuribenzoic acid (PCMB) with a lag time of 2 d. In contrast, A. ferrooxidans AP19-3 which is sensitive to Hg2+ did not grow in the medium. Nine strains of A. ferrooxidans, including seven strains of the American Type Culture Collection grew in the medium with a lag time ranging from 5 to 12 d. The resting cells of MON-1, which has NADPH-dependent mercuric reductase activity, could volatilize Hg0 when incubated in acidic water (pH 3.0) containing 0.1 microM PCMB. However, the resting cells of AP19-3, which has a similar level of NADPH-dependent mercuric reductase activity compared with MON-1, did not volatilize Hg0 from the reaction mixture with 0.1 microM PCMB. The activity level of the 11 strains of A. ferrooxidans to volatilize Hg0 from PCMB corresponded well with the level of growth inhibition by PCMB observed in the growth experiments. The resting cells of MON-1 volatilized Hg0 from phenylmercury acetate (PMA) and methylmercury chloride (MMC) as well as PCMB. The cytosol prepared from MON-1 could volatilize Hg0 from PCMB (0.015 nmol mg(-1) h(-1)), PMA (0.33 nmol mg(-1) h(-1)) and MMC (0.005 nmol mg(-1) h(-1)) in the presence of NADPH and beta-mercaptoethanol.

Organic mercury compounds and autoimmunity.

Based on in vitro studies and short-term in vivo studies, all mercurials were for a long time considered as prototypic immunosuppressive substances. Recent studies have confirmed that organic mercurials such as methyl mercury (MeHg) and ethyl mercury (EtHg) are much more potent immunosuppressors than inorganic mercury (Hg). However, Hg interacts with the immune system in the presence of a susceptible genotype to cause immunostimulation, antinucleolar antibodies targeting fibrillarin, and systemic immune-complex (IC) deposits, a syndrome called Hg-induced autoimmunity (HgIA). Recent studies in mice with a susceptible genotype has revealed that the immunosuppressive effect of MeHg and EtHg will within 1-3 weeks be superseded by immunostimulation causing an HgIA-like syndrome. At equimolar doses of Hg, MeHg has the weakest immunostimulating, autoimmunogen, and IC-inducing effect, while the effect of thimerosal is similar to that of inorganic mercury. The immunosuppression is caused by the organic mercurials per se. Since they undergo rapid transformation to inorganic Hg, studies are being undertaken to delineate the importance of the organic substances per se and the newly formed inorganic Hg for induction of autoimmunity.

Basigin (CD147) is the target for organomercurial inhibition of monocarboxylate transporter isoforms 1 and 4: the ancillary protein for the insensitive MCT2 is EMBIGIN (gp70).

Translocation of monocarboxylate transporters MCT1 and MCT4 to the plasma membrane requires CD147 (basigin) with which they remain tightly associated. However, the importance of CD147 for MCT activity is unclear. MCT1 and MCT4 are both inhibited by the cell-impermeant organomercurial reagent p-chloromercuribenzene sulfonate (pCMBS). Here we demonstrate by site-directed mutagenesis that removal of all accessible cysteine residues on MCT4 does not prevent this inhibition. pCMBS treatment of cells abolished co-immunoprecipitation of MCT1 and MCT4 with CD147 and enhanced labeling of CD147 with a biotinylated-thiol reagent. This suggested that CD147 might be the target of pCMBS, and further evidence for this was obtained by treatment of cells with the bifunctional organomercurial reagent fluorescein dimercury acetate that caused oligomerization of CD147. Site-directed mutagenesis of CD147 implicated the disulfide bridge in the Ig-like C2 domain of CD147 as the target of pCMBS attack. MCT2, which is pCMBS-insensitive, was found to co-immunoprecipitate with gp70 rather than CD147. The interaction between gp70 and MCT2 was confirmed using fluorescence resonance energy transfer between the cyan fluorescent protein- and yellow fluorescent protein-tagged MCT2 and gp70. pCMBS strongly inhibited lactate transport into rabbit erythrocytes, where MCT1 interacts with CD147, but not into rat erythrocytes where it interacts with gp70. These data imply that inhibition of MCT1 and MCT4 activity by pCMBS is mediated through its binding to CD147, whereas MCT2, which associates with gp70, is insensitive to pCMBS. We conclude that ancillary proteins are required to maintain the catalytic activity of MCTs as well as for their translocation to the plasma membrane.

Mode of action of 2-furylmercury chloride, an anti-rhinovirus compound.

2-Furylmercury chloride (2-FMC), an organic mercury derivative, has been found to inhibit the replication of all tested human rhinovirus (HRV) serotypes belonging to the antiviral group B and a limited number of HRV serotypes belonging to the antiviral group A. The mechanism of action of 2-FMC was tested against HRV-2 (antiviral group B, minor receptor group), and compared with an antiviral compound for which the viral target was already determined (enviroxime). 2-FMC was found to bind reversibly to virus particles. However, time-dependent plaque reduction assays revealed that 2-FMC did not interfere with early events of HRV-2 replication. Using a quantitative RT-PCR ELISA assay, we were able to prove that 2-FMC inhibits the synthesis of viral RNA. However, the mode of action of 2-FMC is not identical to that of enviroxime, another inhibitor of viral RNA synthesis. Time-of-addition and time-of-withdrawal experiments demonstrated that 2-FMC acted during a broader time interval than enviroxime.

Human organic anion transporter 1 mediates cellular uptake of cysteine-S conjugates of inorganic mercury.

BACKGROUND: The epithelial cells lining the renal proximal tubule have been shown to be the primary cellular targets where mercuric ions gain entry, accumulate, and induce pathologic effects in vivo. Recent data have implicated at least one of the organic anion transport systems in the basolateral uptake of inorganic mercury (Hg(2+)). METHODS: Using a line of Madin-Darby canine kidney (MDCK) II cells transfected stably with the human organic anion transporter 1 (hOAT1), and oocytes from Xenopus laevis microinjected with cRNA for hOAT1, we tested the hypothesis that hOAT1 can transport biologically relevant mercuric conjugates of cysteine (Cys). RESULTS: Indeed, MDCK II cells expressing a functional form of hOAT1 gained the ability to transport the mercuric conjugate 2-Amino-3-(2-amino-2-carboxy-ethylsulfanyl-mercuricsulfanyl)-propionic acid (Cys-S-Hg-S-Cys), but not the corresponding di-glutathione S-conjugate of Hg(2+) (G-S-Hg-S-G). Moreover, p-aminohippurate (PAH), adipate, and glutarate (but not succinate or malonate) inhibited individually the uptake of Cys-S-Hg-S-Cys in a dose-dependent manner. Uptake of Cys-S-Hg-S-Cys, but not G-S-Hg-S-G, was also documented in Xenopus oocytes expressing hOAT1. CONCLUSION: These data represent ostensibly the most direct line of evidence implicating a specific membrane protein (i.e., hOAT1) in the transport of a biologically relevant molecular species of Hg(2+) in a mammalian cell. Moreover, these data indicate that the organic anion transporter(s) likely play a prominent role in the basolateral transport of mercuric ions by proximal tubular cells and in the nephropathy induced by Hg(2+).

Molecular cloning of a cDNA for the human phospholysine phosphohistidine inorganic pyrophosphate phosphatase.

We previously reported the isolation from bovine liver of a novel 56-kDa inorganic pyrophosphatase named phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPPase). It is a unique enzyme that hydrolyzes not only oxygen-phosphorus bonds in inorganic pyrophosphate but also nitrogen-phosphorus bonds in phospholysine, phosphohistidine and imidodiphosphate in vitro. In this study, we determined the partial amino acid sequence of the purified bovine LHPPase. To investigate whether humans have the same enzyme, we isolated a cDNA clone from a HeLa cell cDNA library that encodes for the human homologue of LHPPase. Although its sequence does not include the consensus sequence of a typical inorganic pyrophosphatase, it does contain a similar sequence of the active site in other phosphatases such as protein-tyrosine phosphatase, dual-specific phosphatase and low molecular weight acid phosphatase. Human LHPPase was highly expressed in the liver and kidney, and moderately in the brain. The recombinant protein was produced in E. coli. Its ability to hydrolyze oxygen-phosphorus bonds and nitrogen-phosphorus bonds was confirmed. The enzymatic characteristics of this human protein were similar to those of purified bovine LHPPase. Thus, we concluded that the cDNA encoded the human counterpart of bovine LHPPase.

Diffusion pathways to critical cysteines in the vesicular acetylcholine transporter of Torpedo.

Previous work had demonstrated that organomercurial-mediated modification of two cysteine residues in the vesicular acetylcholine transporter (VAChT) from Torpedo californica inhibits binding of vesamicol. The cysteines are protected by acetylcholine and vesamicol (Keller et al. 2000. J. Neurochem. 74:1739-1748). Modified "cysteine 1" is accessible to glutathione from the cytoplasmic surface, whereas modified "cysteine 2" is not. Different organomercurials and aqueous environments were used here to characterize diffusion pathway(s) leading to the cysteines. para-Chloromercuriphenylsulfonate modifies VAChT much more slowly than do more hydrophobic p-chloromercuribenzoate and phenylmercury chloride. Permeabilization of vesicles with cholate detergent increases the rate of modification by p-chloromercuriphenylsulfonate. Permeabilization does not affect the ability of glutathione to reverse modification by p-chloromercuriphenylsulfonate. Higher ionic strength causes about four-fold increase in the rate of modification. The results suggest that hydrophobic and electrostatic barriers inhibit modification of Torpedo VAChT by negatively charged organomercurials and glutathione cannot reach cysteine 2 from either side of the membrane.

Polyphosphate produced in recombinant Escherichia coli confers mercury resistance.

An Escherichia coli strain was generated by fusion of a merA-deleted broad-spectrum mer operon from Pseudomonas K-62 with a bacterial polyphosphate kinase gene (ppk) from Klebsiella aerogenes in vector pUC119. A large amount of the ppk-specified polyphosphate was identified in the mercury-induced bacterium with the fusion plasmid designated pMKB18 but not in the cells without mercury induction. These results suggest that the synthesis of polyphosphate as well as the expression of the mer genes is mercury-inducible and regulated by merR. The E. coli strain with pMKB18 was more resistant to both Hg2+ and C6H5Hg+ than its isogenic strain with cloning vector pUC119. The recombinant strain accumulated more mercury from Hg2+- and C6H5Hg+-contaminated medium. Hg2+ transported into the cytoplasm appeared to be bound by chelation with the polyphosphate produced by the recombinant cells. The transported phenylmercury was degraded to Hg2+ before the chelation since polyphosphate did not directly chelate with C6H5Hg+. These results indicate that polyphosphate is capable of reducing the cytotoxicity of the transported Hg2+ probably via chelation between polyphosphate and Hg2+.

Synthesis and characterization of an immobilized phenylethanolamine N-methyltransferase liquid chromatographic stationary phase.

Norepinephrine is N-methylated to epinephrine by the catalytic effect of the terminal enzyme in catecholamine biosynthesis, phenylethanolamine N-methyltransferase (PNMT). PNMT has been covalently immobilized onto a silica-based liquid chromatographic support, glutaraldehyde-P (Glut-P). The resulting PNMT-Glut-P stationary phase (PNMT-SP) was enzymatically active, stable, and reusable. Standard Michaelis-Menten kinetic studies were performed with both free and immobilized PNMT and known substrates and inhibitors were examined. The results demonstrate that the PNMT-SP can be utilized for the rapid screening of potential PNMT substrates as well as the screening of compounds for PNMT inhibitory activity.

Modulation of the calmodulin-induced inhibition of sarcoplasmic reticulum calcium release channel (ryanodine receptor) by sulfhydryl oxidation in single channel current recordings and [(3)H]ryanodine binding.

The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl-sulfonic acid (4-CMPS) and 4, 4'-dithiodipyridine (4,4'-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [(3)H]ryanodine binding to HSR vesicles. 0.1 microm CaM reduced the open probability (P(o)) of the calcium release channel at maximally activating calcium concentrations (50-100 microm) from 0.502 +/- 0.02 to 0.137 +/- 0.022 (n = 28), with no effect on unitary conductance. 4-CMPS (10-40 microm) and 4,4'-DTDP (0.1-0.3 mm) induced a concentration dependent increase in P(o) (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4'-DTDP to higher concentrations in single channel recordings and [(3)H]ryanodine binding. 40 microm 4-CMPS induced a near maximal (P(o) > 0.9) and 0.3 mm 4,4'-DTDP a submaximal (P(o) = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4'-DTDP affected Ca-[(125)I]calmodulin binding to HSR. 1 mm MgCl(2) reduced P(o) from 0.53 to 0.075 and 20-40 microm 4-CMPS induced a near maximal channel activation (P(o) > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4'-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel.