Bis( -cysteinato)gold(I): Chemical characterization and identification in renal cortical cytoplasma

-Cysteinatogold(I) was prepared by the reaction of -cysteine with KAuBr₄ in acidic media and its solubility determined from pH 4 to 10. The solubility at pH 7.4 and 37° C is 1 μM. In the presence of excess cysteine, the solubility increases because of formation of bis( -cysteinato)gold(I). The equilibrium constant for formation of the bis complex is 2.1 ± 0.4 × 10⁻³, which at pH 7.4 corresponds to an apparant formation constant of 4.4 × 10⁴. The formation of the bis adduct was confirmed by chromatographic separation of the products of the reaction between [³⁵S]- -cysteine and Na₂AuTM. This complex elutes with K_av = 1.15 which allows it to be distinguished from other gold thiolates that might form in vivo. The bis(cysteinato)gold(I) complex is shown to be present in kidney cytosol isolated from rats given Na₂AuTM in vivo. When additional cysteine is added to the cytosol in vitro, the peak at 1.15 is increased, but if glutathione is added, the low molecular weight gold elutes at K_av = 1.00, which is taken as evidence for the existence of bis(cysteinato)gold(I) in the cytosol preparation. The amount of gold present as bis(cysteinato)gold(I) after 4 different dose schedules has been measured and found to increase with the total cytosol gold concentration. -Cysteinatogold(I) does not dissolve in the presence of bovine serum albumin to form an adduct.     

The binding of gold(I) to metallothionein

The binding of gold(I) to metallothionein, MT, has been unambiguously established by the reaction of Na₂AuTM with purified horse kidney MT. Zinc was displaced more readily than cadmium although the latter could be displaced using large Au/Cd ratios. The metal exchange reactions were complete within 2 hr of mixing. Further evidence that such reactions might be physiologically significant were obtained by studying in vitro metal displacements in the liver cytosol of in vivo metal treated rats: When Na₂AuTM was added to the cytosol of rats administered CdCl₂ in vivo, zinc, copper and cadmium were displaced in 2/1/1 ratios from the metallothionein fraction. The zinc and cadmium displacement provide direct evidence that the gold was binding to MT. Addition of Cd⁺² to liver cytosol of gold-treated rats resulted in displacement of copper and zinc, but not gold, from the MT fractions. When liver MT is prepared from rats exposed to Au or Cd, the Cd/protein ratio increased during the preparation, but the Au/protein ratio decreased. The Mt-bound metals account for 95% of the cytosolic Cd but only 15%–30% of the cytosolic gold in these studies. Thus, the nonspecific binding of gold to MT in vivo should be considered as one aspect in its equilibration among protein binding sites, which include, inter alia, metallothionein. Gold was found to coelute with zinc and cadmium in the MT fraction of rat kidney cytosol, when both Cd and Na₂AuTM were administered to the rats. The possible significance of gold binding to MT in the treatment of rheumatoid arthritis-chrysotherapy-is briefly discussed.     

Covalent binding of 4-nitrobenzyl mercaptan S-sulfate to the sulfhydryl groups of hepatic cytosolic proteins and bovine serum albumin with mixed disulfide bond formation

4-Nitrobenzyl [35S]mercaptan S-sulfonic acid ([35S]NBM S-sulfate), a new type of reactive metabolite of the thiol [35S]NBM in rat liver cytosol fortified with 3'-phosphoadenosine 5'-phosphosulfate, bound rapidly and covalently at pH 7.4 and 37 degrees C to the sulfhydryl groups of rat liver cytosolic proteins with formation of disulfide bonds. From the radioactive proteins was isolated and identified the sole amino acid adduct, S-([35S]NBM)cysteine, after their acid hydrolysis under the anaerobic conditions. Bovine serum albumin (BSA), a model protein with a single SH group, also reacted readily with radioactive NBM S-sulfate to form a disulfide bond in stoichiometric manner. S-([35S]NBM)-cysteine was also isolated and identified as the sole amino acid adduct from the well-washed, radioactive BSA after the same anaerobic acid hydrolysis. A normal hepatic level of GSH not only retarded the BSA-NBM adduct formation completely, but also detached the radioactivity from BSA by the reduction of the disulfide bond with formation of [35S]NBM and its disulfide. Of twenty-one amino acids examined at pH 7.4 and 37 degrees C, only cysteine reacted with NBM S-sulfate and afforded S-(NBM)cysteine with concomitant formations of S-sulfocysteine, cystine, NBM, and its disulfide.     

Adverse immune reactions to gold. I. Chronic treatment with an Au(I) drug sensitizes mouse T cells not to Au(I), but to Au(III) and induces autoantibody formation

Upon weekly i.m. injections of disodium gold thiomalate (Na2AuTM) 100% of A.SW mice produced IgG autoantibodies to antinuclear Ag and nucleolar Ag, respectively; about 70% of C57BL/6 mice produced IgG antinuclear Ag, whereas DBA/2 mice were resistant. Moreover, C57BL/6 mice, but not DBA/2 mice, showed increased mesangial deposits of IgG. These alterations were due not to disodium thiomalate, but to the gold ion of Na2AuTM. An assumed T cell reactivity of susceptible mouse strains to Na2AuTM was tested by means of the direct popliteal lymph node (PLN) assay. However, no distinct PLN reaction to Na2AuTM was detectable. Likewise, AuCl did not induce a PLN reaction. Both Na2AuTM and AuCl contain gold in the Au(I) state. The poor PLN responses to Au(I) contrasted with the strong PLN responses to Au(III) compounds. PLN reactions to Au(III) were dose dependent, T cell dependent, and specific. When Au(III) was reduced to Au(I) by addition of Na2TM or methionine before testing in the PLN assay its sensitizing capacity was significantly decreased. Thus, the oxidation state of gold, i.e., Au(III) vs Au(I), plays a major role for its sensitizing capacity. Therefore, we propose that the Au(I) of Na2AuTM is oxidized to Au(III) before T cells are sensitized and adverse immunologic reactions develop. Results obtained with the adoptive transfer PLN assay indicated that, indeed, repeated i.m. injections of Na2AuTM sensitized A.SW and C57BL/6 splenic T cells to Au(III).     

S-(N-methylcarbamoyl)glutathione: a reactive S-linked metabolite of methyl isocyanate

S-(N-methylcarbamoyl)glutathione, a chemically-reactive glutathione conjugate, has been isolated from the bile of rats administered methyl isocyanate and characterized, as its N-benzyloxycarbonyl dimethylester derivative, by tandem mass spectrometry. The ability of this glutathione adduct to donate an N-methylcarbamoyl moiety to the free -SH group of cysteine was evaluated in vitro with the aid of a highly specific thermospray LC/MS assay procedure. The glutathione adduct reacted readily with cysteine in buffered aqueous media (pH 7.4, 37 degrees C) and after 2 hr, 42.5% of the substrate existed in the form of S-(N-methylcarbamoyl)cysteine. The reverse reaction, i.e. between the cysteine adduct and free glutathione, also took place readily under these conditions. It is concluded that conjugation of methyl isocyanate with glutathione in vivo affords a reactive S-linked product which displays the potential to carbamoylate nucleophilic amino acids. The various systemic toxicities associated with exposure of animals or humans to methyl isocyanate could therefore be due to release of the isocyanate from its glutathione conjugate, which thus may serve as a vehicle for the transport of methyl isocyanate in vivo.     

The 13C NMR study of the binding of gold(I) thiomalate with ergothionine in aqueous solution.

The interaction of gold(I) thiomalate (Autm) (Myocrysine) with ergothionine (ErSH) has been studied in aqueous solution at pH 7.4. It was found that ErSH forms a ternary complex of the type ErS-Au-tm at a 1:1 mole ratio; unlike other thiols (e.g., cysteine and glutathione) it does not eject thiomalate (Htm) as a free ligand. However, in the presence of glutathione (GtSH), both ligands, ErSH as well as Htm, from the ErS-Au-tm complex were freed. The 13C NMR chemical shifts of C-2 resonance of ergothionine in the presence of Autm shifts greater than imidazolidine-2-thione (Imt) and 1,3-Diazinane-2-thione (Diaz), indicating the stronger complexation of ErS-Au-tm compared to Imt-Au-tm and Diaz-Au-tm.     

Formation of S-[2-(N7-guanyl)ethyl] adducts by the postulated S-(2-chloroethyl)cysteinyl and S-(2-chloroethyl)glutathionyl conjugates of 1,2-dichloroethane

The formation of S-[2-(N7-guanyl)ethyl]glutathione (GEG) from dihaloethanes is postulated to occur through two intermediates: the S-(2-haloethyl)glutathione conjugate and the corresponding episulfonium ion. We report the formation of GEG when deoxyguanosine (dG) was incubated with chemically synthesized S-(2-chloroethyl)glutathione (CEG). The depurination of GEG was shown to be first order with a half-life of 7.4 +/- 0.4 h at 27 degrees C. Evidence is also presented for the formation of S-[2-(N7-guanyl)ethyl]-L-cysteine (GEC) in incubation mixtures containing dG and S-(2-chloroethyl)-L-cysteine (CEC), the corresponding cysteine conjugate of CEG. This finding demonstrates that this (haloethyl)cysteine conjugate does not require activation by enzymatic action of cysteine conjugate beta-lyase but, instead, can directly alkylate DNA. The half-life of the depurination of GEC was 6.5 +/- 0.9 h, which is no different from that of GEG. Of the two conjugates, CEC is a somewhat more active alkylating agent toward dG than CEG as N7-guanylic adduct was detected in reaction mixtures with lower concentrations of CEC than with CEG.     

Some complexes of 2.3-Dimercaptopropanol (BAL) with gold(I) and gold(III)

2, 3-Dimercaptopropanol (BAL) reacted with the tetrachloroaurate ion in solution to form a series of insoluble polymers of definite stoichiometry. Both gold(I) and gold(III) have been identified in these compounds. Reaction of BAL with the tetrabromoaurate ion and with thiomalic acid and D-penicillamine complexes of gold also produced insoluble precipitates. However, with an L-cysteine complex of gold no precipitate was isolated, although there was evidence of replacement of cysteine with BAL. The implications of these results for the use of BAL in cases of gold toxicity are discussed     

Non-enzymatic glutathione conjugation of 2-nitroso-6-methyldipyrido [1,2-a: 3',2'-d] imidazole (NO-Glu-P-1) in vitro: N-hydroxy-sulfonamide, a new binding form of arylnitroso compounds and thiols

In order to study the possible detoxification mechanisms of the carcinogenic arylamine, 2-amino-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (Glu-P-1), the in vitro non-enzymatic reaction of 2-nitroso-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (NO-Glu-P-1) with reduced glutathione (GSH) was examined at pH 7.4 under both aerobic and anaerobic conditions. Two GSH-arylamine adducts were isolated and found to contain the Glu-P-1 and GSH moieties in a 1:1 molar ratio via an N-S linkage. Their structures were assigned as sulfinamide (-NH-SO-) and N-hydroxy-sulfonamide (-N(OH)-SO2-) by their behaviour under acidic and basic conditions and by UV-VIS, 1H-NMR, infrared and mass spectrometries. Also, a N-hydroxy-sulfonamide adduct was produced when NO-Glu-P-1 and cysteine were reacted at pH 7.4. The N-hydroxy-sulfonamide structure is a new binding form between arylnitroso compounds and thiols. The formation of these adducts may also take place in vivo as a detoxification of toxic arylamines since GSH is abundant in organs such as liver or kidney.     

Dissociable and non-dissociable cytoplasmic protein-thyroid hormone interactions

Dog kidney cytosol contains a high molecular weight (50 000–70 000) and a low molecular weight (approx. 6000) thyronine-binding protein. Low molecular weight cytosol thyronine-binding protein has not been previously recognized in cytoplasm. Binding of thyroxine (tetraiodothyronine, T₄) by the low molecular weight protein has a half-time of association of more than 24 h and accounts for 32% of bound cytoplasmic tetraiodothyronine after 48 h of incubation. Binding of labeled tetraiodothyronine and triiodothyronine by this moiety is non-dissociable in the presence of 1 · 10^?5 M unlabeled tetra- or triiodothyronine. The low molecular weight protein exists in a dispersed and apparently aggregated form; the latter elutes in the void volume on Sephadex G-100 and its generation is minimized by 2 mM Ca²⁺. This binding protein elutes in a fraction which has a high A_260nm : A_280nm ratio, is pentose enriched (orcinol method) and which, because of these characteristics and low susceptibility to digestion by nuclease, is postulated to be a ribosylated cytoplasmic protein or polypeptide.Binding of tetra- and triiodothyronine by the high molecular weight protein has a half-time of association of 2 h and is saturable. Displacement of labeled triiodothyronine from this cytosol thyronine-binding protein is more readily effected with excess unlabeled tetra- than with triiodothyronine, indicating the absence of a triiodothyronine-specific cytosol thyronine-binding protein site. 3,3′,5′-Triiodothyronine (reverse triiodothyronine) is bound with low avidity. Uptake of high molecular weight protein by isolated kidney cell nuclei cannot be demonstrated.Binding of tetraiodothyronine by cytosol proteins is independent of pH in the pH range 6.8–8.9, but binding of triiodothyronine is minimized at pH 7.4 and enhanced at alkaline pH to the point of equivalency of tetra- and triiodothyronine binding at pH 8.9.At concentrations of tetraiodothyronine calculated to exist intracellularly, essentially all soluble fraction tetraiodothyronine is bound to cytosol thyronine-binding protein, restricting access of this iodothyronine to binding sites in nucleus and mitochondria. Cytosol removes labeled tetra- and triiodothyronine previously reacted in vitro with isolated cell nuclei; such removal is a linear function of cytosol protein concentration and is blocked by saturation of cytosol thyronine-binding protein with unlabeled iodothyronines. Only the high molecular weight protein accounts for unbinding by cytosol of nuclear hormone.     

Platinum(II) binding to metallothioneins

The reaction of equine renal metallothionein (MT) with excess K2PtCl4 at pH 2 results in a polymeric adduct containing 17 +/- 2 mol Pt/mol MT. A monomeric adduct containing 7 mol Pt/mol MT is obtained at neutral pH. Rates of reaction of Pt7MT with DTNB and iodoacetic acid are consistent with Pt2+ to cysteine thiolate coordination, and the extent of reaction in both cases is 11 +/- 2 mol cys/mol MT. Adducts from the reaction of K2PtCl4 with apoMT chemically modified at the N-terminal methionine residue, Cd7MT, and native MT are also reported. A structural model of Pt7MT is proposed in which the square planar tetrathiolate Pt(II) unit is incorporated into a three-metal beta cluster. Implications for the metabolism of platinum anticancer drugs are discussed.     

Acid-base properties and copper(II) complexes of dipeptides containing histidine and additional chelating bis(imidazol-2-yl) residues

Copper(II) complexes of dipeptides of histidine containing additional chelating bis(imidazol-2-yl) agent at the C-termini (PheHis-BIMA [N-phenylalanyl-histidyl-bis(imidazol-2-yl)methylamine] and HisPhe-BIMA [N-histidyl-phenylalanyl-bis(imidazol-2-yl)methylamine]) were studied by potentiometric, UV-Visible and Electron Paramagnetic Resonance (EPR) techniques. The imidazole nitrogen donor atoms of the bis(imidazol-2-yl)methyl group are described as the primary metal binding sites forming stable mono- and bis(ligand) complexes at acidic pH. The formation of a ligand-bridged dinuclear complex [Cu2L2]4+ is detected in equimolar solutions of copper(II) and HisPhe-BIMA. The coordination isomers of the dinuclear complex are described via the metal binding of the bis(imidazol-2-yl)methyl, amino-carbonyl and amino-imidazole(His) functions. In the case of the copper(II)-PheHis-BIMA system the [NH2, N-(amide), N(Im)] tridentate coordination of the ligand is favoured and results in the formation of di- and trinuclear complexes [Cu2H(-1)L]3+ and [Cu3H(-2)L2]4+ in equimolar solutions. The presence of these coordination modes shifts the formation of "tripeptide-like" ([NH2, N-, N-, N(Im)]-coordinated) [CuH(-2)L] complexes into alkaline pH range as compared to other dipeptide derivatives of bis(imidazol-2-yl) ligands. Although there are different types of imidazoles in these ligands, the deprotonation and coordination of the pyrrole-type N(1)H groups does not occur below pH 10.     

Studies on the heat-precipitated phosphoenzyme complex of eel electric organ (Na + K).ATPase.

When the hydrolytic reaction between eel electric organ (Na + K) · ATPase and [γ-³²P]ATP is terminated at neutral pH by heat precipitation, a phosphoenzyme complex is formed which reaches maximal levels in the simultaneous presence of Mg, Na, and K. After formation of a steady-state level of phosphoenzyme in the presence of Mg and Na, a pulse of K increases the level of the heat-precipitated phosphoenzyme (while decreasing the level of the acid-precipitated phosphoenzyme). The formation of the heat-precipitated phosphoenzyme is clearly inhibited by ouabain only when the phosphoenzyme is formed in the presence of Mg, Na, and K. Inorganic phosphate decreases the level of the heat-precipitated phosphoenzyme, but not that of the acid-precipitated phosphoenzyme (in the presence of Mg and Na or in the presence of Mg, Na, and K). Moreover, a heat-precipitated, ouabain-sensitive phosphoenzyme forms in the reaction between the eel (Na + K) · ATPase and ³²P_i with or without ATP. The pH stability of the heat-precipitated phosphoenzyme complex is maximal at pH 6 to 8, and this complex shows little or no reactivity with neutral hydroxylamine, suggesting that the phosphate is not bound to an acyl residue of the protein. These experiments indicate that both heat-resistant and acid-resistant phosphoenzymes are formed during the (Na + K) · ATPase reaction at pH 7.4.     

Synthesis and X-ray crystal structure determination of two pseudopolymorphic forms of μ-[1,2-bis(diphenylphosphino)ethane] bis [chlorogold(I)]: a digold(I) DNA binder

An improved synthetic route to the linearly coordinated digold(I) complex, μ-[1,2-bis(diphenylphosphino)ethane]bis[chlorogold(I)], is reported. This complex crystallizes in two pseudopolymorphic forms from a chloroform/methylene chloride solution; the crystal and molecular structures of both are discussed and compared. In both crystal forms the potentially chelating diphenylphosphinoethane (dppe) ligand instead coordinates to two separate gold atoms. The coordination environment of each gold atom is linear in both pseudopolymorphs and the structures display normal goldchloride and goldphosphorus bond distances. On the molecular level, the pseudopolymorphs differ fundamentally by a twist about one of the gold phosphorous bonds with the phosphorous atoms of the dppe ligand adopting a transoid orientation relative to one another in both polymorphs. These conformations thus place the intramolecular gold atoms 6 Å apart and preclude intramolecular AuAu bonding interactions. As has been observed for related gold(I) complexes there are short intermolecular AuAu contacts of the order of 3.2 Å present in both structures. The conformational flexibility of the gold complex relative to its observed biological activity as a DNA binder is discussed.     

Generation of reactive oxygen species by photolysis of the ruthenium(II) complex Ru(NH3)5(pyrazine)2+ in oxygenated solution.

Metal-to-ligand charge transfer photolysis of the ruthenium(II) pyrazine complex Ru(NH3)5pz2+ (I) in pH 7.4 oxygenated phosphate buffer solution generates the Ru(III) analog Ru(NH3)5pz3+ plus the reactive oxygen species singlet oxygen and superoxide. Based on the very short MLCT lifetime (re-measured as approximately 250 ps in D2O) of I* and the quantum yield for singlet oxygen formation (0.01 for aerated D2O) the rate constant for oxygen quenching of I* was calculated to be approximately (3+/-1)x10(10) M-1 s-1.     

Synthesis by Schwann cells of basal lamina and membrane-associated heparan sulfate proteoglycans

Primary cultures that contain only Schwann cells and sensory nerve cells synthesize basal lamina. The assembly of this basal lamina appears to be essential for normal Schwann cell development. In this study, we demonstrate that Schwann cells synthesize two major heparan sulfate-containing proteoglycans. Both proteoglycans band in dissociative CsCl gradients at densities less than 1.4 g/ml, and therefore, presumably, have relatively low carbohydrate-to-protein ratios. The larger of these proteoglycans elutes from Sepharose CL-4B in 4 M guanidine hydrochloride (GuHCl) at a Kav of 0.21 and contains heparan sulfate and chondroitin sulfate chains of Mr 21,000 in a ratio of approximately 3:1. This proteoglycan is extracted from cultures by 4 M GuHCl but not Triton X-100 and accumulates only when Schwann cells are actively synthesizing basal lamina. The smaller proteoglycan elutes from Sepharose CL-4B at a Kav of 0.44 and contains heparan sulfate and chondroitin sulfate chains of Mr 18,000 in a ratio of approximately 4:1. This proteoglycan is extracted by 4 M GuHCl or by Triton X-100. The accumulation of this proteoglycan is independent of basal lamina production.     

The spin trapping of superoxide with M4PO (3,3,5,5-tetramethylpyrroline-N-oxide)

The spin trap 3,3,5,5-tetramethylpyrroline-N-oxide (M4PO) reacts with superoxide to produce a short-lived spin adduct (M4PO/.OOH, AN = 14.0 G, AH = 6.5 G) that decays by a first-order process (t1/2 approximately 35 s at pH 7.4). This short lifetime may limit its usefulness in studies of superoxide.     

Complex formation of p-boronophenylalanine with some monosaccharides

To increase the solubility of p-boronophenylalanine (p-bpa) in neutral pH solution, the complex formation of p-bpa with some monosaccharides has been studied by 11B-NMR and UV spectroscopy. The complex formation constants (log K) obtained by the UV method in pH 7.4 solution are 2.43 (fructose), 2.19 (mannitol), 1.28 (galactose), 1.10 (mannose), and 0.85 (glucose), respectively. One hundred milligrams of p-bpa is able to dissolve in 3 ml of 0.3 M fructose solution at pH 7.98. Based on the results obtained, the behavior of p-bpa-monosaccharide complexes in vivo after injection of the complex solution is described.     

A (1)H NMR study of the interaction of antitumor metallocenes with glutathione

The interaction of the antitumor active metallocene dihalides Cp(2)MCl(2) (M=Ti, Nb, Mo) and 1 equiv. of glutathione was studied by (1)H NMR spectroscopy at pD 2-7 in 4 mM NaCl solutions. No interaction between glutathione and titanocene dichloride was detected at pD 2, while at pD 5-7 competitive hydrolysis of the cyclopentadienyl ligands occurred. With niobocene dichloride formation of approximately 20% of an adduct was observed at pD 2 and 5, but hydrolysis of the Cp ligands in the adduct occurred over 24 h. Molybdocene dichloride formed two stable adducts at pD 6 which were tentatively assigned as a Cp(2)Mo-glutathione chelate involving coordination of the cysteine thiol and glycine carboxylate to the metal centre, and a thiol centred 1:2 Cp(2)Mo-glutathione complex. The implications for the mechanism of antitumor action of the metallocene dihalides is discussed.     

The S-sulfate formation from 4-nitrobenzyl mercaptan in rat liver cytosol

4-Nitrobenzyl mercaptan (NBM) was enzymatically transformed at pH 6.0 into its S-sulfate in rat liver cytosol fortified with 3'-phosphoadenosine 5'-phosphosulfate. At pH 7.4, the S-sulfate was not detected from the incubation mixture. 4-Nitrobenzyl alcohol was also transformed under the same incubation conditions into the corresponding O-sulfate at a higher rate at pH 6.0 than at pH 7.4. Under the incubation conditions used, NBM S-sulfate reacted with the substrate NBM at a significant rate to afford 4-nitrobenzyl disulfide. The disulfide formation from NBM and the S-sulfate occurred more readily at pH 7.4 than at pH 6.0, so that biologically formed NBM S-sulfate was strongly suggested not to remain unchanged in the incubation mixture at pH 7.4.