Sulfhydryl Reagents P-chloromercuribenzoate Research Articles

Copper Efflux from Murine Microvascular Cells Requires Expression of the Menkes Disease Cu-ATPase

Previously, we showed that the transport of Cu by PC12 pheochromocytoma cells and C6 glioma cells correlated with the expression of a Cu-transporting ATPase (Atp7a) that has been linked to Menkes disease. Here, we show that cerebrovascular endothelial (CVE) cells that comprise the blood-brain barrier (BBB) also express the gene for the Cu-ATPase. By using reverse transcription-polymerase chain reaction (RT-PCR) and primers designed from mouse Atp7a cDNA, we amplified a 925-bp and a 760-bp cDNA fragment from two extreme regions of Atp7a mRNA from murine CVE cells; 777 bp of the 925-bp fragment and 677 bp of the 760-bp fragment had a 99.7 and 100% sequence homology, respectively, with mouse Atp7a cDNA. The 777-bp sequences covered the heavy metal binding (Hmb) domain and the 677-bp fragment coded for residues at the −COOH terminus of Atp7a. A functional analysis showed that Cu efflux was blocked by the sulfhydryl reagent p-chloromercuribenzoate (p-CMB), a potential inhibitor of Atp7a function. This study provides strong evidence that a Cu-ATPase in the BBB controls the penetration of Cu into the brain and that lesions to the Cu-ATPase in CVE cells are a primary cause of low brain Cu levels in Menkes disease.

A Menkes P-type ATPase involved in copper homeostasis in the central nervous system of the rat.

We previously reported that copper efflux from C6 rat glioma cells was blocked by a brief exposure to sulfhydryl reagents p-chloromercuribenzoate (PCMB) and iodoacetamide as well as dicyclohexylcarbodiimide, suggesting the possible involvement of a Cu-transporting ATPase in the efflux mechanism. In this report, we show that copper efflux from PC12 cells, a neuron-like cell line established from rat adrenal pheochromocytoma, is also inhibited by PCMB exposure. Furthermore, we show that both C6 and PC12 cells express a homolog of the Menkes gene (MNK) as detected by RT-PCR with primers designed from a mouse cDNA and confirmed by sequence analysis of the amplified product. An expected 760-bp fragment representing the transduction and phosphorylation domains and a 925-bp fragment encoding the heavy metal-binding domain of Atp7a were amplified from a RNA extract of C6 and PC12 cells. Sequence data revealed that 690 bp of the 760-bp fragment from C6 cells were an identical match to a similar fragment from PC12 cells. Both fragments encoded a 229 amino-acid polypeptide that had a 98.7% sequence homology to mouse Atp7a. In addition, 880 bp from the 925-bp fragment of the two cell lines were identical and encoded a 293 amino-acid polypeptide with 94.5% sequence homology to mouse Atp7a. These data establish that a Menkes-type Cu-transporting ATPase is expressed in rat C6 and PC12 cells and strongly support the hypothesis that both neurons and glia are involved in maintaining Cu homeostasis in the central nervous system.

Activation of a NADH dehydrogenase in the human erythrocyte by beta-adrenergic agonists: possible involvement of a G protein in enzyme activation.

NADH dehydrogenase in the plasma membrane transfers electrons from NADH to external oxidants like ferricyanide, through pathways which are linked to metabolic processes in the cell. Hormone binding to specific sites (receptors) can modify the enzyme activity, suggesting a direct or indirect coupling between the redox system and the hormone receptors. Reduction of external ferricyanide to ferrocyanide by human erythrocytes was stimulated by beta-adrenergic agonists (adrenaline, ritodrine and isoxsuprine), this effect being dependent upon concentration and pH. The agonist-stimulatory effect was attenuated in the presence of metoprolol (10(-4) M), a beta-adrenergic antagonist, and was not modified in the presence of prazosin, an alpha-adrenergic antagonist, suggesting that modification of the redox activity is mediated by binding of the agonists to beta-adrenergic receptors present in the human erythrocytes. Basal and agonist-dependent activities were inhibited in the presence of sulfhydryl reagents p-chloromercuribenzoate (PCMB, 10(-5) M) and N-ethylmaleimide (NEM, 10(-3) M), indicating the involvement of -SH groups. Inactivation by NEM was reversed by washing the cells with GTP (10(-3) M) and GTP gamma S (10(-4) M), suggesting that the specific alkylated -SH group(s) is located on a G protein in the hormone-receptor-G-protein complex. The human erythrocytes contain G proteins, displaying both guanine-nucleotide-binding properties and GTPase activity. Fluoride (10(-2) M) and fluoroaluminate (AlF4- (F-, 10(-2) M + Al3+, 10(-5) M), G protein activators, enhanced the basal and agonist-dependent activities, suggesting the involvement of G proteins in this system. The overall results indicated that one of the coupling components between the hormonal receptors and the redox system is probably a G protein, and the mechanism of enzyme activation after hormone binding to the receptor is based on the redox state of cysteine residues probably within the receptor-G-protein complex.

An arginine/ornithine exchange system in Spiroplasma melliferum

Spiroplasma melliferum cells utilize arginine via the arginine dihydrolase pathway. L-Arginine uptake by intact cells was a saturable process both as a function of time and arginine concentration (Km = 40 μM). Uptake was not affected by pH in the range pH 5.0-8.0, or by L-citrulline, D-arginine, L-histidine or L-canavanine at concentrations tenfold higher than that of L-arginine. In contrast, L-arginine uptake was markedly inhibited by L-ornithine and partially inhibited by L-lysine. Uptake was neither affected by protonophores nor by cation ionophores, but was inhibited by protease treatment or by the sulfhydryl reagents p-chloromercuribenzoate or N-ethylmaleimide. Sealed membrane vesicles prepared by fusing isolated S. melliferum membranes with asolectin-cholesterol vesicles catalysed a rapid exchange (t1/2 = 1 min) between arginine and ornithine. Exchange did not require ATP and could be demonstrated in both directions, i.e. with either arginine or ornithine trapped within vesicles. These observations suggest that the driving forces for arginine uptake by whole cells are the concentration gradients of arginine and ornithine formed by arginine metabolism.

Role of carboxyl and sulfhydryl residues on rabbit small intestinal brush-border membrane Na(+)-glucose cotransporter.

The role of sulfhydryl (SH) and carboxylic acid residues in Na(+)-dependent glucose uptake, Na(+)-dependent phlorizin binding, and substrate exchange by the rabbit small intestinal brush-border membrane (BBM) Na(+)-glucose cotransporter was examined in sodium dodecyl sulfate-BBM vesicles. The sulfhydryl reagent p-chloromercuribenzoate (PCMB) inhibited all three measures of cotransporter function in a dithiothreitol-sensitive manner with similar K0.5 values (concn of PCMB resulting in 50% inhibition). PCMB sulfonate had no effect on Na(+)-glucose cotransporter function < 250 microM. The carboxylic acid reagent 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide no effect on Na(+)-glucose cotransporter function. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited all three measures of cotransporter function with similar K0.5 values for inhibition. Inhibition by DCCD did not require addition of a nucleophile. In contrast, PCMB-pretreated cotransporter was insensitive to DCCD in the absence of added nucleophile with respect to substrate transport (Na(+)-dependent glucose uptake) but not Na(+)-dependent phlorizin binding. These results indicate an intravesicular or lipophilic environment for both the PCMB-reactive SH residue and the DCCD-reactive carboxylic acid residues, suggesting that a SH residue may act as an endogenous nucleophile for interaction of DCCD with the Na(+)-glucose cotransporter and suggesting that different carboxylic acid residues may be involved in Na(+)-dependent glucose uptake and Na(+)-dependent phlorizin binding.

Characteristics of sorbitol uptake in rat glial primary cultures.

Uptake of [U-14C]sorbitol was studied in astrogliarich rat primary cultures. Initial rate of sorbitol uptake is proportional to sorbitol concentration between 20 microM and 400 mM. Sorbitol transport is not inhibited by glucose, fructose, and a variety of structurally related polyols, or by cytochalasin B, an inhibitor of glucose transport. Phloretin, phlorizin, filipin, and n-hexanol, all compounds that alter the properties of biological membranes, and the sulfhydryl reagent p-chloromercuribenzoate inhibit sorbitol uptake to various degrees. Variation in the concentrations of extracellular Na+ and K+ does not affect transfer of sorbitol across the cell membrane. It is concluded that sorbitol is taken up into glial cells by a diffusion process, not involving a carrier and probably not through the lipid bilayer, but through a proteinaceous channel-like structure.

Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Characterization of the membrane-associated component.

Sodium dodecyl sulfate (SDS) was shown to elicit NADPH-dependent superoxide (O2-) production by a cell-free system derived from sonically disrupted resting guinea pig macrophages (Bromberg, Y., and Pick, E. (1985) J. Biol. Chem. 260, 13539-13545). O2- production was absolutely dependent on the cooperation between a membrane-associated component, sedimenting with the 48,000 X g pellet and a cytosolic factor, nonsedimentable at 265,000 X g. The present report describes the solubilization and characterization of the membrane-associated component of the SDS-activable O2(-)-forming NADPH oxidase (operationally termed pi). Treatment of the 48,000 X g pellet with 30 mM octyl glucoside resulted in complete transfer of pi to the soluble fraction. The solubilized pellet produced an average of 0.92 mumol of O2-/mg of protein/min upon reduction of octyl glucoside content below the critical micellar concentration and in the presence of cytosol, 100 microM SDS, and 0.2 mM NADPH. The activity of solubilized pellet-cytosol combinations was also expressed as NADPH-dependent, azide-resistant oxygen consumption and hydrogen peroxide production. pi was inactivated by the sulfhydryl reagent p-chloromercuribenzoate. Solubilized pellet contained spectroscopically detectable cytochrome b559 (225.6 +/- 15.0 pmol/mg mg protein). Both pi and cytochrome b559 were bound by Cibacron Blue Sepharose and could be eluted by a gradient of octyl glucoside (0-30 mM) in the presence of 1 M KCl. On high performance gel filtration on Superose 12, both pi and cytochrome b559 eluted in the excluded volume; when 25 mM octyl glucoside was present in the elution buffer, pi was partially dissociated from cytochrome b559. Sequential purification of pi on Blue Sepharose followed by gel filtration on Superose 12 in the presence of 25 mM octyl glucoside lead to complete resolution of pi from cytochrome b559 (pi was found in the Mr = 28,000 - 11,000 range while the bulk of cytochrome b559 eluted in the Mr = 113,000 - 71,000 range). We propose that pi is distinct from cytochrome b559 and represents a membrane-associated component in an amphiphile-activated electron transport chain from NADPH to oxygen.

Role of sulfhydryl groups in catalytic activity of purified cholesterol 7α-hydroxylase system from rabbit and rat liver microsomes

Electrophoretically homogeneous preparations of cytochrome P-450 LM 4 from cholestyramine-treated rabbits catalyzed 7α-hydroxylation of cholesterol, 12α-hydroxylation of 5β-cholestane-3α,7α-diol and 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol. Dithiothreitol, a disulfide reducing agent, specifically stimulated the cholesterol 7α-hydroxylase activity severalfold. The 7α-hydroxylase activity was much more sensitive to the sulfhydryl reagents p-chloromercuribenzoate, N-ethylmaleimide and iodoacetamide than the 12α- and 25-hydroxylase activities. Cholesterol 7α-hydroxylase activity, inactivated by these reagents, could be reactivated by treatment with dithiothreitol. Similar results were obtained with purified cytochrome P-450 from rat liver microsomes. The results indicate that sulfhydryl groups are more important for cholesterol 7α-hydroxylation than for other C 27-steroid hydroxylations.

Ultrastructural demonstration of ferricyanide reductase (Diaphorase) activity in the envelopes of the plastids of etiolated barley (Hordeum vulgare L.) leaves

Electron-dense precipitate was found consistently in the plastid envelope compartment in etiolated barley (Hordeum vulgare L.) leaves, incubated prior to fixation with succinate or malate as substrates and ferricyanide as the electron acceptor. Sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide abolished this reaction, while KCN did not affect it. Prefixation with 0.1% glutaraldehyde followed by incubation in basic media did not change the fine structural localization of precipitate, whereas pretreatment with 1.25% glutaraldehyde resulted in aspecific precipitation. Omission of the subtrate from the medium brought about diminished or negative reaction. Our results indicate that a (possibly not yet assembled) nitrate reductase complex is present in the plastid envelope compartment, the diaphorase part of which is responsible for the observed precipitation.

Effects of sulfhydryl reagents on the binding and release of penicillin G by D-alanine carboxypeptidase IA of Escherichia coli.

Purified D-alanine carboxypeptidase IA of Escherichia coli is inhibited by penicillin G and binds penicillin G reversibly. The binding of penicillin to the enzyme is relatively insensitive to sulfhydryl reagents, while release of penicillin from the enzyme is severely inhibited by these reagents. The inhibition of release parallels the inhibition of carboxypeptidase activity by the sulfhydryl reagents. In the presence of the sulfhydryl reagent p-chloromercuribenzoate, an acyl-enzyme intermediate, produced by the reaction of carboxypeptidase IA with diacetyl-L-lysyl-D-alanyl-D-alanine, accumulates and can be isolated. These results indicate that binding of penicillin to carboxypeptidase IA occurs by an acylation step of the carboxypeptidase reaction, while penicillin release occurs by a deacylation step of the reaction. Only the latter is inhibited by sulfhydryl reagents.

Inactivation of Soluble Bovine Milk Galactosyltransferase (Lactose Synthetase) by Sulfhydryl Reagents and Trypsin: PROTECTION BY SUBSTRATES AND PRODUCTS

Abstract Galactosyltransferase exhibited a time-dependent loss of activity when incubated with sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide. Though incubations were continued for periods 20-fold greater than the half-time of inactivation, neither reagent eliminated all catalytic activity, and generally 5 to 10% of the initial activity remained. p-Chloromercuribenzoate inactivated the enzme more rapidly then N-ethylmaleimide, but the inhibition by p-chloromercuribenzoate was reversed by mercaptoethanol. Substrates and substrate analogs protected against inhibition by p-chloromercuribenzoate and N-ethylmaleimide. Ligands giving the most protection against inhibition were analogs of the nucleotide substrate and product, uridine diphosphate galactose and UDP, respectively, in the presence of manganous chloride. When incubated with 10 mm N-ethylmaleimide, galactosyltransferase exhibited an apparent rate constant of inactivation of 0.023 min-1; adding analogs of UDP at concentrations of 2 mm reduced this rate by 30% and the same analogs in the presence of manganous chloride reduced the rate constant of inactivation by 98%. Inhibition of galactosyltransferase by N-ethylmaleimide resulted in a dramatic decrease in Vmax whereas the apparent Km for UDP-galactose remained constant. A similar pattern of protection against inactivation of galactosyltransferase by trypsin was observed suggesting that a conformational change in the protein is responsible for the observed protection.

Nature of the specificity of alcohol coupling to L-alanine transport into isolated membrane vesicles of a marine pseudomonad.

Ethanol stimulated the uptake of l-alanine into isolated membrane vesicles of a marine pseudomonad at a rate and to an extent comparable with that obtained with reduced nicotinamide adenine dinucleotide (NADH) or the artificial electron donor ascorbate-N, N, N', N'-tetramethyl-p-phenylenediamine (ascorbate-TMPD). Methanol and branched-chain alcohols had little or no capacity to energize transport. No quantitative relationship was found between the ability of a compound to induce oxygen uptake and to energize transport, since with ethanol initial rates of oxygen uptake were approximately 4% of that obtained with NADH or ascorbate-TMPD. Cytochrome analysis revealed that NADH and ethanol reduced cytochromes b and c, whereas ascorbate-TMPD coupled primarily at the level of cytochrome c. Approximately 25% of the cytochromes reduced by dithionite were reducible by ethanol. Ethanol reduction of both cytochromes b and c was prevented by 2-heptyl-4-hydroxyquinoline-N-oxide, p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetate. The ethanol- and NADH-energized transport systems for l-alanine were subject to quantitatively similar inhibition by cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, 2, 4-dinitrophenol, and the sulfhydryl reagents p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetate. In contrast, for ascorbate-TMPD-driven transport, only cyanide and 2, 4-dinitrophenol remained fully effective as inhibitors, p-chloromercuribenzoate was only half as effective, and the other compounds stimulated transport. Inhibition of ethanol oxidation strikingly paralleled the inhibition of ethanol-driven transport for each of the inhibitors, including 2, 4-dinitrophenol. Marked differences between inhibition of oxygen uptake and inhibition of transport were observed when NADH or ascorbate-TMPD were the electron donors. The data indicate that only a small proportion of the respiratory chain complexes in the membrane vesicles are involved in transport and these are efficiently coupled to ethanol oxidation. The results also suggest that when 2, 4-dinitrophenol inhibits transport it is not acting as an uncoupling agent.

Histochemical detection of in vitro DNA polymerase activity.

A significant level of nuclear DNA polymerase activity (deoxyribonucleoside-triphosphate: DNA deoxyribonucleotidyltransferase EC 2.7.7.7) can be detected in (a) cryostat sections and (b) toluene treated tissues of onion seed radicles by autoradiography with 3H-TTP. Incorporation was optimal in the presence of all four deoxyribonucleoside triphosphates and dithiothreitol but sensitive to (a) DNase (b) high concentrations of KCl and (c) the sulfhydryl reagents p-chloromercuribenzoate and N-ethyl maleimide. Attempts to activate incorporation with the inclusion of (a) small concentrations of DNase and (b) ATP in the incubating medium failed. The percentage of cells displaying enzyme activity in vitro was similar to the number observed in vivo suggesting that incorporation was associated with S-phase cells and is genuine DNA replication as opposed to repair synthesis.

Cofactor Requirements of γ-Butyrobetaine Hydroxylase from Rat Liver

Abstract The hydroxylation of γ-butyrobetaine (4-trimethylaminobutyrate) to carnitine (3-hydroxy-4-trimethylaminobutyrate) is catalyzed by a soluble enzyme from rat liver which has been partially purified. The enzyme which previously has been shown to require molecular oxygen and ferrous ion has a specific requirement for 2-ketoglutarate. Several reductants stimulate the formation of hydroxylated product; the most active ones are ascorbate and isoascorbate, whereas reduced 2,6-dichlorophenolindophenol and 2-amino-5,6-dimethyl-4-hydroxy-5,6,7,8-tetrahydropteridine are less active. The previously observed stimulation both by NADPH, isocitrate, isocitrate dehydrogenase (EC 1.1.1.42), and by microsomes has been found to be related to the formation of 2-ketoglutarate. One mole of 2-ketoglutarate is degraded per mole of carnitine formed. Carbon dioxide and succinate are products of 2-ketoglutarate degradation; no free succinic semialdehyde can be detected. Several compounds which are chemically or biologically related to γ-butyrobetaine and to 2-ketoglutarate have been tested as inhibitors in the reaction. Only succinic semialdehyde and 3-trimethylaminopropyl-1-sulfonate are effective inhibitors. The sulfhydryl reagents p-chloromercuribenzoate and p-chloromercuriphenylsulfonate in 0.1 mm concentration completely inhibit the formation of carnitine after preliminary incubation with the enzyme for 20 min at 37°. N-Ethylmaleimide, iodosobenzoate, and iodoacetate are less effective inhibitors under these conditions, whereas sodium arsenite, carbarsone, and acetarsone cause appreciable inhibition only in 10 mm concentration. Preliminary incubation of the enzyme with ferrous ion and ascorbate results in significantly lower yield of carnitine than when catalase has also been included in the preliminary incubation medium. It is suggested that γ-butyrobetaine is hydroxylated to carnitine simultaneously with the oxidative decarboxylation of 2-ketoglutarate in a reaction sequence which involves the intermediate formation of a peroxide of the two substrates. Ferrous ion might act as oxygen-activating agent. Free sulfhydryl groups are apparently necessary for enzymic activity and ascorbate and catalase probably act by maintaining these groups as well as ferrous ion in the reduced state.

Effects of sulfhydryl reagents on infectivity of some viruses

A number of viruses have been treated with the sulfhydryl reagents p-chloromercuribenzoate and iodoacetamide under standard conditions. This treatment reduces the infectivity of pleuropneumonia and psittacosis-lymphogranuloma organisms and also that of most groups of viruses. Arborviruses and enteroviruses differed in susceptibility to the reagents. Some, such as GDVII and group A arborviruses, were completely resistant to thiol reagents under the conditions stated, whereas others, including other arborviruses, poliovirus, and ECHO viruses, were susceptible in various degrees.

The inactivation of enterovirus infectivity by the sulfhydryl reagent p-chloromercuribenzoate.

The infectivity of several enteroviruses was inactivated by the sulfhydryl reagent p-chloromercuribenzoate (PCMB). The rate of inactivation was dependent on the ionic environment in which the reaction was carried out. Inactivation of infectivity was reversed by the thiol compound, reduced glutathione. Under certain conditions, PCMB prevented the adsorption of some enteroviruses to monolayer cultures of monkey kidney cells. The results suggest that enterovirus sulfhydryl groups are involved in the establishment of infection, and that they play a role in the adsorption of virus to host cells.