Thiol Reagent N-ethylmaleimide Research Articles

The Role of Cysteines and Histidins of the Norepinephrine Transporter

The thiol reagent N-ethylmaleimide (NEM) is known to inhibit irreversibly ligand binding by the norepinephrine transporter (NET), while the simultaneous presence of NET substrates or ligands protects from this inhibition. Therefore, cysteine residues located within the substrate binding pocket of the NET were assumed to play an important role in ligand binding. To examine which (if any) of the 10 cysteines (Cys) of the human (h) NET might be involved in transport and/or binding function, we mutated all hNET cysteines to alanine. Using transfected HEK293 cells we studied NEM effects on the hNET with respect to [(3)H]nisoxetine binding. Two cysteines (Cys176 and Cys185) within the extracellular loop of the NET have been proposed to form a disulfide bond. We could demonstrate that this is of crucial importance as corresponding hNET mutants, in which these cysteines have been replaced, showed a lack of plasma membrane expression. However, due to their oxidized state in the native NET protein, Cys176 and Cys185 may not be targets for NEM. All other Cys-to-Ala hNET mutants were fully active and showed no change in inhibition of [(3)H]nisoxetine binding by NEM. These observations clearly exclude cysteines as being involved in hNET ligand binding. Since NEM also interacts with histidin (His), we mutated all 13 histidins of the hNET to alanine and examined the NET mutants in functional and binding assays. His222 within the large extracellular loop of the transporter was identified as an interaction partner of NEM since in the corresponding hNET mutant NEM exhibited a significantly reduced inhibitory potency. Furthermore, we could show that histidins in position 296, 370 and 372 are important for nisoxetine binding, while His220, 441, 598 and 599 are crucial for plasma membrane expression of the hNET.

Release of NO from a nitrosyl ruthenium complex through oxidation of mitochondrial NADH and effects on mitochondria

Nitrosyl ruthenium complexes are promising NO donor agents with numerous advantages for the biologic applications of NO. We have characterized the NO release from the nitrosyl ruthenium complex [Ru(NO2)(bpy)2(4-pic)]+ (I) and the reactive oxygen/nitrogen species (ROS/RNS)-mediated NO actions on isolated rat liver mitochondria. The results indicated that oxidation of mitochondrial NADH promotes NO release from (I) in a manner mediated by NO2 formation (at neutral pH) as in mammalian cells, followed by an oxygen atom transfer mechanism (OAT). The NO released from (I) uncoupled mitochondria at low concentrations/incubation times and inhibited the respiratory chain at high concentrations/incubation times. In the presence of ROS generated by mitochondria NO gave rise to peroxynitrite, which, in turn, inhibited the respiratory chain and oxidized membrane protein–thiols to elicit a Ca2+-independent mitochondrial permeability transition; this process was only partially inhibited by cyclosporine-A, almost fully inhibited by the thiol reagent N-ethylmaleimide (NEM) and fully inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). These actions correlated with the release of cytochrome c from isolated mitochondria as detected by Western blotting analysis. These events, typically involved in cell necrosis and/or apoptosis denote a potential specific action of (I) and analogs against tumor cells via mitochondria-mediated processes.

Thiol - Dependent Sensitization of Mitochondria and Tumor Cells to Doxorubicin

The problem of multidrug resistance has been intensively studied during the last decade. It is known that some synthetic progestins can chemosensitize tumor cells to cytostatic drugs; however, the mechanisms of the sensitization are not clearly understood. In this study, the effect of synthetic analogue of progesterone (17α-acetoxy- 3b-butanoyloxy-6-methyl-pregna-4, 6-diene-20-on, buterol) on the survival of doxorubicin-resistant MCF-7 cells and the coupling of isolated mitochondria was investigated in comparison with the thiol reagent N-ethylmaleimide (NEM). Both compounds increased the doxorubicin-induced cell death and decreased the oxidative phosphorylation and the coupling in mitochondria; their presence was necessary for the direct action of doxorubicin on the mitochondrial membrane potential. Possible specific targets for NEM and buterol are the SH-groups of mitochondrial ATPase and the nucleotide-binding domain of MDR proteins, owing to which they inhibit the activity of both ATPase and MDR proteins and increase the accumulation of drugs in the cell. In addition, buterol can form hydrogen bonds between its ether carbonyl at carbon C3 and some other thiols, thereby inducing oxidative stress. By contrast to NEM, which irreversibly blocks thiol groups, buterol binds thiols non-covalently, which provides a new useful approach to the enhancement of the anticancer activity of cytostatics in tumor cells.

Functional Importance of GGXG Sequence Motifs in Putative Reentrant Loops of 2HCT and ESS Transport Proteins

The 2HCT and ESS families are two families of secondary transporters. Members of the two families are unrelated in amino acid sequence but share similar hydropathy profiles, which suggest a similar folding of the proteins in membranes. Structural models show two homologous domains containing five transmembrane segments (TMSs) each, with a reentrant or pore loop between the fourth and fifth TMSs in each domain. Here we show that GGXG sequence motifs present in the putative reentrant loops are important for the activity of the transporters. Mutation of the conserved Gly residues to Cys in the motifs of the Na(+)-citrate transporter CitS in the 2HCT family and the Na(+)-glutamate transporter GltS in the ESS family resulted in strongly reduced transport activity. Similarly, mutation of the variable residue "X" to Cys in the N-terminal half of GltS essentially inactivated the transporter. The corresponding mutations in the N- and C-terminal halves of CitS reduced transport activity to 60 and 25% of that of the wild type, respectively. Residual activity of any of the mutants could be further reduced by treatment with the membrane permeable thiol reagent N-ethylmaleimide (NEM). The X to Cys mutation (S405C) in the cytoplasmic loop in the C-terminal half of CitS rendered the protein sensitive to the bulky, membrane impermeable thiol reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AmdiS) added at the periplasmic side of the membrane, providing further evidence that this part of the loop is positioned between the transmembrane segments. The putative reentrant loop in the C-terminal half of the ESS family does not contain the GGXG motif, but a conserved stretch rich in Gly residues. Cysteine-scanning mutagenesis of a stretch of 18 residues in the GltS protein revealed two residues important for function. Mutant N356C was completely inactivated by treatment with NEM, and mutant P351C appeared to be the counterpart of mutant S405C of CitS; the mutant was inactivated by AmdiS added at the periplasmic side of the membrane. The data support, in general, the structural and mechanistic similarity between the ESS and 2HCT transporters and, more particularly, the two-domain structure of the transporters and the presence and functional importance of the reentrant loops present in each domain. It is proposed that the GGXG motifs are at the vertex of the reentrant loops.

Properties of Arsenite Efflux Permeases (Acr3) from Alkaliphilus metalliredigens and Corynebacterium glutamicum

Members of the Acr3 family of arsenite permeases confer resistance to trivalent arsenic by extrusion from cells, with members in every phylogenetic domain. In this study bacterial Acr3 homologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum were cloned and expressed in Escherichia coli. Modification of a single cysteine residue that is conserved in all analyzed Acr3 homologues resulted in loss of transport activity, indicating that it plays a role in Acr3 function. The results of treatment with thiol reagents suggested that the conserved cysteine is located in a hydrophobic region of the permease. A scanning cysteine accessibility method was used to show that Acr3 has 10 transmembrane segments, and the conserved cysteine would be predicted to be in the fourth transmembrane segment.

Reversible Inactivation of Human Dipeptidyl Peptidases 8 and 9 by Oxidation

Hydrogen peroxide (H2O2) can act as an intracellular messenger by oxidizing sulfhydryl groups in cysteines that can be oxidized at neutral pH. The oxidizing agents H2O2 and pyrroloquinoline quinone and the large thiol reagents N-ethylmaleimide and 4-(hydroxymercuri) benzoate each inhibited dipeptidyl peptidase (DP) activity in the intracellular DPIV-related proteins DP8 and DP9 at pH 7.5. In contrast, these treatments did not alter activity in DPIV and fibroblast activation protein. Peptidase inhibition was completely reversed by 2-mercaptoethanol or reduced glutathione. Alkylation of DP8 by the small thiol reagent iodoacetamide prevented inhibition by H2O2, N-ethylmaleimide or pyrroloquinoline qui- none. Two cysteines were reactive per peptidase monomer. We exploited these properties to highly purify DP8 by thiol affinity chromatography. Homology modelling of DP8 and DP9 was consistent with the proposal that the mechanism in- volves decreased protein flexibility caused by intramolecular disulfide bonding. These novel data show that DP8 and DP9 are reversibly inactivated by oxidants at neutral pH and suggest that DP8 and DP9 are H2O2 sensing proteins.

N-Ethylmaleimide differentially inhibits substrate uptake by and ligand binding to the noradrenaline transporter

Using transfected HEK293 cells that express the human (h) noradrenaline transporter (hNAT), we show differential inhibitory effects of the thiol reagent N-ethylmaleimide (NEM) on [(3)H]NA uptake and [(3)H]nisoxetine binding. Irreversible inhibition of uptake by NEM was complete, faster, and occurred at lower concentrations. Furthermore, hNAT ligands (substrates and inhibitors) prevented NEM-induced inhibition of binding but not that of uptake, indicating different underlying mechanisms of inhibition. NEM-induced uptake inhibition was not primarily due to inhibition of the Na(+)/K(+)-ATPase since ouabain caused only partial inhibition. For the first time, we show that NEM at low concentrations causes a rapid and complete depletion of cellular adenosine triphosphate (ATP) not only in HEK293 cells but also in several other eukaryotic cell lines. Thus, while high NEM concentrations alkylate the NAT protein in a ligand-protectable manner, low concentrations inhibit substrate uptake through a loss of the Na(+) and K(+) gradient as a driving force by depleting cellular ATP.

Mitochondrial transport in proline catabolism in plants: the existence of two separate translocators in mitochondria isolated from durum wheat seedlings

Abiotic stresses, such as high salinity or drought, can cause proline accumulation in plants. Such an accumulation involves proline transport into mitochondria where proline catabolism occurs. By using durum wheat seedlings as a plant model system, we investigated how proline enters isolated coupled mitochondria. The occurrence of two separate translocators for proline, namely a carrier solely for proline and a proline/glutamate antiporter, is shown in a functional study in which we found the following: (1) Mitochondria undergo passive swelling in isotonic proline solutions in a stereospecific manner. (2) Externally added L: -proline (Pro) generates a mitochondrial membrane potential (Delta Psi) with a rate depending on the transport of Pro across the mitochondrial inner membrane. (3) The dependence of the rate of generation of Delta Psi on increasing Pro concentrations exhibits hyperbolic kinetics. Proline transport is inhibited in a competitive manner by the non-penetrant thiol reagent mersalyl, but it is insensitive to the penetrant thiol reagent N-ethylmaleimide (NEM). (4) No accumulation of proline occurs inside the mitochondria as a result of the addition of proline externally, whereas the content of glutamate increases both in mitochondria and in the extramitochondrial phase. (5) Glutamate efflux from mitochondria occurs at a rate which depends on the mitochondrial transport, and it is inhibited in a non-competitive manner by NEM. The dependence of the rate of glutamate efflux on increasing proline concentration shows saturation kinetics. The physiological role of carrier-mediated transport in the regulation of proline catabolism, as well as the possible occurrence of a proline/glutamate shuttle in durum wheat seedlings mitochondria, are discussed.

Loop VIII/IX of the Na+-Citrate Transporter CitS ofKlebsiella pneumoniaeFolds into an Amphipathic Surface Helix

The sodium ion-dependent citrate transporter CitS of Klebsiella pneumoniae is a member of the 2-hydroxycarboxylate transporter (2HCT) family whose members transport divalent citrate in symport with two sodium ions. Profiles of the hydrophobic moment suggested the presence of an amphipathic helical structure in the cytoplasmic loop between transmembrane segments (TMSs) VIII and IX (the AH loop) in all members of the family. Cysteine-scanning mutagenesis was used to study the secondary structure of the AH loop. We have mutated 20 successive residues into cysteine residues, characterized each of the mutants for its transport activity, and determined the accessibility of the residues. Three of the mutants, G324C, F331C, and F332C, had very low citrate transport activity, and two others, I321C and S333C, exhibited significantly decreased activity after treatment of right-side-out membranes with membrane permeable thiol reagent N-ethylmaleimide (NEM), but not with membrane impermeable 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AmdiS) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). No protection against NEM was observed with citrate or sodium ions. Labeling of the cysteine residues in the 20 mutants with the fluorescent probe fluorescein 5-maleimide, in membrane vesicles with an inverted orientation, resulted in a clear periodicity in the accessibility of the residues. Residues expected to be at the hydrophobic face of the putative alpha-helix were not accessible for the label, whereas those at the hydrophilic face were easily accessed and labeled. Pretreatment of whole cells and inside-out membranes expressing the mutants with the membrane impermeable reagent AmdiS confirmed the cytoplasmic localization of the AH region. It is concluded that the loop between TMSs VIII and IX folds into an amphipathic surface helix.

Identification of Organelles in Bacteria Similar to Acidocalcisomes of Unicellular Eukaryotes

Acidocalcisomes are acidic calcium storage compartments described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds. In this work, we report that the volutin granules of Agrobacterium tumefaciens possess properties similar to the acidocalcisomes. Transmission electron microscopy revealed that each intracellular granule was surrounded by a membrane. X-ray microanalysis of the volutin granules showed large amounts of phosphorus, magnesium, potassium, and calcium. Calcium in the volutin granules increased when the bacteria were incubated at high extracellular calcium concentration. Immunofluorescence and immunoelectron microscopy, using antisera raised against peptide sequences conserved in the A. tumefaciens proton pyrophosphatase, indicated localization in intracellular vacuoles. Purification of the volutin granules using iodixanol density gradients indicated a preferential localization of the pyrophosphatase activity in addition to high concentrations of phosphate, pyrophosphate, short- and long-chain polyphosphate, but lack of markers of the plasma membrane. The pyrophosphatase activity was potassium-insensitive and inhibited by the pyrophosphate analogs, amynomethylenediphosphonate and imidodiphosphate, by dicyclohexylcarbodiimide, and by the thiol reagent N-ethylmaleimide. Polyphosphate was also localized to the volutin granules by 4',6'-diamino-2-phenylindole staining. The organelles were acidic, as demonstrated by staining with LysoSensor blue DND-167, a dye especially used to detect very acidic compartments in cells, and cycloprodigiosin, a compound isolated from a marine bacterium that has been shown to uncouple proton pyrophosphatase activity acting as a chloride/proton symport. The results suggest that acidocalcisomes arose before the prokaryotic and eukaryotic lineages diverged.

Identification of steroid sulfate transport processes in the human mammary gland.

Circulating hormones and local biotransformation of steroid precursors are both sources of estrogen in human mammary tissue. Estrone-3-sulfate (E(1)S) is an important estrogenic form in premenopausal women, and dehydroepiandrosterone sulfate (DHEAS) constitutes a major adrenal precursor. Membrane transport systems that govern delivery of these anionic steroid conjugates to the mammary gland were investigated. RNA was screened by RT-PCR and Northern blotting for expression of organic anion transporting polypeptide (OATP) (solute carrier family 21A) and organic anion transporter (OAT) (solute carrier family 22A) gene families. OATP-B (SLC21A9) was the major carrier expressed; OATP-D (SLC21A11) and OATP-E (SLC21A12) were less abundant. In normal sections, OATP-B immunolocalized to the myoepithelium that surrounds the ductal epithelial cells. In invasive carcinoma, ductal epithelial cells were positive. OATP-B was characterized in stable transfected Chinese hamster ovary cells. E(1)S affinity constant (K(m)) [K(m) = 5 micro mol/liter, maximum velocity (V(max)) V(max) = 777 pmol/mg.min] and DHEAS (K(m) = 9 micro mol/liter, V(max) = 85 pmol/mg.min) were substrates. The prostaglandins (PG) A(1) and PGA(2) stimulated uptake of E(1)S and DHEAS by increasing V(max) 2-fold but not changing K(m). The effect of PGA was selectively blocked by the lipophilic thiol reagent N-ethylmaleimide but not by the hydrophilic acetamido-4'(iodoacetyl)aminostilbene-2,2'-disulfonic acid, suggesting an interaction between the electrophilic cyclopentenone ring and specific cysteine residues of OATP-B.

Down-modulation of nuclear localisation and pro-fibrogenic effect of 4-hydroxy-2,3-nonenal by thiol- and carbonyl-reagents

Among the oxidative breakdown products of ω-6 unsaturated fatty acids, the aldehyde 4-hydroxy-2,3-nonenal (HNE) is receiving increasing attention for its potential pathophysiological implication, which at least partly lies on the demonstrated ability to modulate gene expression of a number of genes. Here we show that a marked down-modulation of HNE nuclear localisation in cells of a macrophage line (J774-A1) can be afforded by treatment with sulfydryl and carbonyl reagents without significantly interfering with cell viability. As regards the addition of thiol-group reagents to the cell suspension, N-ethylmaleimide (NEM) led to a sustained decrease of HNE nuclear localisation, while 4-(chloromercuri)-benzene-sulfonic acid (PCMBS) gave a similar but more transient effect. Hydroxylamine (HYD), a carbonyl-group reagent, was also able to inhibit HNE nuclear localisation. The actual efficacy of the inhibitors used was then tested on the HNE-induced stimulation of transforming growth factor β1 (TGFβ1) production by J774-A1 cells. Indeed, the thiol reagents NEM and PCMBS, both markedly down-modulating HNE nuclear localisation, were able to inhibit HNE-induced increase of TGFβ1 protein synthesis. The carbonyl reagent HYD was less effective on this respect, producing strong but incomplete protection against HNE-induced TGFβ1 increase. Taken together, the results indicate that sulfydryl groups are involved in the process of HNE cellular internalisation, while both sulfydryl and carbonyl groups are involved in the process of HNE nuclear translocation, and consequently in the modulation of gene expression by the aldehyde. Further, an actual demonstration is provided that HNE-induced effect on gene regulation can be efficiently counteracted by suitable interference with HNE biochemistry.

Vacuolar proton pyrophosphatase activity and pyrophosphate (PPi) in Toxoplasma gondii as possible chemotherapeutic targets.

The addition of PP(i) promoted the acidification of a subcellular compartment in cell homogenates of Toxoplasma gondii tachyzoites, implying the presence of a proton-translocating pyrophosphatase. The proton gradient was collapsed by addition of the K(+)/H(+) antiporter nigericin, and was also inhibited by addition of the PP(i) analogue aminomethylenediphosphonate (AMDP). Both proton transport and PP(i) hydrolysis were dependent upon K(+), but Na(+) caused partial inhibition of these activities. PP(i) hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, NaF and to the thiol reagent N-ethylmaleimide. This activity was unaffected by common inhibitors of phosphohydrolases, except that NaO(3)V (sodium orthovanadate) stimulated the activity by 87%. Immunofluorescence microscopy, using antisera raised against conserved peptide sequences of a plant vacuolar pyrophosphatase, suggested that the pyrophosphatase in T. gondii tachyzoites was located in the plasma membrane and intracellular vacuoles of the parasite. High-field (31)P-NMR spectroscopy showed that PP(i )was more abundant than ATP in tachyzoites. Bisphosphonates (PP(i) analogues), drugs that are used in the treatment of bone diseases, inhibited proton transport and PP(i) hydrolysis in tachyzoite homogenates, and also inhibited intracellular proliferation of tachyzoites in tissue culture cells.

The Multiple Carrier Model of Nonribosomal Peptide Biosynthesis at Modular Multienzymatic Templates

Gramicidin S synthetase 1 and 2 were affinity-labeled at their thiolation centers either by thioesterification with the amino acid substrate or by specific alkylation with the thiol reagent N-ethylmaleimide in combination with a substrate protection technique. The labeled proteins were digested either chemically by cyanogen bromide or by proteases. An efficient multistep high pressure liquid chromatography methodology was developed and used to isolate the active site peptide fragments of all five thiolation centers of gramicidin S synthetase in pure form. The structures of these fragments are investigated by N-terminal sequencing, mass spectrometry, and amino acid analysis. Each of the active site peptide fragments contains the consensus motif LGG(H/D)S(L/I), which is specific for thioester formation in nonribosomal peptide biosynthesis. It was demonstrated that a 4'-phosphopantetheine cofactor is attached to the central serine of the thiolation motif in each amino acid-activating module of the gramicidin S synthetase multienzyme system forming the thioester binding sites for the amino acid substrates and catalyzing the elongation process. Our data are strong support for a "multiple carrier model" of nonribosomal peptide biosynthesis at multifunctional templates, which is discussed in detail.

H2O2 activates red blood cell K-Cl cotransport via stimulation of a phosphatase.

K-Cl cotransport is involved in volume regulation in a number of cell types. Cell swelling stimulates K-Cl cotransport, probably by inhibition of a volume-sensitive kinase. K-Cl cotransport can also be activated by oxidants and thiol reagents. We investigated the effect of H2O2 on K-Cl cotransport of LK sheep red blood cells in an attempt to identify the target of oxidants. H2O2 stimulated K-Cl cotransport. The stimulation was virtually abolished by subsequent incubation with calyculin, a protein phosphatase inhibitor. This suggests that H2O2 stimulates a calyculin-sensitive phosphatase and activates K-Cl cotransport by causing a decrease in phosphorylation of the transporter or a regulatory protein. The thiol reagent N-ethylmaleimide, which stimulates K-Cl cotransport, did not stimulate cotransport further in cells with cotransport activated by staurosporine but did stimulate cotransport further in cells with cotransport activated by H2O2. These results suggest that there are at least two distinct phosphorylation sites on the transporter or a regulator. The results also suggest that the phosphatase is associated with the membrane.

Selective Inhibition of Auxin-Stimulated NADH Oxidase Activity and Elongation Growth of Soybean Hypocotyls by Thiol Reagents.

The NADH oxidase activity of isolated vesicles of soybean (Glycine max cv Williams 82) plasma membranes and elongation growth of 1-cm-long hypocotyl segments were stimulated by auxins (indole-3-acetic acid or 2,4-dichlorophenoxyacetic acid [2,4-D]). The auxin-induced stimulations of both NADH oxidase and growth were prevented by the thiol reagents N-ethylmaleimide, p-chloromercuribenzoate, 5,5[prime]-dithiobis(2-nitrophenylbenzoic acid), dithiothreitol, and reduced glutathione. These same reagents largely were without effect on or stimulated slightly the basal levels of NADH oxidase and growth when assayed in the absence of auxins. In the presence of dithiothreitol or reduced glutathione, both 2,4-D and indole-3-acetic acid either failed to stimulate or inhibited the NADH oxidase activity. The rapidity of the response at a given concentration of thiol reagent and the degree of inhibition of the 2,4-D-induced NADH oxidase activity were dependent on order of reagent addition. If the thiol reagents were added first, auxin stimulations were prevented. If auxins were added first, the inhibitions by the thiol reagents were delayed or higher concentrations of thiol reagents were required to achieve inhibition. The results demonstrate a fundamental difference between the auxin-stimulated and the constitutive NADH oxidase activities of soybean plasma membranes that suggest an involvement of active-site thiols in the auxin-stimulated but not in the constitutive activity.

Evidence for inhibitory SH groups in the thiol activated K:Cl cotransporter of low K sheep red blood cells

The monofunctional thiol reagents N-ethylmaleimide (NEM) and methyl methanethiosulfonate (MMTS) stimulate ouabain resistant (OR) electroneutral K:Cl cotransport in LK sheep red blood cells at low, but not at high concentrations. Diamide (DM), on the other hand, only stimulates OR K:Cl flux (Lauf, P.K., J. Memb. Biol. 101: 179-188, 1988). The DM stimulated K:Cl cotransport was decreased toward the control value prior to DM stimulation when NEM or MMTS were added, subsequently. The inhibitory effect was dependent on the compound's concentration and exposure time and, in the case of MMTS, was reversed upon addition of dithiothreitol (DTT). The inhibition was more prominent when NEM treatment was performed at pH 8.0 and disappeared at pH 6.0. In contrast the NEM stimulatory effect was most effective when the pH of NEM treatment was 6.0 (Bauer, J. & Lauf, P.K., J. Memb. Biol. 73: 257-261, 1983). The results suggest the existence of additional, however, inhibitory thiol groups in the already thiol-activated K:Cl cotransporter, with a different pKa value and a lower affinity for NEM or MMTS as compared to the stimulatory thiol groups. Like the activating thiols, the inhibitory sulfhydryls appeared to be inaccessible to non-penetrating thiol reagents and hence, must be located deeper within the red cell membrane.

Glutathione-gated K+ channels of Escherichia coli carry out K+ efflux controlled by the redox state of the cell

The kinetics of K+ efflux across the membranes of i) wild-type Escherichia coli poisoned by the thiol reagent N-ethylmaleimide, ii) K+ retention mutants and iii) glutathione-deficient mutants, have revealed a common "K+ leaky phenotype"; it is characterized by a very high rate of K+ efflux. The results suggest that the products of kefB and kefC genes could encode two K+ channels, both gated by glutathione. The possible function of these K+ channels seems to be a K+ exit controlled by the redox state of the cell; indeed, it can be inferred from the effects of several oxidants and reductants that turning on and off of the K+ efflux mediated by the channels can be correlated with the redox state of glutathione.

Partial Purification and Characterization of a 3′- Phosphoadenosine 5′ -Phosphosulfate: Desulfoglucosinolate Sulfotransferase from Cress (Lepidium sativum)

A 3' -phosphoadenosine 5' -phosphosulfate (PAPS):desulfoglucosinolate sulfotransferase (EC 2.8.2-) was extensively purified from light-grown cress (Lepidium sativum L.) seedlings by gel filtration and concanavalin A-Sepharose 4B, Matrex Gel Green A, and Mono Q fast protein liquid chromatography. The purified enzyme, which required bovine serum albumin for stabilization, had a native molecular weight of 31,000 +/- 5,000 and an apparent isoelectric point of 5.2. Using PAPS (K(m) 60 micromolar) as sulfur donor, it catalyzed the sulfation of desulfobenzylglucosinolate (K(m) 82 micromolar), desulfo-p-hydroxybenzylglucosinolate (K(m) 670 micromolar), and desulfoallylglucosinolate (K(m) 6.5 millimolar) at an optimal pH of 9.0. All other potential substrates tested, including flavonoids, flavonoid glycosides, cinnamic acids, and phenylacetaldoxime, were not sulfated. Sulfotransferase activity was stimulated by MgCl(2), MnCl(2) and reducing agents and inhibited by ZnCl(2), PbNO(3) NiCl(2) and the reaction product PAP. The thiol reagents N-ethylmaleimide, p-chloromercuriphenylsulfonic acid, and 5,5' -dithio-bis-(2-nitrobenzoic acid) were also potent inhibitors, but the enzyme was protected from covalent modification by beta-mercaptoethanol. The kinetics of desulfobenzylglucosinolate sulfation were consistent with a rapid equilibrium ordered mechanism with desulfobenzylglucosinolate binding first and PAPS second.

Presence of haemin-controlled eIF-2 alpha kinases in both undifferentiated and differentiating mouse erythroleukaemia cells.

In rabbit reticulocytes, globin synthesis is regulated by a haemin-controlled translational inhibitor (HCI) which acts by phosphorylating the alpha-subunit of eukaryotic initiation factor 2 (eIF-2). With purified eIF-2 as substrate, haemin-controlled eIF-2 alpha kinases could be partially purified from cultured mouse erythroleukaemia cells (MEL cells), which can be induced in vivo to erythroid differentiation. The eIF-2 alpha kinases from both uninduced and induced MEL cells are clearly distinct from the double-stranded-RNA-activated eIF-2 alpha kinase described for many mammalian cell types. A rough quantitative estimation indicates that, on a per-cell basis, induced MEL cells contain the same amount of haemin-controlled eIF-2 alpha kinase activity as rabbit reticulocytes, whereas uninduced MEL cells contain about one-tenth as much. As to their chromatographic behavior on CM-Sephadex and DEAE-cellulose and their sensitivity towards physiological concentrations of haemin (5-10 microM), the eIF-2 alpha kinases from MEL cells are indistinguishable from HCI. They differ from HCI with respect to their response towards activating stimuli such as prolonged incubation at 37 degrees C or brief exposure to the thiol reagent N-ethylmaleimide.