Sulfhydryl Reagents Research Articles

Influence of analgesic active 3-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl]-dihydrofuran-2-one on the antioxidant status, glucose utilization and lipid accumulation in some in vitro and ex vivo assays

Purpose: Earlier we demonstrated that 3-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl]-dihydrofuran-2-one (LPP1) elevates nociceptive thresholds in the mouse model of diabetic neuropathic pain. Since drug-induced impairments of glucose and lipid metabolism and the oxidative stress might diminish benefits from analgesia achieved by analgesic drugs used in diabetic neuropathy, the effect of LPP1 on glucose utilization, lipid accumulation and its antioxidant and cytotoxic potential were assessed in some in vitro and ex vivo tests.Methods: Total antioxidant capacity was evaluated spectrophotometrically using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical method, whereas the activities of glutathione (GSH) peroxidase and reductase were measured using methods based on the oxidation of NADPH to NADP. The spectrophotometric method for the evaluation of GSH level in mouse brain tissue homogenates involved the oxidation of GSH by the sulfhydryl reagent 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) to form a yellow derivative, 5′-thio-2-nitrobenzoic acid (TNB), measurable at 412 nm. Cytotoxicity and glucose utilization were measured in hepatoma HepG2 cells and in 3T3-L1 adipocytes. Lipid accumulation was measured in 3T3-L1 cell lines.Results: LPP1 had dose-dependent antioxidant properties in DPPH radical assay (14–22% versus control; p < 0.001). Its single administration caused an increase in GSH concentration in brain tissue homogenates of mice by 34% (versus control group; p < 0.05). LPP1 was not cytotoxic and it did not increase glucose utilization or lipid accumulation in cell cultures.Conclusions: Previously demonstrated antinociceptive properties of LPP1 are accompanied by a lack of cytotoxicity. LPP1 does not impair glucose or lipid metabolism and is an antioxidant. All these properties might be advantageous for its use in diabetic neuropathy.

Electrostatic charge at position 552 affects the activation and permeation of FMRFamide-gated Na+ channels

The FMRFamide-gated Na(+) channel (FaNaC) is a unique peptide-gated sodium channel and a member of the epithelial sodium channel/degenerin family. Previous studies have shown that an aspartate residue (Asp(552)) in the second transmembrane domain is involved in activation of the FaNaC. To examine the significance of a negative charge at position 552, we used a cysteine-modification method. Macroscopic currents of a cysteine mutant (D552C) were potentiated or inhibited by use of positively or negatively charged sulfhydryl reagents ([2-(trimethylammonium)ethyl]methanethiosulfonate bromide, MTSET, and sodium (2-sulfonatoethyl)methanethiosulfonate, MTSES, respectively). Dose-response analysis showed that treatment with MTSET increased the potency of the FMRFamide in the FaNaC whereas treatment with MTSES reduced the maximum response. Negative charge at position 552 was necessary for the characteristic inward rectification of the FaNaC. These results suggest that negative electric charge at position 552 is important to the activation and permeation properties of the FaNaC.

Mutational and crystallographic analysis of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813: Interconversion between oxidase and monooxygenase activities

In this study, it was shown for the first time that l-amino acid oxidase of Pseudomonas sp. AIU813, renamed as l-amino acid oxidase/monooxygenase (l-AAO/MOG), exhibits l-lysine 2-monooxygenase as well as oxidase activity. l-Lysine oxidase activity of l-AAO/MOG was increased in a p-chloromercuribenzoate (p-CMB) concentration-dependent manner to a final level that was five fold higher than that of the non-treated enzyme. In order to explain the effects of modification by the sulfhydryl reagent, saturation mutagenesis studies were carried out on five cysteine residues, and we succeeded in identifying l-AAO/MOG C254I mutant enzyme, which showed five-times higher specific activity of oxidase activity than that of wild type. The monooxygenase activity shown by the C254I variant was decreased significantly. Moreover, we also determined a high-resolution three-dimensional structure of l-AAO/MOG to provide a structural basis for its biochemical characteristics. The key residue for the activity conversion of l-AAO/MOG, Cys-254, is located near the aromatic cage (Trp-418, Phe-473, and Trp-516). Although the location of Cys-254 indicates that it is not directly involved in the substrate binding, the chemical modification by p-CMB or C254I mutation would have a significant impact on the substrate binding via the side chain of Trp-516. It is suggested that a slight difference of the binding position of a substrate can dictate the activity of this type of enzyme as oxidase or monooxygenase.

Effect of external conditions on the stability of enzymically synthesized carthamin

Enzymically synthesized carthamin was subjected to testing of its stability under various conditions. It easily turned to unknown reddish-yellow or orange-yellow substances in water. This instability was further accelerated by the presence of a sulfhydryl reagent and or at higher temperature. In a typical result, the rate of the discolouration was calculated to be 21 nmol carthamin h&lt;sup&gt;-1&lt;/sup&gt; at 20 °C in water and rose to 44.0 nmol carthamin h&lt;sup&gt;-1&lt;/sup&gt; at 60 °C in water containing 1.0 mM 2-mercaptoethanol. Organic solvents such as methanol, ethanol and acetone showed little effect on carthamin discolouration, while the stability of carthamin in these solvents was gradually decreased by rising temperature. In various buffers, in the acidic range. carthamin was relatively stable, whereas, it turned unstable on the basic side. All of the thiol reagents tested were unfavourable for retaining stability of carthamin in solutions. 2-Mercaptoethanol, reduced glutathione and thioglycollic acid exhibited the strongest degrading effect on the pigment. At the 1.0 mM level, 2-mercaptoethanol reduced the absorption peak at 517 nm by 0.02 min&lt;sup&gt;-1&lt;/sup&gt;, which corresponded to 0.12 nmol carthamin cm&lt;sup&gt;-3&lt;/sup&gt; min&lt;sup&gt;-1&lt;/sup&gt; L-Methionine, L-ascorbic and D-araboascorbic acids were found to be strong bathochromic effectors For example, L-methionine rised AA 517 nm up to 0.01 min&lt;sup&gt;-1&lt;/sup&gt;, whose value equaled to 0-1 nmol carthamin cm&lt;sup&gt;-3&lt;/sup&gt; min&lt;sup&gt;-1&lt;/sup&gt; at 30 °C Carthamin was absorbed and adsorbed and stabilized by many macro-molecular Substances. Among the compounds tested, cellulose was found to be the most effective stabilizer. The stabilization effect of cellulose was specific for carthamin and the term "SAITO EFFECT" is proposed for it. This effect was reduced by hydrolysis of the carthamin-adsorbed cellulose with Asperqillus cellulase in an incubation system

Crosslinking of Cys-mutated human galectin-1 to the model glycoprotein ligands asialofetuin and laminin by using a photoactivatable bifunctional reagent.

Galectins are a group of animal lectins characterized by their specificity for β-galactosides. In our previous study, we showed that a human galectin-1 (hGal-1) mutant, in which a cysteine residue was introduced at Lys(28), forms a covalently cross-linked complex with the model glycoprotein ligands asialofetuin and laminin by using the photoactivatable sulfhydryl reagent benzophenone-4-maleimide (BPM). In the present study, we used several hGal-1 mutants in which single cysteine residues were introduced at different positions and examined their ability to form a covalent complex with asialofetuin or laminin by using BPM. We found that the efficiency of formation of the cross-linked products differed depending on the positions of the cysteine introduced and also on the ligand used for crosslinking. Therefore, by using different cysteine hGal-1 mutants, the chances of isolating different ligands for hGal-1 should increase depending on the systems and cells used.

Subunit Disassembly Pathway of Human Hemoglobin Revealing the Site-Specific Role of Its Cysteine Residues

Cysteine residues play a unique role in human hemoglobin (Hb) by affecting its cooperative oxygen binding behavior and the stability of its tetrameric structure. However, how these cysteine residues fulfill their biophysical functions from the molecular level is yet unclear. Here we study the subunit disassembly pathway of human hemoglobin using the sulfhydryl reagent, p-hydroxymercuribenzoate (PMB) and investigate the functional roles of cysteine residues in human hemoglobin. We show evidence from the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry that all three types of cysteine residues, including the surface-exposed βCys93 and the shielded αCys104 and βCys112 are reactive to PMB, resolving an issue long under debate. It is demonstrated that all three types of cysteine residues must be blocked by PMB to accomplish the subunit disassembly, and the PMB-cysteine reactions proceed in a stepwise manner with an order of βCys93, αCys104, and βCys112. The PMB reactions with the three different cysteine residues demonstrate strong site-specificity. The possible influence of PMB-cysteine reactions to the stability of various intersubunit salt bridges has been discussed based on the crystallographic structure of hemoglobin, providing insights in understanding the hemoglobin subunit disassembly pathway and the site-specific functional role of each cysteine residue in hemoglobin.

Structural Analysis of the N-terminal Domain of Subunit a of the Yeast Vacuolar ATPase (V-ATPase) Using Accessibility of Single Cysteine Substitutions to Chemical Modification

The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V1 domain that hydrolyzes ATP and an integral V0 domain that translocates protons. Subunit a is a 100-kDa integral membrane protein (part of V0) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. Although the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V1V0 complex and the free V0 domain to identify residues protected from modification by the presence of V1. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V1 domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.

Quercetin antagonism of GABAAρ1 receptors is prevented by ascorbic acid through a redox-independent mechanism

Quercetin is a natural flavonoid widely distributed in plants that acts as a neuroprotective agent and modulates the activity of different synaptic receptors and ion channels, including the ionotropic GABA receptors. GABAAρ1 receptors were shown to be antagonized by quercetin, but the mechanisms underlying these antagonistic actions are still unknown. We have analyzed here if the antagonistic action produced by quercetin on GABAAρ1 receptors was related to its redox activity or due to alternative mechanism/s.Homomeric GABAAρ1 receptors were expressed in frog oocytes and GABA-evoked responses electrophysiologically recorded. Quercetin effects on GABAAρ1 receptors were examined in the absence or presence of ascorbic acid. Chemical protection of cysteines by selective sulfhydryl reagents and site directed mutagenesis experiments were also used to determine ρ1 subunit residues involved in quercetin actions.Quercetin antagonized GABAAρ1 receptor responses in a dose-dependent, fast and reversible manner. Quercetin inhibition was prevented in the presence of ascorbic acid, but not by thiol reagents that modify the extracellular Cys-loop of these receptors. H141, an aminoacidic residue located near to the ρ1 subunit GABA binding site, was involved in the allosteric modulation of GABAAρ1 receptors by several agents including ascorbic acid. Quercetin similarly antagonized GABA-evoked responses mediated by mutant H141DGABAAρ1 and wild-type receptors, but prevention exerted by ascorbic acid on quercetin effects was impaired in mutant receptors. Taken together the present results suggest that quercetin antagonistic actions on GABAAρ1 receptors are mediated through a redox-independent allosteric mechanism.

Partial purification, characterization and cDNA cloning of aminoaldehyde dehydrogenase in germinated soybean (Glycine max L.)

γ-Aminobutyric acid (GABA) is a non-proteinous amino acid with some functions for human health. In this study, aminoaldehyde dehydrogenase (EC 1.2.1.19, AMADH) which has been proven to be an essential enzyme responsible for the GABA bioformation via polyamine degradation pathway was purified from 2-day germinated soybeans and characterized. The partially purified enzyme showed a single major band with a relative molecular mass of approximately 53.72 kDa-subunit using sodium dodecyl sulfate gel electrophoresis. AMADH exhibited the optimal activity at pH 8.0 and at 45 °C, respectively. The activity of purified AMADH was significantly inhibited by some heavy metal ions and sulfhydryl reagents. The cDNA of AMADH was sequenced by RT-PCR, which indicated 1,677 bp long and contained a 1,515 bp open reading frame that encoded 504 amino-acid peptides (GenBank accession number: KC478661).

Propofol Binding to the Resting State of the Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Induces Structural Changes in the Inter- and Intrasubunit Transmembrane Domain (TMD) Cavities

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428-431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.

Regulatory Phosphorylation Induces Extracellular Conformational Changes in a CLC Anion Channel

CLH-3b is a CLC-1/2/Ka/Kb channel homolog activated by meiotic cell cycle progression and cell swelling. Channel inhibition occurs by GCK-3 kinase-mediated phosphorylation of serine residues on the cytoplasmic C-terminus linker connecting CBS1 and CBS2. Two conserved aromatic amino acid residues located on the intracellular loop connecting membrane helices H and I and α1 of CBS2 are required for transducing phosphorylation changes into changes in channel activity. Helices H and I form part of the interface between the two subunits that comprise functional CLC channels. Using a cysteine-less CLH-3b mutant, we demonstrate that the sulfhydryl reagent reactivity of substituted cysteines at the subunit interface changes dramatically during GCK-3-mediated channel inhibition and that these changes are prevented by mutation of the H-I loop/CBS2 α1 signal transduction domain. We also show that GCK-3 modifies Zn2+ inhibition, which is thought to be mediated by the common gating process. These and other results suggest that phosphorylation of the cytoplasmic C-terminus inhibits CLH-3b by inducing subunit interface conformation changes that activate the common gate. Our findings have important implications for understanding CLC regulation by diverse signaling mechanisms and for understanding the structure/function relationships that mediate intraprotein communication in this important family of Cl− transport proteins.

The cardiac Na + -Ca 2+ exchanger has two cytoplasmic ion permeation pathways

The Na(+)-Ca(2+) exchanger (NCX) is a ubiquitously expressed plasma membrane protein. It plays a fundamental role in Ca(2+) homeostasis by moving Ca(2+) out of the cell using the electrochemical gradient of Na(+) as the driving force. Recent structural studies of a homologous archaebacterial exchanger, NCX_Mj, revealed its outward configuration with two potential ion permeation pathways exposed to the extracellular environment. Based on the symmetry of NCX_Mj structure, an atomic model of an inward-facing conformation was generated showing similar pathways but directed to the cytoplasm. The presence of these water-filled cavities has yet to be confirmed experimentally, and it is unknown if the mammalian exchanger adopts the same structure. In this study, we mutated multiple residues within transmembrane segments 2 and 7 of NCX1.1 (cardiac isoform) to cysteines, allowing us to investigate their sensitivity to membrane-impermeable sulfhydryl reagents as exchanger current block. By trapping NCX1.1 in the inward-facing configuration, we have mapped two differently sized cytoplasmic aqueous cavities, the access of which is modified during exchange. This data reveals movements of the protein associated with ion transport. Electrophysiological characterization shows that the conserved residues within transmembrane segments 2 and 7, coordinating Na(+) and Ca(2+) ions in NCX_Mj, play a fundamental role in NCX1.1. Our results suggest a similar architecture between the mammalian and archaebacterial exchangers.

The effects of para-chloromercuribenzoic acid and different oxidative and sulfhydryl agents on a novel, non-AT1, non-AT2 angiotensin binding site identified as neurolysin

A novel, non-AT1, non-AT2 brain binding site for angiotensin peptides that is unmasked by p-chloromercuribenzoate (PCMB) has been identified as a membrane associated variant of neurolysin. The ability of different organic and inorganic oxidative and sulfhydryl reactive agents to unmask or inhibit 125I-Sar1Ile8 angiotensin II (SI-Ang II) binding to this site was presently examined. In tissue membranes from homogenates of rat brain and testis incubated in assay buffer containing losartan (10μM) and PD123319 (10μM) plus 100μM PCMB, 5 of the 39 compounds tested inhibited 125I-SI Ang II binding in brain and testis. Mersalyl acid, mercuric chloride (HgCl2) and silver nitrate (AgNO3) most potently inhibited 125I-SI Ang II binding with IC50s ~1–20μM. This HgCl2 inhibition was independent of any interaction of HgCl2 with angiotensin II (Ang II) based on the lack of effect of HgCl2 on the dipsogenic effects of intracerebroventricularly administered Ang II and 125I-SI Ang II binding to AT1 receptors in the liver. Among sulfhydryl reagents, cysteamine and reduced glutathione (GSH), but not oxidized glutathione (GSSG) up to 1mM, inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis. Thimerosal and 4-hydroxymercuribenzoate moderately inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis at 100μM; however, they also unmasked non-AT1, non-AT2 binding independent of PCMB. 4-Hydroxybenzoic acid did not promote 125 I-SI Ang II binding to this binding site indicating that only specific organomercurial compounds can unmask the binding site. The common denominator for all of these interacting substances is the ability to bind to protein cysteine sulfur. Comparison of cysteines between neurolysin and the closely related enzyme thimet oligopeptidase revealed an unconserved cysteine (cys650, based on the full length variant) in the proposed ligand binding channel (Brown et al., 2001) [45] near the active site of neurolysin. It is proposed that the mercuric ion in PCMB and closely related organomercurial compounds binds to cys650, while the acidic anion forms an ionic bond with a nearby arginine or lysine along the channel to effect a conformational change in neurolysin that promotes Ang II binding.

Gene Cloning, Expression, and Characterization of a Cyclic Nucleotide Phosphodiesterase from Arthrobacter sp. CGMCC 3584

Based on thermal asymmetric interlaced polymerase chain reaction, the arpde gene encoding a cyclic nucleotide-specific phosphodiesterase was cloned from Arthrobacter sp. CGMCC 3584 for the first time. The 930-bp region encoded a 309-amino-acid protein with a molecular weight of 33.6 kDa. The recombinant ArPDE was able to hydrolyze 3',5'-cAMP, 3',5'-cGMP, and 2',3'-cAMP. The K m values of ArPDE for 3',5'-cAMP and 3',5'-cGMP were 6.82 and 12.82 mM, respectively. ArPDE was thermostable and displayed optimal activity at 45 °C and pH 7.5. The enzyme did not require any metal cofactors, although its activity was stimulated by 2 mM Co(2+) and inhibited by Zn(2+). Nucleotides, reducing agents, and sulfhydryl reagents had different inhibitory effects on the activity of ArPDE. NaF, the actual compound used to improve the industrial yield of cAMP, exhibited 62 % inhibitions at concentrations of 10 mM.

Cysteine Scanning Mutagenesis of Transmembrane Helix 3 of a Brain Glutamate Transporter Reveals Two Conformationally Sensitive Positions

Glutamate transporters in the brain remove the neurotransmitter from the synapse by cotransport with three sodium ions into the surrounding cells. Recent structural work on an archaeal homolog suggests that, during substrate translocation, the transport domain, including the peripheral transmembrane helix 3 (TM3), moves relative to the trimerization domain in an elevator-like process. Moreover, two TM3 residues have been proposed to form part of a transient Na3' site, and another, Tyr-124, appears close to both Na3' and Na1. To obtain independent evidence for the role of TM3 in glutamate transport, each of its 31 amino acid residues from the glial GLT-1 transporter was individually mutated to cysteine. Except for six mutants, substantial transport activity was detected. Aqueous accessibility of the introduced cysteines was probed with membrane-permeant and membrane-impermeant sulfhydryl reagents under a variety of conditions. Transport of six single cysteine mutants, all located on the intracellular side of TM3, was affected by membrane-permeant sulfhydryl reagents. However, only at two positions could ligands modulate the reactivity. A120C reactivity was diminished under conditions expected to favor the outward-facing conformation of the transporter. Sulfhydryl modification of Y124C by 2-aminoethyl methanethiosulfonate, but not by N-ethylmaleimide, was fully protected in the presence of sodium. Our data are consistent with the idea that TM3 moves during transport. Moreover, computational modeling indicated that electrostatic repulsion between the positive charge introduced at position 124 and the sodium ions bound at Na3' and Na1 underlies the protection by sodium.

Molecular determinants of human dipeptidyl peptidase III sensitivity to thiol modifying reagents

Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49, involved in protein metabolism and oxidative stress response. DPPIII crystal structure shows the two lobe-like domains separated by a wide cleft. The human enzyme has a total of six cysteines, three in the lower (Cys19, Cys147,and Cys176) and three in the upper (Cys509, Cys519,and Cys654), catalytic, domain containing the active site zinc ion. To elucidate the molecular basis of this enzyme ’ s susceptibility to sulfhydryl reagents, biochemical analysis of a set of Cys to Ala mutants was used, supported by mass spectrometry. Cys176, a residue 44 A apart from the catalytic center of the ligand-free enzyme, was found responsible for the inactivation with the submicromolar concentration of an organomercurial compound, and three additional cysteines contributed to sensitivity to aromatic disulfides. Upon treatment with oxidized glutathione [glutathione disulfide(GSSG)], cysteine residues at positions 147, 176, and 654 were found glutathionylated. The mutational analysis confirmed the involvement of Cys176 and Cys654 inhuman DPP III inactivation by GSSG. Observation that Cys176, a residue quite distant from the active center,contributes to enzyme inactivation, indicates that the substrate-binding site of human DPP III comprises both lower and upper protein domain.

Nitric oxide potentiation of the homomeric ρ1 GABACreceptor function

NO is a highly diffusible and reactive gas produced in the nervous system, which acts as a neuronal signal mediating physiological or pathological mechanisms. NO can modulate the activity of neurotransmitter receptors and ion channels, including NMDA and GABA(A) receptors. In the present work, we examined whether GABA(C) receptor function can also be regulated by NO. Homomeric ρ1 GABA(C) receptors were expressed in oocytes and GABA-evoked responses electrophysiologically recorded in the presence or absence of the NO donor DEA. Chemical protection of cysteines by selective sulfhydryl reagents and site-directed mutagenesis were used to determine the protein residues involved in the actions of NO. GABAρ1 receptor responses were significantly enhanced in a dose-dependent, fast and reversible manner by DEA and the specific NO scavenger CPTIO prevented these potentiating effects. The ρ1 subunits contain only three cysteine residues, two extracellular at the Cys-loop (C177 and C191) and one intracellular (C364). Mutations of C177 and C191 render the ρ1 GABA receptors non-functional, but C364 can be safely exchanged by alanine (C364A). NEM, N-ethyl maleimide and (2-aminoethyl) methanethiosulfonate prevented the effects of DEA on GABAρ1 receptors. Meanwhile, the potentiating effects of DEA on mutant GABAρ1(C364A) receptors were similar to those observed on wild-type receptors. Our results suggest that the function of GABA(C) receptors can be enhanced by NO acting at the extracellular Cys-loop.

Functional Consequences of Sulfhydryl Modification of the γ-Aminobutyric Acid Transporter 1 at a Single Solvent-Exposed Cysteine Residue

The aims of this study were to optimize the experimental conditions for labeling extracellularly oriented, solvent-exposed cysteine residues of γ-aminobutyric acid transporter 1 (GAT1) with the membrane-impermeant sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) and to characterize the functional and pharmacological consequences of labeling on transporter steady-state and presteady-state kinetic properties. We expressed human GAT1 in Xenopus laevis oocytes and used radiotracer and electrophysiological methods to assay transporter function before and after sulfhydryl modification with MTSET. In the presence of NaCl, transporter exposure to MTSET (1–2.5 mM for 5–20 min) led to partial inhibition of GAT1-mediated transport, and this loss of function was completely reversed by the reducing reagent dithiothreitol. MTSET treatment had no functional effect on the mutant GAT1 C74A, whereas the membrane-permeant reagents N-ethylmaleimide and tetramethylrhodamine-6-maleimide inhibited GABA transport mediated by GAT1 C74A. Ion replacement experiments indicated that MTSET labeling of GAT1 could be driven to completion when valproate replaced chloride in the labeling buffer, suggesting that valproate induces a GAT1 conformation that significantly increases C74 accessibility to the extracellular fluid. Following partial inhibition by MTSET, there was a proportional reduction in both the presteady-state and steady-state macroscopic signals, and the functional and pharmacological properties of the remaining signals were indistinguishable from those of unlabeled GAT1. Therefore, covalent modification of GAT1 at C74 results in completely nonfunctional as well as electrically silent transporters.

Glycerol-3-Phosphate Acyltransferase-2 Is Expressed in Spermatic Germ Cells and Incorporates Arachidonic Acid into Triacylglycerols

BackgroundDe novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. In this work we show that mitochondrial GPAT2 overexpression in CHO-K1 cells increased TAG content and both GPAT and AGPAT activities 2-fold with arachidonoyl-CoA as a substrate, indicating specificity for this fatty acid.Methods and ResultsIncubation of GPAT2-transfected CHO-K1 cells with [1-14C]arachidonate for 3 h increased incorporation of [14C]arachidonate into TAG by 40%. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2's role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein.ConclusionsThese results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.

Cysteine Residues in the Transmembrane (TM) 9 to TM11 Region of the Human Equilibrative Nucleoside Transporter Subtype 1 Play an Important Role in Inhibitor Binding and Translocation Function

Inhibitor and substrate interactions with equilibrative nucleoside transporter 1 (ENT1; SLC29A1) are known to be affected by cysteine-modifying reagents. A previous study from our laboratory established Cys222 in transmembrane (TM) 6 as the residue responsible for methyl methanethiosulfonate (a membrane-permeable sulfhydryl modifier)-mediated enhancement of the binding of the ENT1 inhibitor nitrobenzylmercaptopurine riboside (NBMPR) in intact cells. However, the capacity of charged sulfhydryl reagents to inhibit the binding of NBMPR in broken cell preparations (allowing cytoplasmic access) was not affected by mutation of any of the cysteines (Cys87, 193, 213, or 222) in the N-terminal half of the protein. We thus hypothesized that the inhibitory effects of the modifiers were due to the one or more of the six cysteine residues in the C-terminal half of ENT1, particularly one or both of those in the fifth intracellular loop (Cys414 and Cys416). Each of the cysteines were mutated to serine or alanine and expressed in nucleoside transport-deficient PK15 cells and probed with a series of methanethiosulfonate sulfhydryl-modifying reagents. Transporter function was assessed by the site-specific binding of [(3)H]NBMPR and the cellular uptake of [(3)H]2-chloroadenosine. These studies established that Cys378 is an extracellular-located residue modified by [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET) to inhibit the binding of NBMPR to intact cells. Mutation of Cys414 led to an enhancement of the ability of MTSET to inhibit NBMPR binding, and this enhancement was eliminated by the comutation of Cys378, indicating that disruption of the fifth intracellular loop modifies the conformation of TM10 and its extracellular extension. Mutation of Cys416 led to the loss of the ability of the charged sulfhydryl reagents to inhibit NBMPR binding in isolated membranes and also led to the loss of transport function. This finding further supports an allosteric interaction between the fifth intracellular loop and the extracellular NBMPR binding domain and implicates this region in the translocation function of human ENT1.