Sulfhydryl-modifying Reagent N-ethylmaleimide Research Articles

Biochemical Characterization of Allantoinase from Escherichia coli BL21

Bacterial allantoinase (ALLase; EC 3.5.2.5), which catalyzes the conversion of allantoin into allantoate, possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carboxylated lysine. Here, we characterized ALLase from Escherichia coli BL21. Purified recombinant ALLase exhibited no activity but could be activated when preincubating with some metal ions before analyzing its activity, and was in the order: Mn(2+)-≫Co(2+)->Zn(2+)->Ni(2+)->Cd(2+)- ~Mg(2+)-activated enzyme; however, activity of ALLase (Mn(2+)-activated form) was also significantly inhibited with 5mM Co(2+), Zn(2+), and Cd(2+) ions. Activity of Mn(2+)-activated ALLase was increased by adding the reducing agent dithiothreitol (DTT), but was decreased by treating with the sulfhydryl modifying reagent N-ethylmaleimide (NEM). Inhibition of Mn(2+)-activated ALLase by chelator 8-hydroxy-5-quinolinesulfonic acid (8-HQSA), but not EDTA, was pH-dependent. Analysis of purified ALLase by gel filtration chromatography revealed a mixture of monomers, dimers, and tetramers. Substituting the putative metal binding residues His59, His61, Lys146, His186, His242, and Asp315 with Ala completely abolished the activity of ALLase, even preincubating with Mn(2+) ions. On the basis of these results, as well as the pH-activity profile, the reaction mechanism of ALLase is discussed and compared with those of other cyclic amidohydrolases.

The 11β-hydroxysteroid dehydrogenase type 2 activity in human placental microsomes is inactivated by zinc and the sulfhydryl modifying reagent N-ethylmaleimide

Proper glucocorticoid exposure in utero is vital to normal fetal organ growth and maturation. The human placental 11β-hydroxysteroid dehydrogenase type 2 enzyme (11β-HSD2) catalyzes the unidirectional conversion of cortisol to its inert metabolite cortisone, thereby controlling fetal exposure to maternal cortisol. The present study examined the effect of zinc and the relatively specific sulfhydryl modifying reagent N-ethylmaleimide (NEM) on the activity of 11β-HSD2 in human placental microsomes. Enzyme activity, reflected by the rate of conversion of cortisol to cortisone, was inactivated by NEM (IC 50=10 μM), while the activity was markedly increased by the sulfhydryl protecting reagent dithiothreitol (DTT; EC 50=1 mM). Furthermore, DTT blocked the NEM-induced inhibition of 11β-HSD2 activity. Taken together, these results suggested that the sulfhydryl (SH) group(s) of the microsomal 11β-HSD2 may be critical for enzyme activity. Zn 2+ also inactivated enzyme activity (IC 50=2.5 μM), but through a novel mechanism not involving the SH groups. In addition, prior incubation of human placental microsomes with NAD + (cofactor) but not cortisol (substrate) resulted in a concentration-dependent increase (EC 50=8 μM) in 11β-HSD2 activity, indicating that binding of NAD + to the microsomal 11β-HSD2 facilitated the conversion of cortisol to cortisone. Thus, this finding substantiates the previously proposed concept that a compulsorily ordered ternary complex mechanism may operate for 11β-HSD2, with NAD + binding first, followed by a conformational change allowing cortisol binding with high affinity. Collectively, the present results suggest that cellular mechanisms of SH group modification and intracellular levels of Zn 2+ may play an important role in regulation of placental 11β-HSD2 activity.

Glutathione-dependent regulation of nitric oxide production in isolated rat hepatocyte suspensions.

Freshly isolated suspensions of rat parenchymal liver cells (hepatocytes) spontaneously produce large amounts of nitrite following collagenase isolation. Our previous studies indicate that nitrite production is associated with the expression of inducible nitric oxide synthase (iNOS) and reflects NO production. Depletion of glutathione (GSH) with diethylmaleate (DEM) inhibited nitrite production, and this inhibition was time-dependent. DEM was more effective in blocking nitrite production if it was added within the first 1 hr of the start of the incubation. The reducing agent dithiothreitol (DTT) and the alkylating agent ethyl methanesulfonate (EMS) also inhibited hepatocyte nitrite production, and this inhibition was also greatest if they were added within 1 hr of initiating the incubation. However, EMS added at 3 hr still reduced 6-hr nitrite production by about 70%. This reduction in nitrite production by EMS added at 3 hr may be due to the direct modification of thiol groups on the iNOS protein because we have determined that iNOS activity is inhibited by the sulfhydryl modifying reagent N-ethylmaleimide (NEM). Western blots also indicate that the iNOS protein is expressed when EMS is added at 3 hr. The addition of DEM, DTT, or EMS at 0 time greatly reduced the levels of cellular iNOS mRNA relative to controls as determined by quantitative RT-PCR. Based on our results with mRNA levels, both DTT and depletion of cellular GSH appear to inhibit the early signaling events leading to iNOS expression and suggest that the control of iNOS induction in hepatocytes is sensitive to the thiol redox status of the cell.

The intermolecular disulfide bridge of human glial cell line-derived neurotrophic factor: its selective reduction and biological activity of the modified protein.

Recombinant human glial cell line-derived neurotrophic factor has been implicated to have therapeutic potential in the treatment of neurodegenerative diseases. The mature protein is a single polypeptide of 134 amino acid residues and functions as a disulfide-linked dimer. Reduction of the protein with dithiothreitol at pH 7.0 and in the absence of denaturant showed that the single intermolecular cystine bridge was reduced preferentially. Direct alkylation of the generated free sulfhydryl group using iodoacetamide or iodoacetate without denaturant was incomplete. Unfolding the protein in 6 M guanidine hydrochloride prior to the modification showed rapid disulfide scrambling. However, the sulfhydryl-modifying reagent N-ethylmaleimide was able to label quantitatively the free cysteinyl residue in the absence of any added chaotropic agent. By a combination of peptide mapping, Edman degradation, and mass spectrometric analysis, the labeled residue was identified to be Cys101, hence verifying the location of the intermolecular disulfide bond. The modified protein behaved as a noncovalent dimer when chromatographed through a Superdex 75 column under nondenaturing conditions and was comparable in biological activity to an unmodified control sample. The results therefore indicate that the intermolecular disulfide bridge of the protein is not essential for its biological function.

Conformational changes of an Hsp70 molecular chaperone induced by nucleotides, polypeptides, and N-ethylmaleimide.

Hsp70 molecular chaperones are highly conserved ATPases that guide the folding and assembly of proteins in many cellular pathways. They use the energy of ATP binding and hydrolysis to regulate their interactions with hydrophobic regions of unfolded proteins. The activities and the conformations of the N-terminal nucleotide- and C-terminal polypeptide-binding domains of Hsp70s are coupled. We recently reported that the sulfhydryl-modifying reagent N-ethylmaleimide (NEM) inactivates the yeast Hsp70 Ssa1p by reacting with its three cysteine residues which are located in the nucleotide-binding domain. To further characterize conformational changes associated with interdomain coupling and to determine whether NEM alters Ssa1p's conformation, the structures of Ssa1p and NEM-modified Ssa1p (NEM-Ssa1p) were compared using a variety of biophysical techniques. Size exclusion chromatography revealed that NEM-Ssa1p is more oligomeric and more resistant to nucleotide- or polypeptide-dependent depolymerization than Ssa1p. Measurement of the thermal stability indicated that NEM modification has an effect very similar to that of binding of nucleotides to the unmodified protein. Circular dichroism demonstrated small differences in the secondary structure of Ssa1p and NEM-Ssa1p, and in their complexes with nucleotides. NEM modification increased the ANS fluorescence of Ssa1p and exposed numerous trypsin-sensitive sites in its nucleotide-binding domain. The intrinsic fluorescence of Ssa1p's only tryptophan residue, which is located in a C-terminal alpha-helical region adjacent to the polypeptide-binding cleft, was quenched in the presence of ATP, but not ADP. NEM modification altered nucleotide-dependent changes in the intrinsic fluorescence of Ssa1p. Together, these results demonstrate that NEM alters the conformation of Ssa1p and disrupts, but does not eliminate, interdomain communication. Furthermore, the results provide evidence for a model in which the polypeptide-binding cleft of Hsp70s is covered by an alpha-helical lid that is open in the presence of ATP, but closed in the presence of ADP.

Possible target components for the inhibitory effect of N-ethylmaleimide on the activation of neutrophil NADPH oxidase.

Potential target components for the inhibitory effect of covalent sulfhydryl-modifying reagent N-ethylmaleimide (NEM) on the activation of NADPH oxidase in human neutrophils was studied in a cell-free system. The capacity of both cytosol and membrane fractions to induce the translocation of cytosolic components and O2-generation in the cell-free activation system was affected by NEM. The phosphorylation of p47phox, which mediates the translocation of cytosolic complex, by protein kinase C was not inhibited by NEM and NEM-treated p47phox was as effective as untreated p47phox both in the kinase-dependent and in the amphiphile-dependent cell-free activation systems. In addition, phosphatidic acid-dependent phosphorylation of cytosol including p47phox was not affected by NEM. The inhibition of cytosol's capacity to activate NADPH oxidase was partially reversed by an addition of the fraction containing G-protein rac. Taken together, the data suggest that membrane component cytochrome b558 and cytosolic component rac may be the potential targets for the NEM effect on the activation of NADPH oxidase.

The ruminant parasite Haemonchus contortus expresses an alpha1,3-fucosyltransferase capable of synthesizing the Lewis x and sialyl Lewis x antigens.

Glycoproteins from the ruminant helminthic parasite Haemonchus contortus react with Lotus tetragonolobus agglutinin and Wisteria floribunda agglutinin, which are plant lectins that recognize alpha1,3-fucosylated GlcNAc and terminal beta-GalNAc residues, respectively. However, parasite glycoconjugates are not reactive with Ricinus communis agglutinin, which binds to terminal beta-Gal, and the glycoconjugates lack the Lewis x (Le(x)) antigen or other related fucose-containing antigens, such as sialylated Le(x), Le(a), Le(b) Le(y), or H-type 1. Direct assays of parasite extracts demonstrate the presence of an alpha1,3-fucosyltransferase (alpha1,3FT) and beta1,4-N-acetylgalactosaminyltransferase (beta1,4GalNAcT), but not beta1,4-galactosyltransferase. The H. contortus alpha1,3FT can fucosylate GlcNAc residues in both lacto-N-neotetraose (LNnT) Galalpha1-->4GlcNAcbeta1-->3Galbeta1-->4Glc to form lacto-N-fucopentaose III Galbeta1-->4[Fuca1-->3]GlcNAc beta1-->3Galbeta1-4GIc, which contains the Le(x) antigen, and the acceptor lacdiNAc (LDN) GalNAcbeta1-->4GlcNAc to form GalNAc beta1-->4[Fualpha1-->3]GlcNAc. The alpha1,3FT activity towards LNnT is dependent on time, protein, and GDP-Fuc concentration with a Km 50 microM and a Vmax of 10.8 nmol-mg(-1) h(-1). The enzyme is unusually resistant to inhibition by the sulfhydryl-modifying reagent N-ethylmaleimide. The alpha1,3FT acts best with type-2 glycan acceptors (Galbeta1-->4GlcNAcbeta1-R) and can use both sialylated and non-sialylated acceptors. Thus, although in vitro the H. contortus alpha1,3FT can synthesize the Le(x) antigen, in vivo the enzyme may instead participate in synthesis of fucosylated LDN or related structures, as found in other helminths.

Covalent Modification of the Regulatory Domain Irreversibly Stimulates Cystic Fibrosis Transmembrane Conductance Regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is regulated by three cytosolic domains, the regulatory domain (R domain) and two nucleotide binding domains. To learn more about how the cytosolic domains regulate channel activity, we used chemical modification to probe their structure. When we applied the sulfhydryl-modifying reagent N-ethylmaleimide (NEM) and other N-substituted maleimides to the cytosolic domains, we found that they rapidly and irreversibly stimulated channel activity. CFTR contains 14 intracellular cysteine residues that might be targets for NEM modification. We identified one, Cys832, that was essential for the response. Cys832 is located in the R domain. Single channel studies showed that NEM stimulated CFTR by increasing the duration of bursts of activity and by shortening the closed interval between bursts. At the single channel level, CFTR in which Cys832 was mutated to alanine behaved identically to wild-type CFTR, except that it failed to respond to NEM. Additional studies showed that NEM modification increased the potency of ATP-mediated stimulation. Previous work has shown that modification of the R domain by phosphorylation, which introduces negative charge, or replacement of multiple serines by negatively charged aspartates stimulates the channel. Our current data show that covalent modification of the R domain with a neutral, hydrophobic adduct at a site that is not phosphorylated can also stimulate CFTR. This finding suggests that an alteration in the conformation of the R domain may be a key feature that regulates channel activity.

N-Ethylmaleimide Inactivates a Nucleotide-free Hsp70 Molecular Chaperone

Hsp70 molecular chaperones are ATPases that bind to hydrophobic regions of proteins and guide their folding, assembly, and translocation across membranes. The ability of purified Hsp70s to uncoat clathrin-coated vesicles or to stimulate the post-translational translocation of precursor proteins into the endoplasmic reticulum, mitochondria, and the nucleus was previously shown not to be sensitive to the sulfhydryl-modifying reagent N-ethylmaleimide (NEM). During purification of factors required for protein folding in the cytosol, we found that the ATP-agarose binding activity of the yeast Hsp70 Ssa1p in postribosomal supernatants was inhibited by NEM. We also found that completely removing nucleotides from purified Ssa1p rendered its ATP-agarose binding activity, ATPase activity, and post-translational translocation-stimulating activity sensitive to NEM. We modified nucleotide-free Ssa1p with [14C]NEM and then digested it with proteases. Purification and sequencing of the radiolabeled proteolytic fragments revealed that each of Ssa1p's three cysteine residues (Cys-15, Cys-264, and Cys-303) was modified with [14C]NEM. ADP protected each of the cysteine residues from modification and protected Ssa1p from inactivation. The cysteine residues are the reactive centers of three NEM-reactive sites (NRS1-3). A comparison of Ssa1p's NRSs to sequences of other Hsp70s and actin revealed that Cys-15 of NRS1 is highly conserved and that sensitivity to NEM may be a property of many Hsp70s. Based on the three-dimensional structure of Hsc70, the predicted locations of Ssa1p's cysteine residues suggest that NEM may disrupt the conformation of Ssa1p or interfere with its ability to bind nucleotides. Together the results demonstrate that Ssa1p is an NEM-sensitive factor in cytosolic extracts from yeast that stimulates post-translational translocation of proteins into organelles.

Regulatory effects of zinc and copper on the calcium transport system in rat liver nuclei: Relation to SH groups in the releasing mechanism

In isolated hepatic nuclei, the heavy metals Zn 2+ and Cu 2+ (10 μM) inhibited Ca 2+ uptake and caused a prompt release of Ca 2+ from preloaded nuclei in a concentration-dependent manner, with Zn 2+ being more effective than Cu 2+. The sulfhydryl group reducing agent dithiothreitol (DTT) protected the nuclei from the effects of heavy metals; DDT (1 mM) almost completely blocked Zn 2+- or Cu 2+-induced Ca 2+ release and inhibition ofCa 2+ uptake. The sulfhydryl modifying reagent N-ethylmaleimide (NEM; 0.2 mM) also caused Ca 2+ release, but it did not have an appreciable effect on Ca 2+ uptake. Furthermore, in the presence of NEM heavy metals did not evoke Ca 2+ release. The present study demonstrates that Zn 2+ and Cu 2+ have a stimulatory effect on Ca 2+ release from isolated rat liver nuclei, and that the SH group may play an important role in the Ca 2+-releasing mechanism in liver nuclei.

Chloroplast Protein Import

The first step of chloroplast protein import is binding of a precursor protein to the surface of the organelle. Precursor binding for the small subunit of ribulose-1,5-bisphosphate carboxylase to isolated pea chloroplasts was investigated using a receptor-ligand binding assay. Translocation of precursors was blocked by conducting the binding assays at 0 degrees C. Binding of precursor was judged to be receptor mediated by the following criteria: (a) precursor binding was saturable at between 1500 and 3500 molecules per chloroplast; (b) binding is a high affinity interaction with a dissociation constant of 6 to 10 nanomoles; (c) binding is physiologically productive since most of the bound precursors could be imported from the bound state; and (d) precursor binding was sensitive to both protease and the sulfhydryl modifying reagent N-ethylmaleimide. The effects of these two reagents differed in that protease reduced the total number of binding sites from the surface of chloroplasts but had little effect on binding affinity, whereas N-ethylmaleimide reduced the binding affinity but had little or no effect on receptor density.

Rapid Deactivation of NADPH Oxidase in Neutrophils: Continuous Replacement by Newly Activated Enzyme Sustains the Respiratory Burst

The cell-free system for activation of the neutrophil NADPH oxidase allowed us to examine activation of the oxidase in the absence of its NADPH-dependent turnover. The covalent sulfhydryl-modifying reagent N-ethylmaleimide completely inhibited the activation step (Ki = 40 mumol/L) in the cell-free system but had no effect on turnover of the preactivated particulate NADPH oxidase (up to 1 mmol/L). When N-ethylmaleimide was added to intact neutrophils during the period of maximal O2 generation in response to stimuli that activate the respiratory burst (phorbol myristate acetate, f-Met-Leu-Phe, opsonized zymosan, arachidonic acid), O2- generation ceased within seconds. Study of components of the cell-free activation system indicated that the cytosolic cofactor was irreversibly inhibited by N-ethylmaleimide whereas the N-ethylmaleimide-treated, membrane-associated oxidase could be activated by arachidonate and control cytosolic cofactor. Likewise, the cell-free system prepared from intact neutrophils that had been briefly exposed to N-ethylmaleimide and then washed reflected the effects of N-ethylmaleimide on the isolated cell-free components: cytosolic cofactor activity was absent, but the membrane oxidase remained fully activatable. Thus inhibition of oxidase activation by N-ethylamaleimide unmasked a rapid deactivation step that was operative in intact neutrophils but not in isolated particulate NADPH oxidase preparations. The demonstrated specificity of N-ethylmaleimide for oxidase activation and lack of effect on turnover of the NADPH oxidase suggested that sustained O2- generation by intact neutrophils was a result of continued replenishment of a small pool of active oxidase. The existence of an inactive pool of NADPH oxidase molecules in particulate preparations from stimulated neutrophils was supported more directly by activating these preparations again in the cell-free system.

Euglena gracilis chloroplast elongation factor Tu. Interaction with guanine nucleotides and aminoacyl-tRNA.

The interaction of the chloroplast elongation factor Tu (EF-Tuchl) from Euglena gracilis with guanine nucleotides and aminoacyl-tRNA has been investigated. The apparent dissociation constant at 37 degrees C for the EF-Tuchl X GDP complex is about 3 X 10(-7) M and for the EF-Tuchl X GTP complex, it is about 1 order of magnitude higher. The sulfhydryl modifying reagent N-ethylmaleimide severely inhibits the polymerization activity of Euglena EF-Tuchl. In the presence of N-ethylmaleimide, the dissociation constant for the modified EF-Tuchl X GDP complex is increased by an order of magnitude. Conversely, both GDP and GTP protect EF-Tuchl from the modification. The polymerization activity of EF-Tuchl is also sensitive to the antibiotic kirromycin. In the presence of kirromycin, the apparent dissociation constant for the EF-Tuchl X GTP complex is lowered 10-fold. The interaction of aminoacyl-tRNA with EF-Tuchl was investigated by examining the ability of EF-Tuchl to prevent the spontaneous hydrolysis of Phe-tRNA and by gel filtration chromatography. The binding of aminoacyl-tRNA to EF-Tuchl occurs only in the presence of GTP indicating the formation of the ternary complex EF-Tuchl X GTP X Phe-tRNA. The effect of kirromycin on the interaction was also investigated. In the presence of kirromycin, no interaction between EF-Tuchl and Phe-tRNA is observed, even in the presence of GTP.