Inactivation of Trypanosoma cruzi and Crithidia fasciculata topoisomerase I by Fenton systems

Fenton systems (H(2)O(2)/Fe(II) or H(2)O(2)/Cu(II)) inhibited Trypanosoma cruzi and Crithidia fasciculata topoisomerase I activity. About 61-71% inactivation was produced by 25 microM Fe(II) or Cu(II) with 3.0 mM H(2)O(2). Thiol compounds and free radical scavengers prevented Fenton system effects, depending on the topoisomerase assayed. With the T. cruzi enzyme, reduced glutathione (GSH), dithiothreitol (DTT), cysteine and N-acetyl-L-cysteine (NAC) entirely prevented the effect of the H(2)O(2)/Fe(II) system; mannitol protected 37%, whereas histidine and ethanol were ineffective. With C. fasciculata topoisomerase, GSH, DTT and NAC protected 100%, cysteine, histidine and mannitol protected 28%, 34% and 48%, respectively, whereas ethanol was ineffective. With the H(2)O(2)/Cu(II) system and T. cruzi topoisomerase, DTT and histidine protected 100% and 60%, respectively, but the other assayed protectors were less effective. Similar results were obtained with the C. fasciculata enzyme. Topoisomerase inactivation by the H(2)O(2)/Fe(II) or H(2)O(2)/Cu(II) systems proved to be irreversible since it was not reversed by the more effective enzyme protectors. It is suggested that topoisomerases could act either as targets of 'reactive oxygen species' (ROS) generated by Fenton systems or bind the corresponding metal ions, whose redox cycling would generate reactive oxygen species in situ.     

Distinctive iron requirement of tryptophan 5-monooxygenase: TPH1 requires dissociable ferrous iron

A peripheral type of tryptophan 5-monooxygenase (EC 1.14.16.4), TPH1, is very unstable in vitro, but the inactivation was reversible and full reactivation occurs upon anaerobic incubation with a high concentration of dithiothreitol (DTT, 15 mM). In this study, distinctive iron requirement of TPH1 was revealed through analysis of the enzyme's inactivation and activation by DTT. For this purpose, all the glasswares, plastics, Sephadex G-25 gels, and reagents including protein solutions had been treated with metal chelators, and apo-TPH was prepared by treatment with EDTA. Apo-TPH thus prepared exclusively required free Fe2+ for its catalytic activity; 10(-8) M was enough under the strict absence of Fe3+ but 10(-12) M was too low. No other metal ions including Fe3+ were effective. It appeared that Fe3+ bound to the enzyme with a higher affinity than Fe2+, resulting in the inactivation. Ascorbate, a non-thiol reducing agent, did not substitute DTT in the activation of TPH1, but enhanced the Fe2+-dependent activity of apo-TPH as effectively as DTT. Thus, the DTT-activation was essentially substituted by preparation of apo-TPH by the EDTA treatment and the assay of apo-TPH in the presence of Fe2+ and ascorbate. The activation of TPH1 by incubation with DTT was accompanied by exposure of 9 sulfhydryls out of the total 10 cysteine residues, but the cleavage of disulfide bonds seemed not to be crucial, even if it occurred. The effect of DTT was substituted by some other sulfhydryls whose structure was analogous to that of commonly used metal chelators. Based on these observations, the following dual roles of DTT are proposed: (1) in the activation of TPH, DTT removes inappropriate bound iron (Fe3+) as a chelator, keeping Fe3+ away from the enzyme's binding site which needs to bind Fe2+ for the catalytic activity, and (2) in both the activation and reaction processes, DTT prevents oxidation of Fe2+ to Fe3+ as a reducing agent.     

Manganese is essential for catalytic activity of Escherichia coli agmatinase.

Purified Escherichia coli agmatinase (EC 3.5.3.11) expressed the same activity in the absence or presence of added Mn2+ (0-5mM). However, it was strongly inhibited by Co2+, Ni2+, and Zn2+ and almost half inactivated by EDTA. Partial inactivation by EDTA yielded enzyme species containing 0.85 +/- 0.1 Mn2+/subunit, and it was accompanied by a decrease in intensity of fluorescence emission and a red shift from the emission maximum of 340 nm to 346 nm, indicating the movement of tryptophane residues to a more polar environment. The activity and fluorescence properties of fully activated agmatinase were restored by incubation of dialysed species with Mn2+. Manganese-free species, obtained by treatment with EDTA and guanidinium chloride (3 M), were active only in the presence of added Mn2+. Results obtained, which represent the first demonstration of the essentiality of Mn2+ for agmatinase activity, are discussed in connection with a possible binuclear metal center in the enzyme.     

The role of disulfide bonds in the conformational stability and catalytic activity of phytase.

Previous studies have predicted five disulfide bonds in Aspergillus niger phytase (phy A). To investigate the role of disulfide bonds, intrinsic fluorescence spectra, far-ultraviolet circular dichroism (CD) spectra, and an enzyme activity assay were used to compare the differences of catalytic activity and conformational stability of phytase during denaturation in urea in the presence and absence of dithiothreitol (DTT). In the presence of 2 mM DTT, the inactivation and unfolding were greatly enhanced at the same concentration of denaturant. The fluorescence emission maximum red shift and decreases of ellipticity at 222 nm were in accord with the changes of catalytic activity. The kinetics of the unfolding courses were a biphasic process consisting of two first-order reactions in the absence of DTT and a monophasic process of a first-order reaction in the presence of DTT. The results suggested that the loss of enzymatic activity was most likely because of a conformational change, and that disulfide bonds played an important role in three-dimensional structure and catalytic activity.     

Redox modification of sodium-calcium exchange activity in cardiac sarcolemmal vesicles

Na-Ca exchange activity in bovine cardiac sarcolemmal vesicles was stimulated up to 10-fold by preincubating the vesicles with 1 microM FeSO4 plus 1 mM dithiothreitol (DTT) in a NaCl medium. The increase in activity was not reversed upon removing the Fe and DTT. Stimulation of exchange activity under these conditions was completely blocked by 0.1 mM EDTA or o-phenanthroline; this suggests that the production of reduced oxygen species (H2O2, O2-.,.OH) during Fecatalyzed DTT oxidation might be involved in stimulating exchange activity. In agreement with this hypothesis, the increase in exchange activity in the presence of Fe-DTT was inhibited 80% by anaerobiosis and 60% by catalase. H2O2 (0.1 mM) potentiated the stimulation of Na-Ca exchange by Fe-DTT under both aerobic and anaerobic conditions; H2O2 also produced an increase in activity in the presence of either FeSO4 (1 microM) or DTT (1 mM), but it had no effect on activity by itself. Superoxide dismutase did not block the effects of Fe-DTT on exchange activity; however, the generation of O2-. by xanthine oxidase in the presence of an oxidizable substrate stimulated activity more than 2-fold. Hydroxyl radical scavenging agents (mannitol, sodium formate, sodium benzoate) did not attenuate the stimulation of activity observed with Fe-H2O2. Exchange activity was also stimulated by the simultaneous presence of glutathione (GSH; 1-2 mM) and glutathione disulfide (GSSG; 1-2 mM). Neither GSH nor GSSG was effective by itself and either 0.1 mM EDTA or o-phenanthroline blocked the effects on transport activity of the combination of GSH + GSSG. Treatment of the GSH and GSSG solutions with Chelex ion-exchange resin to remove contaminating transition metal ions reduced (by 40%) the degree of stimulation observed with GSH + GSSG. Full stimulating activity was restored to the Chelex-treated GSH and GSSG solutions by the addition of 1 microM Fe2+; Cu2+ was less effective than Fe2+ whereas Co2+ and Mn2+ were without effect. In the presence of 1 microM Fe2+, GSH alone produced a slight increase in transport activity, but this was markedly enhanced by the addition of Chelex-treated GSSG. The results indicate that stimulation of exchange activity requires the presence of both a reducing agent (DTT, GSH, O-.2, or Fe2+) and an oxidizing agent (H2O2, GSSG, and perhaps O2) and that the effects of these agents are mediated by metal ions (e.g. Fe2+).(ABSTRACT TRUNCATED AT 400 WORDS)     

E. coli propionyl-CoA synthetase is regulated in vitro by an intramolecular disulfide bond

The E. coli propionyl-CoA synthetase (PCS) was cloned, expressed, purified, and analyzed. Kinetic analyses suggested that the enzyme preferred propionate as substrate but would also use acetate. The purified, stored protein had relatively low activity but was activated up to about 10-fold by incubation with dithiothreitol (DTT). The enzyme activation by DTT was reversed by diamide. This suggests that the protein contains a regulatory disulfide bond and that the reduction to two sulfhydryl groups activates PCS while the oxidation to a disulfide leads to its inactivation. This idea was tested by sequential mutagenesis of the 9 Cys in the protein to Ala. It was revealed that the C128A and C315A mutants had wildtype enzyme activity but were no longer activated by DTT or inhibited by diamide. The data obtained indicate that two Cys residues could be involved in redox-regulated system through formation of an intramolecular disulfide bridge in PCS.     

E. coli propionyl-CoA synthetase is regulated in vitro by an intramolecular disulfide bond

The E. coli propionyl-CoA synthetase (PCS) was cloned, expressed, purified, and analyzed. Kinetic analyses suggested that the enzyme preferred propionate as substrate but would also use acetate. The purified, stored protein had relatively low activity but was activated up to about 10-fold by incubation with dithiothreitol (DTT). The enzyme activation by DTT was reversed by diamide. This suggests that the protein contains a regulatory disulfide bond and that the reduction to two sulfhydryl groups activates PCS while the oxidation to a disulfide leads to its inactivation. This idea was tested by sequential mutagenesis of the 9 Cys in the protein to Ala. It was revealed that the C128A and C315A mutants had wildtype enzyme activity but were no longer activated by DTT or inhibited by diamide. The data obtained indicate that two Cys residues could be involved in redox-regulated system through formation of an intramolecular disulfide bridge in PCS.     

Properties of latent and thiol-activated rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and regulation of enzyme activity

The effect of the thiols glutathione (GSH), dithiothreitol (DTT), and dithioerythritol (DTE) on the conversion of an inactive, latent form (El) of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) to a catalyticaly active form (Ea) is examined. Latent hepatic microsomal HMG-CoA reductase is activated to a similar degree of activation by DTT and DTE and to a lower extent by GSH. All three thiols affect both Km and Vmax values of the enzyme toward HMG-CoA and NADPH. Studies of the effect of DTT on the affinity binding of HMG-CoA reductase to agarose-hexane-HMG-CoA (AG-HMG-CoA) resin shows that thiols are necessary for the binding of the enzyme to the resin. Removal of DTT from AG-HMG-CoA-bound soluble Ea (active enzyme) does not cause dissociation of the enzyme from the resin at low salt concentrations. Substitution of DTT by NADPH does not promote binding of soluble El (latent enzyme) to AG-HMG-CoA. The enzymatic activity of Ea in the presence of DTT and GSH indicates that these thiols compete for the same binding site on the enzyme. Diethylene glycol disulfide (ESSE) and glutathione disulfide (GSSG) inhibit the activity of Ea. ESSE is more effective for the inhibition of Ea than GSSG, causing a higher degree of maximal inhibition and affecting the enzymatic activity at lower concentrations. A method is described for the rapid conversion of soluble purified Ea to El using gel-filtration chromatography on Bio-Gel P-4 columns. These combined results point to the importance of the thiol/disulfide ratio for the modulation of hepatic HMG-CoA reductase activity.     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

Effect of a disulfide bond on mevalonate kinase

Mevalonate kinase is one of ATP-dependent enzymes in the mevalonate pathway and catalyzes the phosphorylation of mevalonate to form mevalonate 5-phosphate. In animal cells, it plays a key role in regulating biosynthesis of cholesterol, while in microorganisms and plants, it is involved in the biosynthesis of isoprenoid derivatives that are one of the largest groups of natural products. Crystal structure and sequence alignment show that a unique disulfide bond exists in mevalonate kinase of thermostable species Methanococcus jannaschii, but not in rat mevalonate kinase. In the present study, we investigated the effect of the disulfide bond in M. jannaschii mevalonate kinase and an engineered disulfide bond in rat mevalonate kinase mutant A141C on the properties of enzymes through characterization of their wild-type and variant enzymes. Our result suggests that the Cys107-Cys281 disulfide bond is important for maintaining the conformation and the thermal activity of M. jannaschii mevalonate kinase. Other interactions could also have contributions. The thiol-titration and fluorescence experiment further indicate that rat mevalonate kinase A141C variant enzyme has a new disulfide bond, which makes the variant protein enhance its thermal activity and resist to urea denaturation.     

The disulfide linkage and the free sulfhydryl accessibility of acyl-coenzyme A:cholesterol acyltransferase 1 as studied by using mPEG5000-maleimide

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is a membrane protein located in the endoplasmic reticulum (ER). It plays important roles in cellular cholesterol homeostasis. Human ACAT1 (hACAT1) contains nine cysteines (C). To quantify and map its disulfide linkage, we performed thiol-specific modifications by mPEG(5000)-maleimide (PEG-mal) and iodoacetamide (IA) under denatured condition, using extracts that contain wild-type or various single C to A mutant hACAT1s. With the wild-type enzyme, seven Cs could be modified before dithiothreitol (DTT) treatment; nine Cs could be modified after DTT treatment. With the C528A or the C546A enzyme, all eight Cs could be modified before or after DTT treatment. With all other remaining single C to A mutant enzymes, six Cs could be modified before DTT treatment, and eight Cs could be modified after DTT treatment. We next performed Lys-C protease digestion on hACAT1 with a hemagglutinin (HA) tag at the C-terminus. The digests were treated with or without DTT and analyzed by SDS-PAGE and Western blotting. The two predicted C-terminal fragments (K496-K531 and N532-F550-HA tag) were trapped as a single peptide band, but only when the digests were treated without DTT. Thus, C528 and C546 near the enzyme's C-terminus form a disulfide. PEG-mal is impermeable to ER membranes. We used PEG-mal to map the localizations of the seven free sulfhydryls and the disulfide bond of hACAT1 present in microsomal vesicles. The results show that C92 is located on the cytoplasmic side of the ER membrane and the disulfide is located in the ER lumen, while all other free Cs are located within the hydrophobic region(s) of the enzyme.     

Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme

The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E. C. 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo.     

Influence of disulfide bonds on the conformational changes and activities of refolded phytase

Aspergillus sp. phytase contains five disulfide bonds. In order to elucidate their role, the reactivation and refolding of phytase in the absence and presence of dithiothreitol (DTT) was investigated. The results indicated that the disulfide bonds play an important role in the catalytic activity and conformational stability of the enzyme.     

Reversible inactivation of AT(2) angiotensin II receptor from cysteine-disulfide bond exchange

Dithiothreitol (DTT) treatment of angiotensin II (Ang II) type 2 (AT(2)) receptor potentiates ligand binding, but the underlying mechanism is not known. Two disulfide bonds proposed in the extracellular domain were examined in this report. Based on the analysis of ligand affinity of cysteine (Cys, C) to alanine (Ala, A) substitution mutants, we provide evidence that Cys(35)-Cys(290) and Cys(117)-Cys(195) disulfide bonds are formed in the wild-type AT(2) receptor. Disruption of the highly conserved Cys(117)-Cys(195) disulfide bond linking the second and third extracellular segments leads to inactivation of the receptor. The Cys(35)-Cys(290) bond is highly sensitive to DTT. Its breakage results in an increased binding affinity for both Ang II and the AT(2) receptor-specific antagonist PD123319. Surprisingly, in the single Cys mutants, C35A and C290A, a labile population of receptors is produced which can be re-folded to high-affinity state by DTT treatment. These results suggest that the free -SH group of Cys(35) or Cys(290) competes with the disulfide bond formation between Cys(117) and Cys(195). This Cys-disulfide bond exchange results in production of the inactive population of the mutant receptors through formation of a non-native disulfide bond.     

Recombinant human tyrosine hydroxylase isozymes. Reconstitution with iron and inhibitory effect of other metal ions.

Human tyrosine 3-monooxygenase (tyrosine hydroxylase) exists as four different isozymes (TH1-TH4), generated by alternative splicing of pre-mRNA. Recombinant TH1, TH2 and TH4 were expressed in high yield in Escherichia coli. The purified isozymes revealed high catalytic activity [when reconstituted with Fe(II)] and stability at neutral pH. The isozymes as isolated contained 0.04-0.1 atom iron and 0.02-0.06 atom zinc/enzyme subunit. All three isozymes were rapidly activated (13-40-fold) by incubation with Fe(II) salts (concentration of iron at half-maximal activation = 6-14 microM), and were inhibited by other divalent metal ions, e.g. Zn(II), Co(II) and Ni(II). They all bind stoichiometric amounts of Fe(II) and Zn(II) with high affinity (Kd = 0.2-3 microM at pH 5.4-6.5). Similar time courses were observed for binding of Fe(II) and enzyme activation. In the absence of any free Fe(II) or Zn(II), the metal ions were released from the reconstituted isozymes. The dissociation was favoured by acidic pH, as well as by the presence of metal chelators and dithiothreitol. The potency of metal chelators to remove iron from the hydroxylase correlated with their ability to inhibit the enzyme activity. These studies show that tyrosine hydroxylase binds iron reversibly and that its catalytic activity is strictly dependent on the presence of this metal.     

Recombinant purple acid phosphatase isoform 3 from sweet potato is an enzyme with a diiron metal center

Purple acid phosphatases (PAPs) from sweet potato (sp) have been classified on the basis of their primary structure and the dinuclear metal center into isoforms spPAP1 [Fe(III)-Zn(II)] and spPAP2 [Fe(III)-Mn(II)]; for spPAP3 only the cDNA is known. With the aim of unraveling the character of the dinuclear metal center we report here the characterization of this isoform at the protein level. We cloned spPAP3 cDNA in a baculovirus and overexpressed this enzyme in Sf9 insect cells. Preparation of recombinant spPAP3 in two steps afforded pure enzyme with yields of 4.5 mg.L(-1) culture medium. This enzyme is a dimeric, disulfide-linked PAP of 110 kDa, similar to known PAP isoforms from higher plants. Enzymatic studies and spectroscopic properties (max. absorption at 550-565 nm) indicated a diiron enzyme; quantitative and semiquantitative metal analysis using ICP-OES and TOF-SIMS, respectively, revealed the presence of only iron in purified spPAP3. Metal replacement in the second metal-binding site upon preparation of the semiapo-enzyme with Fe(II), Zn(II), or Mn(II) showed highest activities with Fe(II). The data show that recombinant spPAP3 has a diiron metal center. Site-directed mutagenesis was conducted to check catalytic efficiency at the atomic level. Tyr291 at the substrate-binding site in spPAP3 was mutated to His and Ala, the respective residues found in spPAP1 and spPAP2. Kinetic analysis showed that conversion of Tyr291 to His further optimized the performance of this protein as a diiron enzyme, whereas the Ala mutation weakened the catalytic efficiency regardless of the metal present in the second binding site.     

Manganese-containing superoxide dismutase from Escherichia coli: reversible resolution and metal replacements.

Exposure of the manganese-containing Superoxide dismutase of Escherichia coli to pH 3.2, in the presence of 0.7 m guanidinium chloride, causes a rapid loss of manganese and of activity. The apoenzyme so produced can be reconstituted by addition of MnCl₂ followed by neutralization. In contrast, manganese cannot be restored to the apoenzyme by adding MnCl₂after neutralization. The reconstituted enzyme is indistinguishable from the native enzyme in terms of its catalytic activity or electrophoretic behavior on polyacrylamide gels. Co(II), Ni(II), Zn(II), Fe(II), or Cu(II) could compete with Mn(II) during reconstitution of the apoenzyme. In the cases of Co(II), Ni(II), and Zn(II), it was shown that, in preventing reconstitution by Mn(II), they were themselves bound to the enzyme in stoichiometric amounts, in place of Mn(II). The binding of Fe(II) was also explored and was distinct in that the enzyme could bind more than stoichiometric amounts of this metal. None of the derivatives, in which Mn(II) had been replaced by another metal, were catalytically active. Nevertheless, these derivatives could be again resolved by exposure to acid guanidinium chloride and could then be converted back into the active holoenzyme by neutralization after addition of MnCl₂. It appears that the active site of this enzyme can accommodate and can tightly bind several metals other than manganese, but exhibits activity only with manganese. It also appears that movement of metal out of or into this site is only feasible at low pH and in the presence of a chaotropic agent. A substantial amount of the cobalt-substituted enzyme was prepared and its optical properties were recorded.     

Reactive nitrogen species derived activation of rat liver microsomal glutathione S-transferase

The effect of reactive nitrogen species on rat liver microsomal glutathione S-transferase (MGST1) was investigated using microsomes and purified MGST1. When microsomes or the purified enzyme were incubated with peroxynitrite (ONOO(-)), the GST activity was increased to 2.5-6.5 fold in concentration-dependent manner and a small amount of the MGST1 dimer was detected. MGST1 activity was increased by ONOO(-) in the presence of high amounts of reducing agents including glutathione (GSH) and the activities increased by ONOO(-) or ONOO(-) plus GSH treatment were decreased by 30-40% by further incubation with dithiothreitol (DTT, reducing disulfide) or by sodium arsenite (reducing sulfenic acid). Furthermore, GSH was detected by HPLC from the MGST1 which was incubated with ONOO(-) plus GSH or S-nitrosoglutathione followed by DTT treatment. In addition, the MGST1 activity increased by nitric oxide (NO) donors such as S-nitrosoglutathione, S-nitrosocysteine or the non-thiol NO donor 1-hydroxy-2-oxo-3 (3-aminopropyl)-3-isopropyl was restored by the DTT treatment. Since DTT can reduce S-nitrosothiol and disulfide bond to thiol, S-nitrosylation and a mixed disulfide bond formation of MGST1 were suggested. Thus, it was demonstrated that MGST1 is activated by reactive nitrogen species through a forming dimeric protein, mixed disulfide bond, nitrosylation and sulfenic acid.     

The methionyl aminopeptidase from Escherichia coli can function as an iron(II) enzyme.

The identity of the physiologically relevant metal ions for the methionyl aminopeptidase (MetAP) from Escherichia coli was investigated and is suggested to be Fe(II). The metal content of whole cells in the absence and presence of expression of the type I MetAP from E. coli was determined by inductively coupled plasma (ICP) emission analysis. The observed change in whole cell concentrations of cobalt, cadmium, copper, nickel, strontium, titanium, and vanadium upon expression of MetAP was negligible. On the other hand, significant increases in the cellular metal ion concentrations of chromium, zinc, manganese, and iron were observed with the increase in iron concentration being 4.4 and 6.2 times greater than that of manganese and zinc, respectively. Activity assays of freshly lysed BL21(DE3) cells containing the pMetAAP plasmid revealed detectable levels (>2 units/mg) of MetAP activity. Control experiments with BL21(DE3) without the MetAP plasmid showed no detectable enzymatic activity. Since MetAP is active upon expression, these data strongly suggest that cobalt is not the in vivo metal ion for the MetAP from E. coli. The MetAP from E. coli as purified was found to be catalytically inactive (相似文献