Roles for the two cysteine residues of AhpC in catalysis of peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium

The catalytic properties of cysteine residues Cys46 and Cys165, which form intersubunit disulfide bonds in the peroxidatic AhpC protein of the alkyl hydroperoxide reductase (AhpR) system from Salmonella typhimurium, have been investigated. The AhpR system, composed of AhpC and a flavoprotein reductase, AhpF, catalyzes the pyridine nucleotide-dependent reduction of organic hydroperoxides and hydrogen peroxide. Amino acid sequence analysis of the disulfide-containing tryptic peptide demonstrated the presence of two identical disulfide bonds per dimer of oxidized AhpC located between Cys46 on one subunit and Cys165 on the other. Mutant AhpC proteins containing only one (C46S and C165S) or no (C46,165S) cysteine residues were purified and shown by circular dichroism studies to exhibit no major disruptions in secondary structure. In NADH-dependent peroxidase assays in the presence of AhpF, the C165S mutant was fully active in comparison with wild-type AhpC, while C46S and C46,165S displayed no peroxidatic activity. In addition, only C165S was oxidized by 1 equiv of hydrogen peroxide, giving a species that was stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF. Oxidized C165S also reacted rapidly with a stoichiometric amount of the thiol-containing reagent 2-nitro-5-thiobenzoic acid to generate a mixed disulfide, and was susceptible to inactivation by hydrogen peroxide, strongly supporting its identification as a cysteine sulfenic acid (Cys46-SOH). The lack of reactivity of the C46S mutant toward peroxides was not a result of inaccessibility of the remaining thiol as demonstrated by its modification with 5, 5'-dithiobis(2-nitrobenzoic acid), but could be due to the lack of a proximal active-site base which would support catalysis through proton donation to the poor RO- leaving group. Our results clearly identify Cys46 as the peroxidatic center of AhpC and Cys165 as an important residue for preserving the activity of wild-type AhpC by reacting with the nascent sulfenic acid of the oxidized protein (Cys46-SOH) to generate a stable disulfide bond, thus preventing further oxidation of Cys46-SOH by substrate.     

Subunit-specific interactions of cyanide with the N-methyl-D-aspartate receptor

Cyanide can potentiate N-methyl-D-aspartate receptor-mediated physiological responses in neurons. Here we show that this phenomenon may be attributable to a subunit-specific chemical modification of the receptor directly by the toxin. N-Methyl-D-aspartate (30 microM)-induced whole cell responses in mature (22-29 days in vitro) rat cortical neurons were potentiated nearly 2-fold by a 3-5-min treatment with 2 mM potassium cyanide, as did a similar treatment with 4 mM dithiothreitol. A 1-min incubation with the thiol oxidant 5,5'-dithiobis(2-nitrobenzoic acid) (0.5 mM) readily reversed the potentiation induced by either cyanide or dithiothreitol. Cyanide did not increase further currents previously potentiated by dithiothreitol nor was it able to potentiate responses during brief co-application with the agonist. Transient expression studies in Chinese hamster ovary cells with wild-type and mutated recombinant N-methyl-D-aspartate subunits (NR) demonstrated that cyanide selectively potentiated NR1/NR2A receptors, presumably via the chemical reduction of NR2A. In contrast, currents mediated by NR1/NR2B receptors were somewhat diminished by the metabolic inhibitor. Some of the effects of cyanide on NR1/NR2B receptors may be mediated by the formation of a thiocyanate adduct with a cysteine residue located in NR1. Cyanide thus is able to distinguish chemically between two different N-methyl-D-aspartate receptor subtypes and produce diametrically opposing functional effects.     

Carboxymethylation of thiol groups in ovalbumin: implications for proteins that contain both thiol and disulfide groups

The cysteine residues of hen ovalbumin were S-carboxymethylated with non-radioactive iodoacetic acid under various conditions by altering the pH at which the protein was denatured in 8 M urea, by using different molar ratios of non-radioactive iodoacetic acid to cysteine and by varying the time at which carboxymethylation was commenced after denaturing conditions had been applied. Under the various conditions, the thiol groups were carboxymethylated to different extents, the residual thiol groups being measured by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of sodium dodecyl sulfate. When ovalbumin is carboxymethylated in alkaline urea, it unfolds slowly and the carboxymethylation is incomplete even with 150-fold excess iodoacetic acid. The known rapid thiol-disulfide exchange that occurs at alkaline pH values makes this method of carboxymethylation unsuitable as a preliminary step for blocking the native cysteine residues of ovalbumin before reduction and labelling the thiol groups formed by reduction of the disulfide bonds. Titration of the thiol groups of ovalbumin in 6 M guanidine hydrochloride or 1% (w/v) sodium dodecyl sulfate at pH 8.2 with 5,5'-dithiobis(2-nitrobenzoic acid) is more rapid than in 8 M urea and these solvents would be preferable for studies of the disulfide-bonded sequences. Denaturation of ovalbumin in acidic 8 M urea is a very rapid process, and under mild acid conditions thiol-disulfide interchange is much slower. Subsequent carboxymethylation of the cysteine residues at alkaline pH with 150-fold excess iodoacetic acid results in complete carboxymethylation and the carboxymethylated ovalbumin can be reduced and labelled with radioactive iodoacetic acid with specific labelling of the half-cystine residues involved in the disulfide bond. The results are discussed in relation to the allocation of half-cystine residues in other protein systems that contain both thiol and disulfide groups.     

A truncated version of an ADP-glucose pyrophosphorylase promoter from potato specifies guard cell-selective expression in transgenic plants

ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme in starch biosynthesis in higher plants. A 3.2-kb promoter of the large subunit gene of the AGPase from potato has been isolated and its activity analyzed in transgenic potato and tobacco plants using a promoter-beta-glucuronidase fusion system. The promoter was active in various starch-containing cells, including guard cells, tuber parenchyma cells, and the starch sheath layer of stems and petioles. No expression was observed in mesophyll cells. Analysis of various promoter derivatives showed that with respect to expression in petioles and stems, essential elements must be located in the 5' distal region of the promoter, whereas elements important for expression in tuber parenchyma cells are located in an internal fragment comprising nucleotides from positions -500 to -1200. Finally, a 0.3-kb 5' proximal promoter fragment was identified that was sufficient to obtain exclusive expression in guard cells of transgenic potato and tobacco plants. The implications of our observations are discussed with respect to starch synthesis in various tissues and the use of the newly identified promoter as a tool for stomatal biology.     

Growth hormone therapy in Silver Russell syndrome: 5 years experience of the Australian and New Zealand Growth database (OZGROW)

BACKGROUND: Tetrachlorohydroquinone dehalogenase catalyzes the reductive dehalogenation of tetrachlorohydroquinone to trichlorohydroquinone and then to 2,6-dichlorohydroquinone. This enzyme undergoes oxidative damage during purification which causes it to form aberrant products. The damage is reversible by treatment with dithiothreitol. Possible types of oxidative damage include an inappropriate disulfide bond, a cysteine sulfenic acid, or a methionine sulfoxide. RESULTS: Using electrospray liquid chromatography / mass spectrometry, we have demonstrated that oxidation of tetrachlorohydroquinone dehalogenase with H2O2 results in formation of a sulfenic acid at Cys13. Further oxidation to a sulfinic acid was also observed. CONCLUSIONS: Oxidation of Cys 13 to a sulfenic acid prevents the normal reductive dehalogenation reaction from being completed. This finding is consistent with previous work which suggested that Cys 13 acts as a nucleophile during the conversion of tetrachlorohydroquinone to trichlorohydroquinone. The technique described for identification and localization of the cysteine sulfenic acid should be applicable to a wide variety of biological systems.     

Properties and subunit structure of pig heart pyruvate dehydrogenase

Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.     

Two-step autocatalytic processing of the glutaryl 7-aminocephalosporanic acid acylase from Pseudomonas sp. strain GK16

The glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 is an (alphabeta)2 heterotetramer of two nonidentical subunits. These subunits are derived from nascent polypeptides that are cleaved proteolytically between Gly198 and Ser199 after the nascent polypeptides have been translocated into the periplasm. The activation mechanism of the GL-7-ACA acylase has been analyzed by both in vivo and in vitro expression studies, site-directed mutagenesis, in vitro renaturation of inactive enzyme precursors, and enzyme reconstitution. An active enzyme complex was found in the cytoplasm when its translocation into the periplasm was suppressed. In addition, the in vitro-expressed GL-7-ACA acylase was processed into alpha and beta subunits, and the inactive enzyme aggregate of the precursor was also processed and became active during the renaturation step. Mutation of Ser199 to Cys199 and enzyme reconstitution allowed us to identify the secondary processing site that resides in the alpha subunit and to show that Ser199 of the beta subunit is essential for these two sequential processing steps. Mass spectrometry clearly indicated that the secondary processing occurs at Gly189-Asp190. All of the data suggest that the enzyme is activated through a two-step autocatalytic process upon folding: the first step is an intramolecular cleavage of the precursor between Gly198 and Ser199 for generation of the alpha subunit, containing the spacer peptide, and the beta subunit; the second is an intermolecular event, which is catalyzed by the N-terminal Ser (Ser199) of the beta subunit and results in a further cleavage and the removal of the spacer peptide (Asp190 to Gly198).     

A formal system of approval for monographs in the pharmacopoeia of culture media--statement from the IUMS-ICFMH working party on culture media

By site-directed mutagenesis on human cytidine deaminase (CDA), five mutant proteins were obtained: C65A, C99A, C102A, E67D and E67Q. The three cysteine mutants were completely inactive, whereas E67D and E67Q showed a specific activity about 200- and 200000-fold lower, respectively, than the wild-type CDA. Zinc analysis revealed that only E67D, E67Q and C65A contained 1 mol Zn2+/mol subunit as in the wild-type CDA. Kinetic measurements with the specific carboxylic group reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline performed on wild-type CDA suggest that Glu67 is essential for the catalytic process. Furthermore, when both native and denatured CDA was titrated with 5,5'-dithiobis(2-nitrobenzoic acid) six sulfhydryl groups were detected, whereas in the denatured and reduced enzyme nine such groups were found, according to the sequence data. When p-hydroxymercuriphenyl sulfonate was used, nine sulfhydryl groups were detectable and the release of 1 mol of zinc per mole of CDA subunit was revealed by the metal indicator dye 4-(2-pyridylazo)resorcinol. It seems plausible that the limiting step for the maintenance of zinc in the active site is the formation of coordination between Cys99 and Cys102, whereas Cys65 could lead the zinc to the correct position and orientation within the active site.     

Characterization of a mammalian peroxiredoxin that contains one conserved cysteine

A new type of peroxidase enzyme, named thioredoxin peroxidase (TPx), that reduces H2O2 with the use of electrons from thioredoxin and contains two essential cysteines was recently identified. TPx homologs, termed peroxiredoxin (Prx), have also been identified and include several proteins, designated 1-Cys Prx, that contain only one conserved cysteine. Recombinant human 1-Cys Prx expressed in and purified from Escherichia coli has now been shown to reduce H2O2 with electrons provided by dithiothreitol. Furthermore, human 1-Cys Prx transiently expressed in NIH 3T3 cells was able to remove intracellular H2O2 generated in response either to the addition of exogenous H2O2 or to treatment with platelet-derived growth factor. The conserved Cys47-SH group was shown to be the site of oxidation by H2O2. Thus, mutation of Cys47 to serine abolished peroxidase activity. Moreover, the oxidized intermediate appears to be Cys-SOH. In contrast to TPx, in which one of the two conserved cysteines is oxidized to Cys-SOH and then immediately reacts with the second conserved cysteine of the second subunit of the enzyme homodimer to form an intermolecular disulfide, the Cys-SOH of 1-Cys Prx does not form a disulfide. Neither thioredoxin, which reduces the disulfide of TPx, nor glutathione, which reduces the Cys-SeOH of oxidized glutathione peroxidase, was able to reduce the Cys-SOH of 1-Cys Prx and consequently could not support peroxidase activity. Human 1-Cys Prx was previously shown to exhibit a low level of phospholipase A2 activity at an acidic pH; the enzyme was thus proposed to be lysosomal, and Ser32 was proposed to be critical for lipase function. However, the mutation of Ser32 or Cys47 has now been shown to have no effect on the lipase activity of 1-Cys Prx, which was also shown to be a cytosolic protein. Thus, the primary cellular function of 1-Cys Prx appears to be to reduce peroxides with the use of electrons provided by an as yet unidentified source; the enzyme therefore represents a new type of peroxidase.     

Regulation of the cyanide-resistant alternative oxidase of plant mitochondria. Identification of the cysteine residue involved in alpha-keto acid stimulation and intersubunit disulfide bond formation

The cyanide-resistant alternative oxidase of plant mitochondria is a homodimeric protein whose activity can be regulated by a redox-sensitive intersubunit sulfhydryl/disulfide system and by alpha-keto acids. After determining that the Arabidopsis alternative oxidase possesses the redox-sensitive sulfhydryl/disulfide system, site-directed mutagenesis of an Arabidopsis cDNA clone was used to individually change the two conserved Cys residues, Cys-128 and Cys-78, to Ala. Using diamide oxidation and chemical cross-linking of the protein expressed in Escherichia coli, Cys-78 was shown to be: 1) the Cys residue involved in the sulfhydryl/disulfide system; and 2) not required for subunit dimerization. The C128A mutant was stimulated by pyruvate, while the C78A mutant protein had little activity and displayed no stimulation by pyruvate. Mutating Cys-78 to Glu produced an active enzyme which was insensitive to pyruvate, consistent with alpha-keto acid activation occurring through a thiohemiacetal. These results indicate that Cys-78 serves as both the regulatory sulfhydryl/disulfide and the site of activation by alpha-keto acids. In light of these results, the previously observed effects of sulfhydryl reagents on the alternative oxidase of isolated soybean mitochondria were re-examined and were found to be in agreement with a single sulfhydryl residue being the site both of alpha-keto acid activation and of the regulatory sulfhydryl/disulfide system.     

Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase

The free cysteine residues in the extremely thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized using selective chemical modification with the stable nitroxyl biradical bis(1-oxy-2,2,5,5-tetramethyl-3-imidazoline-4-yl)disulfide, via a thiol-disulfide exchange reaction and with 2[14C]iodoacetic acid, via S-alkylation. The respective reactions were monitored by electron paramagenetic resonance (EPR) and by the incorporation of the radioactive label. In native TBADH, the rapid modification of one cysteine residue per subunit by the biradical and the concomitant loss of catalytic activity was reversed by DTT. NADP protected the enzyme from both modification and inactivation by the biradical. RPLC fingerprint analysis of reduced and S-carboxymethylated lysyl peptides from the radioactive alkylated enzyme identified Cys 203 as the readily modified residue. A second cysteine residue was rapidly modified with both modification reagents when the catalytic zinc was removed from the enzyme by o-phenanthroline. This cysteine residue, which could serve as a putative ligand to the active-site zinc atom, was identified as Cys 37 in RPLC. The EPR data suggested a distance of < or 10 A between Cys 37 and Cys 203. Although Cys 283 and Cys 295 were buried within the protein core and were not accessible for chemical modification, the two residues were oxidized to cystine when TBADH was heated at 75 degrees C, forming a disulfide bridge that was not present in the native enzyme, without affecting either enzymatic activity or thermal stability. The status of these cysteine residues was verified by site directed mutagenesis.     

Alternate amino terminal processing of surfactant protein A results in cysteinyl isoforms required for multimer formation

The biological functions of rat surfactant protein A (SP-A), an oligomer composed of 18 polypeptide subunits derived from a single gene, are dependent on intact disulfide bonds. Reducible and collagenase-reversible covalent linkages of as many as six or more subunits in the molecule indicate the presence of at least two NH2-terminal interchain disulfide bonds. However, the reported primary structure of rat SP-A predicts that only Cys6 in this region is available for interchain disulfide formation. Direct evidence for a second disulfide bridge was obtained by analyses of a set of three mutant SP-As with telescoping deletions from the reported NH2-terminus. Two of the truncated recombinant proteins formed reducible dimers despite deletion of the domain containing Cys6. Edman degradation revealed that each mutant protein was a mixture of two isoforms with and without an isoleucine-lysine-cysteine (IKC) extension at the NH2-terminus, which was derived from the COOH-terminal end of the reported signal peptide. Large variations in the abundance of the IKC isoforms between truncated SP-As suggested that the amino acid sequences located downstream from the signal peptide modulated alternate-site cleavage by signal peptidase. Elution of the newly identified cysteine in the position of DiPTH-Cys indicated participation in disulfide linkage, which was interchain based on the direct correlation between prevalence of the IKC variant and the extent of dimerization for each truncated protein. Sequencing of both native rat SP-A and human SP-A also revealed isoforms with disulfide-forming NH2-terminal extensions. The extended rat SP-A isoforms were enriched in the more fully glycosylated and multimeric SP-A species separated on SDS-PAGE gels. Thus, a novel post translational modification results in naturally occurring cysteinyl isoforms of rat SP-A, which are essential for multimer formation.     

A review of the effects of manipulation of the cysteine residues of rat aryl sulfotransferase IV

Aryl sulfotransferase IV from rat liver has the broad substrate range that is characteristic of the enzymes of detoxication. With the standard assay substrates, 4-nitrophenol and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), sulfation is optimum at pH 5.4 whereas the reaction is minimal in the physiological pH range. These properties preclude a physiological function for this cytosolic enzyme. Partial oxidation of the enzyme, however, results not only in an increase in the rate of sulfation but also in a shift of the pH optimum to the physiological pH range. The mechanism for this dependence on the redox environment involves oxidation at Cys66, the cysteine residue that is conserved throughout the phenol sulfotransferase family. As documented by mass spectroscopic methods, oxidation by GSSG leads to the formation of an internal disulfide between Cys66 and Cys232; for mutants at Cys232, the oxidation product is a mixed disulfide of Cys66 and glutathione. Both of these disulfide species activate the enzyme and allow it to function at a pH optimum in the physiological range. The activated enzyme differs from the reduced form by a more circumscribed substrate spectrum. All five mutants, in which each of the cysteines of the sulfotransferase subunit have been changed to serine, are catalytically active. Only Cys66 is required for the redox response.     

Assignment of the inter- and intramolecular disulfide linkages in recombinant human macrophage colony stimulating factor using fast atom bombardment mass spectrometry

The disulfide bridges in recombinant human macrophage colony stimulating factor (rhM-CSF), a 49-kDa homodimeric protein, were assigned. The 18 cysteines in the dimer form three intermolecular and two sets of three intramolecular disulfide bonds. The intermolecular disulfide bridges hold the dimer together and form symmetric bonds in which Cys31 and Cys157/Cys159 from one monomer unit are linked to the corresponding cysteines of the second monomer. The intramolecular disulfide bonds are located between Cys7-Cys90, Cys48-Cys139, and Cys102-Cys146, respectively. The resistance of native M-CSF to proteolytic cleavage was overcome by an initial chemical cleavage reaction using BrCN. The close proximity of four cysteines (Cys139, Cys146, Cys157, and Cys159) results in a tight core complex that makes the protein undigestable for most proteases. Digestion using endoprotease Asp-N resulted in cleavage at Asp156 near the C-terminal end of this region, thereby opening the complex structure.     

O-Acetylserine and O-acetylhomoserine sulfhydrylase of yeast. Subunit structure

The molecular weight of O-acetylserine (OAS)-O-acetylhomoserine (OAH) sulfhydrylase purified from yeast was estimated to be about 200,000 by Sephadex G-200 gel chromatography in various buffers. The S20, w value of this protein was determined to be about 9.0 by sucrose density gradient centrifugation. The calculated molecular weight based on this value was similar to that estimated by gel chromatography. Treatment with 1% sodium dodesylsulfate (SDS) or 6 M urea dissociated the enzyme into 4 subunits; these had a molecular weight estimated to be 51,000 by SDS-poly-acrylamide gel electrophoresis and to be 57,000 by Sephadex G-100 gel chromatography in the presence of 6 M urea and 0.5% beta-mercaptoethanol. The 4 subunits appeared to be identical, based on the symmetric subunit elution pattern from a Sephadex column, a single peptide band on SDS-polyacrylamide gel, and the detection of histidine as the sole N-terminal amino acid in the native enzyme. Since dissociation into the subunits occurred without the use of reducing agents, the association of the subunits seems to require no disulfide linkage. One mole of the subunit contained one mole of sulfhydryl group which appeared to be buried inside the molecule. Partial restoration of the catalytic activity was observed when the urea-denatured enzyme was dialyzed to remove urea, especially in the presence of reducing agents such as dithiothreitol. The urea-denatured enzyme showed a tendency in the absence of reducing agents to form a subunit dimer linked by a disulfide bond between the cystine residues exposed by denaturation. The amino acid composition of the enzyme was determined; it contained one half-cystine residue per subunit, and the content of acidic residues was much higher than that of basic residues. Based on these findings, the subunit structure of the enzyme is discussed.     

Direct cochlear nerve monitoring: first report on a new atraumatic, self-retaining electrode

Lyophilized dimeric recombinant bovine growth hormone (r-bGH) produced through incubation of r-bGH at 37 degrees C and 96% relative humidity for 8 days was examined by Raman spectroscopy. The secondary structure of the dimeric material is comparable to that of nonincubated r-bGH, due to the high similarity of the amide I, III, and V vibrational envelopes of the two samples. The dimeric material exhibits disulfide stretching that is indicative of the presence of only one disulfide bond (Cys53-Cys164). No sulfhydryl S-H stretching vibrations are observed, suggesting that cysteines from the cleaved disulfide bridge (Cys181-Cys189) are bound to nonsulfur atoms. Either high humidity (96%) or mild heat (37 degrees C) alone will cleave only one disulfide bond, but the final products are different. Incubation at ambient temperature and high humidity leads to a significant secondary structural change, while mild heat at very low humidity does not alter r-bGH secondary structure. Spectral data for incubations solely in mild heat are consistent with r-bGH structures that have lost the small loop (Cys181-Cys189) disulfide bridge, while incubations under only high humidity conditions are compatible with what would be expected if the large loop (Cys53-Cys164) cystine link was broken. Mild heat and high humidity are both present in dimer formation, yet only the small loop bridge is severed. The data suggest that heat may be the primary factor in determining which cystine link is broken. More severe heating (75 degrees C) cleaves both cystines and alters both secondary and tertiary structure.     

Solubilization of hydrophobic peptides by reversible cysteine PEGylation

"PEG-a-Cys" reagent, synthesized by the esterification of monomethoxy-poly(ethylene glycol) (avg. MW = 5 kDa) to Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid)], is shown to "PEGylate" reversibly the cysteine residue of a 25-residue synthetic hydrophobic peptide (H2N-REAAALAAAAALAAWAALCPARRRR-CO2H) designed to model a transmembrane segment of a membrane protein. A mixed disulfide bond was formed between the reagent and the peptide that was readily cleaved with the mild reducing agent tricarboxyethylphosphine hydrochloride (TCEP.HCl). Carboxypeptidase B digestion of the charged carboxyl terminus of the peptide through to the Ala residue--which mimics the enzymatic cleavage of a TM segment from a fusion protein--releases a highly hydrophobic peptide. A time-dependent decrease in the amplitude of the digested peptide circular dichroism (CD) spectra was attributed to the aggregation and/or precipitation of the peptide. While PEGylation of the peptide with PEG-a-Cys had a negligible effect on conformation, it inhibited the loss of CD amplitude in both intact and digested peptides, suggesting that it was effective in solubilization of hydrophobic peptides.     

Oxidative regeneration and selective reduction of native disulfide bonds in the N-terminal half-molecule of ovotransferrin

The denatured, disulfide-reduced form of the N-terminal half-molecule of ovotransferrin was reoxidized with either oxidized dithiothreitol or GSSG and analyzed for the localization of disulfide bonds. Chemical analyses of the reoxidized proteins revealed that the disulfide peptides corresponding to the six native protein disulfides (SS-I, SS-II, SS-III, SS-IV, SS-V, and SS-VI) are all regained in the reoxidized protein. The peptide recoveries from the reoxidized proteins were, however, about half of those from the native protein with respect to the two inner disulfides (SS-IV and SS-V) in the kringle bridges, but all the disulfide peptides corresponding to the remaining disulfides (SS-I, SS-II, SS-III, and SS-VI) were recovered at almost equivalent yields in the native and reoxidized proteins. In addition, on searching for a nonnative disulfide peptide, the two disulfides, Cys171-Cys174 and Cys174-Cys182, which can be accounted for by mispaired bridges of sulfhydryls in SS-IV and SS-V, were detected in the protein reoxidized with oxidized dithiothreitol. Upon disulfide reduction of the native protein with reduced dithiothreitol, both SS-IV and SS-V were selectively cleaved under the same buffer and temperature conditions as in the oxidative refolding. The lower stabilities of the two inner disulfide bonds in the kringle may be related to the lower recoveries of the disulfide peptides from SS-IV and SS-V and the generation of the nonnative disulfide bonds.     

Activation of the plant alternative oxidase by high reduction levels of the Q-pool and pyruvate

This report describes the activation of the alternative oxidase (AOX) of higher plant mitochondria by a high reduction level of the ubiquinone pool in the presence of pyruvate. In mitochondria from both thermogenic (Arum italicum spadices) and nonthermogenic (Glycine max cotyledons) tissues AOXis activated when the Q-pool becomes highly reduced in the presence of pyruvate. Pyruvate is essential for this activation. The enzyme is not activated when pyruvate is added after a transient high reduction level of the Q-pool, but is when pyruvate is added before the transient reduction. Pyruvate also protects the enzyme against inhibition during catalytic turnover. Although this activation is not accompanied by a reduction of the covalent disulfide bond, the same activation can be achieved with dithiothreitol (DTT). It is suggested that a part of the activation by DTT is not the result of reducing the covalent disulfide bond, and the relation between these types of activation is discussed. The importance of this activation for the in vivo regulation and its relation to previously reported activators is discussed. A mechanism is proposed in which it is suggested that AOX is inactivated by its product (oxidized ubiquinone) during catalysis and that this inhibition is prevented in the presence of pyruvate. The inhibition can be reversed by a reductive process, achieved by high levels of reduction of the Q-pool or by DTT, but not by pyruvate. This restoration of activity is not related to the redox process involved in reducing the covalent disulfide bond.     

Tau self-association: stabilization with a chemical cross-linker and modulation by phosphorylation and oxidation state

tau is a major component of paired helical filaments found in the neurofibrillary tangles of Alzheimer's diseased brain. However, the mechanism or mechanisms responsible for the association of tau to form these aggregates remains unknown. In this study, the role of intermolecular disulfide bonds in the formation of higher order oligomers of bovine tau and the human recombinant tau isoform T3 was examined using the chemical cross-linking agent disuccinimidylsuberate (DSS). In addition, the role of phosphorylation and oxidation state on the in vitro self-association of tau was studied using this experimental model. Stabilization of tau-tau interactions with DSS indicated that intermolecular disulfide bonds probably play a predominant role in dimer formation, but the formation of higher order oligomers of tau cannot be attributed to these bonds alone. tau-tau interactions were significantly decreased either by blocking Cys residues or by exposing the tau to a reducing (nitrogen and dithiothreitol), instead of an oxidizing, environment. tau self-association was also significantly decreased by prior phosphorylation with calcium/calmodulin-dependent protein kinase II. Phosphorylation by cyclic AMP-dependent protein kinase or dephosphorylation by alkaline phosphatase did not alter tau self-assembly. These data suggest a role for several factors that may modulate tau self-association in vivo.