Thiol-specific Reagents Research Articles

Probing the role of cysteine residues in glucosamine-1-phosphate acetyltransferase activity of the bifunctional GlmU protein from Escherichia coli: site-directed mutagenesis and characterization of the mutant enzymes.

The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme from Escherichia coli was lost when GlmU was stored in the absence of beta-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. The Km values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.

Activation of G protein-coupled receptors entails cysteine modulation of agonist binding

The increase in the affinity of agonists with an increase in pH and experiments using thiol-specific reagents indicate that G protein-coupled receptors contain an ionizable cysteine residue at the ligand binding site. Since treatment of receptors with reducing agents produces functional activation and potentiates agonist stimulation, it is likely that this free sulfhydryl modulates receptor activation. We have derived a two-state acid-base model for cysteine modulation of ligand binding which leads to a description of ligand efficacy. We have shown that pH-dependent binding of agonists is closely correlated with measurements of ligand efficacy at the 5-HT2A receptor. In general, efficacy is determined by the preference of a ligand for the base form of the receptor. Efficacy may also be described in thermodynamic terms as the coupling free energy involving a ligand and the acid and base states of the receptor. Molecular modeling of the third transmembrane domain of the 5-HT2A receptor, which contains a conserved cysteine, shows that efficacy is determined by the difference between the electrostatic interaction energies of a ligand with the acid and base forms of the receptor model. The difference in interaction energy between the two forms of cysteine makes the largest contribution to this electrostatic interaction energy difference. Therefore, the cysteine makes the largest contribution to ligand efficacy. Using this approach, we can distinguish between the efficacies of agonists with varying molecular structures and account for the difference between the properties of agonists and antagonists.

Activation of G protein-coupled receptors entails cysteine modulation of agonist binding

The increase in the affinity of agonists with an increase in pH and experiments using thiol-specific reagents indicate that G protein-coupled receptors contain an ionizable cysteine residue at the ligand binding site. Since treatment of receptors with reducing agents produces functional activation and potentiates agonist stimulation, it is likely that this free sulfhydryl modulates receptor activation. We have derived a two-state acid-base model for cysteine modulation of ligand binding which leads to a description of ligand efficacy. We have shown that pH-dependent binding of agonists is closely correlated with measurements of ligand efficacy at the 5-HT2A receptor. In general, efficacy is determined by the preference of a ligand for the base form of the receptor. Efficacy may also be described in thermodynamic terms as the coupling free energy involving a ligand and the acid and base states of the receptor. Molecular modeling of the third transmembrane domain of the 5-HT2A receptor, which contains a conserved cysteine, shows that efficacy is determined by the difference between the electrostatic interaction energies of a ligand with the acid and base forms of the receptor model. The difference in interaction energy between the two forms of cysteine makes the largest contribution to this electrostatic interaction energy difference. Therefore, the cysteine makes the largest contribution to ligand efficacy. Using this approach, we can distinguish between the efficacies of agonists with varying molecular structures and account for the difference between the properties of agonists and antagonists.

Reverse transcriptase of mouse mammary tumour virus: expression in bacteria, purification and biochemical characterization.

We have constructed a plasmid that induces in bacteria the synthesis of an enzymically active reverse transcriptase (RT) of mouse mammary tumour virus (MMTV), a retrovirus with a typical B-type morphology. The highest catalytic activity was detected only when 27 residues from the C-terminus of the protease were included in the N-terminus of the recombinant RT, after an extra deoxyadenosine was added between the pro and pol genes to overcome the -1 frameshift event (which occurs naturally in virus-infected cells). The recombinant protein with a six-histidine tag was purified to homogeneity by a two-column purification procedure, Ni2+ nitriloacetic acid/agarose followed by carboxymethyl-Sepharose chromatography. Unlike most RTs, the purified MMTV RT is enzymically active as a monomer even after binding a DNA substrate. Like all RTs studied, the recombinant MMTV RT possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity, all of which show a preference for Mg2+ over Mn2+ ions. Other features of these enzymic activities, such as extension of DNA primers, processivity of DNA synthesis, pH dependence, steady-state kinetic constants, effects of Na+ or K+ ions and sensitivity to a thiol-specific reagent and to a zinc chelator, have been evaluated. The catalytic properties of MMTV RT were compared with those of the well-studied RT of HIV-1, the causative agent of AIDS. Interestingly, MMTV RT exhibits a high sensitivity to nucleoside triphosphate analogues (which are known to be potent inhibitors of HIV RTs and are being used as the major anti-AIDS drugs), as high as that of HIV-1 and HIV-2 RTs. Furthermore the recombinant MMTV RT shows a processivity of DNA synthesis higher than that of HIV-1 RT.

Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: a prototype for the prolyl oligopeptidase family.

The proline iminopeptidase from Xanthomonas campestris pv. citri is a serine peptidase that catalyses the removal of N-terminal proline residues from peptides with high specificity. We have solved its three-dimensional structure by multiple isomorphous replacement and refined it to a crystallographic R-factor of 19.2% using X-ray data to 2.7 A resolution. The protein is folded into two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet flanked by two helices and the 11 N-terminal residues on one side, and by four helices on the other side. The smaller domain is placed on top of the larger domain and essentially consists of six helices. The active site, located at the end of a deep pocket at the interface between both domains, includes a catalytic triad of Ser110, Asp266 and His294. Cys269, located at the bottom of the active site very close to the catalytic triad, presumably accounts for the inhibition by thiol-specific reagents. The overall topology of this iminopeptidase is very similar to that of yeast serine carboxypeptidase. The striking secondary structure similarity to human lymphocytic prolyl oligopeptidase and dipeptidyl peptidase IV makes this proline iminopeptidase structure a suitable model for the three-dimensional structure of other peptidases of this family.

Carbonyl reductase purified from rabbit liver is not the product of a carbonyl reductase gene (RCBR5 or RCBR6) cloned from the rabbit liver cDNA library.

Six peptides were obtained by the digestion of carbonyl reductase purified from rabbit liver. The amino acid sequences of the six peptides were virtually identical to the corresponding regions in amino acid sequences deduced from two cloned carbonyl reductase genes (RCBR5 and RCBR6). However, there was a difference of one amino acid residue between the sequences of peptides from the purified enzyme and the corresponding region in the amino acid sequences deduced from the two cDNAs. The purified carbonyl reductase was confirmed to exhibit no reactivity towards menadione, even though the transient expression of the two cDNA for rabbit liver carbonyl reductase has been reported to cause a marked increase of menadione reductase activity in COS7 cells. The enzyme purified from rabbit liver was inactivated by thiol-specific reagents, 5,5'-dithiobis(2-nitrobenzoic acid) and sodium tetrathionate, suggesting that menadione probably interacts with the functional cysteine residue(s), and cannot serve as a substrate of the purified enzyme. Based on these results, it is concluded that the carbonyl reductase purified from rabbit liver is not the product of cloned carbonyl reductase gene (RCBR5 or RCBR6).

Avidin–FITC Topological Studies with Three Cysteine Mutants of Equinatoxin II, a Sea Anemone Pore-Forming Protein

Equinatoxin II (EqtII) is a cysteinless pore-forming protein from sea anemoneActinia equina.Three cysteine mutants were produced in anE. coliexpression system in order to study the topology of lysine 77, arginine 126, and alanine 179. Accessibility of an introduced thiol group in the water soluble mutants was studied by using the thiol specific reagent fluorescein maleimide. In aqueous solution all three mutants were readily modified with the probe, indicating their accessibility to the solvent. Mutants were also biotinylated with biotin maleimide, enabling coupling with avidin–fluorescein isothiocyanate (avidin-FITC). After binding and insertion of biotinylated toxins into liposomes, avidin-FITC, which is unable to enter intravesicular compartment through toxin-created pores, was used to discriminate intra- or extravesicularly located thiols. All the mutated residues are found to be located on the outside of the lipid vesicles. The results proved the biotin-avidin system as suitable for topological studies of proteins creating pores in membranes.

A Mercuric Ion Uptake Role for the Integral Inner Membrane Protein, MerC, Involved in Bacterial Mercuric Ion Resistance

Bacterial detoxification of mercuric ion depends on the presence of one or more integral membrane proteins (MerT and/or MerC) whose postulated function is in transport of Hg2+ from a periplasmic Hg2+-binding protein (MerP) to cytoplasmic mercuric reductase. In this study, MerC from the Tn21-encoded mer operon was overexpressed and studied in vesicles and in purified form to clarify the role played by this protein in mercuric ion resistance. MerC-containing vesicles were found to take up mercuric ion independently of MerP. Since uptake correlated with the level of MerC expression was unaffected by osmotic pressure, and was only partially decreased in the presence of 0.05% Triton X-100, the observed uptake appears to represent mainly binding to MerC. Binding was inhibited by thiol-specific reagents, consistent with an essential role for cysteine residues. The essential thiol groups were inaccessible to hydrophilic thiol reagents, whereas hydrophobic reagents completely abolished Hg2+ binding. These observations are consistent with the predicted topology of the protein, wherein all 4 cysteine residues are either in the cytoplasm or the bilayer. A role for MerC in Hg2+ transport is thus also likely. Based on these results, a modified model for bacterial Hg2+ transport is proposed.

The Na/Ca-K exchanger of rod photoreceptor exists as dimer in the plasma membrane.

The oligomeric state of the Na/Ca-K exchanger in the plasma membrane of bovine photoreceptors was investigated using chemical cross-linking techniques. In the natural membrane, virtually all Na/Ca-K exchanger could be cross-linked mainly to a complex having an apparent molecular mass of 490 kDa by cupric phenanthroline catalyzed disulfide bonding as evidenced by Western blotting. Stable cross-links of the exchanger were also obtained with the thiol-specific reagent N,N'-p-phenylidenedimaleimide. Neuraminidase treatment reduced the apparent molecular mass of the highly glycosylated Na/Ca-K exchanger and of the 490 kDa cross-link product by 50 and 85 kDa, respectively. DL-1,4-Bismaleimido-2,3-butanediol (BMBD), a novel cleavable dimaleimide, was synthesized in order to produce cross-links that were stable to reductive conditions. Purification of the BMBD cross-linked exchanger followed by two-dimensional SDS polyacrylamide electrophoresis identified the cross-linked homodimers of the exchanger. There was no indication of higher oligomers, suggesting that the exchanger exists as a dimer in the plasma membrane. Hydrodynamic properties of the detergent-solubilized exchanger were determined by velocity sedimentation and gel filtration chromatography. The Triton X-100-solubilized exchanger ran as a single species having a Stokes radius of 10.0 nm, a sedimentation coefficient of 5.4 S, and a partial specific volume of 0.74 mL/g in Triton X-100. Similar results were obtained for the CHAPS-solubilized exchanger. A molecular mass of 236 and 205 kDa was calculated for the exchanger-detergent complex and the detergent-free protein, respectively. Neuraminidase treatment further reduced the molecular mass of the exchanger indicating that glycosylation contributes significantly to the mass of the exchanger. Cross-links of the exchanger were not detected if cross-linking was attempted after solubilization in 10 mM CHAPS. However, after reconstitution of the purified exchanger into soybean phosphatidylcholine vesicles, chemical cross-linking yielded again dimers. On the basis of these cross-linking and hydrodynamic studies, we conclude that the exchanger exists as a homodimer in the rod outer segment plasma membrane but dissociates into a monomer when solubilized in detergent.

Localization of the labile disulfide bond between SU and TM of the murine leukemia virus envelope protein complex to a highly conserved CWLC motif in SU that resembles the active-site sequence of thiol-disulfide exchange enzymes.

Previous studies have indicated that the surface (SU) and transmembrane (TM) subunits of the envelope protein (Env) of murine leukemia viruses (MuLVs) are joined by a labile disulfide bond that can be stabilized by treatment of virions with thiol-specific reagents. In the present study this observation was extended to the Envs of additional classes of MuLV, and the cysteines of SU involved in this linkage were mapped by proteolytic fragmentation analyses to the CWLC sequence present at the beginning of the C-terminal domain of SU. This sequence is highly conserved across a broad range of distantly related retroviruses and resembles the CXXC motif present at the active site of thiol-disulfide exchange enzymes. A model is proposed in which rearrangements of the SU-TM intersubunit disulfide linkage, mediated by the CWLC sequence, play roles in the assembly and function of the Env complex.

Redox regulation of large conductance Ca(2+)-activated K+ channels in smooth muscle cells.

The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca(2+)-activated K+ (maxi-K) channels were examined in excised patches from tracheal myocytes. Channel activity was modified by sulfhydryl redox agents applied to the cytosolic surface, but not the extracellular surface, of membrane patches. Sulfhydryl reducing agents dithiothreitol, beta-mercaptoethanol, and GSH augmented, whereas sulfhydryl oxidizing agents diamide, thimerosal, and 2,2'-dithiodipyridine inhibited, channel activity in a concentration-dependent manner. Channel stimulation by reduction and inhibition by oxidation persisted following washout of the compounds, but the effects of reduction were reversed by subsequent oxidation, and vice versa. The thiol-specific reagents N-ethylmaleimide and (2-aminoethyl)methanethiosulfonate inhibited channel activity and prevented the effect of subsequent sulfhydryl oxidation. Measurements of macroscopic currents in inside-out patches indicate that reduction only shifted the voltage/nP0 relationship without an effect on the maximum conductance of the patch, suggesting that the increase in nP0 following reduction did not result from recruitment of more functional channels but rather from changes of channel gating. We conclude that redox modulation of cysteine thiol groups, which probably involves thiol/disulfide exchange, alters maxi-K channel gating, and that this modulation likely affects channel activity under physiological conditions.

Characterization of recombinant yeast dolichyl mannosyl phosphate synthase and site-directed mutagenesis of its cysteine residues.

Dolichyl mannosyl phosphate synthase is associated with membranes of the rough endoplasmic reticulum and catalyzes mannosyl transfer from GDP-mannose to the hydrophobic long-chain acceptor dolichyl-phosphate. The gene for the yeast enzyme encodes a protein with a molecular mass of 30.36 kDa containing three cysteine residues, at positions 93, 172 and 259 [Orlean, P., Albright, C. & Robbins, P. W. (1988) J. Biol. Chem. 263, 17499-17507]. Inhibition of the synthase by thiol-specific reagents, including N-ethylmaleimide, p-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2), and lucifer yellow iodoacetamide (LYI), suggests that sulfhydryl groups might play a role in the catalytic mechanism of the enzyme. Titration of the synthase with Nbs2 or LYI indicated that 1 mol sulfhydryl/mol protein was accessible to these reagents, and that saturation of this site completely inhibited enzyme activity. To ascertain the reactive group and its possible function in enzyme catalysis, each of the cysteine residues was replaced individually by site-directed mutagenesis. The mutant enzymes had specific activities comparable to that of the wild-type enzyme, demonstrating that none of the cysteine residues were essential for catalytic activity. All of the mutant proteins except those containing a substitution at Cys93 were inhibited by thiol-blocking reagents, indicating that Cys93 might be physically located near the catalytic site of the enzyme. GDP-mannose, dolichyl phosphate and substrate analogs were found to protect against Nbs2 inactivation, further suggesting that Cys93 was physically near, or within, the substrate-binding site of the enzyme.

Site-directed mutagenesis of the cysteine residues in the Pichia stipitis xylose reductase

Xylose reductase catalyzes the reduction of xylose to xylitol and is known to play a pivotal role in pentose metabolism in yeasts. We previously showed that a cysteine residue may be involved in binding of the coenzyme NADPH to the Pichia stipitis xylose reductase through chemical modification studies. The question arose as to which of the three cysteine residues in this enzyme may be involved in coenzyme binding. We cloned the XYL1 gene encoding xylose reductase from P. stipitis into the phagemid pEMBL18(+) suitable for site-directed mutagenesis. Each of the three cysteine residues (Cys 19, Cys 27 and Cys 130) was individually mutated to serine. All three Cys→Ser variants remained functional, but with reduced catalytic activity. Sensitivity of the P. stipitis xylose reductase to thiol-specific reagents was attributed to both Cys 27 and Cys 130 residues as substitution of either residue with Ser resulted in a significant but incomplete loss of sensitivity to PCMBS. The apparent K m values of the Cys variants for NADPH, NADH and xylose did not differ from those of the wild-type enzyme isolated from yeast by more than 4-fold. Our results suggest that none of the Cys residues are directly involved in NADPH binding, although Cys 130 may reside in or near the coenzyme binding region and might play a role in coenzyme specificity.

Purification and characterization of two components of epoxypropane isomerase/carboxylase from Xanthobacter Py2.

Epoxypropane isomerase from Xanthobacter Py2 has been resolved into at least two components (A and B) by ion-exchange chromatography. Both components were required for the degradation of epoxypropane and were purified further. Component A was apparently homohexameric with a subunit M(r) of about 44,000, and possessed NAD(+)-dependent dihydrolipoamide dehydrogenase activity and lipoamide reductase activity. It was sensitive to inhibition by o-phenanthroline and the thiol-specific reagents N-ethylmaleimide(NEM)and p-chloromercuribenzoate. Component B was homodimeric with a subunit M(r), of 62,170 and contained 2 mol.mol-1 FAD. It had an NADPH-dependent lipoamide reductase activity which was sensitive to NEM and p-chloromercuribenzoate. The N-terminal amino acid sequences and monomer sizes of components A and B correspond to those of ORF1 and ORF3 respectively (ORF = open reading frame) of a recently published sequence of a clone which complements mutants unable to degrade epoxypropane. NADPH was found to replace the need for a low-M(r), fraction in epoxypropane degradation assays containing components A and B and NAD+. The predicted amino acid sequence of component A (ORF1) has been analysed and shown to contain a potential ADP binding site near the N-terminus and putative cofactor binding domain near the C-terminus, with sequence similarity to the biotinyl and lipoyl binding domains of biotin-dependent carboxylases and 2-oxoacid dehydrogenases respectively. A reaction mechanism for epoxypropane degradation, incorporating recent evidence for combined isomerization and carboxylation to acetoacetate, is discussed.

Recombinant Anti-Human Melanoma Antibodies Are Versatile Molecules

The low cost, high versatility, and reliable production of bacterially produced recombinant antibody fragments speeds up the development of tumor-targeting agents. High-quality recombinant anti-melanoma antibodies are much sought after in the scientific community. We cloned the murine antibody 225.28S, currently used in radioimmunoimaging of human melanoma lesions, in single-chain Fv configuration (scFv) for soluble expression in bacteria. The recombinant antibody fragment conserved the binding specificity of the parental antibody. In order to arm the scFv(225.28S) with biologically useful effector functions, we developed vectors for soluble expression of scFv(225.28S) in bacteria that allow both covalent and noncovalent chemical antibody modification at positions that do not interfere with antigen binding. An expression vector was developed that appends a cysteine residue at the C-terminal extremity of the recombinant antibody, thus allowing reaction with thiol-specific reagents, including 99mTc labeling, at a position that does not interfere with antigen binding. The scFv(225.28S) was also successfully expressed with a casein kinase II substrate tag that enables efficient and stable 32P labeling. For noncovalent antibody modification, we developed an expression vector that appends the human calmodulin gene at the C-terminal extremity of scFv(225.28S). The calmodulin domain is poorly immunogenic and can be targeted with chemically modified high-affinity calmodulin ligands. The recombinant anti-human melanoma antibodies described in this article should prove useful "building blocks" for the development of anti-melanoma diagnostic and therapeutic strategies.

Redox modifications of spinach chloroplast fructose-1, 6-bisphosphatase.

Complete activation of chloroplast fructose-1, 6-bisphosphatase by dithiothreitol involves the reduction of its four disulfide bonds as revealed by thiol titration and activity measurement. Both before and after reduction, the enzyme is inhibited by the thiol-specific reagent 5,5'-dithiobis(2-nitro-benzoic acid) with complete inactivation upon modifications of the four accessible thiols. However, oxidative modification of the enzyme facillitates the reduction of the four mentioned disulfide bonds as the process of activation by DTT is accelerated.

Use of the Thiol-specific Derivatizing Agent N-Iodoacetyl-3-Iiodotyrosine to Demonstrate Conformational Differences between the Unbound and hsp90-bound Glucocorticoid Receptor Hormone Binding Domain

The hormone binding domain (HBD) of the glucocorticoid receptor (GR) contains five cysteine residues, with three of them being spaced close to one another in the steroid binding pocket. The HBD also contains the contact region for the chaperone protein hsp90, which must be bound to the GR for it to have a steroid binding conformation. Binding of hsp90 to the receptor through its HBD inactivates the DNA binding domain (DBD). The DBD contains a number of cysteines essential to its DNA binding activity. Here, we assess the effects of hsp90 binding on the accessibility of cysteine residues in both the HBD and DBD to derivatization by a thiol-specific reagent. We report that N-iodoacetyltyrosine (IAT) inactivates steroid binding activity of the immunopurified, untransformed GR.hsp90 complex in a manner that is prevented by the sulfhydryl reagents cysteine and dithiothreitol but is not reversed by them. The 125I-labeled IAT derivative N-iodoacetyl-3-[125I]iodotyrosine ([125I]IAIT) covalently labels the immunopurified, hsp90-bound receptor in a thiol-specific manner. Dissociation of hsp90 leads to an approximately 2-fold increase in [125I]IAIT labeling of the full-length, 100-kDa GR. The increase in thiol labeling is related to the presence of hsp90 because it is blocked by molybdate, which prevents hsp90 dissociation. Cleavage of the [125I]IAIT-labeled receptor with trypsin yields a 15-kDa labeled fragment containing the DBD and a 30-kDa labeled fragment containing all of the cysteines in the HBD and the contact region for hsp90. Dissociation of hsp90 from the GR results in a 2.3-fold increase in [125I]IAIT labeling of the 15-kDa fragment and a 50% decrease in labeling of the 30-kDa fragment. These data are consistent with the proposal that dissociation of hsp90 from the GR produces a conformational change in the HBD such that some of the thiols that are exposed in the GR*hsp90 complex become buried and are no longer accessible to the [125I]IAIT probe. In contrast, binding of the GR to hsp90 restricts access of cysteines in the DBD to this small thiol-derivatizing agent, a restriction that is relieved as a result of unmasking or conformational change accompanying hsp90 dissociation.

Malonate decarboxylase of Klebsiella pneumoniae catalyses the turnover of acetyl and malonyl thioester residues on a coenzyme-A-like prosthetic group.

During aerobic growth of Klebsiella pneumoniae on malonate, a soluble malonate decarboxylase is induced. Malonate decarboxylation consumes a proton (not H2O) and forms acetate and CO2 (not HCO3-) as products. The enzyme was purified 56-fold to apparent homogeneity. It has a native molecular mass of 142 kDa and consists of four subunits alpha, beta, gamma and delta with molecular masses of 65, 34, 30, and 12 kDa, respectively. Two different forms of the enzyme were recognised: a catalytically inactive SH-enzyme and the catalytically active acetyl-S-enzyme which is formed by post-translational acetylation of the SH-enzyme with ATP, acetate and a specific ligase. The acetyl-S-enzyme was converted into the SH-enzyme by incubation with hydroxylamine or dithioerythritol. Chemical reacylation of the SH-enzyme, which restores catalytic activity, was achieved with acetic anhydride or more efficiently with malonyl-CoA. This acylation of the SH group was prevented after incubation with various thiol-specific reagents. After incubation of the SH-enzyme with iodo[1-14C]acetate, the delta subunit became specifically labelled. This subunit was also labelled after incubation of the acetyl-S-enzyme with [2-14C]malonate. The radioactivity was completely liberated from the protein upon malonate addition. These results indicate that the delta subunit is the acyl-carrier protein of the complex and that malonate decarboxylation proceeds in two steps: the acetyl residue on the ACP is first replaced by a malonyl residue which subsequently undergoes decarboxylation thereby regenerating the acetyl-S-ACP. The binding site for the acyl residues on the acyl-carrier protein was shown to be 2'-(5"-phosphoribosyl)-3'-dephospho-CoA after alkaline cleavage of this prosthetic group from the enzyme and chromatographic as well as mass spectroscopic analyses.

GLUTATHIONE AND PROTEIN THIOL STATUS IN RENAL INJURY BY AMPHOTERICIN B (AMPH) IN RATS. † 2133

The mechanisms responsible for AMPH-induced renal injury are not known. Because oxidant stresses appear to cause or exacerbate other examples of renal injury, we investigated oxidative mechanisms in AMPH nephrotoxicity. Fischer-344 (F344) rats are more suceptible to oxidant-induced injury than are Sprague-Dawley rats, and we found F344 rats to be slightly more sensitive to AMPH nephrotoxicity. Female F344 rats given single doses of 20 mg/kg of AMPH, i.p., developed elevated plasma BUN and Cr concentrations (59.7±0.6 and 1.03±0.05 mg/dl, respectively; vehicle-treated controls, 13.8±0.7 and 0.46±0.03) that peaked by 48 h, declining to baseline values by day 6 post dose. Tubular injury and progress through initial stages of tissue repair were observed by histology. Plasma and tissue levels of AMPH were measured by HPLC. Plasma concentrations showed an initial decline, with an apparent half-life of 23 h, through day 4, but no further decrease was observed through day 7 (peak 0.27; days 4-7 0.03 μg/ml). The concentrations of AMPH in renal and hepatic tissues were not closely correlated with plasma levels or tissue injury. Despite a 10-fold greater concentration of AMPH in liver (range of days 1-5: 16.9-59.4 μg/g tissue) than in kidney (1.11-3.53), plasma ALT activities did not increase. Biochemical and histologic evidence of recovery of renal injury was observed, even in animals with persistently high renal concentrations of AMPH. The redox status of renal proteins was assessed by fluorescence labeling with the thiol-specific reagent monobromobimane, followed by separation by SDS-PAGE. No substantive changes in total or in any individual protein thiol was observed, even in animals that showed marked renal damage. Further, biliary efflux of total glutathione did not increase as would be expected during an oxidant stress. Conclusions: 1) Hepatic vs renal injury do not correlate with tissue AMPH concentrations; 2) Recovery of renal function is observed despite the continued presence of AMPH; 4) Protein thiol status and biliary glutathione efflux do not support oxidant stress as the primary mechanism of AMPH nephrotoxicity.

Single-cell investigation by laser scanning confocal microscopy of cytochemical alterations resulting from extracellular oxidant challenge

Confocal laser scanning fluorescence microscopy coupled to image analysis was employed in order to develop and evaluate procedures for the appraisal at the single-cell level of: (1) protein-bound 4-hydroxynonenal, the specific product of membrane peroxidation (by means of immunocytochemistry with biotin-avidin revelation); (2) protein oxidation (by reaction of protein carbonyls with 2, 4-dinitrophenyl-hydrazine followed by immunocytochemistry of dinitrophenyl moieties); and (3) cellular protein thiols (by direct alkylation of sulfhydryl groups with thiol-specific fluorescent reagents possessing different cell permeabilities). The procedures proved able to reveal the subcellular distribution of cytochemical parameters useful as indices of oxidative stress conditions, and may allow "redox phenotyping" of isolated cells, which would provide an efficient tool in selected experimental models.