The S-sulfate formation from 4-nitrobenzyl mercaptan in rat liver cytosol

4-Nitrobenzyl mercaptan (NBM) was enzymatically transformed at pH 6.0 into its S-sulfate in rat liver cytosol fortified with 3'-phosphoadenosine 5'-phosphosulfate. At pH 7.4, the S-sulfate was not detected from the incubation mixture. 4-Nitrobenzyl alcohol was also transformed under the same incubation conditions into the corresponding O-sulfate at a higher rate at pH 6.0 than at pH 7.4. Under the incubation conditions used, NBM S-sulfate reacted with the substrate NBM at a significant rate to afford 4-nitrobenzyl disulfide. The disulfide formation from NBM and the S-sulfate occurred more readily at pH 7.4 than at pH 6.0, so that biologically formed NBM S-sulfate was strongly suggested not to remain unchanged in the incubation mixture at pH 7.4.     

Covalent binding of a mercaptan S-sulfate to hepatic cytosolic proteins and its inhibition by glutathione

4-Nitrobenzyl mercaptan (NBM) S-sulfate, a new type of the sulfate conjugate enzymatically formed from NBM in the presence of 3'-phosphoadenosine 5'-phosphosulfate in rat liver cytosol, bound covalently to rat liver cytosolic proteins at pH 7.4. The protein binding of NBM S-sulfate was strongly retarded by GSH. GSH not only played a role as a scavenger for NBM S-sulfate with formation of NBM and GSSG via S-(4-nitrobenzyl)thioglutathione, but also cleaved the covalent bonds, possibly disulfides formed from NBM S-sulfate and sulfhydryl groups of the cytosolic proteins. Thus, evidence was provided that NBM S-sulfate be a new type of the reactive metabolite.     

Labeling of proteins with [35S]methionine and/or [35S]cysteine in the absence of cells

Incubation of [35S]methionine and [35S]cysteine with bovine albumin, globulin, catalase, hemoglobin, or human globulin resulted in incorporation of the 35S label into each of these proteins. Trichloroacetic acid (TCA) precipitation revealed that the percentage of label incorporated ranged from 1 to 15%. The 35S labeling was resistant to dissociation by reducing SDS-PAGE, prolonged dialysis against 4 M urea, heating, TCA precipitation, and dilution by gel filtration. The labeling effect was more efficient with [35S]cysteine than [35S]methionine. Incubation of 35S label with proteins differing in methionine and cysteine content revealed no requirement for sulfur-containing amino acids in the target protein. Protein carboxymethylation reduced but did not prevent 35S label incorporation. Amino acid analysis of labeled proteins revealed that the radioactive label was not consistently associated with an individual amino acid. Differences in the ability of various proteins to spontaneously label with these amino acids suggest caution in the interpretation of metabolic labeling experiments and the necessity for inclusion of additional controls. Alternatively, our experience indicates a potentially useful method for labeling proteins in the absence of cells.     

Binding site of cerulenin in fatty acid synthetase

An antibiotic cerulenin, (2R, 3S)-2,3-epoxy-4-oxo-7,10-trans,trans- dodecadienamide, irreversibly inhibits fatty acid synthetase from Saccharomyces cerevisiae. Three moles of cerulenin were bound to 1 mol of the enzyme with concomitant loss of its activity. Pretreatment of the enzyme with iodoacetamide reduced the amount of cerulenin bound to the enzyme. Since iodoacetamide is known to specifically bind to the cysteine residue on the condensing reaction domain, cerulenin is considered to bind to the same domain. Tryptic digestion of the [3H] cerulenin-treated enzyme gave a radioactive peptide; its amino acid composition was Asx 1, Thr 1, Ser 1, Glx 2, Pro 1, Gly 1, Ala 1, Val 1, Ile 1, and Leu 2. This composition included all the amino acids of the condensing reaction site (Thr-Pro-Val-Gly-Ala-Cys) previously reported by Kresze et al. (Eur. J. Biochem., 79, 181 [1977] except for Cys. When the enzyme was treated with [3H]cerulenin and digested successively with trypsin and carboxypeptidase P, a [3H] cerulenin-cysteine adduct was isolated as the sole product. This was identified with the adduct chemically synthesized from non-labeled cerulenin and cysteine, and its structure was elucidated by 1H-, 13C-NMR, and fast atom bombardment mass spectrometry. These results indicate that cerulenin, forming a hydroxylactam ring, reacts at its epoxide carbon (C-2 position) with the SH-group of the cysteine residue in the condensing reaction domain of yeast fatty acid synthetase.     

Structural characterization of the major covalent adduct formed in vitro between acetaminophen and bovine serum albumin

The structure of the covalent adduct formed in vitro between [14C]-acetaminophen ([14C]APAP) and bovine serum albumin (BSA) has been investigated with the aid of new analytical methodology. The APAP-BSA adduct, isolated from mouse liver microsomal incubations to which the radiolabeled drug and BSA had been added, was cleaved using a combination of specific (cyanogen bromide) and non-specific (acid hydrolysis) procedures, following which the mixture of amino acids obtained was derivatized, in aqueous solution, with ethyl chloroformate. The resulting ethoxycarbonyl derivatives were recovered by extraction into ethylacetate, methylated and subjected to profile analysis using both reverse-phase and normal-phase HPLC techniques. In each HPLC step, one major radioactive amino acid adduct was detected and was identified by mass spectrometry as the derivative of 3-cystein-S-yl-4-hydroxyaniline. Based on this finding, and with a knowledge of the behavior under acidic hydrolysis conditions of the 3-cysteinyl conjugate of APAP, it could be concluded that the major APAP-BSA adduct is one in which the drug is bound, via a thioether linkage at the C-3 position, to a sulfhydryl group on the protein. Furthermore, it could be established that this -SH function almost certainly is that associated with the cys-34 residue of BSA.     

Mechanism of the irreversible inactivation of mouse ornithine decarboxylase by alpha-difluoromethylornithine. Characterization of sequences at the inhibitor and coenzyme binding sites.

Mouse ornithine decarboxylase (ODC) was expressed in Escherichia coli and the purified recombinant enzyme used for determination of the binding site for pyridoxal 5'-phosphate and of the residues modified in the inactivation of the enzyme by the enzyme-activated irreversible inhibitor, alpha-difluoromethylornithine (DFMO). The pyridoxal 5'-phosphate binding lysine in mouse ODC was identified as lysine 69 of the mouse sequence by reduction of the purified holoenzyme form with NaB[3H]4 followed by digestion of the carboxymethylated protein with endoproteinase Lys-C, radioactive peptide mapping using reversed-phase high pressure liquid chromatography and gas-phase peptide sequencing. This lysine is contained in the sequence PFYAVKC, which is found in all known ODCs from eukaryotes. The preceding amino acids do not conform to the consensus sequence of SXHK, which contains the pyridoxal 5'-phosphate binding lysine in a number of other decarboxylases including ODCs from E. coli. Using a similar procedure to analyze ODC labeled by reaction with [5-14C]DFMO, it was found that lysine 69 and cysteine 360 formed covalent adducts with the inhibitor. Cysteine 360, which was the major adduct accounting for about 90% of the total labeling, is contained within the sequence -WGPTCDGL(I)D-, which is present in all known eukaryote ODCs. These results provide strong evidence that these two peptides form essential parts of the catalytic site of ODC. Analysis by fast atom bombardment-mass spectrometry of tryptic peptides containing the DFMO-cysteine adduct indicated that the adduct formed in the enzyme was probably the cyclic imine S-(2-(1-pyrroline)methyl)cysteine. This is readily oxidized to S-((2-pyrrole)methyl)cysteine or converted to S-((2-pyrrolidine)methyl)cysteine by NaBH4 reduction. This adduct is consistent with spectral evidence showing that inactivation of the enzyme with DFMO does not entail the formation of a stable adduct between the pyridoxal 5'-phosphate, the enzyme, and the inhibitor.     

Formation of S-[2-(N7-guanyl)ethyl] adducts by the postulated S-(2-chloroethyl)cysteinyl and S-(2-chloroethyl)glutathionyl conjugates of 1,2-dichloroethane

The formation of S-[2-(N7-guanyl)ethyl]glutathione (GEG) from dihaloethanes is postulated to occur through two intermediates: the S-(2-haloethyl)glutathione conjugate and the corresponding episulfonium ion. We report the formation of GEG when deoxyguanosine (dG) was incubated with chemically synthesized S-(2-chloroethyl)glutathione (CEG). The depurination of GEG was shown to be first order with a half-life of 7.4 +/- 0.4 h at 27 degrees C. Evidence is also presented for the formation of S-[2-(N7-guanyl)ethyl]-L-cysteine (GEC) in incubation mixtures containing dG and S-(2-chloroethyl)-L-cysteine (CEC), the corresponding cysteine conjugate of CEG. This finding demonstrates that this (haloethyl)cysteine conjugate does not require activation by enzymatic action of cysteine conjugate beta-lyase but, instead, can directly alkylate DNA. The half-life of the depurination of GEC was 6.5 +/- 0.9 h, which is no different from that of GEG. Of the two conjugates, CEC is a somewhat more active alkylating agent toward dG than CEG as N7-guanylic adduct was detected in reaction mixtures with lower concentrations of CEC than with CEG.     

Unique characteristics of rat spleen lymphocyte, L1210 lymphoma and HeLa cells in glutathione biosynthesis from sulfur-containing amino acids

Suspensions of rat spleen lymphocyte, murine L1210 lymphoma and HeLa cells were partially depleted of glutathione (GSH) with diethyl maleate and allowed to utilize either [35S]methionine, [35S]cystine or [35S]-cysteine for GSH synthesis. Lymphocytes preferentially utilized cysteine, compared to cystine, at a ratio of about 30 to 1, which was not related to differences in the extent of amino acid uptake. Only HeLa cells displayed a slight utilization of methionine via the cystathionine pathway for cysteine and GSH biosynthesis. HeLa and L1210 cells readily utilized either cystine or cysteine for GSH synthesis. The three cell types accumulated detectable levels of intracellular cysteine glutathione mixed disulfide when incubated in a medium containing a high concentration of cystine. Various enzyme activities were measured including gamma-glutamyl transpeptidase, GSH S-transferase and gamma-cystathionase. These results support the concept of a dynamic interorgan relationship of GSH to plasma cyst(e)ine that may have importance for growth of various cell types in vivo.     

Intracellular folding pathway of human chorionic gonadotropin beta subunit.

Few experimental models have been used to investigate how proteins fold inside a cell. Using the formation of disulfide bonds as an index of conformational changes during protein folding, we have developed a unique system to determine the intracellular folding pathway of the beta subunit of human chorionic gonadotropin (hCG). Three folding intermediates of the beta subunit were purified from [35S]cysteine-labeled JAR choriocarcinoma cells by immunoprecipitation and by reverse-phase high performance liquid chromatography (HPLC). To identify unformed disulfide bonds, nonreduced folding intermediates were treated with trypsin to liberate non-disulfide-bound, [35S]cysteine-containing peptides from the disulfide-linked peptides. Released peptides were purified by HPLC and identified by amino acid sequencing. The amount of a peptide that was released indicated the extent of disulfide bond formation involving the cysteine in that peptide. Of the six disulfide bonds in hCG-beta, bonds 34-88 and 38-57 form first. The rate-limiting event of folding involves the formation of the S-S bonds between cysteines 23 and 72 and cysteines 9 and 90. Disulfide bond 93-100, the formation of which appears to be necessary for assembly with the alpha subunit of the hCG heterodimer, forms next. Finally, disulfide bond 26-110 forms after assembly with the alpha subunit, suggesting that completion of folding of the COOH terminus in the beta subunit occurs after assembly with the alpha subunit.     

S-(N-methylcarbamoyl)glutathione: a reactive S-linked metabolite of methyl isocyanate

S-(N-methylcarbamoyl)glutathione, a chemically-reactive glutathione conjugate, has been isolated from the bile of rats administered methyl isocyanate and characterized, as its N-benzyloxycarbonyl dimethylester derivative, by tandem mass spectrometry. The ability of this glutathione adduct to donate an N-methylcarbamoyl moiety to the free -SH group of cysteine was evaluated in vitro with the aid of a highly specific thermospray LC/MS assay procedure. The glutathione adduct reacted readily with cysteine in buffered aqueous media (pH 7.4, 37 degrees C) and after 2 hr, 42.5% of the substrate existed in the form of S-(N-methylcarbamoyl)cysteine. The reverse reaction, i.e. between the cysteine adduct and free glutathione, also took place readily under these conditions. It is concluded that conjugation of methyl isocyanate with glutathione in vivo affords a reactive S-linked product which displays the potential to carbamoylate nucleophilic amino acids. The various systemic toxicities associated with exposure of animals or humans to methyl isocyanate could therefore be due to release of the isocyanate from its glutathione conjugate, which thus may serve as a vehicle for the transport of methyl isocyanate in vivo.     

Structure of the cytochrome c oxidase complex: labeling by hydrophilic and hydrophobic protein modifying reagents

Beef heart cytochrome c oxidase has been reacted with [35S]diazobenzenesulfonate ([35S]DABS), [35S]-N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate ([35S]NAP-taurine), and two different radioactive arylazidophospholipids. The labeling of the seven different subunits of the enzyme with these protein modifying reagents has been examined. DABS, a water-soluble, lipid-insoluble reagent, reacted with subunits II, III, IV, V, and VII but labeled I or VI only poorly. The arylazidophospholipids, probes for the bilayer-intercalated portion of cytochrome c oxidase, labeled I, III, and VII heavily and II and IV lightly but did not react with V or VI. NAP-taurine labeled all of the subunits of cytochrome c oxidase. Evidence is presented that this latter reagent reacts with the enzyme from outside the bilayer, and the pattern of labeling with the different hydrophilic and hydrophobic labeling reagents is used to derive a model for the arrangement of subunits in cytochrome c oxidase.     

Subunit structure and amino acid composition of xylose isomerase from Streptomyces albus.

The subunit structure and amino acid composition of xylose isomerase from Streptomyces albus have been examined. A native molecular weight of 165,000 determined by sedimentation equilibrium was reduced to 43,000 when the protein was treated with 6 M guanidine hydrochloride. No further reduction in molecular weight was observed when potential disulfide bridges of xylose isomerase were reduced and alkylated, indicating that the protein was devoid of interchain disulfide bonds. NH2-terminal analysis using [3H]dansyl chloride showed 0.86 residues of methionine per Mr equals 41,500 unit. Analysis of the native protein with an automated protein sequenator revealed the presence of only one degradable polypeptide chain. Fractionation of the soluble tryptic peptides of S-[14C]carboxymethyl xylose isomerase by ion exchange chromatography and one-dimensional paper electrophoresis yielded 37 to 43 peptides. When the acid-insoluble tryptic peptides were dissolved and analyzed using gel filtration techniques, and additional four peptides were found. A unique radioactive tryptic peptide containing S-carboxymethylcysteine was found among the soluble peptides, confirming cysteine as the limiting amino acid residue in the amino acid composition of xylose isomerase. On the basis of its lysine and arginine content, the number of tryptic peptides is consistent with the hypothesis that the native xylose isomerase is a tetramer of four very similar or identical subunits of Mr equals 41,500, associated by noncovalent bonds.     

Cysteine and methionine-precursors of methyl sulphone metabolites of 2,4',5-trichlorobiphenyl in mice

In order to account for the origin of the sulphur atom in methyl sulphones derived from polychlorinated biphenyls (PCB), groups of mice were treated with 2,4'-5-trichlorobiphenyl and [35S]cysteine or [35S]methionine, respectively. Control animals received the labelled amino acids only. The radioactive substances extracted from lung tissue were characterized by partition between hexane and sulphuric acid, thin-layer radiochromatography (TLRC), gas chromatography (GC) and mass fragmentography (MF). In lung extracts of both experimental groups sulphuric acid soluble metabolites were present: Their Rf-values on TLRC were identifical with that of 4-methylsulphonyl-2,4'-5-trichlorobiphenyl and their identity was confirmed by GC and GC-MF, which also indicated the presence of a minor sulphone isomer. The study shows that both cysteine and methionine could function as donors of the sulphur atom in the methyl sulphone metabolites derived from PCB.     

Non-enzymatic glutathione conjugation of 2-nitroso-6-methyldipyrido [1,2-a: 3',2'-d] imidazole (NO-Glu-P-1) in vitro: N-hydroxy-sulfonamide, a new binding form of arylnitroso compounds and thiols

In order to study the possible detoxification mechanisms of the carcinogenic arylamine, 2-amino-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (Glu-P-1), the in vitro non-enzymatic reaction of 2-nitroso-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (NO-Glu-P-1) with reduced glutathione (GSH) was examined at pH 7.4 under both aerobic and anaerobic conditions. Two GSH-arylamine adducts were isolated and found to contain the Glu-P-1 and GSH moieties in a 1:1 molar ratio via an N-S linkage. Their structures were assigned as sulfinamide (-NH-SO-) and N-hydroxy-sulfonamide (-N(OH)-SO2-) by their behaviour under acidic and basic conditions and by UV-VIS, 1H-NMR, infrared and mass spectrometries. Also, a N-hydroxy-sulfonamide adduct was produced when NO-Glu-P-1 and cysteine were reacted at pH 7.4. The N-hydroxy-sulfonamide structure is a new binding form between arylnitroso compounds and thiols. The formation of these adducts may also take place in vivo as a detoxification of toxic arylamines since GSH is abundant in organs such as liver or kidney.     

The affinity labeling of amino acids in or about the active center of DNA-dependent DNA polymerase I.

The use of an affinity label and an inhibitor that shows relative specificity for one amino acid has led to the identification of two amino acid residues in or near the active center of DNA-dependent DNA polymerase I. [35S]-beta-D-Ribosyl-6-methylthiopurine periodate oxidation product ([35S]MMPR-OP) and [14C]phenylglyoxal ([14C]PG) were used to elucidate the presence of a single lysine and arginine in or about the active center of the enzyme.     

Changes in the ribonucleic acid metabolism of aging mouse tissues with particular reference to the prostate gland

1. Mouse mast-cell tumours P815 Y and HC were shown to contain glycoprotein material composed of glucosamine, galactosamine, sialic acid, galactose and mannose. 2. The major amino acids released after acid hydrolysis of Pronase-treated digests of the glycoprotein are aspartic acid, glutamic acid, serine, threonine, proline, glycine and alanine. The Pronase-digested material is not degraded in alkaline solution. 3. On incubation of mast cells with [(35)S]sulphate, heparin is the major radioactive product. However, [1-(14)C]glucosamine and d-[(14)C]glucose are incorporated largely into the glycoprotein. 4. The fate of [(35)S]sulphate-labelled and [1-(14)C]glucosamine-labelled material was studied. In each case high-molecular-weight radioactive material is released from the cells into the culture medium. The t((1/2)) of [(35)S]sulphate-labelled material in cells is 70hr. and that of [1-(14)C]-glucosamine-labelled material in cells is 40hr. 5. About 60% of the [(35)S]sulphate-labelled material is present in the mitochondrial and granular fraction. [1-(14)C]-Glucosamine-labelled material is present in both microsomal and mitochondrial and granular fractions, [(14)C]sialic acid being concentrated in the microsomal fraction.     

Comparison of Cysteine and Tryptophan Content of Insoluble Proteins Derived from Wild-Type and mi-1 Strains of Neurospora crassa

The possibility of an amino acid substitution (cysteine for tryptophan) in a membrane protein of the [mi-1] strain of Neurospora crassa has been investigated in detail by using a double radioactive labeling procedure. Auxotrophic strains of Neurospora having wild-type [+] or [mi-1] cytoplasm have been grown under conditions which result in the specific labeling of protein tryptophan with (3)H and protein cysteine with (35)S. Although the least soluble 1 to 20% of the [mi-1] mitochondrial membrane protein was usually found to have a higher Cys/Trp ratio (ratio of cysteine plus half-cystine to tryptophan) than the corresponding [+] fraction, it has been shown that these differences were due mainly to the presence of differential amounts of a very insoluble, cysteine-rich (Cys-rich) material. The same Cys-rich material was found in variable amounts in both [+] and [mi-1] cultures, but the concentration was usually higher in the [mi-1] cultures. The Cys-rich material is clearly distinct from "structural protein" on the basis of amino acid composition and appears to have no direct relationship to the [mi-1] phenotype. In the absence of the Cys-rich material, no difference between the Cys/Trp ratios of corresponding [+] and [mi-1] membrane proteins could be detected. We conclude, therefore, that the previously postulated amino acid substitution of cysteine for tryptophan in [mi-1] membrane protein is incorrect.     

Assembly of functional rhodopsin requires a disulfide bond between cysteine residues 110 and 187

Cysteine residues 110 and 187 are essential for the formation of the correct bovine rhodopsin structure (Karnik, S. S., Sakmar, T. P., Chen, H.-B., and Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8459-8463). We now show that the sulfhydryl groups of these 2 cysteine residues interact to form a disulfide bond. Rhodopsin mutants containing cysteine----serine substitutions were prepared as follows. In one mutant, CysVII, all the 10 cysteine residues of rhodopsin were replaced by serines. A second mutant, CysVIII, contained only C110 and C185; a third mutant, CysIX, contained only C185 and C187 while the fourth mutant, CysX, contained only C110 and C187. Only mutant CysX formed functional rhodopsin. Mutants CysVIII and CysIX reacted with [3H]iodoacetic acid showing the presence of free sulfhydryl groups while mutant CysX was inert to this reagent. CysX reacted with cyanide ion to form a thiocyanate derivative showing the presence of a disulfide bond. The C110-C187 disulfide bond is buried in rhodopsin because reactions with disulfide reducing agents and cyanide ion require prior treatment with denaturants.     

L-cystine inhibits aspartate-beta-semialdehyde dehydrogenase by covalently binding to the essential 135Cys of the enzyme

Aspartate-beta-semialdehyde dehydrogenase (ASADH) from Escherichia coli is inhibited by L- and D-cystine, and by other cystine derivatives. Enzyme inhibition is quantitatively reversed by addition of dithiothreitol (DTT), dithioerythrytol, beta-mercaptoethanol, di-mercaptopropanol or glutathione to the cystine-inactivated enzyme. Cystine labeling of the enzyme is a pH dependent process and is optimal at pH values ranging from 7.0 to 7.5. Both the cysteine incorporation profile and the inactivation curve of the enzyme as a function of pH suggest that a group(s) with pK(a) of 8.5 could be involved in cystine binding. Stoichiometry of the inactivation reaction indicates that one cysteine residue from the enzyme subunit is reactive against cystine, as found by direct incorporation of radioactive cystine into the enzyme and by free-thiol titration of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) before and after the cystine treatment. One mole of cysteine is released from each mol of cystine after reaction with the enzyme. ASA, NADP and NADPH did not prevent cystine inhibition. The [35S]cysteine-labelled enzyme can be visualized after electrophoresis in polyacrylamide gels and further detection by autoradiography. After pepsin treatment of the [35S]cysteine-inactivated enzyme, a main radioactive peptide was isolated by HPLC. The amino acid sequence of this peptide was determined as FVGGN(Cys)(2)TVSL, thus demonstrating that the essential 135Cys is the amino acid residue modified by the treatment with cystine.     

Cysteine and methionine content of the Escherichia coli ribonucleic acid polymerase subunits.

We describe a procedure that allows cysteine and methionine content to be determined on microgram amounts of partially purified protein. The only requirements are that the protein can be obtained as a pure band after electrophoresis on a polyacrylamide gel and that some data on amino acid content be available. This method involves double labeling by growing bacterial cells with [3H]leucine and [35S]SO4 and determining the ratio of these radioisotopes incorporated into the ribonucleic acid polymerase subunits. The relative specific activities of [3H]leucine and [35S]cysteine and methionine are determined from the ratio of these isotopes incorporated into beta-galactosidase, the leucine, cysteine, and methionine contents of which are known. We have used this procedure to determine the sulfur content of the subunits of Escherichia coli ribonucleic acid polymerase. These new data are necessary to quantitate the rates of synthesis of these subunits by in vivo labeling with [35S]SO4.