Search

This manuscript concerns the tissue-specific transcription of mouse and cattle glutamate decarboxylase-like protein 1 (GADL1) and the biochemical activities of human GADL1 recombinant protein. Bioinformatic analysis suggested that GADL1 appears late in evolution, only being found in reptiles, birds, and mammals. RT-PCR determined that GADL1 mRNA is transcribed at high levels in mouse and cattle skeletal muscles and also in mouse kidneys. Substrate screening determined that GADL1, unlike its name implies, has no detectable GAD activity, but it is able to efficiently catalyze decarboxylation of aspartate, cysteine sulfinic acid, and cysteic acid to β-alanine, hypotaurine, and taurine, respectively. Western blot analysis verified the presence of GADL1 in mouse muscles, kidneys, C2C12 myoblasts, and C2C12 myotubes. Incubation of the supernatant of fresh muscle or kidney extracts with cysteine sulfinic acid resulted in the detection of hypotaurine or taurine in the reaction mixtures, suggesting the possible involvement of GADL1 in taurine biosynthesis. However, when the tissue samples were incubated with aspartate, no β-alanine production was observed. We proposed several possibilities that might explain the inactivation of ADC activity of GADL1 in tissue protein extracts. Although β-alanine-producing activity was not detected in the supernatant of tissue protein extracts, its potential role in β-alanine synthesis cannot be excluded. There are several inhibitors of the ADC activity of GADL1 identified. The discovery of GADL1 biochemical activities, in conjunction with its expression and activities in muscles and kidneys, provides some tangible insight toward establishing its physiological function(s).

Hydrogen peroxide (H2O2) triggers a redox cycle between ferric and ferryl hemoglobin (Hb) leading to the formation of a transient protein radical and a covalent hemeprotein cross-link. Addition of H2O2 to highly purified human hemoglobin (HbA0) induced structural changes that primarily resided within β subunits followed by the internalization of the heme moiety within α subunits. These modifications were observed when an equal molar concentration of H2O2 was added to HbA0 yet became more abundant with greater concentrations of H2O2. Mass spectrometric and amino acid analysis revealed for the first time that βCys-93 and βCys-112 were oxidized extensively and irreversibly to cysteic acid when HbA0 was treated with H2O2. Oxidation of further amino acids in HbA0 exclusive to the β-globin chain included modification of βTrp-15 to oxyindolyl and kynureninyl products as well as βMet-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the β subunits as a result of the H2O2 attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two α-peptide fragments (α128 - α139) and a heme moiety with the loss of iron, cross-linked between αSer-138 and the porphyrin ring. The novel oxidative pathway of HbA0 modification detailed here may explain the diverse oxidative, toxic, and potentially immunogenic effects associated with the release of hemoglobin from red blood cells during hemolytic diseases and/or when cell-free Hb is used as a blood substitute.

The effect of H(2)O(2) on smooth muscle heavy meromyosin (HMM) and subfragment 1 (S1) was examined. The number of molecules that retained the ability to bind ATP and the actinactivated rate of P(i) release were measured by single-turnover kinetics. H(2)O(2) treatment caused a decrease in HMM regulation from 800- to 27-fold. For unphosphorylated and phosphorylated heavy meromyosin and for S1, approximately 50% of the molecules lost the ability to bind to ATP. H(2)O(2) treatment in the presence of EDTA protected against ATPase inactivation and against the loss of total ATP binding. Inactivation of S1 versus time correlated to a loss of reactive thiols. Treatment of H(2)O(2)-inactivated phosphorylated HMM or S1 with dithiothreitol partially reactivated the ATPase but had no effect on total ATP binding. H(2)O(2)-inactivated S1 contained a prominent cross-link between the N-terminal 65-kDa and C-terminal 26-kDa heavy chain regions. Mass spectral studies revealed that at least seven thiols in the heavy chain and the essential light chain were oxidized to cysteic acid. In thiophosphorylated porcine tracheal muscle strips at pCa 9 + 2.1 mM ATP, H(2)O(2) caused a approximately 50% decrease in the amplitude but did not alter the rate of force generation, suggesting that H(2)O(2) directly affects the force generating complex. Dithiothreitol treatment reversed the H(2)O(2) inhibition of the maximal force by approximately 50%. These data, when compared with the in vitro kinetic data, are consistent with a H(2)O(2)-induced loss of functional myosin heads in the muscle.

We used a proteomic approach to identify proteins that associate with keratins 8 or 18 (K8/K18) in a pervanadate-dependent manner. Pervanadate triggers Ran-K8/K18 binding and a gel-migration-shift of Ran from 25 to 27 kDa, which does not occur upon exposure to H2O2 or vanadate or if pervanadate is excluded during cell solubilization. Generation of 27-kDa Ran is not related to hyperphosphorylation, is heat-insensitive, but occurs upon conversion of Ran cysteines to cysteic acid. The pervanadate-mediated Ran cysteine --> cysteic acid oxidation and its related gel migration shift affects other proteins including actin. Mutation of the three Ran cysteines (Cys-85, -112, and -120) showed that Ran Cys-112 oxidation generates 27-kDa Ran and accounts for its keratin binding. Proteasome inhibition accentuates Ran-keratin binding after cell exposure to pervanadate. Therefore, cell-free exposure to pervanadate causes cysteine to cysteic acid oxidation of Ran and several other proteins and Ran-K8/K18 association. In cells, stabilization of oxidized Ran by proteasome inhibition promotes Ran-keratin interaction. Keratin sequestration of oxidized Ran may provide a back-up protective mechanism in some cases of oxidative injury.

The proteomics analysis reported here shows that a major cellular response to oxidative stress is the modification of several peroxiredoxins. An acidic form of the peroxiredoxins appeared to be systematically increased under oxidative stress conditions. Peroxiredoxins are enzymes catalyzing the destruction of peroxides. In doing so, a reactive cysteine in the peroxiredoxin active site is weakly oxidized (disulfide or sulfenic acid) by the destroyed peroxides. Cellular thiols (e.g. thioredoxin) are used to regenerate the peroxiredoxins to their active state. Tandem mass spectrometry was carried out to characterize the modified form of the protein produced in vivo by oxidative stress. The cysteine present in the active site was shown to be oxidized into cysteic acid, leading to an inactivated form of peroxiredoxin. This strongly suggested that peroxiredoxins behave as a dam upon oxidative stress, being both important peroxide-destroying enzymes and peroxide targets. Results obtained in a primary culture of Leydig cells challenged with tumor necrosis factor α suggested that this oxidized/native balance of peroxiredoxin 2 may play an active role in resistance or susceptibility to tumor necrosis factor α-induced apoptosis.

The presence of a cysteine residue(s) near the active site of acylpeptide hydrolase was suggested by inactivation of the enzyme with sulfhydryl-modifying agents and by the substantial protection against inactivation afforded by the competitive inhibitor acetylmethionine. 5,5'-dithiobis-(2-nitrobenzoate) titrations of the native and the denatured enzyme together with analysis for cysteic acid after performic acid oxidation showed that the enzyme contained 12 free SH groups and three disulfide bonds/monomer. Chemical modification with radiolabeled iodoacetamide led to the labeling of Cys-30 and Cys-64 suggesting that one or both of these Cys residues are close to the active site. Modification of one or both of them probably inhibits the enzyme either because of a distortion of the active site or because the adducts present a barrier to the efficient diffusion of substrates into and products out of the active site. Studies on the mechanism of action of acylpeptide hydrolase have employed p-nitrophenyl-N-propyl carbamate as a potent active site-directed inhibitor. Enzyme inactivation, which follows pseudo first-order kinetics, is diminished by the competitive inhibitor acetylmethionine. The inhibited enzyme slowly regains activity at a rate that is increased in the presence of the nucleophile hydroxylamine. A general mechanism involving an acyl-enzyme intermediate is supported by evidence for the formation of acetyl-alanyl hydroxamate during hydrolysis of acetyl-alanine p-nitroanilide in the presence of hydroxylamine. The effect on Vmax and Km during this reaction indicate that hydrolysis of the acyl-enzyme intermediate is rate-limiting.

Protein HC, an extremely charge-heterogeneous lipocalin, carries a yellow-brown fluorescent chromophore of unknown structure covalently bound at an unidentified site of its polypeptide chain. Two chromophore-carrying peptides with 60% of the chromophore material (defined as material absorbing light at 330 nm) were isolated from pepsin-digested native human protein HC by reversed-phase high performance liquid chromatography. Sequence analysis of these peptides indicated that the chromophore was bound to the cysteine residue at position 34 of the protein HC polypeptide chain. Sequence analysis of a native chromophore-tripeptide complex, isolated from pronase digests of the pepsin-produced peptides, identified the sequence Thr33-X34-Pro35, corroborating the position of the chromophore linkage. Quantitative amino acid analysis of the hydrolyzed, performic acid-oxidized, chromophore-tripeptide complex demonstrated approximately equal amounts of threonine, cysteic acid, and proline in the complex. Reduction and carboxymethylation of the native chromophore-tripeptide complex did not remove the chromophore from the peptide. The absorption spectrum of the chromophore-tripeptide complex was similar to that of native protein HC, implying that all of the heterogeneity of protein HC resides in its chromophore.

We have found that a chicken egg lysozyme derivative (beta-101-lysozyme) containing an L-isoaspartyl residue at position 101 has a Km for methylation by the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77) of 183 microM, about 30 times higher than that expected from previous studies with isoaspartyl-containing peptides. In the course of investigating the reasons for this poor enzyme recognition, we found that charged residues on the carboxyl side of isoaspartyl residues had a large effect on the affinity of the enzyme for synthetic peptides. This is best illustrated by the lysozyme-related peptide YVSisoDGDG, which has a Km for methylation of 469 microM. When the penultimate aspartyl residue is replaced by a cysteinyl residue, the Km drops to 4.6 microM, comparable to other peptides of similar size. Furthermore, replacing it with a cysteic acid residue results in a Km of 104 microM, suggesting that a negative charge at this position may lead to a weaker affinity of the peptide substrate for the methyltransferase. Assays with additional synthetic peptides indicate that moving the negative charge to the first or third residue on the carboxyl side of the isoaspartyl residue has a similar but less severe effect in reducing its affinity for the methyltransferase. Enzymatic methylation has recently been proposed to be the first step in the conversion of abnormal isoaspartyl residues to aspartyl residues. The results reported here, however, along with previous evidence that protein tertiary structure can inhibit isoaspartyl methylation, suggest that only a subclass of damaged sites are capable of efficiently entering a putative repair pathway; the sites not recognized by the methyltransferase may accumulate in vivo.

Cystine binding protein isolated from Escherichia coli W3092 in high yield was used to develop a binding assay for the disulfide amino acid cystine. Filtration of a mixture of [14C]cystine and cystine binding protein through nitrocellulose filters separated the [14C]cystine-cystine binding protein complex from the unbound cystine. The binding of [14C]cystine-cystine binding protein complexes to the filter was optimal (100%) at pH 4 to 5, and was insensitive to Mg2+ (10 mm), EDTA (10 mm), or concentrations of NaCl to 0.5 m. Bovine serum albumin in excess of 30 µg decreased binding of the 14C complex to the filter. The binding of [14C]cystine to the cystine binding protein was inhibited by lanthionine, much less by diaminopimelic acid, cystathionine, and d-cystine, and was not inhibited by reduced or oxidized glutathione, penicillamine disulfide, cysteic acid, methionine, taurine, homocystine, carboxymethyl cysteine, or the N-ethylmaleimide adduct of cysteine. Nonradioactive cystine competed with [14C]cystine resulting in the expected diminution of the bound radioactivity, permitting the direct calculation of nonradioactive cystine concentrations of unknown samples. With the cystine binding protein assay, the cystine concentrations of human plasma were in the range of 87 to 100% of the values obtained by ion exchange chromatography, and the cystine contents of cultured skin fibroblasts from patients with nephropathic cystinosis were in the range of 89 to 121% of the values obtained by ion exchange chromatography. As little as 10 pmoles of cystine were measured.

A soluble ribonuclease inhibitor from the human placenta has been purified 4000-fold by a combination of ion exchange and affinity chromatography. The inhibitor has been isolated in 45% yield (about 2 mg/placenta) as a protein that is homogeneous by sodium dodecyl sulfate-gel electrophoresis. In common with the inhibitors of pancreatic ribonuclease from other tissues that have been studied earlier, the placental inhibitor is an acidic protein of molecular weight near 50,000; it forms a 1:1 complex with bovine pancreatic RNase A and is a noncompetitive inhibitor of the pancreatic enzyme, with a Ki of 3 X 10(-10) M. The amino acid composition of the protein has been determined. The protein contains 30 half-cystine plus cysteine residues determined as cysteic acid after performic acid oxidation. At pH 8.6 the nondenatured protein alkylated with iodoacetic acid in the presence of free thiol has 8 free sulfhydryl groups. The inhibitor is irreversibly inactivated by sulfhydryl reagents and also by removal of free thiol from solutions of the protein. Inactivation by sulfhydryl reagents causes the dissociation of the RNase - inhibitor complex into active RNase and inactive inhibitor.

Previous reports (Drescher, D.G., and Lee, K.S. (1978) Anal. Biochem. 84, 559-569; Lee, K.S., and Drescher, D.G. (1978) Int. J. Biochem. 9, 457-467) have shown that high performance liquid chromatographic analysis of amino acids with the o-phthaldialdehyde/2-mercaptoethanol reagent (OPA/2-ME) is one of the most sensitive procedures currently available for micro amino acid analysis. In the present paper, methods are presented for the modification of cysteine and cystine in proteins for micro amino acid analysis using OPA/2-ME. Cysteine and cystine, which both show low fluorescence with OPA/2-ME, are converted to cysteic acid with performic acid directly, or to S-3-sulfopropylcysteine with 1,3-propane sultone after reduction of cystine with tri-n-butylphosphine. Cysteic acid and S-3-sulfopropylcysteine form highly fluorescent adducts with OPA/2-ME. The formation of S-3-sulfopropylcysteine in proteins and the subsequent hydrolysis of the proteins with methanesulfonic acid are particularly useful for complete amino acid analysis at the picomole level using a single sample.

SUMMARY A method is described for purification of sulfhydryl oxidase from bovine milk which consistently yields preparations with greater than 3000-fold purification over skim milk. A concentration-dependent association-dissociation of the enzyme was adapted to the development of an isolation procedure. Purified preparations exhibited two zones, both of which displayed activity, upon polyacrylamide disc gel electrophoresis, but only one zone following disc gel electrophoresis in sodium dodecyl sulfate. Its mobility indicated a subunit weight of 89,000. Several lines of evidence suggest that iron is an integral part of the enzyme. Treatment of the enzyme with EDTA resulted in complete loss of activity which could be subsequently restored by dialysis against 1 FM ferrous sulfate. Furthermore, atomic absorption analysis and neutron activation analysis of separate enzyme preparations each indicated 0.5 atom of iron per subunit. Chemical analyses of sulfhydryl oxidase accounted for 97 % of the sample weight, of which 89% could be attributed to amino acid residues and 11% to carbohydrate residues. Five half-cystine residues per subunit were indicated by cysteic acid analysis and by sulfhydryl group determination following reaction with sodium borohydride. Comparison of this value to the total sulfhydryl groups without reduction tentatively suggests the presence of one disulfide bond. Sulfhydryl oxidase was found to catalyze the oxidation of sulfhydryl groups in both small compounds and proteins, using O2 as oxidant and producing, in equimolar quantities, HzOz and the corresponding disulfide. A Michaelis constant of 90 pM was obtained using reduced glutathione as substrate, under conditions of optimal pH and temperature, viz., pH 7.0 and 35”. Substrate inhibition was apparent at GSH concentrations above 0.8 mu. In the presence of sulfhydryl oxidase, reductively denatured RNase was reoxidized and fully reactivated within 1 hour, whereas in the absence of the oxidase under otherwise identical conditions, full recovery of RNase activity required 24 hours. The presence of reducing agent was not required for this activity, nor was prior reduction of the sulfhydryl oxidase. Based on the observed activity, it appears

The conditions and utility of the N-chlorosuccinimide/urea (NCS/urea) reagent for the selective cleavage of tryptophanyl peptide bonds in proteins is demonstrated with cytochrome c. At low concentrations of NCS/urea the oxidation of thioether side chains in cytochrome c is the predominant reaction. Methionyl residues are oxidized to sulfoxide and the heme-thioether bridge is partially cleaved. At 10-fold excess of NCS/urea reagent, cleavage of the tryptophanyl peptide bond is optimal at approximately 50% yield in several species of cytochrome c studied. Analytical data on isolated horse cytochrome c peptide fragments demonstrate lack of modification and cleavage at tyrosyl and histidyl residues. However, at high concentrations of NCS/urea reagent (30-fold) unexpected conversions of methionine to sulfone and cysteine to cysteic acid in intact proteins are observed. This is in contradistinction to the absence of sulfone in NCS/urea-reacted amino acid mixtures. The mechanisms of halogenation and cleavage by N-bromosuccinimide, N-iodosuccinimide, and N-chlorosuccinimide are discussed. It is porposed that the selectivity with respect to halogenation by N-chlorosuccinimide is due to the insignificant participation of molecular chlorine in the NCS/urea reaction. A mechanism of halogenation and cleavage by NCS at tryptophan is also offered.

Nontoxic abrus lectin has been prepared by a new purification procedure. The method is accomplished by 45% saturation ammonium sulfate fractionation from a 5% acetic acid extract of the seeds of Abrus precatorius followed by diethylaminoethyl-Sephadex A-50 and Sepharose 4B affinity chromatography. The abrus lectin appeared homogeneous as judged by electrophoresis, analytical ultracentrifugation, and isoelectric focusing. The lectin molecule has a weight of 126,000 as determined by sedimentation equilibrium. It is composed of four subunits, of which two pairs have either identical or closely similar molecular sizes (33,800 and 32,200 daltons) as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results of amino acid analyses are given; none of the cysteic acid appears to arise from cysteine. An electrofocusing experiment indicated the isoelectric point to be 5.0. Crystals large enough for x-ray investigation were obtained by a vapor diffusion technique. X-ray precession photographs revealed that abrus lectin crystallizes in a tetragonal unit cell of symmetry P41212 and dimensions a = 140 and c = 210 A. The asymmetric unit contains 2 protein molecules of molecular weight 126,000 and has a solvent content of approximately 41% by volume.

Phosphoribosylpyrophosphate synthetase from Salmonella typhimurium contains four cysteine residues per subunit. Three of these react readily with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB), forming an active derivative with kinetic and physical properties similar to the native enzyme, but one reacts only under denaturing conditions. Stoichiometric amounts of KMnO4 inactivate the DTNB-treated enzyme. The loss of activity is correlated with the oxidation of the remaining cysteinyl group to cysteic acid by KMnO4. Amino acid analysis indicates that no other residues are altered. The rate of inactivation of the enzyme is decreased 30-fold by saturatin g concentrations of the substrate ATP. Inorganic phosphate also protects substantially against KMnO4. Titration of the native enzyme with limiting amounts of KMnO4 shows that the sulfhydryl group essential for activity competes effectively with the other sulfhydryl groups for KMnO4. These results suggest that the essential sulfhydryl group is near the active site, and that KMnO4, a phosphate analogue, can act as an active site-directed reagent at the ATP binding site of the enzyme. The KMnO4-oxidized enzyme is more highly aggregated than untreated enzyme and fails to bind ATP appreciably.

Proteolytic digests of biologically active fractions of recombinant human leukocyte interferon A expressed in large quantities in Escherichia coli were analyzed by fast atom bombardment mass spectrometry and high-performance liquid chromatography. The values observed in the mass spectra of digests of the major fraction of recombinant human leukocyte interferon A with trypsin and Staphylococcus aureus protease V8 accounted for 93% of the amino acid sequences of human leukocyte interferon A predicted from the nucleotide sequence of the gene encoding the protein, indicating that the major fraction of recombinant human leukocyte interferon A was expressed with the same amino acid sequence as that translated from the nucleotide sequence of the gene encoding the protein. Mass spectrometry of proteolytic digests of two minor fractions of recombinant human leukocyte interferon A and mass and amino acid analyses of their high-performance liquid chromatography fractions showed that the amino group of the N-terminal amino acid residue of interferon was in part acetylated, and the Cys-1 and Cys-98 residues were oxidized to cysteic acid or linked to glutathione. These findings suggest that amino acid residues in recombinant proteins prepared in large quantities in E. coli are modified post-translationally.

The two protomers of the purified regulatory subunit from porcine cAMP-dependent protein kinase I have been shown to be covalently cross-linked by interchain disulfide bonding. Limited proteolysis which cleaves the polypeptide chain into two fragments demonstrated that the disulfide bonding was associated exclusively with the fragment that corresponded to the NH2-terminal region of the polypeptide chain. This NH2-terminal fragment accounted for approximately 15 to 20% of the molecule. The disulfide bonding was further characterized by alkylating the cysteines in the native regulatory subunit. Following oxidation with performic acid, each regulatory subunit contained 7 cysteic acid residues; however, under denaturing conditions, but without prior reduction, only 5 cysteine residues could be alkylated with iodoacetic acid. Following limited proteolysis, all five of these cysteines were associated with the larger COOH-terminal, cAMP binding domain. In contrast, if the denatured subunit was first reduced prior to alkylation, all 7 cysteine residues were alkylated. The 2 cysteines that were only accessible to alkylation after prior reduction were both associated with the NH2-terminal end of the polypeptide chain ultimately with a 5,400 peptide. Alkylation of the isolated, denatured NH2-terminal domain with iodoacetic acid resulted in no covalent modification unless the fragment was first reduced with dithiothreitol. The NH2-terminal and COOH-terminal domains were shown to be linked by a region of the polypeptide chain that is rich in both proline and arginine. It is the arginine-rich site that is readily prone to proteolytic cleavage.

Rat chondrosarcoma chondrocytes were labeled with [3H]serine or [3H]mannose as a precursor. Intracellular proteoglycan core protein precursor was purified from cell lysates by immunoprecipitation with polyclonal antibodies against the hyaluronic acid-binding region, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The core precursor was eluted from the gels and treated with alkaline borohydride in order to convert serine residues substituted with xylose or N-acetylgalactosamine to alanine (or with alkaline sulfite to convert them to cysteic acid). After acid hydrolysis, the proportions of labeled serine and alanine (or cysteic acid) were determined by high performance liquid chromatography, and the results were compared with those obtained for the completed proteoglycan molecules isolated from the same cultures. In the completed proteoglycans, about 55% of the serine residues were substituted with xylose or N-acetylgalactosamine, while the corresponding figure for the intracellular precursor molecules was less than 5%. These results indicate, in agreement with our previous kinetic data, that the major part of the xylosyl transfer to the chondrosarcoma proteoglycan core protein precursor must occur late in the processing sequence, i.e. after about 85% of its intracellular lifetime and no more than 7 min before the addition of the rest of the chondroitin sulfate chain. The ratio of [3H]mannose to [3H]fucose in the core precursor was about 19, while that for the complete proteoglycan was about 2. This indicates the presence of high mannose, N-linked oligosaccharides on the core protein precursor which are converted to the complex forms on the completed proteoglycan. These data provide further support that the core precursor resides mainly in the pre-Golgi compartment and that xylosylation occurs mainly in a Golgi compartment.

Sickle erythrocytes are known to undergo excessive auto-oxidation, resulting in the generation of increased intracellular levels of several species of free radical oxidants. This environment is likely to enhance the accumulation of oxidative lesions by membrane components, although, as yet, this has been shown directly only for the sickle membrane phospholipids. We examined the oxidative status of protein 4.1, a major component of the human erythrocyte protein skeleton. We found that protein 4.1 isolated from sickle erythrocytes bound approximately 4-fold less to protein 4.1-stripped membranes than did the normal protein. The binding defect was inherent in the sickle protein and not in its membrane-binding site(s) since normal protein 4.1 bound to sickle protein 4.1-stripped inside-out vesicles similar to normal protein 4.1-stripped inside-out vesicles. Sickle membranes, in particular spectrin-depleted inside-out vesicles, contained less protein 4.1 than normal membranes. Purified sickle protein 4.1 contained 20-40% high molecular weight aggregated protein (Mr greater than 200,000), whereas the purified normal protein contained approximately 10% high molecular weight protein. The high molecular weight protein was immunoreactive with antibodies to protein 4.1 but not with antibodies to spectrin, ankyrin, band 3, glycophorin, or hemoglobin, suggesting that the high molecular weight protein was cross-linked protein 4.1 and not a complex of protein 4.1 and some other membrane protein(s). Purified sickle protein 4.1 was eluted from an anion-exchange resin at a higher salt concentration than normal protein 4.1. Oxidizing normal protein 4.1 with diamide resulted in an anion-exchange elution pattern similar to the sickle protein, suggesting that oxidation can affect protein surface charge. Activated thiol beads bound one-half as much sickle protein 4.1 as normal protein 4.1 when both were solubilized directly from membranes, demonstrating that thiol oxidation had occurred in vivo. Direct quantification of protein thiols revealed that the sickle protein contained 1-2 mol% fewer cysteines/protein 4.1 monomer than did the normal protein. By amino acid analysis, sickle protein 4.1 was found to contain less methionine and tyrosine than did the normal protein and contained approximately 1 mol% cysteic acid, whereas the normal protein did not contain any cysteic acid. Taken together, our results strongly suggest that sickle protein 4.1 has sustained oxidative damage in vivo. This damage can alter the functional properties of the sickle protein and may be an underlying factor in the myriad of membrane abnormalities reported in sickle erythrocytes.

A group of 23-29-kDa polypeptides in the membranes of bovine rod outer segments are substrates for S-adenosylmethionine-dependent methylation reactions. The bulk of the methyl group incorporation is in base-labile ester-like linkages, and does not appear to be due to the widespread D-aspartyl/L-isoaspartyl methyltransferase (EC 2.1.1.77). To determine the site(s) of methylation, 3H-methylated proteins separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate were eluted and digested with papain, leucine aminopeptidase-M, and prolidase. After performic acid oxidation of the digest, a base-labile radioactive material was recovered that coeluted with a synthetic standard of cysteic acid methyl ester upon cation exchange and G-15 gel filtration chromatography, as well as in two thin-layer electrophoresis and two thin-layer chromatography systems. These results provide direct evidence for the methylation of the alpha-carboxyl group of a carboxyl-terminal cysteinyl residue, a modification that has been proposed for the 21-kDa Ha-ras product and other cellular proteins (Clarke, S., Vogel, J. P., Deschenes, R. J., and Stock, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4643-4647).

The three disulfide bonds of bovine pancreatic secretory trypsin inhibitor were assigned on the basis of the structures of cystine peptides isolated from thermolysin hydrolysates of the native inhibitor prepared at pH 6.5. The peptides were isolated by chromatographic procedures with Sephadex G-75, Sephadex G-25, and Dowex 50-X2. Five major cystine peptides were oxidized with performic acid and the cysteic acid fragments were separated by chromatography on Dowex 50-X2 and by high voltage paper electrophoresis. The cysteic acid peptides were located in the amino acid sequence of the inhibitor (Greene, L. J., and Bartelt, D. C., J. Biol. Chem., 244, 2646 (1969)) on the basis of amino acid composition and end group determination. Cystine peptides corresponding to disulfide bonds I–V, II–IV, and III–VI were isolated in 55, 44, and 91% yield, respectively. Intermediates in the degradation of the inhibitor by thermolysin having either one or two peptide bonds hydrolyzed per molecule were isolated. The sites of hydrolysis were identified as the peptide bonds Ile-Leu (residues 2 to 3) and Glu-Val (residues 12 to 13).

The amino acid sequence of the human κ Bence-Jones protein Ag containing 214 residues has been determined by a combination of four procedures: sequence analysis of the tryptic and of the chymotryptic peptides of the carboxymethylated protein described earlier and partial sequence analysis of the tryptic peptides of the aminoethylated protein and of the peptic peptides of the oxidized protein described herein. The tryptic peptides from the aminoethylated protein account for the total amino acid content and provide some necessary overlaps. Analysis of two of these peptides and of one of the peptic cysteic acid peptides has yielded the previously missing sequence for positions 19 through 32. There are two analogous disulfide bridges: one connecting Cys-23 and Cys-88 and one connecting Cys-134 and Cys-194. Comparison to the provisional sequences of other human κ Bence-Jones proteins has verified the concept that the NH2-terminal half of κ light chains is prone to variation in sequence whereas the COOH-terminal half has a fixed sequence except for one hereditary variation. Many of the differences in sequence in the NH2-terminal portion of κ-chains are not attributable to single point mutations. The various genetic mechanisms proposed to explain the variability in sequence of immunoglobulin light chains are discussed.

When solutions of cystine and cysteine are treated with dithiothreitol and then with sodium tetrathionate, quantitative yields of S-sulfocysteine can be obtained. This reaction has been applied to protein hydrolysates and the resulting S-sulfocysteine has been determined by ion exchange chromatography on an amino acid analyzer. With ribonuclease and reduced glutathione this procedure has given accurate values for the cystine or cysteine content of the protein or peptide. When tryptophan is also present, the addition of a small amount of sodium tetrathionate to the 6 n HCl used for the hydrolysis has usually proved to be desirable; the function of the tetrathionate appears to be to react with the indole nucleus. With this modification, the procedure has given precise results for the cysteine plus cystine contents of streptococcal proteinase, lysozyme, and wool. The results indicate that, in terms of their primary structures, cysteine and cystine are not destroyed during acid hydrolysis under these conditions; treatment with dithiothreitol and tetrathionate serves to convert any altered forms of the amino acids in the hydrolysates to S-sulfocysteine and to permit quantitative recovery in the form of this derivative. This method for determining the half-cystine content of proteins provides an alternative to the determinations of the amino acids as cysteic acid or S-carboxymethylcysteine. The treatment does not affect the recoveries of other amino acids in the hydrolysate, with the possible exception of tyrosine.

Incubation of the TPN-dependent isocitrate dehydrogenase from pig heart with 1-14C-N-ethylmaleimide (NEM) leads to inactivation and altered Michaelis constants for isocitrate and oxalosuccinate concomitant with incorporation of 2 moles of reagent. Reaction of enzyme with radioactive NEM in the presence of isocitrate and Mn++ yields a catalytically functional enzyme, with the same altered Michaelis constants, which also contains 2 moles of reagent. Inactive enzyme with a single carboxymethyl methionyl residue exhibits the same ability to bind radioactive NEM as does native enzyme; however, prior inactivation by reaction of 5 sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) prevents binding of NEM. Cysteine is the sole type of amino acid modified in the inactive NEM-enzyme, as determined by paper chromatographic identification of radioactive cysteinosuccinic acid and S-(N-ethylsuccinimido)cysteine in acid and proteolytic digests of 14C-labeled modified enzyme. Thirteen moles of cysteic acid are found after performic acid oxidation of native enzyme, the same as the number of free —SH groups observed by reaction with DTNB after denaturation; therefore, no disulfide bonds are present in isocitrate dehydrogenase. A decrease in the number of measurable —SH groups after reaction with NEM is consistent with modification of cysteinyl residues. By contrast, the recovery of NH2-terminal alanine after the Edman reaction was approximately the same for native and modified enzyme. Inactive enzymes with a single carboxymethyl methionyl residue, with 2 or 5 altered sulfhydryl groups, give a reaction of antigenic identity to that of native enzyme as measured against rabbit antibody to isocitrate dehydrogenase. The molecular size of the inactive NEM-enzyme is the same as that of native enzyme, as determined by gel filtration on Sephadex G-150, and the rates of proteolytic digestion by Pronase are not significantly different. These data argue against a generalized structural change in the modified enzyme. However, a small conformational change is indicated by a decrease in the reaction rate of DTNB with the residual —SH groups of the inactive NEM-enzyme. The magnitude of conformational change is not directly related to loss in enzymatic activity, since an observed decrease in the amplitude of the optical rotatory dispersion curves is greater for active NEM-enzyme ([α]234 -5610°) than it is for inactive NEM-enzyme ([α]234 -7430°), as compared to native isocitrate dehydrogenase ([α]234 -8050°). These results, in conjunction with those of the preceding paper, suggest that, in the absence of isocitrate and MnSO4, modification of Sulfhydryl Group A produces inactivation, whereas modification of Sulfhydryl Group B leads to a small conformational change which is reflected in altered Michaelis constants. In the presence of substrates, NEM reacts with 2 sulfhydryl groups, including Group B but not Group A, which produces a further change in the conformation of the enzyme but no concomitant change in the kinetic properties.

All of the cysteic acid residues (4.6 per subunit) found by amino acid analysis of performic acid-oxidized Escherichia coli glutamine synthetase are present as cysteine in the native enzyme but are buried in the interior of the molecule. They become exposed and accessible to titration by sulfhydryl reagents only when the enzyme is disaggregated into subunits or when divalent cation is removed with ethylenediaminetetraacetate. From the total number of peptides and the number of arginine-containing and 14C-carboxymethylated peptides that are produced upon tryptic digestion of 14C-carboxymethylated glutamine synthetase, it is suggested that the enzyme is composed of identical subunits. However, until an explanation is found for the wide variation in the observed specific radioactivity of the 14C-carboxymethylated peptides thus obtained, this conclusion is still uncertain. Glutamine synthetase can be inactivated by organic mercurials, and the extent of inactivation depends upon the amount of divalent cation in association with the enzyme. Two states of the enzyme are thus defined: "relaxed enzyme," without metal, which is susceptible to inactivation and disaggregation by p-chloromercuriphenylsulfonate, and "taut enzyme," which has bound manganese and is resistant to such treatment. The metabolic effectors of glutamine synthetase may cause alterations in the rate and extent of inactivation of the enzyme by mercurials. Of the inhibitors that are partially competitive with respect to glutamate, tryptophan diminishes the inactivation by mercurials, cytidine triphosphate enhances this inactivation, and glycine has little effect. Of the compounds that are partially competitive inhibitors with respect to ammonia, histidine is protective against inactivation and glucosamine-6-P has no effect. Similarly, with the noncompetitive inhibitors, AMP decreases the rate of inactivation while carbamyl phosphate and alanine have no influence. These results provide additional support for the occurrence in glutamine synthetase of separate binding sites for each inhibitor, as would be expected from the independent nature of the cumulative feedback inhibition phenomenon.

Formyltetrahydrofolate synthetase, a tetrameric protein, contains 23 half-cystine residues as determined by cysteic acid analysis and 24 free sulfhydryl groups as determined by four different methods thus indicating absence of disulfide bonds. All of the cysteine residues react with sulfhydryl reagents in the absence of denaturing agents. Titration of the enzyme with p-chloromercuribenzoate (CMB) caused a loss in activity which was a linear function of the amount of thiol groups reacted. A complete loss in activity occurred after 11 CMB molecules had reacted per mole of enzyme. The loss in activity was accompanied by the appearance of the monomeric form of the protein; however, the presence of tetramer which contained CMB was demonstrated by Sephadex G-200 chromatography of enzyme which had been partially inactivated with 14C-labeled CMB. The reaction of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was different from that with CMB. The relation between thiol groups reacted and loss in activity was not linear, and about 18 to 20 thiol groups had to react before a complete loss in activity occurred. Dissociation to the monomeric form did not occur until about 75% of the cysteine residues had reacted. Titration with HgCl2 showed that a linear decrease in activity occurred until complete inactivation was observed after 8 Hg2+ ions had been bound by the protein. Hg2+ could be bound to virtually all of the cysteine residues without causing dissociation of the enzyme. Reactivation by cysteine of enzyme inactivated by CMB or DTNB was variable and could be achieved only when the enzyme was still in the tetrameric form. Total reactivation of mercury enzyme was achieved with 2-mercaptoethanol or cysteine. The inactivation by CMB and DTNB was retarded by ATP and tetrahydrofolate and facilitated by MgATP. Formate and Mg2+ had no effect on the inactivation. It is concluded that the inactivation of formyltetrahydrofolate synthetase by sulfhydryl reagents results from conformational changes produced in the tetramer as well as from dissociation of the tetramer to the inactive monomer. The effects produced by the substrates probably result from conformational changes in the protein. There is no evidence which suggests that a sulfhydryl group or groups constitute part of the active site of the enzyme.

It is proposed that the transfer of the amidine group from arginine to glycine, catalyzed by transamidinase, occurs through the formation of an S-enzyme isothiourea intermediate formed by reaction between the amidine group of the substrate and a sulfhydryl group of a cysteine residue of the enzyme. The identification of the cysteine residue (isolated as cysteic acid), which appears to be involved in the binding of the amidine group, has been performed with the following procedure. Acid hydrolysis of the enzyme-amidine complex. This results in the release of 2-amino thiazoline 4-carboxylic acid carrying the carbon atom of the amidine group in position 2. Bromine oxidation of 2-amino thiazoline 4-carboxylic acid to carbamylcysteic acid. Alkaline hydrolysis of carbamylcysteic acid to cysteic acid with loss of the carbon atom originally belonging to the amidine group of the substrate.

The enzyme, β-ketoacyl acyl carrier protein (ACP) synthetase, which catalyzes the formation of acetoacetyl-ACP from acetyl-ACP and malonyl-ACP, has been purified to homogeneity from Escherichia coli. The molecular weight, determined from equilibrium centrifugation studies, is 66,000. Amino acid analysis after performic acid oxidation indicates that it contains 8 moles of cysteic acid per mole of protein. Titration with 5,5'-dithiobis(2-nitrobenzoic acid) after protein denaturation with sodium dodecyl sulfate indicates 7.2 —SH groups per mole of protein. The enzyme is inhibited by iodoacetamide when 1 mole of cysteine per mole of protein is alkylated, and acetyl-ACP protects the enzyme against alkylation. The enzyme reacts with acetylACP to form an acetyl enzyme intermediate, and the properties of this intermediate suggest that the acetyl group is bound to a cysteine residue as an acyl thioester. Acetyl enzyme is active in transferring the acetyl group either to ACP to form acetyl-ACP or to malonyl-ACP to form acetoacetyl-ACP.

Reduction of thioredoxin reductase with different reductants has been followed spectrally; by titration with TPNH in the presence of DPNase or with dithionite, it has been shown that this enzyme can accept 4 electrons per FAD. Thus, an additional acceptor group is postulated and is identified as a disulfide group which undergoes reversible reduction to a dithiol. The thioredoxin reductase activity is very sensitive to low concentrations of mercurial, whereas the transhydrogenase activity requires much higher levels for inhibition. Prereduction of the enzyme enhances the former, but not the latter process. Amperometric titrations show that the 4 cysteic acid residues per FAD, determined by amino acid analysis, arise from 2 free sulfhydryls and a disulfide bond which is reducible by TPNH. Mercurials are shown, in spectral experiments, to shift the electrons from the flavin the second redox group by trapping action on the nascent dithiol; after such a treatment, the enzyme is completely reduced by only 2 electrons per FAD and cannot be reoxidized by thioredoxin.

Several lines of evidence are presented which, together with previous data that arginine decarboxylase (mol wt = 820,000; s020,w = 23.3 S) contains an integral multiple of five subunits, indicate that the enzyme is composed of 10 identical or very nearly identical polypeptide chains. (a) Acrylamide gel electrophoresis in sodium dodecyl sulfate and mercaptoethanol gives a single band containing a material having a molecular weight of approximately 96,000. (b) In denaturing solvents, 6.7 moles of sulfhydryl groups react per 82,000 g of protein with 5,5'-dithiobis(2-nitrobenzoic acid). Since 7.0 residues of cysteic acid were found after performic acid oxidation, the molecule contains no disulfide bonds. In the native (23 S) enzyme, none of the sulfhydryl groups are reactive, while in the dissociated (mol wt = 160,000, s020,w = 8.0 S) form of the enzyme, 0.9 mole of sulfhydryl groups reacts per 82,000 g. Similar results were obtained for the dissociated species by reaction with both p-chloromercuribenzoate and N-ethylmaleimide. Thus, the dissociated (8 S) species has 1 mole of uniquely reactive sulfhydryl residue per 82,000 g. (c) Amino-terminal analysis by the isocyanate procedure gives 6 to 8 moles of methionine residues per 820,000 g; this result was confirmed by dinitrophenylation. Digestion with carboxypeptidase A releases 10.3 moles of alanine per 820,000 g and hydrazinolysis gives 5 to 6 moles of alanine. (d) Peptide maps of tryptic hydrolysates have 54 to 58 ninhydrin-positive spots; the expected number, for 10 identical polypeptide chains, is 61. (e) Isoelectric focusing of arginine decarboxylase which had been reassociated from the 8 S to the 23 S species gave a single peak with a pI of 4.44. A revised purification procedure, suitable for preparing large quantities of enzyme, is also presented.

The amino acid composition of Chalaropsis lysozyme (221 residues) is: Lys, 4; His, 4; Arg, 5; Asp, 23; Thr, 18; Ser, 21; Glu, 11; Pro, 12; Gly, 29; Ala, 27; Val, 8; Met, 3; Ile, 12; Leu, 10; Tyr, 14; Phe, 11; Cys, 2; Trp, 7; and amide ammonia, 23. The calculated molecular weight is 23,385. Tryptophan was determined by both spectrophotometric and chemical methods. Cystine was determined as cysteic acid after performic acid oxidation. Chalaropsis lysozyme contains a single disulfide bridge. Threonine is the NH2 terminus. Carboxypeptidase A released, at identical rates, 1 mole per mole each of leucine, threonine, alanine, and glycine from performic acid-oxidized enzyme. Cyanogen bromide cleavage of lysozyme Ch yields the four peptides expected from the methionine content of 3 residues per molecule. The four peptides were separated by gel filtration chromatography and were ordered to give a partial structural formula. The molecule is clearly very different in over-all structure from that of egg white lysozyme or T4 phage lysozyme.

Peroxyacetyl nitrate does not react with the sulfhydryl groups of "native" ovalbumin, but does react with those of reduced glutathione in the presence of ovalbumin. At pH 4.5 all of the sulfhydryl groups of human hemoglobin react with peroxyacetyl nitrate, whereas at pH 7.2 the reaction is slower and is limited to two to three sulfhydryls per mole of hemoglobin. Ribonuclease in the reactive form is not inactivated by peroxyacetyl nitrate even when the oxidant is in 300-fold molar excess. Amino acid analysis of the protein shows a decrease in cystine and increase in cysteic acid. Reduced ribonuclease is prevented from regaining activity if exposed to peroxyacetyl nitrate in the sulfhydryl form. The inactivation of papain by peroxyacetyl nitrate depends on the amount of free protein sulfhydryl at the time of gas exposure.

Soybean trypsin inhibitor could be partially reduced by treatment with 0.25 m sodium borohydride at 0° without loss of inhibitory activity toward trypsin. From the number of —SH groups released and the carboxymethylcysteine content of the carboxymethylated derivative, it was evident that two —SH groups had been produced under these conditions. A comparison of the diagonal electrophoretic maps of peptic digests of unmodified inhibitor, partially reduced and carboxymethylated inhibitor, and fully reduced and carboxymethylated inhibitor indicated that only one of the two disulfide bonds of the partially reduced derivative had been modified. Peptides in which the cystine residues had been converted to either cysteic acid or carboxymethylcysteinesulfone were isolated from the carboxymethylated derivatives of partially and fully reduced inhibitor. A comparison of their composition with the known sequence of amino acids surrounding the cystine residues of the inhibitor permitted the unequivocal identification of the disulfide bond which had been reduced by sodium borohydride.

Peroxyacetyl nitrate reacts with the sulfhydryl groups of reduced glutathione. Thioester formation accounts for as much as 50% of the reacted sulfhydryl groups. As much as 60% of the reacted sulfhydryl groups can be accounted for as oxidized glutathione. The stoichiometry of the reaction is approximately 3 moles of sulfhydryl reacting per mole of peroxyacetyl nitrate, and this stoichiometry is the same over the pH range from 4.0 to 8.6 in acetate, phosphate, and tris-(hydroxymethyl)aminomethane buffers. Amino acid analyses of reduced and oxidized glutathione after treatment with peroxyacetyl nitrate show that cysteic acid is produced, but not in sufficient quantity to account for the decrease in cysine.

Porcine pancreatic ribonuclease contains 8 half-cystine residues located at the same positions in the 124-amino acid residue sequence as in the bovine enzyme. To determine the disposition of the disulfide bonds, the enzyme was subjected to a preliminary proteolysis with pepsin in 5% formic acid and subsequently to the simultaneous action of trypsin and chymotrypsin at pH 6.5. As judged by gel filtration over Sephadex G-25, only negligible quantities of cystine were present in the glycopeptide fraction. Three major cystine-containing fractions obtained by gel filtration were each subjected to fractionation and analysis by zone electrophoresis on paper. They contained five principal cystine peptides. The amino acid compositions of the cysteic acid peptides derived by oxidation of these cystine peptides, together with appropriate sequence analyses, showed that disulfide bonds link half-cystines I–VI, II–VII, III–VIII, and IV–V in the parent molecule.

Previous studies have described the isolation of mutationally altered proteases in Pseudomonas fragi (Noreau, J., and Drapeau, G.R. (1979) J. Bacteriol, 140, 911-916. In the present study, it is shown that one of these proteases cleaves specifically the peptide bonds on the NH2-terminal side of either aspartic acid or cysteic acid residues in oxidized ribonuclease. With myoglobin as the substrate, a similar specificity was observed except that only four out of the six aspartyl bonds present were hydrolyzed.