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Capnine (2-amino-3-hydroxy-15-methylhexadecane-1-sulfonate) and capnoids ( N -fatty acylated capnine derivatives) are sulfonolipids present in the outer membrane of gliding bacteria in the phylum Bacteroidetes and play a role in their unique gliding motility. They are structurally similar to sphingolipids and are thought to be biosynthesized via a similar pathway. Here we report the identification and biochemical characterization of the capnine biosynthetic enzymes cysteate synthase (CapA) and cysteate-C-fatty acyltransferase (CapB) from the pathogenic gliding bacterium Capnocytophaga ochracea and NAD(P)H-dependent dehydrocapnine reductase CapC from the avian pathogen Ornithobacterium rhinotracheale . CapA catalyzes the formation of cysteate from O -phospho-l-serine and sulfite, and CapB catalyzes the formation of dehydrocapnine from cysteate and 13-methyl-myristoyl-CoA, followed by reduction by CapC. CapA is closely related to cystathionine-β-synthase but distantly related to the archaeal cysteate synthase. Close homologues of CapA, CapB, and the CapA isozyme archaeal cysteate synthase are present in many Bacteroidetes bacteria, including environmental, pathogenic, and human oral and intestinal microbiome bacteria, suggesting the widespread ability of these bacteria to biosynthesize capnine and related sulfonolipids.

The high mobility group box 1 (HMGB1), which is released during acute acetaminophen (APAP) overdose, is thought to mediate a subsequent immune response, particularly hepatic infiltration of macrophages. The redox behavior of HMGB1 and the proteoforms of HMGB1 present in oxidative environments has been the subject of a number of confusing and contradictory studies. Therefore, a stable isotope dilution two-dimensional nanoultrahigh-performance liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry method was developed in order to characterize and quantify oxidative modifications to the cysteine (Cys) residues (Cys-23, Cys-45, and Cys-106) that are present in HMGB1. Disulfide linkages were determined using carbamidoethyl derivatization before and after reduction as well as by direct analysis of disulfide cross-linked peptides. A stable isotope labeled form of HMGB1 was used as an internal standard to correct for sample to sample differences in immunoaffinity precipitation, derivatization, and electrospray ionization. Four discrete HMGB1 proteoforms were found to be released from a hepatocarcinoma cell model of APAP overdose after 24 h. Fully reduced HMGB1 with all three Cys-residues in their free thiol state accounted for 18% of the secreted HMGB1. The proteoform with disulfide between Cys-23 and Cys-45 accounted for 24% of the HMGB1. No evidence was obtained for a disulfide cross-link between Cys-106 and the other two Cys-residues. However, 45% of the HMGB1 formed a cross-link with unidentified intracellular proteins via an intermolecular disulfide bond, and 12% was present as the terminally oxidized cysteic acid. Surprisingly, there was no evidence for the formation of HMGB1 disulfides with GSH or other low molecular weight thiols. Secreted plasma HMGB1 Cys-23/Cys45 disulfide proteoform together with the Cys-106/protein disulfide proteoforms could potentially serve as early biomarkers of hepatoxicity after APAP overdose as well as biomarkers of drug-induced liver injury.

Sulfonolipids (SoLs) are a unique class of sphingolipids featuring a sulfonate group compared to other sphingolipids. However, the biological functions and biosynthesis of SoLs in human microbiota have been poorly understood. Here, we report the discovery and isolation of SoLs from a human opportunistic pathogen Chryseobacterium gleum DSM16776. We show for the first time the pro-inflammatory activity of SoLs with mice primary macrophages. Furthermore, we used both in vivo heterologous expression and in vitro biochemical reconstitution to characterize two enzymes, cysteate synthase and cysteate fatty acyltransferase, that are specifically involved in the biosynthesis of SoLs rather than other sphingolipids. Based on these two SoL-specific enzymes, our bioinformatics analysis showed a wider distribution of SoL biosynthetic genes in microbes that had not been reported as SoL producers. We selected four of these strains and verified their cysteate synthase and cysteate fatty acyltransferase activities in SoL biosynthesis. Considering this wider distribution of SoL-specific biosynthetic enzymes in the context of SoLs' activity in mediating inflammation, a common and fundamental biological process, it may suggest a more comprehensive function of SoLs at play.

The species Streptomyces lincolnensis is known as a producer of lincomycin A, a clinically important lincosamide antibiotic with activity against Gram-positive bacteria. Here, we report that S. lincolnensis produces a new cysteate-containing lactone product, cysteoamide (1), which arises from nonribosomal peptide synthetase-programmed sequential assembly of the monomers phenylacetic acid, valine, cysteate, threonine, β-hydroxyleucine, and β-alanine and subsequent intramolecular cyclization to form a lactone ring. The structure of 1 was determined by combined analysis of NMR and MS spectra, while the amino acid absolute configurations in 1 were assigned by Marfey's analysis following acid hydrolysis. The biosynthetic gene cluster of 1 was defined in the genome of S. lincolnensis by bioinformatics analysis and in vivo genetic study. In addition, in vitro assay revealed that OrfA, a pyridoxal 5'-phosphate-dependent protein, is responsible for the formation of the unusual cysteate unit. Cysteate-containing nonribosomal peptides appear to be widely present in various Streptomyces strains, and this study generates interest in their intrinsic functions that remain poorly understood.

Bacillus subtilis 8 is highly efficient at degrading feather keratin. We observed integrated feather degradation over the course of 48 h in basic culture medium while studying the entire process with scanning electron microscopy. Large amounts of ammonia, sulfite, and L -cysteic acid were detected in the fermented liquid. In addition, four enzymes (gamma-glutamyltranspeptidase, peptidase T, serine protease, and cystathionine gamma-synthase) were identified that play an important role in this degradation pathway, all of which were verified with molecular cloning and prokaryotic expression. To the best of our knowledge, this report is the first to demonstrate that cystathionine gamma-synthase secreted by B. subtilis 8 is involved in the decomposition of feather keratin. This study provides new data characterizing the molecular mechanism of feather degradation by bacteria, as well as potential guidance for future industrial utilization of waste keratin.

The UAG termination codon is generally recognized as the least efficient and least frequently used of the three universal stop codons. This is substantiated by numerous studies in an array of organisms. We present here evidence of a translational readthrough of a mutant nonsense UAG codon in the transcript from the cysteine sulfinic acid decarboxylase ( csad ) gene (ENSDARG00000026348) in zebrafish. The csad gene encodes the terminal enzyme in the taurine biosynthetic pathway. Taurine is a critical amino acid for all animals, playing several essential roles throughout the body, including modulation of the immune system. The sa9430 zebrafish strain (ZDB-ALT-130411-5055) has a point mutation leading to a premature stop codon (UAG) 20 amino acids 5' of the normal stop codon, UGA. Data from immunoblotting, enzyme activity assays, and mass spectrometry provide evidence that the mutant is making a CSAD protein identical to that of the wild-type (XP_009295318.1) in terms of size, activity, and amino acid sequence. UAG readthrough has been described in several species, but this is the first presentation of a case in fish. Also presented are the first data substantiating the ability of a fish CSAD to utilize cysteic acid, an alternative to the standard substrate cysteine sulfinic acid, to produce taurine., Competing Interests: The authors declare no conflict of interest.

Poultry feathers, consisting largely of keratin, are a low-value product of the poultry industry. The safety and digestibility of a dietary protein produced from keratin (KER) was compared to a cysteine-supplemented casein-based diet in a growing rat model for four weeks. KER proved to be an effective substitute for casein at 50% of the total dietary protein, with no changes in the rats' food intake, weight gain, organ weight, bone mineral density, white blood cell counts, liver glutathione, or blood glutathione. Inclusion of KER in the diet reduced total protein digestibility from 94% to 86% but significantly increased total dietary cysteine uptake and subsequent liver taurine levels. The KER diet also significantly increased caecum weight and significantly decreased fat digestibility, resulting in a lower proportion of body fat, and induced a significant increase in blood haemoglobin. KER is therefore a safe and suitable protein substitute for casein, and the cysteic acid in keratin is metabolised to maintain normal liver and blood glutathione levels.

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).

Cupriavidus necator H16 grew exponentially with (R)-cysteate, a structural analogue of aspartate, as sole source of nitrogen in succinate-salts medium. Utilization of cysteate was quantitative and concomitant with growth and with the excretion of the deaminated product (R)-sulfolactate, which was identified thoroughly. The deaminative pathway started with transport of (R)-cysteate into the cell, which we attributed to an aspartate transporter. Transamination to sulfopyruvate involved an aspartate/(R)-cysteate:2-oxoglutarate aminotransferase (Aoa/Coa) and regeneration of the amino group acceptor by NADP⁺-coupled glutamate dehydrogenase. Reduction of sulfopyruvate to (R)-sulfolactate was catalyzed by a (S)-malate/(R)-sulfolactate dehydrogenase (Mdh/Sdh). Excretion of the sulfolactate could be attributed to the sulfite/organosulfonate exporter TauE, which was co-encoded and co-expressed, with sulfoacetaldehyde acetyltransferase (Xsc), though Xsc was irrelevant to the current pathway. The metabolic enzymes could be assayed biochemically. Aoa/Coa and Mdh/Sdh were highly enriched by protein separation, partly characterized, and the relevant locus-tags identified by peptide-mass fingerprinting. Finally, RT-PCR was used to confirm the transcription of all appropriate genes. We thus demonstrated that Cupriavidus necator H16 uses a patchwork pathway by recruitment of 'housekeeping' genes and sulfoacetaldehyde-degradative genes to scavenge for (R)-cysteate-nitrogen.

Endothelial nitric oxide synthase (eNOS) is inhibited by hydrogen peroxide (H(2)O(2)), but the mechanism has not been determined. Thus, the purpose of this study was to delineate the mechanism by which H(2)O(2) inhibits eNOS activity. Using mass spectroscopy, we found that the tetrathiolate cysteine residues 94 and 99 were susceptible to oxidation by H(2)O(2). Molecular modeling predicted that these cysteic acid modifications would disrupt the van der Waals interactions and the hydrogen bonding network mediated by the tetrathiolate cysteines 94 and 99 resulting in changes in quaternary structure, zinc release, and dimer collapse. Using recombinant human eNOS (heNOS) to test the predictions of the molecular modeling we found that H(2)O(2) caused disruption of the heNOS dimer and this was accompanied by zinc release and decreased NO generation. We also found that H(2)O(2) increased the oxidation of tetrahydrobiopterin (BH(4)) to dihydrobiopterin (BH(2)), whereas preincubation of heNOS with excess BH(4) prevented the destruction of zinc tetrathiolate and dimer collapse and preserved activity. Interestingly, we found that the dimmer-stabilizing effect of BH(4) is due to its ability to act as a catalase mimetic. Further, we confirmed that, in ovine aortic endothelial cells, H(2)O(2) could also induce dimer collapse and that increasing cellular BH(4) levels could maintain eNOS in its dimeric form and NO signaling when cells were challenged with H(2)O(2). This study links the inhibitory action of H(2)O(2) on heNOS through the destruction of zinc tetrathiolate metal-binding site and dimer collapse both in vitro and in vivo.

The euryarchaeon Methanosarcina acetivorans has no homologues of the first three enzymes that produce the essential methanogenic coenzyme M (2-mercaptoethanesulfonate) in Methanocaldococcus jannaschii. A single M. acetivorans gene was heterologously expressed to produce a functional sulfopyruvate decarboxylase protein, the fourth canonical enzyme in this biosynthetic pathway. An adjacent gene, at locus MA3297, encodes one of the organism's two threonine synthase homologues. When both paralogues from this organism were expressed in an Escherichia coli threonine synthase mutant, the MA1610 gene complemented the thrC mutation, whereas the MA3297 gene did not. Both PLP (pyridoxal 5'-phosphate)-dependent proteins were heterologously expressed and purified, but only the MA1610 protein catalysed the canonical threonine synthase reaction. The MA3297 protein specifically catalysed a new beta-replacement reaction that converted L-phosphoserine and sulfite into L-cysteate and inorganic phosphate. This oxygen-independent mode of sulfonate biosynthesis exploits the facile nucleophilic addition of sulfite to an alpha,beta-unsaturated intermediate (PLP-bound dehydroalanine). An amino acid sequence comparison indicates that cysteate synthase evolved from an ancestral threonine synthase through gene duplication, and the remodelling of active site loop regions by amino acid insertion and substitutions. The cysteate product can be converted into sulfopyruvate by an aspartate aminotransferase enzyme, establishing a new convergent pathway for coenzyme M biosynthesis that appears to function in members of the orders Methanosarcinales and Methanomicrobiales. These differences in coenzyme M biosynthesis afford the opportunity to develop methanogen inhibitors that discriminate between the classes of methanogenic archaea.

Data from the genome sequence of the aerobic, marine bacterium Roseovarius nubinhibens ISM were interpreted such that 3-sulfolactate would be degraded as a sole source of carbon and energy for growth via a novel bifurcated pathway including two known desulfonative enzymes, sulfoacetaldehyde acetyltransferase (EC 2.3.3.15) (Xsc) and cysteate sulfo-lyase (EC 4.4.1.25) (CuyA). Strain ISM utilized sulfolactate quantitatively with stoichiometric excretion of the sulfonate sulfur as sulfate. A combination of enzyme assays, analytical chemistry, enzyme purification, peptide mass fingerprinting, and reverse transcription-PCR data supported the presence of an inducible, tripartite sulfolactate uptake system (SlcHFG), and a membrane-bound sulfolactate dehydrogenase (SlcD) which generated 3-sulfopyruvate, the point of bifurcation. 3-Sulfopyruvate was in part decarboxylated by 3-sulfopyruvate decarboxylase (EC 4.1.1.79) (ComDE), which was purified. The sulfoacetaldehyde that was formed was desulfonated by Xsc, which was identified, and the acetyl phosphate was converted to acetyl-coenzyme A by phosphate acetyltransferase (Pta). The other portion of the 3-sulfopyruvate was transaminated to (S)-cysteate, which was desulfonated by CuyA, which was identified. The sulfite that was formed was presumably exported by CuyZ (TC 9.B.7.1.1 in the transport classification system), and a periplasmic sulfite dehydrogenase is presumed. Bioinformatic analyses indicated that transporter SlcHFG is rare but that SlcD is involved in three different combinations of pathways, the bifurcated pathway shown here, via CuyA alone, and via Xsc alone. This novel pathway involves ComDE in biodegradation, whereas it was discovered in the biosynthesis of coenzyme M. The different pathways of desulfonation of sulfolactate presumably represent final steps in the biodegradation of sulfoquinovose (and exudates derived from it) in marine and aquatic environments.

In this report, we present in vitro and in vivo evaluation of three 111 In-labeled DTPA conjugates of a cyclic RGDfK dimer: DTPA-Bn-SU016 (SU016 = E[c(RGDfK)] 2; DTPA-Bn = 2-( p-isothioureidobenzyl)diethylenetriaminepentaacetic acid), DTPA-Bn-E-SU016 ( E = glutamic acid) and DTPA-Bn-Cys-SU016 (Cys = cysteic acid). The integrin alpha vbeta 3 binding affinities of SU016, DTPA-Bn-SU016, DTPA-Bn-E-SU016, and DTPA-Bn-Cys-SU016 were determined to be 5.0 +/- 0.7 nM, 7.9 +/- 0.6 nM, 5.8 +/- 0.6 nM, and 6.9 +/- 0.9 nM, respectively, against 125 I-c(RGDyK) in binding to integrin alpha vbeta3, suggesting that E or Cys residue has little effect on the integrin alpha vbeta3 affinity of E[c(RGDfK)] 2. It was also found that the 111 In-labeling efficiency of DTPA-Bn-SU016 and DTPA-Bn-E-SU016 is 3-5 times better than that of DOTA analogues due to fast chelation kinetics and high-yield 111 In-labeling under mild conditions (e.g., room temperature). Biodistribution studies were performed using BALB/c nude mice bearing U87MG human glioma xenografts. 111 In-DTPA-Bn-SU016, 111 In-DTPA-Bn-E-SU016, and 111 In-DTPA-Bn-Cys-SU016 all displayed rapid blood clearance. Their tumor uptake was comparable between 0.5 and 4 h postinjection (p.i.) within experimental error. 111 In-DTPA-Bn-E-SU016 had a significantly lower ( p < 0.01) kidney uptake than 111 In-DTPA-Bn-SU016 and 111 In-DTPA-Bn-Cys-SU016. The liver uptake of 111 In-DTPA-Bn-SU016 was 1.69 +/- 0.18% ID/g at 24 h p.i., while the liver uptake values of 111 In-DTPA-Bn-E-SU016 and 111 In-DTPA-Bn-Cys-SU016 were 0.55 +/- 0.11% ID/g and 0.79 +/- 0.15% ID/g at 24 h p.i., respectively. Among the three 111 In radiotracers evaluated in this study, 111 In-DTPA-Bn-E-SU016 has the lowest liver and kidney uptake and the best tumor/liver and tumor/kidney ratios. Results from metabolism studies indicated that there is little metabolism (<10%) for three 111 In radiotracers at 1 h p.i. Imaging data showed that tumors can be clearly visualized at 4 h p.i. with good contrast in the tumor-bearing mice administered with 111 In-DTPA-Bn-E-SU016. It is concluded that using a glutamic acid linker can significantly improve excretion kinetics of the 111 In-labeled E[c(RGDfK)] 2 from liver and kidneys.

Cysteate and sulfolactate are widespread natural products in the environment, while propanesulfonate, 3-aminopropanesulfonate and propane-1,3-disulfonate are xenobiotics. While some understanding of the bacterial assimilation of cysteate sulfur has been achieved, details of the dissimilation of cysteate and sulfolactate by microbes together with information on the degradation of the xenobiotics have only recently become available. This minireview centres on bacterial catabolism of the carbon moiety in these C(3)-sulfonates and on the fate of the sulfonate group. Three mechanisms of desulfonation have been established. Firstly, cysteate is converted via sulfopyruvate to sulfolactate, which is desulfonated to pyruvate and sulfite; the latter is oxidized to sulfate by a sulfite dehydrogenase and excreted as sulfate in Paracoccus pantotrophus NKNCYSA. Secondly, sulfolactate can be converted to cysteate, which is cleaved in a pyridoxal 5'-phosphate-coupled reaction to pyruvate, sulfite and ammonium ions; in Silicibacter pomeroyi DSS-3, the sulfite is excreted largely as sulfite. Both desulfonation reactions seem to be widespread. The third desulfonation mechanism is oxygenolysis of, e.g. propanesulfonate(s), about which less is known.

The methyl-accepting chemotaxis proteins are a family of receptors in bacteria that mediate chemotaxis to diverse signals. To explore the plasticity of these proteins, we have developed a simple method for selecting cells that swim to target attractants. The procedure is based on establishing a diffusive gradient in semi-soft agar plates and does not require that the attractant be metabolized or degraded. We have applied this method to select for variants of the Escherichia coli aspartate receptor, Tar, that have a new or improved response to different amino acids. We found that Tar can be readily mutated to respond to new chemical signals. However, the overall change in specificity depended on the target compound. A Tar variant that could detect cysteic acid still showed a strong sensitivity to aspartate, indicating that the new receptor had a broadened specificity relative to wild-type Tar. Tar variants that responded to phenylalanine or N-methyl aspartate, or that had an increased sensitivity to glutamate showed a strong decrease in their response to aspartate. In at least some of the cases, the maximal level of sensitivity that was obtained could not be attributed solely to substitutions within the binding pocket. The new tar alleles and the techniques described here provide a new approach for exploring the relationship between ligand binding and signal transduction by chemoreceptors and for engineering new receptors for applications in biotechnology.

Paracoccus pantotrophus NKNCYSA utilizes (R)-cysteate (2-amino-3-sulfopropionate) as a sole source of carbon and energy for growth, with either nitrate or molecular oxygen as terminal electron acceptor, and the specific utilization rate of cysteate is about 2 mkat (kg protein)(-1). The initial degradative reaction is catalysed by an (R)-cysteate : 2-oxoglutarate aminotransferase, which yields 3-sulfopyruvate. The latter was reduced to 3-sulfolactate by an NAD-linked sulfolactate dehydrogenase [3.3 mkat (kg protein)(-1)]. The inducible desulfonation reaction was not detected initially in cell extracts. However, a strongly induced protein with subunits of 8 kDa (alpha) and 42 kDa (beta) was found and purified. The corresponding genes had similarities to those encoding altronate dehydratases, which often require iron for activity. The purified enzyme could then be shown to convert 3-sulfolactate to sulfite and pyruvate and it was termed sulfolactate sulfo-lyase (Suy). A high level of sulfite dehydrogenase was also induced during growth with cysteate, and the organism excreted sulfate. A putative regulator, OrfR, was encoded upstream of suyAB on the reverse strand. Downstream of suyAB was suyZ, which was cotranscribed with suyB. The gene, an allele of tauZ, encoded a putative membrane protein with transmembrane helices (COG2855), and is a candidate to encode the sulfate exporter needed to maintain homeostasis during desulfonation. suyAB-like genes are widespread in sequenced genomes and environmental samples where, in contrast to the current annotation, several presumably encode the desulfonation of 3-sulfolactate, a component of bacterial spores.

The role of phenylalanine 4-monooxygenase (PAH) in the S-oxidation of S-carboxymethyl-L-cysteine (SCMC) in the rat has now been well established in rat cytosolic fractions in vitro. However, the role of PAH in the S-oxidation of SCMC in human cytosolic fractions or hepatocytes has yet to be investigated. The aim of this investigation was to analyse the kinetic parameters of PAH oxidation of both L-phenylalanine (Phe) and SCMC in the human HepG2 cell line in order to investigate the use of these cells as a model for the cellular regulation of SCMC S-oxidation. The experimentally determined Km and V(max) were 7.14 +/- 0.32 mM and 0.85 +/- 0.32 nmole Tyr formed min(-1) x mg protein(-1) using Phe as substrate. For SCMC the values were 25.24 +/- 5.91 mM and 0.79 +/- 0.09 nmole SCMC (RIS) S-oxides formed min(-1) x mg protein(-1). The experimentally determined Km and V(max) for the cofactor BH4 were 6.81 +/- 0.21 microM and 0.41 +/- 0.004 nmole Tyr formed min(-1) x mg protein(-1) for Phe and 7.24 +/- 0.19 microM and 0.42 +/- 0.002 nmole SCMC (R/S) S-oxides formed min(-1) x mg protein(-1) for SCMC. The use of various PAH inhibitors confirmed that HepG2 cells contained PAH and that the enzyme was capable of converting SCMC to its (R) and (S) S-oxide metabolites in an in vitro PAH assay. Thus HepG2 cells have become a useful additional tool for the investigation of the cellular regulation of PAH in the S-oxidation of SCMC.

The term "trichothiodystrophy" (TTD) covers several autosomal recessive diseases whose diagnostic hallmark is short, brittle hair low in sulfur and cystine because of impaired synthesis of high-sulfur matrix protein. Clinical symptoms associated with TTD represent a variable range of abnormalities in organs derived from ectoderm and neuroectoderm. Important laboratory tests of the hair for the diagnosis of TTD comprise polarizing microscopy ("tiger-tail" pattern), electron microscopy, and amino acids analysis of hydrolyzed hair with a special focus on cystine. However, only very few institutions determine the amino acid composition of human hair and nail clippings, which requires special sample preparation including hydrolysis. If no special precautions are taken, quantification of cysteine and cystine becomes inaccurate because of decomposition of these residues during hydrolysis. We therefore performed the sample work-up with azide-dependent oxidation which we have for the first time adapted for analysis of hair and nail clippings. With our control and parent data resembling published data on hair and nail samples, we obtained a decreased proportion of cysteine (half cystine, determined as cysteic acid) in materials obtained from a boy with TTD. Clearly, the method for the quantification of cysteine following sodium azide-dependent oxidation is a suitable and rather convenient approach to the quantification of cyst(e)ine and other amino acids in hair and nail proteins, and is a valuable contribution to the diagnosis of TTD.

The enzymatic conjugation of arginine to the N-termini of proteins is a part of the ubiquitin-dependent N-end rule pathway of protein degradation. In mammals, three N-terminal residues-aspartate, glutamate, and cysteine-are substrates for arginylation. The mouse ATE1 gene encodes a family of Arg-tRNA-protein transferases (R-transferases) that mediate N-terminal arginylation. We constructed ATE1-lacking mouse strains and found that ATE1-/- embryos die with defects in heart development and in angiogenic remodeling of the early vascular plexus. Through biochemical analyses, we show that N-terminal cysteine, in contrast to N-terminal aspartate and glutamate, is oxidized before its arginylation by R-transferase, suggesting that the arginylation branch of the N-end rule pathway functions as an oxygen sensor.

Twenty-three Escherichia coli strains were tested for their ability to use taurine, methanesulfonate, L-cysteate and other alkanesulfonates as sole sulfur sources for growth. One strain was unable to use any of the alkanesulfonates offered as sole sulfur sources for growth but grew with sulfate. Seven strains (class I) used alkanesulfonates for this purpose, but not methanesulfonate or L-cysteate. A further seven strains (class II) grew with all compounds tested, except with L-cysteate, and eight strains (class III) utilized all compounds tested as sulfur sources. Sulfur assimilation from methanesulfonate and L-cysteate was absolutely dependent on the ssuEADCB operon that encodes an alkanesulfonate uptake system (SsuABC) and a two-component monooxygenase (SsuDE) involved in the release of sulfite from alkanesulfonates. Long-term exposure of class I strains to methanesulfonate and of class II strains to L-cysteate selected for derivatives that utilized these two sulfur sources as efficiently as sulfate. The nucleotide sequence of the ssuEADCB operon in the methanesulfonate- and L-cysteate-utilizing derivative EC1250Me+ was identical to that in the class I wild-type EC1250. Gain of the ability to utilize methanesulfonate and L-cysteate as sulfur sources thus appears to result from increased expression of ssu genes rather than from a change in the quality of one or several of the Ssu proteins.

Purpose: To determine whether substitution of the potential phosphorylation sites of bovine rhodopsin's carboxyl-terminal region with the acidic residues aspartic acid, glutamic acid, or cysteic acid promotes the activation of arrestin., Methods: Three peptide analogues of the 19-residue carboxyl-terminal region of rhodopsin (330-348) were synthesized: the fully phosphorylated peptide (7P-peptide), the peptide with all potential phosphorylation sites substituted with glutamic acid (7E-peptide), and the peptide with the phosphorylation sites substituted with cysteic acid (7Cya-peptide). The peptides were tested in assays in which the 7P-peptide had previously been shown to have an effect. Rhodopsin with glutamic acid (Etail) or aspartic acid (Dtail) substituted for the phosphorylation sites in rhodopsin were constructed and expressed in COS-7 cells and tested in an in vitro assay., Results: Earlier work has demonstrated that the 7P-peptide activates arrestin, showing induction of arrestin binding to light-activated unphosphorylated rhodopsin, inhibition of the light-induced phosphodiesterase (PDE) activity in rod outer segments (ROS) with excess arrestin, increase in the initial rapid proteolysis of arrestin by trypsin, and enhanced reactivity of one of arrestin's sulfhydryl groups with inhibition of the reactivity of another. None of these effects was observed in the presence of 7E-peptide or 7Cya-peptide. The 7Cya-peptide inhibited the PDE activity in ROS, but the same effect was observed both in the presence and the absence of excess arrestin. Because none of the other effects was observed with the 7Cya-peptide, the authors conclude that the 7Cya-peptide does not activate arrestin, but acts, probably nonspecifically, through some other part of the transduction system. Considerable arrestin-mediated rhodopsin inactivation was observed with both the Etail and the Dtail mutant, although these substitutions did not yield rhodopsins that were equivalent to phosphorylated rhodopsin., Conclusions: These results, taken together, suggest that the negative charge due to phosphates in the carboxyl-terminal region of rhodopsin are required for the full activation of arrestin and that acidic amino acids (carboxyl and sulfonic) do not mimic the negative charge of phosphorylated residues.

Several low-molecular-weight sulfonates were added to microbial mat slurries to investigate their effects on sulfate reduction. Instantaneous production of sulfide occurred after taurine and cysteate were added to all of the microbial mats tested. The rates of production in the presence of taurine and cysteate were 35 and 24 microM HS(-) h(-1) in a stromatolite mat, 38 and 36 microM HS(-) h(-1) in a salt pond mat, and 27 and 18 microM HS(-) h(-1) in a salt marsh mat, respectively. The traditionally used substrates lactate and acetate stimulated the rate of sulfide production 3 to 10 times more than taurine and cysteate stimulated the rate of sulfide production in all mats, but when ethanol, glycolate, and glutamate were added to stromatolite mat slurries, the resulting increases were similar to the increases observed with taurine and cysteate. Isethionate, sulfosuccinate, and sulfobenzoate were tested only with the stromatolite mat slurry, and these compounds had much smaller effects on sulfide production. Addition of molybdate resulted in a greater inhibitory effect on acetate and lactate utilization than on sulfonate use, suggesting that different metabolic pathways were involved. In all of the mats tested taurine and cysteate were present in the pore water at nanomolar to micromolar concentrations. An enrichment culture from the stromatolite mat was obtained on cysteate in a medium lacking sulfate and incubated anaerobically. The rate of cysteate consumption by this enrichment culture was 1.6 pmol cell(-1) h(-1). Compared to the results of slurry studies, this rate suggests that organisms with properties similar to the properties of this enrichment culture are a major constituent of the sulfidogenic population. In addition, taurine was consumed at some of highest dilutions obtained from most-probable-number enrichment cultures obtained from stromatolite samples. Based on our comparison of the sulfide production rates found in various mats, low-molecular-weight sulfonates are important sources of C and S in these ecosystems.

Heparin potentiates the mitogenic activity of FGF-1 by increasing the affinity for its receptor and by extending its biological half-life. During the course of labeling human FGF-1 with Na(125)I and chloramine T, it was observed that the protein lost its ability to bind to heparin. In contrast, bovine FGF-1 retained its heparin affinity even after iodination. To localize the region responsible for the lost heparin affinity, chimeric FGF-1 proteins were constructed from human and bovine FGF-1 expression constructs and tested for their heparin affinity after iodination. The results showed that the C-terminal region of human FGF-1 was responsible for the loss of heparin affinity. This region harbors a single tyrosine residue in human FGF-1 in contrast to a phenylalanine at this position in bovine FGF-1. Mutating this tyrosine residue in the human FGF-1 sequence to phenylalanine did not restore the heparin affinity of the iodinated protein. Likewise, changing the phenylalanine to tyrosine in the bovine FGF-1 did not reduce the ability of the iodinated protein to bind to heparin. In contrast, a mutant human FGF-1 that has cysteine-131 replaced with serine (C131S) was able to bind to heparin even after iodination while bovine FGF-1 (S131C) lost its binding affinity to heparin upon iodination. In addition, the human FGF-1 C131S mutant showed a decrease in homodimer formation when exposed to CuCl(2). Molecular modeling showed that the heparin-binding domain of FGF-1 includes cysteine-131 and that cysteine-131, upon oxidation to cysteic acid during the iodination procedures, would interact with lysine-126 and lysine-132. This interaction alters the conformation of the basic residues such that they no longer bind to heparin.

Anoxic, fresh-water enrichment cultures to oxidize different organosulfonates were set up with nitrate, ferric iron or sulfate as electron acceptors. Pure cultures were easily obtained with two naturally occurring sulfonates, cysteate (2-amino-3-sulfopropionate) and taurine (2-aminoethanesulfonate), under nitrate-reducing conditions. These two sulfonates were also oxidized during reduction of iron(III), though isolation of pure cultures was not successful. One nitrate-reducing cysteate-oxidizing bacterium, strain NKNCYSA, was studied in detail. It was identified as Paracoccus pantotrophus. Eighteen sulfonates were tested, and the organism degraded cysteate, taurine, isethionate (2-hydroxyethanesulfonate), sulfoacetate or 3-aminopropanesulfonate with concomitant reduction of nitrate, presumably to molecular nitrogen. The carbon skeleton of these substrates was converted to cell material and, presumably, CO2. The amino group was released as ammonia and the sulfono moiety was recovered as sulfate. Cell-free extracts of P. pantotrophus NKNCYSA contained constitutive L-cysteate:2-oxoglutarate aminotransferase (EC 2.6.1.-) and glutamate dehydrogenase (EC 1.4.1.4). Taurine:pyruvate aminotransferase, in contrast, was inducible.

Excitatory amino acid transporters in the central and peripheral nervous systems of insects are thought to assist in maintaining glutamate concentrations in the resting synapse below the activation threshold of glutamate receptors. We have isolated a cDNA from the caterpillar Trichoplusia ni which encodes a high-affinity Na+-dependent glutamate transporter, designated TrnEAAT1. The deduced amino acid sequence shows strong identity with known members of the vertebrate Na+- and K+-dependent amino acid transporter family. Expression of the insect transporter mRNA was predominantly localized in the caterpillar brain. The function of the TrnEAAT1 protein was analyzed in cultured insect cells using a baculovirus expression system. Cells infected with the recombinant virus were found to exhibit a 50-fold increase in ability to accumulate labeled L-glutamate compared to mock-infected cultures, and this activity was shown to be Na+-dependent. Transport activity was further demonstrated by chromatographic identification of various glutamate analogues accumulated by infected cells. Various glutamate uptake inhibitors were used to outline the pharmacological properties of the cloned transporter and to compare it with known mammalian transporters. Despite the significant differences between insect and vertebrate physiology, the characteristics of the respective transporters were found to be remarkably similar.

Organosulfonates are important natural and man-made compounds, but until recently (T. J. Lie, T. Pitta, E. R. Leadbetter, W. Godchaux III, and J. R. Leadbetter. Arch. Microbiol. 166:204-210, 1996), they were not believed to be dissimilated under anoxic conditions. We also chose to test whether alkane- and arenesulfonates could serve as electron sinks in respiratory metabolism. We generated 60 anoxic enrichment cultures in mineral salts medium which included several potential electron donors and a single organic sulfonate as an electron sink, and we used material from anaerobic digestors in communal sewage works as inocula. None of the four aromatic sulfonates, the three unsubstituted alkanesulfonates, or the N-sulfonate tested gave positive enrichment cultures requiring both the electron donor and electron sink for growth. Nine cultures utilizing the natural products taurine, cysteate, or isethionate were considered positive for growth, and all formed sulfide. Two clearly different pure cultures were examined. Putative Desulfovibrio sp. strain RZACYSA, with lactate as the electron donor, utilized sulfate, aminomethanesulfonate, taurine, isethionate, and cysteate, converting the latter to ammonia, acetate, and sulfide. Strain RZATAU was identified by 16S rDNA analysis as Bilophila wadsworthia. In the presence of, e.g., formate as the electron donor, it utilized, e.g., cysteate and isethionate and converted taurine quantitatively to cell material and products identified as ammonia, acetate, and sulfide. Sulfite and thiosulfate, but not sulfate, were utilized as electron sinks, as was nitrate, when lactate was provided as the electron donor and carbon source. A growth requirement for 1,4-naphthoquinone indicates a menaquinone electron carrier, and the presence of cytochrome c supports the presence of an electron transport chain. Pyruvate-dependent disappearance of taurine from cell extracts, as well as formation of alanine and release of ammonia and acetate, was detected. We suspected that sulfite is an intermediate, and we detected desulfoviridin (sulfite reductase). We thus believe that sulfonate reduction is one aspect of a respiratory system transferring electrons from, e.g., formate to sulfite reductase via an electron transport system which presumably generates a proton gradient across the cell membrane.

In mammalian cells, the basal Na+-dependent uptake for many of the neutral amino acids is mediated by a transport activity designated System ASC. A cloned human brain cDNA sequence, ASCT1, encodes a Na+-dependent neutral amino acid transport activity that exhibits a substrate specificity similar to that commonly associated with System ASC. However, the characteristics of ASC activity varies significantly between cell types and not all tissues contain detectable levels of ASCT1 mRNA. A unique property of System ASC activity is an altered substrate selectivity such that at pH values below 7.4 anionic amino acids function as inhibitors and substrates. The experiments in this report were designed to determine if the cloned ASCT1 transporter exhibited this pH-dependent anionic transport. Following transfection of HeLa cells with the ASCT1 cDNA, transport strongly favored neutral zwitterionic) amino acids when uptake was measured at a physiologic pH value of 7.5. However, lowering the assay pH to 5.5 significantly enhanced the interaction of the ASCT1 carrier with anionic amino acids such as cysteate, in a pH-dependent manner. The apparent pK for the titratable group was in the range of 6.5-7.0. These results provide evidence that the human brain ASCT1 transporter exhibits the most distinguishing characteristic known for System ASC and provides a model system to investigate the molecular basis for this shift in substrate acceptance.

Regularities of formation of a pool of free amino acids and their derivatives in the liver of rats were studied during L-2-oxothiazoidin-4-carboxylate-induced glutathione synthesis activation. It was shown that an increase in concentrations of sulfur-containing (cysteate, cysteine) and branched-chain (valine, isoleucine, leucine) amino acids was the most typical change. The correlation and linear-discriminant analysis of the whole range of the compounds studied in the dynamics of the experimental process has proved high informativeness of glycogenic amino acid levels, activation of the whole chain of synthesis and degradation of thiol-containing amino acids as well as determination of the obtained shifts in concentrations of reduced glutathione.

The displacement of Na(+)-dependent D-[3H]-aspartate binding by unlabeled D-aspartate or the inhibitors DL-threo-beta-hydroxyaspartate, L-cysteate, L-glutamate, dihydrokainate, DL-alpha-aminoadipate, alpha-methyl-DL-glutamate, and 1-aminocyclobutane-cis-1,3-dicarboxylate was used to characterize the high-affinity glutamate/aspartate uptake site in human cerebral cortex. Synaptosomal membranes were prepared from tissue obtained at autopsy from nondemented control, Alzheimer's disease (AD), and diffuse Lewy body disease (DLBD) cases. Areas that are damaged in AD (midtemporal, frontal, caudal cingulate, and hippocampal cortices) were compared with those that are spared (occipital and motor cortices). Profiles of the affinities (Ka values) of the ligands showed that areas spared from damage in AD cases differed significantly from equivalent areas in control (p < 0.001) and DLBD (p < 0.001) cases and also from areas susceptible to damage in the same AD cases (p < 0.001). Areas susceptible to damage in AD showed comparable profiles across the three case groups (p = 0.980). The glutamate/aspartate uptake site may be regionally variant in AD cases, and this may underlie local excitotoxicity. D-[3H]Aspartate binding site density was significantly lower in both dementia groups (control vs. AD, p < 0.001; control vs. DLBD, p = 0.009; but AD vs. DLBD, p = 0.528); within-group differences were not significant (control, p = 0.874; AD, p = 0.285; DLBD, p = 0.741).

Transport of L-glutamate in purified sarcolemmal vesicles from rat heart was Na+ dependent and characterized by an apparent affinity value of 0.149 +/- 0.04 mM (n = 6). Na+ binding to the carrier was not very specific, since an inwardly directed K+ gradient could also increase vesicular uptake of L-glutamate. Uptake of L-glutamate into sarcolemmal vesicles was inhibited by L-aspartate and L-cysteinate. These characteristics of glutamate uptake are typical of transport via system XAG-. Acidification of either the intravesicular or the extravesicular space led to a significant increase in L-glutamate transport. This increase was more pronounced when the intravesicular space had been acidified. Thus intracellular acidosis of hypoxic or ischemic myocardium is directly linked to an increased uptake of glutamate.

The ability of cats to use dietary cysteic acid as a precursor for taurine was examined. For 39 wk, six groups of four specific-pathogen-free cats each were fed purified diets that provided either 1.25 g taurine or 0, 1.0, 2.0, 10 or 20 g cysteic acid/kg diet. Concentrations of taurine in plasma and whole blood were measured weekly for 13 wk and monthly until 39 wk. Muscle concentrations of taurine at 12 wk were measured on a biopsy sample of the semitendinosus muscle. Fecal and urinary excretions of taurine and cysteic acid at 17 wk were measured. Concentrations of taurine in plasma and whole blood were linearly and positively correlated with cysteic acid in the diet (r2 = 0.88). Urinary taurine, a major route of excess taurine excretion, was positively correlated with the cysteic acid concentration of the diet (r2 = 0.94). Muscle and whole-blood taurine concentrations were also positively correlated (r2 = 0.96) with dietary cysteic acid concentration. Gross and histopathological examinations were performed on cats fed diets containing 20 g cysteic acid/kg diet. No adverse gross clinical signs were observed in cats fed any diets containing cysteic acid; minor histopathologic changes of the pancreas and thyroid were found in three of four cats fed 20 g cysteic acid/kg diet. Results suggest that cats are able to use dietary cysteic acid as a precursor for taurine biosynthesis.

Three sulfonates were tested for their ability to serve as nutrients for Hansenula wingei, Rhodotorula glutinis, Trigonopsis variabilis and Saccharomyces cerevisiae. Cysteate, taurine and isethionate, under aerobic conditions, could be utilized as sources of sulfur, although in some instances final cell yields were less than those obtained with an equimolar amount of sulfate-sulfur. Sulfonate assimilation by S. cerevisiae resembled that of bacteria (reported earlier by us) in several aspects: first, sulfate-S was used in preference to that of sulfonate, when both were present; second, mutants unable to use sulfate as a source of sulfur because of deficiencies in ATP sulfurylase, adenylylsulfate kinase (APS kinase) or PAPS reductase were able to utilize sulfonates; and third, mutants deficient in sulfite reductase were unable to utilize sulfonates.

Excitotoxic amino acids contain two acidic groups, but cysteine represents an exception to this rule. The hypothesis that cysteine toxicity is mediated by the oxidized and diacidic metabolites cysteine sulphinate and/or cysteate was tested in the present study. The issue was approached in three different ways. Firstly, the distribution of brain injury after subcutaneous administration of cysteine (1 mg/g) to 4-day-old rats was compared with that caused by cysteine sulphinate (3 mg/g). Secondly, the effects of excitatory amino acid receptor antagonists on cysteine and cysteine sulphinate toxicity were investigated. Thirdly, the cerebral concentrations of cysteine sulphinate were determined after cysteine administration and compared with those obtained after cysteine sulphinate injection. The cerebral cortex was the region most vulnerable to cysteine toxicity, followed by the hippocampus (especially the medial subicular neurons), amygdala, caudoputamen, cerebellum and septum. Pronounced extravasation of red blood cells was observed in lesioned areas. One day after cysteine administration, the injury was infarction-like and sharply demarcated. Cysteine sulphinate-induced damage resembled cysteine-induced lesions in some respects: the anterior cingulate and retrosplenial cortices, as well as medial subicular cells, were quite vulnerable. However, the differences prevailed. Cysteine sulphinate, but not cysteine, killed neurons of the superficial part of the tectum, the medial habenula, the ventromedial hypothalamus and the arcuate nucleus. Further, while cysteine toxicity was prominent in deep cortical layers, cysteine sulphinate preferentially damaged superficial cortical neurons. Cysteine toxicity was abolished by pretreatment with MK-801, a selective NMDA antagonist, but not by 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, a selective AMPA receptor blocker. In contrast, the considerably smaller lesion seen after cysteine sulphinate administration was only partially prevented by MK-801. Large (19-fold) increases in cortical cysteine sulphinate concentration were noted after injection of a toxic dose of cysteine. This corresponds to 90 nmol cysteine sulphinate/g protein. The cysteate concentration was not increased above the detection limit. Injection of a toxic dose of cysteine sulphinate elevated cysteine sulphinate concentration in the frontomedial cortex (a region consistently injured by cysteine sulphinate) almost three orders of magnitude more than that observed after cysteine administration. Taken together, these results strongly suggest that neither cysteine sulphinate nor cysteate alone mediate cysteine toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

A variety of sulphonates were tested for their ability to serve as nutrients for Escherichia coli, Enterobacter aerogenes and Serratia marcescens. Cysteate, taurine and isethionate could not serve as sole sources of carbon and energy but, under aerobic conditions, could be utilized as sources of sulphur. Both sulphate and sulphonate supported equivalent cell yields, but the generation times varied with the sulphonate being metabolized. The sulphonate-S of HEPES buffer, dodecane sulphonate and methane sulphonate was also utilized by some strains, whereas the sulphonate-S of taurocholate was not. None of the sulphonates tested served as a sulphur source for growth under anaerobic conditions. Sulphonate utilization appears to be a constitutive trait; surprisingly, however, cells of E. coli and Ent. aerogenes utilized sulphate-S in preference to that of sulphonate, when both were present. E. coli mutants unable to use sulphate as a source of sulphur because of deficiencies in sulphate permease, ATP sulphurylase, adenylylsulphate kinase (APS kinase) or glutaredoxin and thioredoxin were able to utilize sulphonates; hence sulphate is not an obligatory intermediate in sulphonate utilization. However, mutants deficient in sulphite reductase were unable to utilize sulphonates; therefore, this enzyme must be involved in sulphonate utilization, though it is not yet known whether it acts upon the sulphonates themselves or upon the inorganic sulphite derived from them.

Protein-thiol mixed disulfide formation has been implicated as a possible mechanism for the protein-protein aggregation in cataractogenesis. Previously we have found that two species of thiols are bound to proteins: GSH (PSSG) and cysteine (PSSC). In this study we found these molecules are ubiquitous in animal lenses with the highest levels in human, dog and rat, and lowest in monkey. However, the relative amount of PSSG to PSSC is quite different in each animal species. The ratio of PSSG/PSSC was 1/10 in rat lens, 4/1 in human and dog lenses and 2/1 in monkey lens. We also studied the effect of aging on the protein-thiol mixed disulfide levels in human donor lenses between 3 months and 88 years. Lens GSH levels were inversely related to age, similar to earlier reports, but PSSC levels increased linearly with age. PSSG levels showed a triphasic pattern with an initial sharp and linear increase from a low content in infants to a highest level at age 20; fell back about 50% to a new steady state level that was maintained for four more decades; finally, above 60 years, the levels in some lenses were two to three-fold higher while some lenses remained at the same low value. PSSC in human lens appeared to concentrate in the nuclear region and in the water insoluble proteins while PSSG was more evenly distributed. Besides the aging effect on the protein-thiol mixed disulfides, oxidative stress also potentiated protein modification in the human lens.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The effect of sulphur-containing analogues of glutamate and aspartate on the membrane current of glial cells was studied by whole-cell clamping Müller cells isolated from the salamander retina. 2. L-Cysteic acid (CA), L-cysteinesulphinic acid (CSA), L-homocysteic acid (HCA), L-homocysteinesulphinic acid (HCSA) and S-sulpho-L-cysteine (SC) all evoked an inward membrane current that was large at negative potentials, and was smaller (but did not reverse) at more positive potentials up to +30 mV. 3. Removal of external sodium ions abolished the amino acid-evoked currents. Whole-cell clamping with pipettes containing no potassium led to a rapid suppression of the currents, that did not occur when potassium was included in the pipette. 4. The dependence of the currents on sulphur-containing amino acid concentration obeyed first-order Michaelis-Menten kinetics. The current evoked by co-application of L-glutamate and a sulphur-containing analogue was smaller than the sum of the currents produced by glutamate alone and by the sulphur analogue alone. 5. These data are consistent with the sulphur amino acid-evoked current being caused by uptake on the electrogenic glutamate uptake carrier, which co-transports an excess of Na+ ions into the cell, and counter-transports one K+ ion out of the cell. 6. The apparent Km (Michaelis-Menten constant) values for activation of uptake by CA (6 microM) and by CSA (60 microM) are low enough for uptake on the glutamate uptake carrier to be a plausible mechanism for terminating the postulated neurotransmitter action of these agents. However, the apparent Km values for uptake of HCA (2.95 mM), HCSA (1.65 mM) and SC (greater than 1 mM) are much higher than the EC50 (half-maximal effective concentration) concentrations for these agents' activation of NMDA (N-methyl-D-aspartate) channels. 7. Comparing the concentrations of sulphur amino acids needed to activate NMDA channels with their rate of uptake suggests that their potency for causing excitotoxic damage should follow the sequence HCA greater than SC greater than HCSA greater than Glu greater than CSA greater than Asp greater than CA.

Uptake of L-[35S]cysteic acid (L-CA) in rat synaptic membrane vesicles was investigated. Preincubation with either 10 mM L-glutamic acid (L-Glu), 25 mM L-CA, 10 mM DL-homocysteic acid, or 25 mM DL-2-amino-4-phosphonobutyrate on membrane vesicles enhanced L-[35S]CA and L-[3H]Glu uptake. Na+ (5 mM) and omission of Cl- from the assay medium decreased L-[35S]CA uptake into both 10 mM L-Glu-loaded and non-loaded membrane vesicles. The anion transport blockers, 4-acetamide-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS) and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), inhibited L-[35S]CA uptake in a dose-dependent manner. The maximal uptake rate for L-[35S]CA was decreased by 50 microM SITS, while the apparent Km value of L-CA was not changed. SITS increased the EC50 value of Cl- for L-[35S]CA uptake from 5 mM to 10 mM with reduction of the maximal effect. These results suggested that L-[35S]CA uptake into synaptic membrane vesicles was mediated by a SITS-sensitive hetero-exchange transport with non-labeled substrates.

Various bile acids were added to cultures of encapsulated strains of Staphylococcus aureus growing in serum-soft agar medium of brain heart infusion broth. We examined effects of these compounds on cellular characteristics such as growth type, cell volume index, clumping factor reaction, slime yield, taurine content, and L-(--)-cysteic acid decarboxylase activity. Upon addition to the medium of either taurochenodeoxycholic acid, taurocholic acid (25 to 50 microgram/ml), or cholic acid (10 to 25 microgram/ml), the colonial morphology of taurine-positive cells (strain S-7) was altered from the diffuse to the compact type in serum-soft agar. Also, the titer of the clumping factor reaction increased, while the cell volume index and slime yield were markedly decreased. Tauro-bile acids, including taurocholic acid, taurochenodeoxycholic acid, taurodehydrocholic acid, and taurodeoxycholic acid (50 microgram/ml) inhibited the synthesis of taurine and resulted in decreased L-(--)-cysteic acid decarboxylase activity. Among all of the derivatives cholic acid itself was found to inhibit slime production and L-(--)-cysteic acid decarboxylase activity to the greatest extent. Glyco-bile acid derivatives and taurolicholic acid (50 to 100 microgram/ml) had no effect on L-(--)-cysteic acid decarboxylase activity. Compounds such as glycodeoxycholic acid (50 to 100 microgram/ml) had no effect upon any of the cellular characteristics tested. No effect was observed upon addition of any of these compounds to cultures of the taurine-negative strain (T-26-B). We did find a correlation between the inhibition of taurine biosynthesis and decreased slime production. Electron micrographs indicated that this encapsulated strain was converted to an unencapsulated state in the presence of bile acids.

In view of its interesting pharmacological and physiological functions, the enzymic synthesis of the taurine or the beta-aminoethan- alpha-sulfonic acid is described. This compound has been prepared by decarboxylation with rat heart homogenate and at room temperature of cysteic acid obtained by KMnO4 cysteine oxidation. The taurine prepared has been identified by paper and thin-layer chromatography with a taurine pure sample.

Specific binding sites for cysteine sulfinic acid, an excitatory amino acid, in crude synaptic membrane fractions of rat cerebral cortex were examined, using L-[35S]cysteic acid as a ligand. Two specific binding systems of [35S]cysteic acid were found, one Na+-dependent and the other Na+-independent. The Na+-independent specific binding of [35S]cysteic acid was saturable, with a Kd of 474 nM and Bmax of 3.29 pmol/mg protein. The binding was optimal at pH 7.4 and at 37 degrees C. Treatment of the membranes with proteases, concanavalin A, or Triton X-100 markedly reduced the binding. Of various compounds related to cysteic acid, L-cysteine sulfinic acid was the most effective competitor of this binding. These results indicate the existence of an Na+-independent specific binding site for cysteic acid in the synaptic membrane of rat cerebral cortex, which may be different from that for glutamate. Possible involvement of cysteine sulfinic acid as an endogenous ligand for this binding site is discussed.

The retinal uptake and metabolism of cysteine, a precursor for taurine biosynthesis, were analysed using the bull frog. The [14C] cysteine uptake into isolated retina had some specific properties: It was rather temperature independent, required Na ions, was inhibited by ouabain but not by dinitrophenol, and exhibited saturation kinetics composed of two components. When retinal homogenate was incubated with 12-30 microM of L-[U-14C]cysteine, the accumulation of labeled alanine, cysteine sulfinic acid (CSA), cysteic acid (CA), hypotaurine, and taurine was detected. The metabolic conversions of [14C] cysteine to labeled alanine, hypotaurine, and taurine were linear over 90 minutes. Prolonged light adaptation (3 weeks) induced a significant reduction in the formation of labeled CA, CSA, hypotaurine, and taurine from [14C] cysteine. On the other hand, it was found that in dark-adapted retinae, the formation of labeled taurine from [14C] cysteine increased significantly in spite of the reduction in the formation of labeled CA. These results indicate that biosynthetic pathways exist for taurine from cysteine in frog retina, and that these metabolic pathways are involved in the regulation of retinal taurine content under continuous visual adaptation.

Single sections of superficial proximal convolutions of rat kidney were microperfused in vivo and in situ. The perfusion fluids contained radioactively labelled L- or D-aspartate, L-glutamate, L-pyroglutamate, or N-methyl-D-aspartate. L-gamma-Carboxyglutamate as well as the other amino acids were added in the unlabelled form. Results. L- and D-Aspartate (0.073 mmol X 1(-1)) are quickly resorbed at about the same rate. D-Aspartate resorption was blocked by L-aspartate (5 mmol X 1(-1)) but not by beta-alanine (5 mmol X 1(-1)). L-Aspartate resorption was inhibited by L-glutamate (2 mmol X 1(-1)) but not by D-glutamate, L-asparagine, L-phenylalanine or by succinate (2 mmol X 1(-1), each). The fast resorption of L-glutamate (0.073 mmol X 1(-1)) was blocked by D-aspartate, L-cysteate (2 mmol X 1(-1)), but not by 3-mercaptopicolinic acid (0.15 mmol X 1(-1)), L-glutamine, 2-oxoglutarate, taurine, N-methyl-L-glutamate or kainic acid (2 mmol X 1(-1), each). L-gamma-Carboxyglutamate (0.66 mmol X 1(-1)) and N-methyl-D-aspartate (2 mumol X 1(-1)) were found to be resorbed only at an extremely small rate. L-Pyroglutamate (0.076 mmol X 1(-1)) resorption was not influenced by L-glutamate (1 mmol X 1(-1)). Fractional excretion of gamma-carboxyglutamate was 7-25% (L-form) or 45-70% (D-form) at an artificially elevated plasma level of 12 mumol X 1(-1). It is concluded that L- and D-aspartate, L-glutamate, L-cysteate and, to a much smaller extent, L-gamma-carboxyglutamate, are accepted by the tubular resorption mechanism highly specific for "acidic" amino acids. N-Substitution, the amidation of the beta- or gamma-carboxyl group, or the removal of the alpha-amino moiety almost completely abolish the ability of such compounds to be resorbed via this carrier; N-methylated or gamma-carboxylated derivatives of "acidic" amino acids are not resorbed at all from the proximal tubule. The resorption of glutamate, but not of aspartate, is highly stereospecific.

beta-Sulfopyruvic acid (2-carboxy-2-oxoethanesulfonic acid) is prepared in greater than 90% yield by reaction of bromopyruvic acid with sodium sulfite. beta-[35S]Sulfopyruvate is prepared by transamination between [35S]cysteinesulfonate (cysteate) and alpha-ketoglutarate using mitochondrial aspartate aminotransferase isolated from rat liver. Following either chemical or enzymatic synthesis, the crude reaction product is conveniently purified by chromatography on Dowex 1; beta-sulfopyruvate is isolated as the stable, water-soluble dilithium salt. beta-Sulfopyruvate is shown to be an alternative substrate of mitochondrial malate dehydrogenase; in the presence of 0.25 mM NADH, beta-sulfopyruvate is reduced with an apparent Km of 6.3 mM and a Vmax equal to about 40% of that observed with oxaloacetate. This finding forms the basis of a convenient spectrophotometric assay of beta-sulfopyruvate.

Gliding bacteria of the genus Cytophaga synthesize sulfonolipids (1,2) that contain capnine (1-deoxy-15-methylhexadecasphinganine-1-sulfonic acid). Studies of the incorporation of radiolabeled compounds by C. johnsonae show that cysteate is utilized preferentially to both cystine and inorganic sulfate as a precursor of capnine sulfur and to both cystine and serine as a precursor of carbons 1 and 2 of capnine. The results are consistent with a pathway in which capnine is formed by condensation of cysteate with a fatty acyl CoA. Cystine, added as the sole sulfur source in the presence of glucose, provides the sulfur but not the carbon for capnine. Hence, these cells form cysteate not by direct oxidation of cystine (or cysteine), but by transfer of its sulfur to a different carbon compound.

Synaptosomes isolated from rat brain accumulated cysteic acid by a high-affinity transport system (Km = 12.3 +/- 2.1 microM; Vmax = 2.5 nmol mg protein-1 min-1). This uptake was competitively inhibited by aspartate (Ki = 13.3 +/- 1.8 microM) and cysteine sulfinate (Ki = 13.3 +/- 2.3 microM). Addition of extrasynaptosomal cysteate, aspartate, or cysteine sulfinate to synaptosomes loaded with [35S]cysteate induced rapid efflux of the cysteate. This efflux occurred via stoichiometric exchange of amino acids with half-maximal rates at 5.0 +/- 1.1 microM aspartate or 8.0 +/- 1.3 microM cysteine sulfinate. Conversely, added extrasynaptosomal cysteate exchanged for endogenous aspartate and glutamate with half-maximal rates at 5.0 +/- 0.4 microM cysteate. In the steady state after maximal accumulation of cysteate, the intrasynaptosomal cysteate concentrations exceeded the extrasynaptosomal concentrations by up to 10,000-fold. The measured concentration ratios were the same, within experimental error, as those for aspartate and glutamate. Depolarization, with either high [K+] or veratridine, of the plasma membranes of synaptosomes loaded with cysteate caused parallel release of cysteate, aspartate, and glutamate. It is concluded that neurons transport cysteate, cysteine sulfinate, aspartate, and glutamate with the same transport system. This transport system catalyzes homoexchange and heteroexchange as well as net uptake and release of all these amino acids.

The biosynthesis of sulphoquinovosyl diacylglycerol in germinating alfalfa seeds has been examined. Some incorporation of [35S]sulphate into the lipid occurs before chlorophyll production anh this is unaffected by chloramphenicol. Cysteic acid, molybdate, sulphite and sulpholactic acid all reduce incorporation of [35S]sulphate into sulphoquinovosyl diacylgylcerol. Some comparisons are made with other seed types. The results indicate that sulphoquinovosyl diacylgylcerol synthesis in alfalfa probably proceeds by a pathway similar to that in Euglena.

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.

Net renal tubular secretion of endogenous beta-amino acids (taurine, beta-alanine, beta-aminoisobutyric acid) and their endogenous analogue (L-cysteic acid) was revealed in the olive sea snake, Aipysurus laevis, and in the garter snake, Thamnophis sirtalis, by renal clearance methods. The net secretory rates ranged from 16 to 795 nmol X kg-1 X h-1, with taurine being secreted at the highest rates. These rates of secretion are comparable to those observed in marine fish, the only other group of vertebrates exhibiting renal tubular secretion of L-amino acids under physiological conditions. However, only some snakes (8-58%) demonstrated net tubular secretion; the others (0-58%) showed net tubular reabsorption of these amino acids. Net tubular secretion was consistently observed whenever the filtered load of the amino acid was low, and net reabsorption was apparent whenever the filtered load was high; the two variables being significantly correlated. An analysis of the net amino acid transport rates, both secretory and reabsorptive, as a function of the filtered load suggests that in snakes the beta-amino acids are reabsorbed and secreted at discrete tubular sites, with the secretory sites located beyond the major reabsorptive sites. Taurine, beta-alanine, and L-cysteic acid appear to share a common transport system in the snake renal tubule cells.