Cysteine Sulfinate Research Articles

Acute glutamate toxicity in cultured cerebellar granule cells: agonist potency, effects of pH, Zn 2+ and the potentiation by serum albumin

Cell death was due to activation of the receptor responsive to N-methyl- D-aspartate (NMDA). Even during a 30-min incubation, glutamate toxicity was unaffected by 5 μM CNQX and was 90% blocked by 160 nM MK-801, a specific antagonist of the NMDA receptor. The extent of toxicity was dependent on the age of the cultured cells, the concentration of KCl, which determined membrane polarization, the duration of exposure to, and the concentration of, glutamate. Toxicity was critically pH-dependent, increasing from a minimal effect at pH 7.0 to an extremely high level at pH 8.0. Among a number of acidic amino acids tested, glutamate showed the highest potency. Aspartate, cysteine sulfinate and homocysteate reached the same maximal toxicity, but at a 25 times higher concentration. Cysteate and quinolinate were of very low potency. Zn 2+, at 0.5 μM, attenuated glutamate toxicity considerably. At concentrations above 5 μM the metal ion showed strong toxicity on its own. Dialysed serum and serum albumin strongly potentiated glutamate and NMDA toxicity in presence and absence of Mg 2+. A concentration of 2 mg/ml bovine serum albumin caused maximal potentiation; fatty acid-free bovine serum albumin and human serum albumin were all about equipotent. To be effective, the serum albumin had to act simultaneously with glutamate. Among a number of proteins and other polymers, only casein showed an ability to potentiate glutamate toxicity, similar to serum albumin. It is concluded that minor effects of glutamate toxicity could be considerably aggravated by the potentiating activity of serum albumin, also in pathological situations in vivo.

Extracellular Acidic Sulfur‐Containing Amino Acids and γ‐Glutamyl Peptides in Global Ischemia: Postischemic Recovery of Neuronal Activity Is Paralleled by a Tetrodotoxin‐Sensitive Increase in Cysteine Sulfinate in the CA1 of the Rat Hippocampus

An excessive activation of the excitatory amino acid system has been proposed as one possible mediator of the ischemia-induced delayed death of CA1 pyramidal cells in the hippocampus. Using dialytrodes in the CA1 of the rat, we have investigated multiple-unit activity and extracellular changes in acidic sulfur-containing amino acids and gamma-glutamyl peptides during ischemia (20-min, four-vessel occlusion) and during 8 h of reflow. Multiple-unit activity was abolished during ischemia and for the following 1 h, but then recovered, gradually reaching preischemic levels after 8 h of reflow. Extracellular cysteate, cysteine sulfinate, and gamma-glutamyltaurine increased (1.5- to threefold) during ischemia, and extracellular glutathione and gamma-glutamylaspartate plus gamma-glutamylglutamine increased during early reflow (two- to threefold). The recovery of neuronal activity at 4-8 h was paralleled by an increase in extracellular cysteine sulfinate (2.5-fold at 8 h of reflow). Perfusion with 10 microM tetrodotoxin at 8 h of reflow abolished the multiple-unit activity and reduced extracellular cysteine sulfinate. Considering the glutamate-like properties of cysteine sulfinate, the observed postischemic increase may be involved in the development of the delayed neuronal death.

Alanine aminotransferase in Leptosphaeria michotii. Isolation, properties and cyclic variations of its activity in relation to polypeptide level

Alanine aminotransferase (EC 2.6.1.2) was obtained from the fungus Leptosphaeria michotii (West) Sacc, and enriched 714‐fold by a 5‐step purification procedure as a dimer of Mr 110000, associated with a polypeptide of Mr 25000. Its isoelectric point was 5.25. The enzyme was active from pH 3.5 to 9.5 with a maximum at pH 7.5. Its specific activity was 6000 nkat (mg protein)−1; the Km was 6.85 mM for L‐alanine and 0.2 mM for 2‐oxoglutarate. The enzyme did not show any detectable activity in the presence of L‐aspartate, cysteine sulfinate, α‐aminobutyrate or cyclic amino acids as substrates. It did not express alanine:glyoxylate aminotransferase activity. Alanine aminotransferase in L. michotii has previously been shown to have an activity rhythm in constant temperature and darkness. The enzyme level was quantified along the activity rhythm by enzyme‐linked immunosorbent assay (ELISA), using a monospecific polyclonal antibody against the purified enzyme. The cyclic variations of alanine aminotransferase activity were correlated with cyclic variations in the enzyme level.

Immunocytochemistry of the taurine biosynthesis enzyme, cysteine sulfinate decar☐ylase, in the cerebellum: Evidence for a glial licalization

Immunocytochemistry of cysteine sulfinate decar☐ylase was developed in the cerebellum with an antiserum that we have recently produced using as immunogen a homogeneous fraction purified about 2000-fold from liver. In the rat, this antiserum immunoprecipitated the enzymatic activity from brain, labeled one band ( molecular weight= 51,000) on immunoblots of an enriched fraction from brain but none with a brain crude extract and thus appeared to be specific. In the cerebellum numerous immunolabeled cells were found in the white matter that were typically arranged in rows like oligodendrocytes. A few immunolabeled cells were scattered in the granular layer. Around the Purkinje cells numerous small satellite cells were immunostained that sent faintly labeled radial fibers through the molecular layer. These cells were thus identified as Golgi epithelial cells with their Bergmann fibers. Purkinje cells were not labeled either at the cell body or at the nerve ending levels. In the molecular layer no cells were found consistently and convincingly immunolabeled that could correspond to the stellate cells. No immunopositive punctae typical of the interneuron nerve endings were ever observed. A significant cysteine sulfinate decar☐ylase activity was also measured in glial cell fractions enriched in oligodendrocytes and astrocytes respectively. The glial localization of taurine biosynthesis enzyme in the cerebellum does not support the hypothesis that it could be involved in neurotransmission, but rather suggests that taurine is manufactured by glial cells and accordingly may play a physiological role in relation to glial function.

Taurine accumulation by the heart of embryonic skates, Raja eglanteria

ABSTRACT The hearts of elasmobranchs, like those of other vertebrates, contain high concentrations of the β-amino acid taurine, which is thought to play an osmoregulatory role in cardiac tissue. Although mammals and certain invertebrates can synthesize taurine from sulfur-containing precursors, e.g. cysteine, fish are unable to do so since they lack at least one enzyme involved in taurine biosynthesis: cysteine sulfinate decarboxylase (Jacobsen and Smith, 1968). Therefore, taurine must be acquired by fish via their diet (King et al. 1986). This poses a problem for the embryos of those fish that lay eggs and, for the early period of development at least, have no direct contact with amino acids in the external environment. Although solutes in the egg-case fluids can exchange with the sea water (Evans, 1981), the taurine concentration gradient between the egg case and the environment actually favors outward movement of taurine rather than uptake (L. Golstein, unpublished results). Therefore, we chose to study the mechanism by which the embryonic skate heart accumulates and maintains elevated trations of taurine in its heart. Concentrations of about 50 mmol l−1 taurine are typically found in cardiac muscle of adult specimens of the little skate Raja erinacea (Boyd et al. 1977).

Specific Antiserum and Monoclonal Antibodies Against the Taurine Biosynthesis Enzyme Cysteine Sulfinate Decarboxylase: Identity of Brain and Liver Enzyme

Cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme for taurine, was purified 1,800-fold with a 1% yield from rat liver, where it was found to be 20-fold enriched compared with brain. The final fraction was homogeneous, as ascertained through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase HPLC. An antiserum was raised in the rabbit that (a) quantitatively immunoprecipitated CSD activity and (b) immunolabeled only one band (MW = 51,000) on an immunoblot from liver homogenate. Monoclonal antibodies were also raised that recognized the CSD protein and immunolabeled the same 51-kilodalton protein on an immunoblot from liver homogenate. In a brain extract, two CSD activities had been previously found and named CSDI and CSDII, according to their chromatographic elution patterns. We have compared the properties of CSDI from brain--the most likely enzyme involved in the biosynthesis of taurine in the brain, according to previous investigations-and CSD from liver: Both activities (a) were similarly eluted on ion-exchange and hydroxyapatite chromatographies, (b) showed the same elution pattern on gel filtration with an apparent native molecular weight of approximately 63,000, and (c) were immunoprecipitated in a strictly identical manner by the antiserum against liver CSD. Moreover, this antiserum as well as the monoclonal antibodies immunolabeled a single band (51 kilodaltons) on an immunoblot from brain CSD-enriched fraction or liver fraction. All these data show that CSDI from brain and liver CSD are the same monomeric enzyme. They also indicate that a specific antiserum against rat liver CSD has been raised that can be used for immunocytochemical visualization of CSD-containing cells in the brain.

Cyclic changes in translation of the mitochondrial isoenzyme account for the rhythm of aspartate aminotransferase activity in Leptosphaeria michotii

Aspartate aminotransferase in Leptosphaeria michotii has previously been shown to have an activity rhythm in constant conditions. The enzyme is present as two isofonns whose levels were quantified along the activity rhythm by ELISA, using specific polyclonal antibodies raised against polypeptides purified to a state of apparent homogeneity. The time-course of the level of the cytosolic isoform remained unchanged along the experiment. On the contrary, the cyclic variations in amount and transaminase activity (using cysteine sulfinate as substrate) of the mitochondrial isofonn gave rise to the aspartate aminotransferase activity rhythm of the fungus. The mRNA levels of the two isofonns, as determined by in vitro heterologous translation, remained monotonous along the daily cycle. These results and the sensitivity of the rhythm towards protein synthesis inhibitors are consistent with the hypothesis that the aspartate aminotransferase activity rhythm in this species is caused by some mechanism controlling the efficiency of translation of mitochondrial isoform mRNA.

Direct Brønsted Analysis of the Restoration of Activity to a Mutant Enzyme by Exogenous Amines

A true Brønsted analysis of proton transfer in an enzyme mechanism is made possible by the chemical rescue of an inactive mutant of aspartate aminotransferase, where the endogenous general base, Lys258, is replaced with Ala by site-directed mutagenesis. Catalytic activity is restored to this inactive mutant by exogenous amines. The eleven amines studied generate a Brønsted correlation with beta of 0.4 for the transamination of cysteine sulfinate, when steric effects are included in the regression analysis. Localized mutagenesis thus allows the classical Brønsted analysis of transition-state structure to be applied to enzyme-catalyzed reactions.

Aspartate aminotransferase isoenzymes in Leptosphaeria michotii. Properties and intracellular location.

Two forms of aspartate aminotransferase were obtained from the fungus Leptosphaeria michotii and purified to a state of apparent homogeneity by a five-step purification procedure ending with blue Ultrogel chromatography. Holoenzyme specific activities were 13430 and 9110 nkat oxalacetate/mg protein-1 and isoelectric points were 7.1 and 7.0 for forms A and B, respectively. Both isoenzymes were isologous dimers of Mr 92,000. They differed mainly in their Km for 2-oxoglutarate and aspartate, their ability to use cysteine sulfinate as a substrate and their ability in vitro to be specifically tightly associated as follows: form A with a malate dehydrogenase monomer of Mr 25,000; form B with an unidentified protein of Mr 40,000-44,000. Rabbit antiserum raised against the form A holoenzyme was not reactive against the form B holoenzyme and vice versa. Association of the holoenzyme with the complex essentially provoked a shift of the isoelectric point to 5.8 for form B [corrected] and to 5.2 for form B, without affecting kinetic parameters. In order to localize in situ the two transaminase forms, ultrastructural detection was carried out by immunogold staining of thin sections of Lowicryl-K4M-embedded colonies. Antiserum against form A essentially labelled cytoplasm and cell wall and, to some extent, mitochondria, while antiserum against form B heavily labelled mitochondria and cell wall and to a lesser extent cytoplasm. Moreover, mitochondria were isolated and purified by Percoll-density-gradient centrifugation. Only form A was identified in this subcellular fraction using ELISA.

Pharmacological alterations of the activity of afferent fibers innervating hair cells

To determine whether some of the substances that may be present in hair-cell sensory organs could affect neural activity in afferent fibers, we examined 56 compounds for the ability to alter the discharge rate of afferent fibers innervating hair cells in the lateral line organ of Xenopus laevis, the African clawed frog. These compounds included amino acids, glutamyl dipeptides, standard neurotransmitter candidates, and other constituents of tissues and body fluids. Substances found to be excitatory included some neutral amino acids (alanine, serine, threonine, asparagine, glutamine, and proline), ATP, camosine, histidine, and barium chloride. Compounds that suppressed discharge included the aromatic amino acids (phenylalanine, tryptophan, and tyrosine), serotonin, and γ-glutamyl dipeptides. GABA and acidic amino acids (glutamate, aspartate, and cysteine sulfinate) produced a brief excitation followed by a suppression of discharge rate. Several of these substances were active at sufficiently low concentrations that their presence in body fluids may affect afferent fiber discharge rate under normal or pathological conditions.

D-Aspartate potentiates the effects of both l-aspartate and l-glutamate on carp horizontal cells

l-Aspartate, l-glutamate and d-aspartate, alone or in combination, were applied by superfusion or by atomization to the isolated carp retina while recording from cone horizontal cells. Each of these agents when applied alone depolarized horizontal cells and reduced the size of their light responses, an action which mimics the effect of the endogenous photoreceptor transmitter. Application of d-aspartate in conjunction with either of the l-amino acids potentiated the effects of the l-amino acids so that the threshold concentration was reduced by about five-fold, compared to when the l-amino acids were applied alone. The potentiating effect of d-aspartate occurred with all types of cone horizontal cells—both l- and C-types. Furthermore, the potentiating effect of d-aspartate occurred not only in the dark but also in the presence of bright light backgrounds or Co 2+ ions, conditions during which the release of photoreceptor transmitter is reduced or blocked. d-Aspartate also potentiated the depolarizing effects of the acidic amino acid cysteine sulfinate. The potentiating effect of d-aspartate can be attributed to its action as an amino acid uptake blocker in the outer retina. Thus, these findings, in themselves, cannot eliminate l-aspartate, l-glutamate or cysteine sulfinate as candidates for the carp cone transmitter. However, other evidence, previously reported, strongly suggests that l-glutamate and not l-aspartate is the cone transmitter.

Inhibition by forskolin of excitatory amino acid-induced accumulation of cyclic AMP in guinea pig hippocampal slices.

The effect of forskolin on the excitatory amino acid-induced accumulation of cyclic AMP was examined in hippocampal preparations of the guinea pig. Forskolin at concentrations of 0.1-10 microM remarkably enhanced the stimulatory effects of histamine and adenosine, whereas it markedly attenuated the stimulation induced by cysteine sulfinate, an excitatory amino acid. Forskolin reduced the maximal response to cysteine sulfinate without affecting the apparent ED50. At a concentration of 1 microM, forskolin also inhibited the stimulatory effects of glutamate, N-methyl-DL-aspartate, and veratridine without affecting those of kainate and quisqualate. Pretreatment of the slices with 0.1 mM N-ethylmaleimide partially prevented the attenuation by forskolin of cysteine sulfinate-induced accumulation of cyclic AMP without affecting the stimulation induced by cysteine sulfinate. Forskolin at concentrations of less than 1 microM did not affect GTP-stimulated activity and Cl- -dependent activity of adenylate cyclase of the hippocampal membranes.

Cysteine sulfinate modulated calcium permeability in synaptosomes from rat brain.

Cysteine sulfinate (CSA) was shown to increase 45Ca2+ permeability of plasma membrane of synaptosomes isolated from rat brain. The 45Ca2+ enter through a system which has properties different from those of voltage dependent Ca2+ channels or Na+/Ca2+ exchange. The effect of CSA appeared to be mediated by highly specific receptors saturable by ligand. The high concentrations of CSA required (mM) suggest this amino acid is not the physiological agonist for the receptors.

Purification and properties of the cytosolic and mitochondrial forms of aspartate aminotransferase and malate dehydrogenase from rat heart

The present work describes the purification from rat heart of the mitochondrial and cytosolic forms of the enzymes of the malate--aspartate shuttle, aspartate aminotransferase (EC 2.6.1.1) and malate dehydrogenase (EC 1.1.1.37), by a single procedure after the preparation of the original crude extract. In 10 purification steps, the four enzymes were obtained electrophoretically pure in yields ranging from 6 to 54% of their respective isoenzyme levels in the crude extract. Apoenzymes were formed from the aminotransferases by reacting them with cysteine sulfinate and dialyzing. Complete reconstitution was obtained after a brief incubation with pyridoxal phosphate. All four enzymes are dimers. The mitochondrial isoenzymes are of slightly lower molecular weight than their respective cytosolic forms. Michaelis constants and maximal velocities were derived by the use of primary and secondary plots. In general, the properties of the enzymes from rat heart are similar to the properties of the enzymes from other animal sources.

Taurine biosynthesis in rat brain: a new specific and sensitive microassay of cysteine sulfinate decarboxylase (CSDI) activity through selective immunotrapping and its use for distribution studies.

Cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme for taurine, has been shown to exist in two forms in rat brain, respectively CSDI and CSDII, one of which (CSDII) is considered to be in fact glutamate decarboxylase (GAD). CSDI assay after immunotrapping was made possible by using an anti-CSD antiserum raised in sheep immunized with a partially purified CSD fraction from liver. This antiserum immunoprecipitated both liver CSD and brain CSDI activities with the same affinity but did not inhibit their enzymatic activities. The immunotrapping of CSDI was selective without any contamination by GAD/CSDII activity. The immunotrapped CSD activity, which corresponded exactly to the amount of CSD not precipitated by a GAD/CSDII antiserum, was not inhibited by a specific irreversible GAD inhibitor. A quantitative, selective and sensitive assay was thus developed by measuring CSD activity on the solid phase after immunotrapping. Kinetic parameters of the immunotrapped enzyme remained unchanged. CSDI activity represented only a fraction, around 20% with saturating concentration of substrate, of the total CSD activity in rat brain homogenate. This indicates that most studies on total CSD activity dealt essentially with CSDII activity that is indeed GAD. Regional and subcellular distributions of CSDI have been determined. CSDI activity was about threefold higher in the richest (cerebellum) compared to the poorest (striatum) region without any correlation with GAD/CSDII distribution. Subcellular distribution showed a fourfold enrichment of CSDI activity in the synaptosomal fraction. The precise role of CSDI and CSDII in the biosynthesis of taurine in vivo remains to be elucidated.

Taurine biosynthesis in rat brain in vivo: lack of relationship with cysteine sulfinate decarboxylase glutamate decarboxylase-associated activity (GAD/CSDII).

Two distinct forms of cysteine sulfinate decarboxylase (CSD), respectively, CSDI and CSDII, have already been separated in rat brain. One of them, CSDII, appeared to be closely associated with glutamate decarboxylase (GAD). We have investigated whether the taurine concentration in brain was dependent on CSDII activity in vivo. CSDI and CSDII activities were specifically measured in crude brain extracts after selective immunotrapping. After 4 days of chronic treatment of mice with gamma-acetylenic gamma-aminobutyric acid, a drastic and identical decrease in CSDII and GAD activities was observed in the brain. Taurine concentration and CSDI activities were not significantly altered. Following striato-nigral pathway lesioning in the rat brain, GAD and CSDII show an identical 80% decrease in the substantia nigra. In contrast, CSDI activity and taurine concentration in the substantia nigra were similarly but only slightly affected with an about 30% decrease. Our results provide further evidence that GAD and CSDII are indeed the same enzyme. They show that CSDII does not play any role in the biosynthesis of taurine in vivo. Our findings suggest that CSDI might be the biosynthetic enzyme for taurine in vivo and that there might be some endings projecting into the substantia nigra that contain CSDI and taurine.

The oxidation of hypotaurine to taurine: bis-aminoethyl-alpha-disulfone, a metabolic intermediate in mammalian tissue.

The primary mammalian metabolic pathway leading to taurine is generally recognized as beginning with the oxidation of cysteine to cysteine sulfinate via cysteine dioxygenase (19). This rate limiting enzyme is, carefully regulated thus setting the rate for the catabolism of sulfur amino acids (4). A major portion of cysteine sulfinate is subject to unidirectional transamination leading to pyruvate and sulfite; a smaller fraction of cysteine sulfinate is decarboxylated by cysteine sulfinate decarboxylase to hypotaurine (8). The ratio of the two pathways appears to be relatively constant; what controls the pathway bifurcation remains unknown. Although a small fraction (10%) of the hypotaurine is subject to unidirectional transamination to acetaldehyde and sulfate (4,6), the bulk of hypotaurine is oxidized to taurine. A possible alternate metabolic route involves initial oxidation of cysteine sulfinate to cysteic acid by an unspecified mechanism followed by decarboxylation of cysteic acid to taurine. The latter is an established capacity of the enzyme cysteine sulfinate decarboxylase (10).

Multiple Cl −-independent binding sites for the excitatory amino acids: glutamate, aspartate and cysteine sulfinate in rat brain membranes

As we have recently reported that Cl −-dependent (GLU) binding reflects GLU accumulation into membrane vesicles, the characteristics, kinetics and pharmacological specificities of l-[ 3H]glutamate ( l-[ 3H]GLU) binding to crude rat brain synaptic membranes, were investigated in Cl −-free medium. l[ 3H]GLU binding was systematically compared to that of l-[ 3H]cysteine sulfinate ( l-[ 3H]CSA) and l-[ 3H]aspartate ( l-[ 3H]ASP), two other putative excitatory amino acids. A high affinity site was determined for each of these radioactive ligands ( l-[ 3H]GLU: K d = 0.14 μM, B m = 3.4 pmol/mg protein; l-[ 3H]CSA: K d = 0.07 μM, B m = 2.2 pmol/mg protein; l-[ 3H]ASP: K d = 5.8 μM, B m = 31.2 pmol/mg protein). The pharmacological specificity of these Cl −- independent binding sites indicate the existence of at least 3 distinct high affinity sites, all different from the Cl −-dependent GLU binding ‘site’: one having a similar affinity for GLU and CSA, a second one preferring CSA, and a third one preferring ASP. Among the large quantity of structural analogs of the neuroexcitatory amino acids tested, only endogenous compounds (GLU, ASP and CSA) (except hydroxylamine- o-sulfate) were able to interact efficiently. No inhibition by classical agonists and antagonists (such as N-methyl- d-aspartate, quisqualate, kainate, 2-amino-4-phosphobutyrate, or 2-amino-5-phosphonovalerate) was found. In addition to their high specificity, these Cl −-independent sites posses most other biochemical characteristics of receptor proteins

Sulfite determination: Fuchsin method

Publisher Summary This chapter discusses the Fuchsin method used in sulfite determination. The method of Grant for the colorimetric determination of sulfur dioxide in extracts of animal and plant tissues has been adapted to the study of microbial enzymes producing sulfite from substrates, such as 3'-phosphoadenosine 5'-phosphosulfate (PAPS), thiosulfate and cysteine sulfinate (CSA), both in vivo and in vitro . Grant's method was also applied to the enzymatic determination of CSA. The method relies on the formation of a red chromophore with basic fuchsin (pararosaniline). Grant's fuchsin reagent is prepared by adding1.6 ml of a 3% ethanolic solution of basic fuchsin (Fisher) to 93.6 ml distilled water containing 4.4 ml concentrated H 2 SO 4 . The solution is decolorized by addition of 200 mg activated carbon. After repeated shaking over a period of 15 min, the solution is clarified by filtration. Fresh reagent is prepared daily. Maximum color intensity is reached at approximately 15 min after addition of fuchsin reagent. Fading occurs slowly after 60 min at room temperature approximately 85% of the color measured at 15 min remains.

Cysteinesulfinic acid, hypotaurine, and taurine: Reversed-phase high-performance liquid chromatography

Publisher Summary This chapter describes the method that allows quantitative analysis of cysteine sulfinate, taurine, and hypotaurine as well as other amino acids in biological samples. The method is used for analysis of these compounds in animal tissues and plasma and for measurement of their production by tissue preparations incubated with cysteine in vitro. Stock standard solutions of cysteinesulfinate, hypotaurine, taurine, and other compounds are prepared in water, dispensed in small volumes suitable for daily use, and stored at -20°. Standards may also be prepared in 5-sulfosalicylic acid and neutralized in a manner that corresponds to that by which samples are prepared. The fluorescence intensity is proportionate to the amino acid content of standard solutions between 0 and 1000 pmol per 10 μl aliquot for cysteine sulfinate, hypotaurine, and taurine. The fluorescence yield for the three compounds is similar. As little as 10 pmol of any one of the three compounds can be measured. The separation is very reproducible; the coefficients of variation for the retention times of cysteine sulfinate, hypotaurine, and taurine on the system are all about 0.25%. Very high concentrations of thiols—that is, cysteine or cysteamine—in samples may decrease fluorescence yield.