Cysteine Sulfinic Acid Decarboxylase Activity Research Articles

Isolation, molecular characterization of cysteine sulfinic acid decarboxylase (CSD) of red sea bream Pagrus major and yellowtail Seriola quinqueradiata and expression analysis of CSD from several marine fish species

Cysteine sulfinic acid decarboxylase catalyzes reaction of decarboxylation of cysteine sulfinic acid into hypotaurine. This step is considered as a rate limiting step of taurine biosynthesis in animals. Because of lower CD activity, marine fish has limited ability of de novo taurine synthesis and requires taurine in food. However, if we can develop a method to control CSD activity in marine fish, supplement of taurine to diet of marine fish is not necessary. In order to develop this technique, we have to isolate CSD from marine fish. This study, thereby, is conducted to isolate CSD from red sea bream Pagrus major and yellowtail Seriola quinqueradiata. We also analyze the expression of CSD in red sea bream, yellowtail, Japanese seabass Lateolabrax japonicus, and barfin flounder Verasper moseri. Total RNA was extracted from liver of red sea bream and yellowtail. Primers are designed against sequence of highly conserved region of reported CSD in other animals for RT-PCR. 776bp and 725bp partial CSD sequences were successfully cloned from red sea bream and yellowtail. By 5′ and 3′ RACE methods for cloned partial CSD sequences, total length of CSD (1882bp and 1821bp) from red sea bream and yellowtail was determined. Classification of deduced amino acid sequence revealed that red sea bream and yellowtail CSD showed 88.8% homology similar to Nile tilapia, platyfish, etc. Domain analysis shows that pyridoxine binding region was conserved in CSD from both species.Expression analysis of CSD from red sea bream, Japanese seabass, barfin flounder and yellowtail indicated that CSD is expressed in a variety of tissues but commonly in the liver and pyloric ceca of all species examined. Additionally, strong CSD expression was observed in the heart in all species examined except for Japanese seabass.

Role of Glutamate Decarboxylase-like Protein 1 (GADL1) in Taurine Biosynthesis

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).

Cysteine sulfinic acid decarboxylase activity of Aedes aegypti aspartate 1‐decarboxylase: the structural basis of its substrate selectivity

This work aimed at characterizing biochemical properties of insect aspartate 1‐decarboxylase and studied its structure regarding to its substrate selectivity. Aspartate 1‐decarboxylase from Aedes aegypti was expressed, purified and screened for substrate specificity. Results showed that this enzyme has high activity to cysteine sulfinic acid as well as aspartate, with the specific activity to cysteine sulfinic acid being higher than that to aspartate under the same condition. In addition, this mosquito enzyme showed activity to cysteic acid, thereby making it an enzyme with both aspartate‐L‐decarboxylase and cysteine sulfinic acid decarboxylase activities. This represents the first report of an enzyme with cysteine sulfinic acid decarboxylase activity from any invertebrate species. It is also the first enzyme identified with both aspartate 1‐decarboxylase and cysteine sulfinic acid decarboxylase activities. Docking experiments suggests that Q377 plays a role in the broad substrate specificity of mosquito aspartate‐L‐decarboxylase.

Cysteine sulfinic acid decarboxylase activity of Aedes aegypti aspartate 1-decarboxylase: The structural basis of its substrate selectivity

Insect aspartate 1-decarboxylase (ADC) catalyzes the decarboxylation of aspartate to β-alanine. Insect ADC proteins share high sequence identity to mammalian cysteine sulfinic acid decarboxylase (CSADC), but there have been no reports indicating any CSADC activity in insect ADC or any ADC activity in mammalian CSADC. Substrate screening of Aedes aegypti ADC (AeADC), however, demonstrates that other than its activity to aspartate, the mosquito enzyme catalyzes the decarboxylation of cysteine sulfinic acid and cysteic acid as efficiently as those of mammalian CSADC under the same testing conditions. Further analysis of Drosophila melanogaster ADC also demonstrated its CSADC activity, suggesting that all insect ADC likely has CSADC activity. This represents the first identification of CSADC activity of insect ADC. On the other hand, HuCSADC displayed no detectable activity to aspartate. Homology modeling of AeADC and substrate docking suggest that residue Q377, localized at the active site of AeADC, could better interact with aspartate through hydrogen bonding, which may play a role in aspartate selectivity. A leucine residue in mammalian CSADC occupies the same position. A mutation at position 377 from glutamine to leucine in AeADC diminished its decarboxylation activity to aspartate with no major effect on its CSADC activity. Comparison of insect ADC sequences revealed that Q377 is stringently conserved among the available insect ADC sequences. Our data clearly established the CSADC activity of mosquito and Drosophila ADC and revealed the primary role Q377 plays in aspartate selectivity in insect ADC.

Cysteine Sulfinic Acid Decarboxylase mRNA Abundance Decreases in Rats Fed a High-Protein Diet

The partitioning of cysteine metabolism between sulfate and taurine biosynthetic pathways may be regulated in part by the activity of cysteine sulfinic acid decarboxylase (CSAD). CSAD activity is repressed by high-protein feeding, and we have previously reported that changes in CSAD activity are correlated with changes in CSAD protein. We conducted experiments to determine the relative expression of CSAD mRNA in rats fed 18 or 60% casein diets. In rats fed a 60% casein diet for 1 wk, hepatic CSAD activity and CSAD protein were 16 and 36%, respectively, of the values measured in rats fed the 18% casein diet. CSAD mRNA abundance in rats fed the 60% casein diet was 14% of the CSAD mRNA abundance in rats fed an 18% casein diet. The time course of the change in CSAD activity and mRNA abundance was examined in rats fed 18 or 60% casein diets for 48 h. Within 6 h of switching rats to a 60% casein diet, CSAD activity was decreased by 20% and after 48 h, activity was decreased 47% compared to activity measured at baseline. CSAD mRNA abundance was decreased 54% within 12 h of feeding rats a high-protein diet and remained depressed at 48 h. In a parallel group of rats fed the 18% casein diet, CSAD activity and CSAD mRNA were not significantly different from baseline values at 48 h. The decreased expression of CSAD mRNA in rats fed a high-protein diet is consistent with decreases in both CSAD enzyme activity and CSAD protein. Our results suggest dietary protein may regulate CSAD at the level of mRNA.

Dietary regulation of hepatic enzymes in taurine biosynthesis in rats

The effect of various dietary fibers, cholesterol, and bile acid on tissue taurine levels and the activity of hepatic enzymes in the taurine synthetic pathway was compared in rats. Compared with water-insoluble dietary fibers (10% in the diet, cellulose, and chitin), water-soluble dietary fibers (10% in the diet, pectin, and konjak mannan) decreased taurine levels in the liver and serum. A diet containing cholesterol (1%), sodium cholate (0.25%), and both of these steroids also reduced taurine levels in the liver and serum accompanying the decrease in urinary excretion of this compound. Cysteine dioxygenase activity was significantly lower in rats fed water-soluble fibers than in those fed water-insoluble fibers. Compared to water-insoluble dietary fibers, water-soluble fibers increased cysteinesulfinic acid decarboxylase activity. Compared with the steroid-free control diet, diets containing cholesterol, bile acid, and both of these steroids reduced cysteine dioxygenase activity. Dietary bile acid also reduced cysteinesulfinic acid decarboxylase activity, in contrast with cholesterol which increased it. Decarboxylase activity in rats fed a diet containing both of these steroids was comparable to that in animals fed a control diet. Aspartate aminotransferase activity was also modified by these dietary factors in some cases, but to a lesser extent. Fiber and steroids thus are dietary factors that demonstrably alter hepatic taurine synthesis in the rat.

Regulation of taurine biosynthesis and its physiological significance in the brain.

Cysteine sulfinic acid decarboxylase (CSAD), the rate-limiting enzyme in taurine biosynthesis, was found to be activated under conditions that favor protein phosphorylation and inactivated under conditions favoring protein dephosphorylation. Direct incorporation of 32P into purified CSAD has been demonstrated with [gamma 32P]ATP and PKC, but not PKA. In addition, the 32P labeling of CSAD was inhibited by PKC inhibitors suggesting that PKC is responsible for phosphorylation of CSAD in the brain. Okadaic acid had no effect on CSAD activity at 10 microM suggesting that protein phosphatase-2C (PrP-2C) might be involved in the dephosphorylation of CSAD. Furthermore, it was found that either glutamate- or high K(+)-induced depolarization increased CSAD activity as well as 32P-incorporation into CSAD in neuronal cultures, supporting the notion that the CSAD activity is endogenously regulated by protein phosphorylation in the brain. A model to link neuronal excitation, phosphorylation of CSAD and increase in taurine biosynthesis is proposed.

Protein Phosphorylation and Taurine BiosynthesisIn VivoandIn Vitro

Taurine is known to be involved in many important physiological functions. Here we report that both in vivo and in vitro the taurine-synthesizing enzyme in the brain, namely cysteine sulfinic acid decarboxylase (CSAD), is activated when phosphorylated and inhibited when dephosphorylated. Furthermore, protein kinase C and protein phosphatase 2C have been identified as the enzymes responsible for phosphorylation and dephosphorylation of CSAD, respectively. In addition, the effect of neuronal depolarization on CSAD activity and 32P incorporation into CSAD in neuronal cultures is also included. A model to link neuronal excitation and CSAD activation by a Ca2+-dependent protein kinase is proposed.

Multiplicity of Brain Cysteine Sulfinic Acid Decarboxylase - Purification, Characterization and Subunit Structure.

Cysteine sulfinic acid decarboxylase (CSAD), the rate-limiting enzyme in taurine biosynthesis, appears to be present in the brain in multiple isoforms. Two distinct forms of CSAD, referred to as CSAD I and CSAD II, were obtained on Sephadex G-100 column. CSAD I and CSAD II differ in: (1) the elution profile on Sephadex G-100 column; (2) the sensitivity towards Mn(2+), methione, and other sulfur-containing amino acids, and (3) their immunologic properties. CSAD II has been purified to about 2,500-fold by a combination of column chromatographies and polyacrylamide gel electrophoresis (PAGE). The purity of the enzyme preparation was established as judged from the following observations: (1) a single protein band was observed under various electrophoretic conditions, e.g., 5-20% nondenaturing PAGE, 7% nondenaturing PAGE and 10% SDS-PAGE, and (2) in nondenaturing PAGE, the protein band comigrated with CSAD activity. CSAD II has a molecular weight of 90 kDa and is a homodimer consisting of two 43 +/- 2 kDa subunits. CSAD appears to require Mn(2+) for its maximum activity. Other divalent cations fail to replace Mn(2+) in activation of CSAD activity. However, the precise role of Mn(2+) in the action of CSAD remains to be determined. Copyright 1996 S. Karger AG, Basel

Cloning and characterization of rat cysteine sulfinic acid decarboxylase

Cysteine sulfinic acid decarboxylase (CSAD) is a key enzyme in taurine biosynthesis. CSAD activity and enzyme protein concentration are both repressed by the action of the steroid family hormones triiodothyronine and estrogen. To characterize this suppression, a cDNA clone for CSAD was isolated from a rat liver cDNA expression library using polyclonal antibodies to CSAD. The cDNA was sequenced in its entirety and confirmed to be a clone of CSAD. In a Northern blot comparing liver and kidney RNA of male and female rats, the CSAD cDNA probe detected a 2.5 kb mRNA band which was present at levels corresponding to the concentration of enzyme protein. Hyperthyroidism decreased CSAD mRNA as compared to euthyroid controls, providing evidence that negative regulation of CSAD activity occurs at the level of mRNA.

Dietary sulfur amino acid modulation of cysteine sulfinic acid decarboxylase

Male rats were fed sulfur and nonsulfur amino acid-supplemented diets, and the response of cysteine sulfinic acid decarboxylase (CSAD) activity was determined. After adaptation to a casein-based basal diet, rats were fed diets containing additions of L-methionine. Hepatic CSAD activity decreased in a dose-dependent manner. Significant depression of CSAD activity in liver was evident within 24 h of feeding rats a methionine-supplemented diet. Depression of enzyme activity was reversed upon refeeding the basal diet. After rats were fed diets supplemented with methionine, cystine, homocystine, S-methyl-L-cysteine, phenylalanine, leucine, or ethionine for 14 days, hepatic CSAD activity in rats fed S-methyl-L-cysteine-, phenylalanine-, or leucine-supplemented diets was not depressed compared with activity in rats fed a basal diet. In contrast, CSAD activity in livers of rats fed cystine-, homocystine-, methionine-, or ethionine-supplemented diets was 60, 40, 40, and 8%, respectively, of the activity in livers from control rats. Immunochemical detection and quantification of CSAD protein in rat liver indicated that CSAD protein concentration was correlated to CSAD activity. CSAD activity may be specifically regulated by sulfur amino acids metabolized by the S-adenosylmethionine-dependent pathway of methionine metabolism.

Cysteine sulfinic acid decarboxylase activity in response to thyroid hormone administration in rats

The modulation of hepatic and renal cysteine sulfinic acid decarboxylase (EC 4.1.1.29) activities by triiodothyronine (T 3) was studied in a series of experiments. In a dose-response study, hepatic cysteine sulfinic acid decarboxylase activity (CSAD) was depressed by 65% and renal activity was increased threefold in rats injected with 100 μg T 3/100 g body wt for 7 days when compared to rats injected with 0.3 μg T 3/100 g body wt. Western blot analysis indicated that these changes in CSAD activity were due to changes in the quantity of CSAD protein. Changes in hepatic and renal activities were not evident until 24 h after T 3 administration. In response to T 3 clearance, hepatic and renal CSAD activities approached euthyroid values 4–7 days after cessation of T 3 injections although serum T 3 concentrations were no different from euthyroid values 48 h after T 3 injections were stopped. These data indicate that thyroid hormone effects persist after T 3 clearance. The response of CSAD to thyroid status may be related to its role in taurine biosynthesis.

Dietary taurine deficiency and dilated cardiomyopathy in the fox

Taurine deficiency has been implicated as a potential cause of dilated cardiomyopathy. However, the relationship between taurine and myocardial function is presently unclear. The purpose of this study was to determine whether dilated cardiomyopathy in the fox is associated with dietary taurine deficiency. A total of 68 foxes from farms with a history of death caused by dilated cardiomyopathy and 14 foxes from a farm with no history of dilated cardiomyopathy were studied. Dilated cardiomyopathy was diagnosed by echocardiography in 48% of the foxes from one farm with a positive history and in none of the foxes from the control farm. Foxes less than 9 months of age were more commonly affected than older foxes (p = 0.03). Plasma taurine concentrations were significantly less (p < 0.01) in foxes that had dilated cardiomyopathy (26.8 ± 16.4 nmol/ml) than in the control foxes (99.3 ± 60.2 nmol/ml). A significantly higher (p < 0.01) incidence of dilated cardiomyopathy was present in foxes with a history of a sibling or offspring that died of dilated cardiomyopathy than in foxes without a family history of cardiac death. In one fox with dilated cardiomyopathy that was tested, the myocardial taurine concentration was lower (1.7 μmol/gm wet weight) than that of control foxes (7.3 ± 1.6 μmol/gm wet weight). Hepatic cysteinesulfinic acid decarboxylase activity was significantly less (p < 0.001) in foxes with dilated cardiomyopathy (0.97 ± 0.2 nmol/mm · mg protein) than in control foxes (2.11 ± 0.07 nmol CO2/mm · mg protein). In a trial with one fox in congestive heart failure, dilated cardiomyopathy was reversed with taurine treatment (plasma taurine increased from 3.4 nmol/ml to 120 nmol/ml). It was concluded that dilated cardiomyopathy is associated with dietary taurine deficiency and that the disease is more likely to occur in young foxes with low hepatic cysteinesulfinic acid decarboxylase activity.

P-127 - Regional distribution of taurine and cysteine sulfinic acid decarboxylase activity in rat kidney

P-127 - Regional distribution of taurine and cysteine sulfinic acid decarboxylase activity in rat kidney

Hepatic Cysteine Sulfinic Acid Decarboxylase Activity in Rats Fed Various Levels of Dietary Casein

A series of experiments was conducted to examine the effects of dietary protein intake on hepatic cysteine sulfinic acid decarboxylase (EC 4.1.1.29) activity and urinary taurine excretion. When rats were fed diets containing 18, 30, 45 or 60% casein for 1 wk, hepatic cysteine sulfinic acid decarboxylase activity (CSAD) decreased in a progressive and significant manner. Enzyme activity in rats fed a 60% casein diet was 25% of the activity measured in rats fed 18% casein. The time course of the change in CSAD activity was examined in rats fed a 60% casein diet. Within 24 h of switching rats from a moderate (18% casein) to high (60% casein) protein diet, enzyme activity decreased by 50% and continued to decline in rats fed the high protein diet for 7 d. The observed decrease in enzyme activity was reversed when rats were refed the 18% casein diet. The half-life of CSAD was calculated to be 2 and 7 d during the diet switch from 18 to 60% casein and from 60 to 18% casein, respectively. The change in enzyme activity was evident after a single high protein meal. In contrast to CSAD activity, urinary taurine excretion increased 140-fold within 2 d of switching rats from an 18 to 60% casein diet. Upon refeeding of the 18% casein diet taurine excretion rapidly decreased. These findings indicate that CSAD responds in a rapid and reversible manner to dietary protein.

Endogenous synthesis of taurine and GABA in rat ocular tissues.

The endogenous production of taurine and gamma-aminobutyric acid (GABA) in rat ocular tissues was investigated. The activities of taurine-producing enzyme, cysteine sulfinic acid decarboxylase (CSAD), and GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), were observed in the retina, lens, iris-ciliary body and cornea. The highest specific activity of CSAD was in the cornea and that of GAD in the retina. The discrepancy between CSAD activity and taurine content within the ocular tissues indicates that intra- or extraocular transport processes may regulate the concentration of taurine in the rat eye. The GAD activity and the content of GABA were distributed in parallel within the rat ocular tissues. The quantitative results suggest that the GAD/GABA system has functional significance only in the retina of the rat eye.

Development of Taurine Metabolism in Beagle Pups: Effects of Taurine-Free Total Parenteral Nutrition

In beagle pups (from 5 to 84 days of age), plasma taurine concentration decreased between 5 and 21 days of age with no change thereafer; cerebral taurine concentration decreased throughout the period of study but cerebral taurine content increased between 5 and 21 days of age; hepatic taurine content (but not concentration) increased throughout. Both hepatic cysteinesulfinic acid decarboxylase (CSAD) activity and the concentration of taurine-conjugated bile acid of gallbladder bile increased during the period of study. Plasma and cerebral taurine pools were not affected by taurine-free total parenteral nutrition (TPN). Hepatic taurine content was also not affected, but taurine concentration decreased; however, this change resulted from an increase in hepatic size. Hepatic CSAD activity of animals that received TPN was greater than that of 35-day control animals while the concentration of taurine-conjugated bile acids in the gallbladder bile was less. Although plasma taurine concentration was not affected by intravenous glucose therapy, both the hepatic taurine concentration and content of these animals were less than those of 35-day control animals. Cerebral taurine concentration of these animals, on the other hand, were greater. Hepatic CSAD activity of the animals that received only intravenous glucose was similar to that of controls, but the taurine-conjugated bile acid concentration in the gallbladder bile, like that of animals that received TPN, was less than that observed in 35-day control animals.

Growth Depression in Taurine-Depleted Infant Monkeys

In order to determine the effect of taurine depletion in primates, two species were selected which differed in their taurine conjugtion of bile acids. Consequently, eight cebus (taurine conjugators) and nine cynomolgus monkeys (glycine conjugators) were raised from birth with soybean infant milk formula lacking taurine. Half the monkeys received a 500 ppm taurine supplement. After 5 months the taurine concentration of plasma, urine and several tissues was greatly reduced in the unsupplemented monkeys. The least depletion occurred in retinal tissue of both species and in bile acids of cebus, whereas cynomolgus monkeys increased the glycine conjugation of their bile acids 125%. Taurine depletion was associated with a significant growth depression (16.8%) in the unsupplemented monkeys, but retinal degeneration was not observed. Neither species demonstrated an appreciable capacity to synthesize taurine as measured by cysteinesulfinic acid decarboxylase activity in liver and brain. The data suggest that dietary taurine is essential for maximum growth, as measured by weight gain, of infant nonhuman primates fed a soy protein milk formula.

Milk protein quantity and quality in low-birth-weight infants: III. Effects on sulfur amino acids in plasma and urine

Well, appropriate-for-gestational age, low-birth-weight infants weighing 2,100 gm or less were divided into three gestational age groups and assigned randomly within each age group to one of five feeding regimens: pooled human milk; formula 1 (F1) = 1.5gm/dl protein, 60 parts bovine whey proteins: 40 parts bovine caseins; F2 = 3.0 gm/dl, 60:40; F3 = 1.5 gm/dl, 18:82; F4=3.0 gm/dl, 18:82. Plasma and urine concentrations of methionine and of cystathionine were higher in the infants fed F1 to F4 than in the infants fed BM. The plasma cystine concentrations of infants fed F2 (which had a cystine content at least twice that of any of the other formulas) were significantly higher than those of infants fed BM. Plasma taurine concentrations of infants fed F1 or F4, which were virtually devoid of taurine, decreased steadily during the course of study becoming lower than those of infants fed BM. Urine taurine concentrations of infants fed F1, F3, or F4 (but not F2 which had more taurine than F1, F3, or F4) were lower than those of infants fed BM. These results provide further evidence for the limited capacity of the preterm human infant to convert methionine to cystine, owing to delayed maturation of cytathionase, and suggest a limited capacity to convert cystine to taurine. The latter suggestion is consistent with low human hepatic cysteinesulfinic acid decarboxylase activity 0.26 (fetal) and 0.32 (adult) nmoles/mg protein/hour vs 468 in rat liver.

Cysteine sulfinic acid decarboxylase in rat brain: Effect of vitamin B 6 deficiency on soluble and particulate components

Cysteine sulfinic acid decarboxylase activity is not affected when measured in vitro in the presence of pyridoxal phosphate in the brains of vitamin B 6-deficient rats. Activity of this enzyme was not detectable in the brains of vitamin B 6-deficient animals when assayed in the absence of pyridoxal phosphate. The activity of cysteine sulfinic acid decarboxylase in crude particulate and soluble fractions of rat brain reflects the distribution of the enzyme in vivo and the distribution of endogenous B 6 vitamers when assayed in the presence and absence of vitamin B 6. This study indicates that virtually no taurine is synthesized by any component of brain when the animal is subjected to a deficiency of vitamin B 6.