![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853
2To whom correspondence should be addressed. E-mail: mhs6{at}cornell.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
-glutamylcysteine synthetase (GCS) play important regulatory roles in the metabolism of cysteine to sulfate, taurine and glutathione. Because glutathione is released by the liver and degraded by peripheral tissues that express -glutamyl transpeptidase, some peripheral tissues may be exposed to relatively high concentrations of cysteine. Rats were fed diets that contained low, moderate or high concentrations of protein or supplemental cysteine or methionine for 2 wk, and CDO, CSD and GCS activities, concentrations and mRNA levels and the concentrations of cysteine, taurine and glutathione were measured in liver, kidney, lung and brain. All three enzymes in liver responded to the differences in dietary protein or sulfur amino acid levels, but only CSD in kidney and none of the three enzymes in lung and brain responded. Renal CSD activity was twice as much in rats fed the low protein diet as in rats fed the other diets. Changes in renal CSD activity were correlated with changes in CSD concentration. Some significant differences in cysteine concentration in kidney and lung and glutathione and taurine concentrations in kidney were observed, with higher concentrations in rats fed higher levels of protein or sulfur amino acids. In liver, the changes in cysteine level were consistent with cysteine-mediated regulation of hepatic CDO activity, and changes in taurine level were consistent with predicted changes in cysteine catabolism due to the changes in cysteine concentration and CDO activity. Changes in renal and lung cysteine, taurine or glutathione concentrations were not associated with a similar pattern of change in CDO, CSD or GCS activity. Overall, the results confirm the importance of the liver in the maintenance of cysteine homeostasis.
KEY WORDS: • cysteine dioxygenase • glutathione • taurine • rats
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). Cysteinesulfinate decarboxylase (CSD) is involved in the further metabolism of cysteinesulfinate to taurine. -Glutamylcysteine synthetase (GCS) catalyzes the rate-limiting step in glutathione synthesis and, thus, competes with CDO for cysteine as a substrate.
|
–4
), yet the level of cysteine must be high enough to allow its use for other purposes, such as the formation of glutathione and the synthesis of proteins. We have shown that both hepatic CDO and GCS activities are tightly regulated and respond rapidly to changes in the amount of dietary protein or sulfur amino acids. An increase in the level of casein, soy protein, methionine or cystine in rat diets or the addition of sulfur amino acids to the culture medium for primary rat hepatocytes results in a dramatic rise in CDO activity and a notable decrease in GCS activity (5
–7
). Previous studies have demonstrated that the regulation of expression and activity of these three hepatic enzymes is complex (5
–7
).
Cysteine metabolism is somewhat unusual in that much of the sulfur amino acid load reaching the liver is incorporated into glutathione and then exported for use by other tissues (8
). This glutathione is broken down by tissues that express -glutamyl transpeptidase to release cysteine in the peripheral circulation. Hence, some peripheral tissues may be exposed to relatively high concentrations of cysteine. However, little is known about possible regulation of CDO, CSD or GCS in nonhepatic tissues in response to dietary intake or cysteine availability. An inconsistent small increase in CDO mRNA in a neuronal cell line when these cells were cultured with additional cysteine and increases in CDO activity in various regions of the brain when rats were given excess methionine in their drinking water have been reported (9
,10
). More strikingly, Beetsch and Olson (11
) demonstrated a 15-fold increase in CDO activity that was associated with an increase in cysteine uptake and a 20-fold increase in cellular cysteine concentration in primary rat cerebral astrocytes as osmolality of the medium was increased from 300 mosmol/kg to 450 mosmol/kg.
Results of studies with cultured rat hepatocytes indicated that CDO and GCS were regulated in response to changes in cellular cysteine levels (7
). Taurine and sulfate, the major catabolites of cysteine had no effect on either CDO or GCS. Inhibition of glutathione synthesis by buthionine sulfoximine, an inhibitor of GCS, did not prevent the effect of methionine or cysteine on CDO activity or concentration but did result in up-regulation of GCS-HS mRNA and GCS-HS protein (inactivated) as would be expected. Methionine and intermediates in the transmethylation/transsulfuration pathway had no effect on either CDO or GCS when propargylglycine, an inhibitor of cystathionase, was added to block cysteine formation.
Because of the unique roles of CDO, CSD and GCS in cysteine metabolism, we further explored the effect of diet on the regulation of these three enzymes in liver and three nonhepatic tissues that also express all three enzymes (i.e., kidney, lung and brain). In these studies, we quantified CDO, GCS-catalytic or heavy subunit (GCS-HS) and CSD protein and mRNA amounts relative to the same standard so that relative abundance could be assessed among tissues as well as among dietary treatments. Also, because of the probable role of cellular cysteine concentration in the regulation of CDO and GCS, we measured tissue cysteine concentrations to see if they were correlated with enzyme levels. In addition, we measured glutathione and taurine levels in tissues because these two compounds are products of the reactions catalyzed by GCS and CDO plus CSD, respectively.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Semipurified diets were prepared and pelleted by Dyets, Inc. (Bethlehem, PA) to contain various levels of protein (casein) or sulfur amino acids (Table 1
). Diets were based on the AIN-93 formulation (12
). Male Sprague-Dawley rats that weighed 
150 g were purchased from Harlan Sprague-Dawley (Indianapolis, IN). They were housed in a room with light from 2000 h to 0800 h and had free access to diet and water at all times. All rats were fed the MP (200 g casein/kg) diet for 1 wk before experimental group assignment. Thirty-two rats were then blocked by weight, and rats within each block were randomly distributed among the five treatment groups. Six rats were allotted to the LP, HP, LP + C, and LP + M diets, and eight to the MP diet. Extra rats were included in the MP group because we sometimes see a very high variance of CDO activity in those fed protein at levels very near the requirement level.
|
Liver, kidneys, lungs and brain were rapidly removed, rinsed with ice-cold saline, blotted, weighed and frozen in liquid nitrogen. Frozen tissues were stored at -140°C until analyses were performed. Preliminary studies demonstrated that no enzyme activity was lost by freezing and storing tissues for up to 2 wk.
Enzyme assays.
Portions of frozen tissue were homogenized in ice-cold 0.05 mol/L 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH. 6.0, or 0.05 mol/L potassium phosphate buffer, pH 6.8, for assay of CDO and CSD activities, respectively. Portions of frozen tissue were homogenized in 20 mmol/L Tris buffer, pH. 8.2; the 100,000 x g supernatant fraction was obtained and used for assay of -glutamylcysteine synthetase activity. Assays of CDO and CSD activities were carried out as described previously (14
). Glutamate at a final concentration of 100 mmol/L was added to assay mixtures for determining CSD activity in brain to block decarboxylation of cysteinesulfinate by glutamate decarboxylase. Assay of GCS activity was carried out as described by Yan and Huxtable (15
) except that both -glutamylcysteine and glutathione were measured as products because some of the -glutamylcysteine was further converted to glutathione using glycine present in the tissue supernatant. Glutathione and -glutamylcysteine were quantitated by the HPLC method of Fariss and Reed (16
) as modified by Stipanuk et al. (17
). Protein concentration was determined by the method of Smith et al. (18
).
Western blot analysis.
The purified IgG fraction from rabbit anti-CDO serum (19
) was a gift from Dr. Yu Hosokawa (National Institute of Health and Nutrition, Tokyo, Japan). Rabbit anti-CSD serum (20
) was a gift from Dr. Owen Griffith (Medical College of Wisconsin, Milwaukee, WI). Rabbit anti-GCS heavy catalytic subunit serum (GCS-HS) (21
) was a gift from Dr. Henry Jay Forman (University of Alabama, Birmingham, AL). Specificity of antibodies has been described previously (6
).
A portion of each frozen tissue sample was homogenized in ice-cold 0.05 mol/L potassium phosphate buffer, pH 6.8. The homogenate was centrifuged at 20,000 x g for 30 min at 4°C, and the supernatant was further centrifuged at 100,000 x g for 60 min at 4°C. Pooled samples were prepared by combining aliquots of 100,000 x g supernatant fractions (equal amounts of total soluble protein from a given tissue and a given dietary treatment group), and the samples were stored at -140°C. Protein concentration in the tissue supernatant fractions was determined by the method of Smith et al. (18
).
Pooled total liver supernatant protein was separated by one-dimensional SDS-PAGE (150, 100, and 120 g/L polyacrylamide for CDO, GCS-HS, and CSD analyses, respectively), and Western blot analysis of CDO, GCS-HS and CSD were done as described previously (5
,22
). Relative standard curves based on pooled liver supernatants from rats fed the moderate protein diet were run on each gel; these curves served to confirm linearity and to allow quantification on a relative basis in the absence of pure enzymes to use as true standards. Relative protein amounts were quantified using these standard curves (integrated density value vs. µg total liver protein loaded); the relative amount of enzyme protein was then divided by the actual amount of total protein loaded on the gel. Amounts of total soluble protein loaded varied between 0.35 µg and 140 µg for the different proteins and different tissues so that all samples fell within the linear range of the standard curve. Pooled samples were used because of time and cost restrictions due to the variability of quantitative Western analysis and the need to run each set of samples on triplicate gels.
Northern blot analysis.
Complementary DNA probes corresponding to bp 459–717 of rat liver CDO (19
), bp 795-1165 of rat liver CSD (23
) and bp 397–863 of GCS-HS (24
) were synthesized using RT-PCR. The sequence of the probes was verified by the BioResource Services (Cornell University, Ithaca, NY). DECAprobe template-18S-mouse (Ambion, Austin, TX) was used to prepare a probe for 18S RNA as a control for variations in loading and transfer. Radiolabeled cDNA probes were prepared using [32P]dATP and random priming.
Approximately 100 to 150 mg of frozen tissue from each sample was homogenized in denaturation solution and total RNA was isolated using the ToTALLY RNA kit (Ambion). Equal amounts of total RNA from each sample were pooled for a given tissue and given dietary group. Pooled samples were then stored at -70°C until Northern blot analysis was performed.
Northern blot analysis was done as described by Brown (25
) with electrophoresis of total tissue RNA (0.25 to 10 µg) on a 12 g/L agarose-formaldehyde gel and blotting onto a nylon transfer membrane (Nytran SuPer Charge; Schleicher & Schuell, Keene, NH). Radioactivity was measured by phosphorimaging using a Super Resolution Cyclone Storage Phosphor Screen (Packard, Meriden, CT). Bands were quantified using the AlphaEase version 5.5 software (Alpha Innotech Corp.). Probes were stripped from the membrane using the Strip-EZ DNA kit (Ambion) between hybridization with the various probes. A relative standard curve was generated on each membrane by loading appropriate amounts of pooled total RNA from liver of rats fed the moderate protein diet. The relative amount of enzyme mRNA for each sample was calculated using a standard curve of integrated density units vs. the amount of total RNA loaded, and the amount was divided by the total µg RNA loaded. The 18S RNA was also determined as a control for variations in loading and transfer. Pooled samples were used because of time and cost restrictions due to the variability of quantitative northern analysis and the need to run each set of samples on triplicate gels.
Cysteine, glutathione and taurine concentrations.
For analysis of total cysteine and glutathione (thiol plus disulfide forms), 100 to 200 mg of frozen tissue was pulverized with a mortar and pestle sitting on dry ice. The powdered tissue was weighed and added to 1 mL of 1 mol/L perchloric acid containing 1 mmol/L bathophenanthroline disulfonate and 1 mmol/L bathocuproine disulfonate. The sample mixture was sonicated with two 20-s bursts separated by a cooling period, and the sonicated mixture was frozen at -20°C and then thawed and centrifuged at 15,000 x g for 10 min to obtain the acid extract. As an internal standard, -glutamylglutamate was added to the acid supernatant to yield a final concentration of 0.5 mmol/L. The sample/internal standard mixture was then derivatized as described by Fariss and Reed (16
). The 2,4-dinitrophenyl derivatives of acidic amino acids, including those of the derivatized thiols (S-carboxymethylcysteine and S-carboxymethylglutathione), were separated by reversed phase ion-exchange HPLC and quantified as described previously (17
). Tissue taurine concentration was determined by measuring taurine in the tissue homogenates prepared for CSD activity assays using the HPLC method of Bagley et al. (26
).
Statistics.
Data were analyzed by one-way ANOVA and Tukey’s 
-procedure using Minitab 13.1. When necessary, values for CDO activity and GCS activity were transformed to log10 before analysis to meet the assumption of equal variance. Pearson product moment coefficients of correlation were calculated using group means and pooled data or values for individual rats with Minitab 13.1. Differences were considered significant at P 
0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
The mean weight of rats at the start of the dietary treatment period was 274 g. Rats fed the LP diet gained less weight than those fed the more adequate diets (Table 2
), with the mean final weight of rats fed the LP diet 316 g and of rats in the other four groups, 343 g. Mean daily food intake did not differ among the groups. Absolute (Table 2)
and relative (not shown) liver and kidney weights differed among groups (Table 2)
. Tissue protein concentration (mg protein per g tissue) did not vary with dietary treatment (data not shown). Results were expressed on a concentration basis (per g tissue, per g protein or per g total RNA) and, hence, do not reflect the contribution of changes in organ weight to metabolic capacities or metabolite contents of the rat. However, increases in liver and kidney weights would magnify the effect of protein or sulfur amino acid supplementation on CDO but would somewhat compensate for the effects of diet on GCS and CSD, whether metabolic capacity was expressed as total capacity per animal or capacity per 100 g body.
|
CDO activity in liver of rats fed the LP diet was only 0.13 ± 0.007 nmol cysteinesulfinate · min-1 · mg protein-1, but CDO activity was 10-, 35-, 25- and 28-times as great in liver of rats fed the MP, HP, LP + C and LP + M diets, respectively (Table 3
). CDO activity in liver of rats fed the LP + C or LP + M diets was not different from that in liver of rats fed the HP diet, which provided a similar amount of sulfur amino acids per kg of diet. CDO concentration was 7- and 12-times as much in liver of rats fed the MP and HP diets, respectively, as in liver of rats fed the LP diet, whereas hepatic CDO activity was 10- and 35-times as much in rats fed the MP and HP diets as in those fed the LP diet. The CDO mRNA level was not different in liver of rats fed the higher levels of protein or sulfur amino acids than in liver of rats fed the LP diet. The hepatic CDO activity in rats fed the various diets followed the same pattern as did CDO concentration (r = 0.97, P = 0.01), but CDO mRNA levels did not respond to dietary treatment with relative concentrations being within a narrow range from 0.77 to 1.07.
|
. The only instance of CDO activity being more than double that in the same tissue of rats fed the LP diet was the activity of CDO in kidney of rats fed the LP + C diet, which was 2.3-times (P 0.10) that in kidney of rats fed the LP diet. This apparently higher CDO activity was associated with a CDO concentration value that was 2.6-times but with a CDO mRNA concentration that was only 0.9-times that in kidney of rats fed the LP diet. In rats fed the basal (MP) diet, CDO activity was highest in liver and much lower in kidney, lung and brain, and CDO protein concentration and CDO mRNA level followed the same pattern. Although CDO mRNA was not correlated with CDO activity or concentration in the liver of rats fed the various diets, CDO mRNA level was closely correlated with mean CDO activity (r = 0.98, P = 0.02) and with CDO concentration (r = 0.98, P = 0.02) when the four tissues were considered.
CSD activity, concentration, and mRNA level in various tissues.
CSD activity was affected by diet in liver and kidney, with the highest activities being in rats fed the LP diet (Table 4
). Activity in liver of rats fed the HP diet was 28% of that in rats fed the LP diet. Activity in kidney of rats fed the HP diet was 40% of that in rats fed the LP diet. CSD activities in liver or kidney of rats fed the MP, LP + C, and LP + M diets were intermediate to those of rats fed the LP and HP diets. CSD activity in lung and brain of rats was not affected by the differences in dietary protein or sulfur amino acid level.
|
. CSD activity and CSD concentration were correlated in both liver (r = 0.94, P = 0.02) and kidney (r = 0.98, P = 0.004). Although CSD mRNA levels in liver and kidney clearly were down-regulated in response to higher intakes of protein, the association of CSD activity or CSD concentration with CSD mRNA in either liver or kidney when all five dietary treatment groups were considered was not strong (r = 0.67 to 0.81, P = 0.10 to 0.22).
In rats fed the basal (MP) diet, CSD activity and protein concentration were highest in liver, intermediate in kidney and brain, and low in lung (Table 4)
. In contrast, CSD mRNA levels were similar in liver and kidney of rats, regardless of diet, whereas brain and lung CSD mRNA levels were low. The high expression of CSD mRNA relative to CSD concentration or activity in kidney is a notable exception to general trends observed in this study. The mean CSD activity in the various tissues of rats fed moderate protein diets was positively correlated with CSD concentration (r = 0.99, P = 0.006) but not with CSD mRNA level (r = 0.58, P = 0.42). The CSD activity relative to CSD mRNA abundance was much higher for liver than for the other tissues.
GCS activity, concentration, and mRNA level in various tissues.
GCS activity was highest in liver of rats fed the LP diet, which was deficient in protein or sulfur amino acids (Table 5
). GCS activity in liver of rats fed the MP, HP, LP + C and LP + M diets was 47, 27, 49 and 43%, respectively, of that in liver of rats fed the LP diet. GCS activity in kidney, lung, and brain did not respond to dietary treatment (Table 5)
.
|
In rats fed the basal (MP) diet, GCS activity was very high in kidney, intermediate in liver at 3 to 13% of the renal level, and somewhat lower in lung and brain (2% of the renal level). As in liver of rats fed the various diets, GCS activity differences across the four tissues were positively associated with both GCS-HS concentration (r = 0.998, P = 0.002) and GCS-HS mRNA level (r = 1.0, P < 0.001).
Tissue concentrations of cysteine, glutathione and taurine.
Hepatic concentrations of cysteine increased in a step-wise fashion with the increases in dietary protein (Table 6
). The total cysteine concentration increased from 0.02 ± 0.001 µmol/g to 0.04 ± 0.002 µmol/g to 0.07 ± 0.004 µmol/g as the casein level in the diet was increased from 100 to 200 to 400 g/kg. The cysteine concentration in liver of rats fed the LP + M diet was not different from that of rats fed the HP diet, but the cysteine concentration in liver of rats fed the LP + C diet was lower and similar to that of rats fed the MP diet. Hepatic glutathione concentration was higher in liver of rats fed the protein- or sulfur amino acid–supplemented diets (3.43 ± 0.09 to 4.36 ± 0.04 µmol/g) than in liver of rats fed the LP diet (1.65 ± 0.010 µmol/g). Hepatic glutathione concentrations in liver of rats fed the MP and HP diets did not differ and the glutathione concentration in liver of rats fed the LP + C and LP + M diets were similar and greater (P 0.05) than that in liver of rats fed the MP and HP diets. Hepatic taurine concentration increased in a step-wise fashion with the increases in dietary protein or sulfur amino acid level and did not differ among rats fed the HP, LP + C, and LP + M diets.
|
Individual hepatic CSD activities were negatively associated with hepatic cysteine (r = -0.70, P < 0.001), glutathione (r = -0.64, P < 0.001) and taurine (r = -0.80, P < 0.001) levels. Hepatic CSD concentration was negatively associated with mean hepatic cysteine (r = -0.82, P = 0.087), glutathione (r = -0.92, P = 0.025) and taurine (r = -0.90, P = 0.037) levels. CSD mRNA was not associated with hepatic cysteine, glutathione or taurine levels.
Individual hepatic GCS activities were negatively associated with hepatic cysteine (r = -0.67, P < 0.001), glutathione (r = -0.73, P < 0.001), and taurine (r = -0.71, P < 0.001) concentrations. Similar associations were observed for GCS-HS concentration and GCS-HS mRNA: GCS-HS concentration was negatively associated with mean cysteine (r = 0.85, P = 0.07), glutathione (r = -0.93, P = 0.02) and taurine (r = -0.88, P = 0.05) concentrations, and GCS-HS mRNA was negatively associated with mean cysteine (r = -0.71, P = 0.018), glutathione (r = -0.87, P = 0.05) and taurine (r = -0.98, P = 0.004) concentrations.
Cysteine concentration in kidney was not different for rats fed the three different levels of protein (Table 6)
. The only renal cysteine concentrations that differed were those in rats fed the HP and LP + M diets, with the renal cysteine concentration in those fed the LP + M diet 39% greater than that in those fed the HP diet. Glutathione concentration in kidney also did not differ among rats fed the three different levels of protein, but it was 25% higher in rats fed the LP + C diet than in rats fed the LP, MP or HP diet and in rats fed the LP + M diet than in rats fed the LP or MP diet. Renal taurine concentration was 2.3 µmol/g in rats fed the LP diet and increased to 3.5 to 5.3 µmol/g in rats fed the other diets. Renal CSD activity was not correlated with renal cysteine or glutathione concentrations, but it was negatively associated with the renal taurine concentration.
In lung, cysteine concentration did not differ among rats fed the LP, MP, HP, and LP + M diets, but cysteine concentration in lung of rats fed the LP + C diet was 35 or 39% greater than that of rats fed the LP + M or LP diet, respectively. Glutathione and taurine concentrations did not differ in lung of rats fed the five diets. Brain cysteine, glutathione and taurine concentrations were unaffected by dietary treatment.
Among the four tissues studied, the concentrations of cysteine, glutathione and taurine in rats fed the basal (MP) diet demonstrate that cysteine concentration was much higher in kidney (1 µmol/g) than in other tissues (0.03 to 0.07 µmol/g). Glutathione concentration was higher in liver (3.8 µmol/g) than in other tissues ( 1.2 µmol/g). Taurine concentration did not differ among the four tissues with values similar in all four tissues (2.3 to 4.4 µmol/g).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
) recommendation that sulfur amino acids comprise 0.6 g/100 g diet. The LP diet provided half of this (3.3 g methionine equivalents) and was inadequate in total sulfur amino acid content. The HP, LP + C and LP + M diets each provided 13.2 g methionine equivalents per kg and thus an excess of sulfur amino acids. Rats fed the LP diet consumed the same amount of food but gained less weight than did rats fed the other diets, demonstrating that the LP diet clearly was limiting in protein. Rats fed the HP, LP + C or LP + M diets did not gain more weight than did those fed the MP diet, demonstrating that the MP diet was adequate for maximal growth and that rats fed the HP, LP + C or LP + M diets clearly had excess dietary sulfur amino acids. Hepatic CDO, GCS and CSD and renal CSD change in response to changes in sulfur amino acid intake.
Cellular cysteine levels must be closely regulated to prevent cysteine toxicity on the one hand and to ensure adequate levels of cysteine for synthesis of protein, glutathione and coenzyme A on the other. Previous work in our laboratory (5
,6
,27
,28
) has shown that hepatic CDO plays a major and robust role in the regulation of body cysteine levels and that hepatic CSD and GCS also play important roles in regulating the rates of taurine and glutathione synthesis, respectively. Liver also is the major tissue involved in the transsulfuration of dietary methionine to form cysteine, largely because of the presence in liver of an isozyme of methionine adenosyltransferase (EC 2.5.1.6) that has a high Km for methionine (29
). However, the role of nonhepatic tissues in the regulation of body cysteine levels has not been studied extensively.
CDO is expressed in a highly tissue-specific manner (30
–32
). In rats and humans, the highest levels of CDO mRNA are in liver. Much lower, but substantial, amounts of CDO mRNA are in kidney and lung, and still lower, but easily detectable amounts, are in brain. Little or no CDO mRNA is in skeletal muscle, heart, testis, small intestine, spleen or pancreas. Because CDO is expressed in kidney, lung and brain, we investigated the possible effects of dietary protein or sulfur amino acid level on CDO, CSD, and GCS in these three tissues in comparison to the effects known to occur in liver.
Hepatic CDO activity increased in a step-wise manner with increases in dietary protein and was high and similar in rats fed diets supplemented with cystine or methionine to yield the same molar concentration of total sulfur amino acids as were contained in the high protein diet. CSD and GCS activities in liver decreased in a step-wise fashion with increases in dietary protein; hepatic CSD and GCS activities in rats fed the diets supplemented with cystine or methionine were intermediate to those of rats fed the moderate and high protein diets, suggesting that protein had a stronger effect than sulfur amino acids alone on these two enzymes. The up-regulation of CDO and the down-regulation of CSD and GCS in liver in response to increases in dietary level of protein or sulfur amino acids was consistent with previously reported results (5
,6
). The dietary treatments had no effect on CDO, CSD or GCS activity in kidney, lung or brain except for CSD activities in kidney. Renal CSD was significantly lower in rats fed protein- or sulfur amino acid-supplemented diets than in rats fed the low protein diet, suggesting an up-regulation of renal CSD activity when cysteine supply was limiting. This effect of dietary protein on renal CSD activity is consistent with results previously reported by Bella and Stipanuk (14
) and by Jerkins and Steele (33
).
In several ways, the changes in hepatic CDO, CSD and GCS in response to dietary protein seem to be a coordinated response to the same or related metabolic signals. The increase in hepatic CDO as the dietary protein level was increased from 100 g to 200 to 400 g/kg was associated with step-wise decreases in both CSD and GCS in liver. Overall, hepatic CDO activity in individual rats was negatively associated with hepatic CSD (r = -0.68, P < 0.001) and GCS (r = -0.56, P = 0.001) activity, whereas hepatic CSD and GCS activities were positively correlated (r = 0.70, P < 0.001). This coordinated regulation of these three hepatic enzymes of cysteine metabolism ensures that cysteine is conserved for glutathione synthesis and that any cysteinesulfinate formed is preferentially converted to taurine under conditions of low cysteine availability. However, the system is also regulated to ensure rapid catabolism of excess cysteine, somewhat favoring its conversion to pyruvate and sulfate rather than to taurine when sulfur amino acid supply exceeds the requirement.
Because CDO, CSD and GCS are not necessarily evenly distributed throughout parenchymal cells in nonhepatic tissues, studies of tissue homogenates, as reported here, may obscure large changes that occur within particular structures or cells within the tissue. Shimada et al. (30
) reported strong localization of CDO mRNA in the proximal tubules of the kidney and in the bronchiolar epithelium of the lung. Parsons et al. (34
) reported CDO mRNA expression in the proximal convoluted tubules of the renal cortex and in the collecting ducts of both the medulla and the papilla. Parsons et al. (35
) reported that CDO mRNA is localized in neurons of the brain, including the pyramidal cells of the hippocampus and the Purkinje cells of the cerebellum, but Beetsch and Olson (11
) found substantial CDO in cultured rat astrocytes. Reymond et al. (36
,37
) reported that CSD mRNA and CSD protein are localized in the proximal straight tubules of the kidney and that CSD protein was strictly localized in astrocytes within the cerebellum and hippocampus. GCS appears to be localized in glial populations throughout the brain and at various levels in discrete neuronal populations, with the highest levels in the hippocampus, cerebellum and olfactory bulb and lower levels of expression in the cortex and substantia nigra (38
,39
). GCS is localized in the proximal straight tubules of the kidney (40
). Clearly, any future studies of CDO, CSD or GCS in nonhepatic tissues should focus on particular cell types.
Association of changes in hepatic CDO, CSD and GCS and renal CSD activities with enzyme concentration and enzyme mRNA abundance.
Changes in hepatic CDO, CSD and GCS activity were associated with parallel changes in CDO, CSD and GCS-HS concentration, respectively, indicating that changes in activity were primarily due to changes in amount of enzyme rather than to changes in the activation state of the enzyme. Changes in renal CSD activity were likewise highly associated with changes in renal CSD concentration. The correlation coefficients given for the associations of enzyme activity with enzyme concentration or enzyme mRNA levels were calculated using mean and pooled values for the five groups and are, hence, only relevant to associations of means. The practice of using means for correlation statistics yields higher r-values and higher P-values compared with those obtained using data for individual animals.
Changes in hepatic CDO activity and concentration were not correlated with changes in CDO mRNA abundance, indicating that regulation of hepatic CDO in response to diet was clearly post-transcriptional. The fold-differences in hepatic CDO activity among rats fed the various diets appeared to be greater than the fold-differences in CDO protein concentration, but these apparent differences may not be meaningful considering that the opposite pattern was observed in a previous experiment (5
). We are continuing our studies of the molecular mechanism(s) by which the hepatic CDO level is regulated.
Changes in hepatic and renal CSD activity and concentration were associated with parallel changes in hepatic and renal CSD mRNA abundance, suggesting that regulation of CSD levels depends upon changes in CSD mRNA abundance. Hepatic CSD regulation at the level of mRNA was reported by Jerkins et al. (41
) and is confirmed by this study. Phosphorylation of brain CSD associated with activation of the enzyme was observed by Tang et al. (42
), but the role of phosphorylation in the regulation of CSD has not been further studied.
Renal CSD mRNA was unusually high relative to CSD concentration or activity in kidney, suggesting something unique about CSD expression in the kidney. It is very unlikely that this high level of CSD mRNA in kidney was a sampling or methodological artifact because a high level of renal CSD mRNA was consistently observed for all dietary treatment groups and because CDO mRNA, which is localized to the proximal tubules and the collecting ducts, and GCS-HS mRNA were present in the same renal total RNA at levels that mirrored the concentrations of the corresponding enzymes in kidney relative to other tissues.
Changes in hepatic GCS activity and GCS-HS concentration were associated with a similar pattern of change in hepatic GCS-HS mRNA abundance, suggesting that hepatic GCS was regulated at the level of mRNA. The effect of dietary protein or sulfur amino acid level on regulation of GCS expression has received little study, but our group has previously reported decreases in hepatic GCS in rats fed diets with high protein or high sulfur amino acid levels (5
,6
). The signaling mechanisms involved in the regulation of GCS-HS in response to dietary protein or sulfur amino acid level have not been investigated, but these seem to be initiated by mechanisms other than the oxidative or chemical stress pathways that have been extensively studied (43
,44
).
Association of changes in hepatic cysteine concentration with changes in hepatic levels of CDO, CSD and GCS.
Hepatic concentrations of cysteine increased in a step-wise fashion with the increases in dietary protein level, from 0.02 ± 0.001 to 0.04 ± 0.002 to 0.07 ± 0.004 µmol/g as the casein level in the diet was increased from 100 to 200 to 400 g/kg. These cysteine concentrations demonstrate that the hepatic cysteine concentration in rats consuming the various diets differed significantly and that cysteine level could be involved in mediation of the regulation of CDO, CSD and GCS concentrations in liver. However, the fact that the hepatic cysteine concentration did not ever exceed 0.10 µmol/g demonstrates that the regulatory changes also acted to dispose of excess cysteine and, thus, maintain hepatic cysteine concentrations at low (albeit higher), nontoxic levels.
Although the hepatic cysteine concentrations in rats fed the cystine- and methionine-supplemented diets were also greater than those in rats fed the basal LP diet, the cysteine concentration of liver of rats fed the LP + C diet was less than that of those fed the LP + M diet. The basis of this difference is not apparent, but it may be due to the particular time point (mid-point of the dark or feeding cycle) used for our measurements. Differences in rates of absorption, uptake or metabolism of methionine vs. cystine could cause hepatic cysteine concentrations to vary somewhat differently over the course of a 24-h cycle; dietary cyst(e)ine may be cleared more rapidly than dietary methionine, which must undergo transmethylation and transsulfuration to yield cysteine. The similar glutathione and taurine concentrations in liver of rats fed the LP + M and LP + C diets suggests that both groups of rats had an excess supply of cysteine.
In general, CDO activity and concentration were positively correlated and CSD and GCS activities, CSD and GCS-HS concentrations and GCS-HS mRNA levels were negatively correlated with hepatic cysteine, glutathione and taurine levels. Although greater taurine or glutathione synthesis may be expected when the activities of CSD or GCS are higher, production of taurine or glutathione is strongly influenced by the availability of substrate for CSD (i.e., cysteinesulfinate produced by CDO) or GCS (i.e., cysteine). Hence, as observed here, hepatic taurine and glutathione levels were higher when cyst(e)ine intake and CDO activity were high despite the lower activities of CSD and GCS. The close relationships among cysteine, taurine and glutathione make it impossible to argue for a primary role for cysteine in the regulation of cysteine metabolism based on this study alone. Nevertheless, the association of hepatic cysteine concentrations with CDO, CSD and GCS levels in intact rats is an important observation because it supports our finding in cultured hepatocytes that cysteine, rather than a precursor or metabolite of cysteine, is responsible for changes in CDO and GCS activities (7
). A detectable change in tissue cysteine concentration would seem crucial to any mechanism based on sensing cysteine availability. Much remains to be learned about signaling pathways involved in the regulation of cysteine catabolism, taurine synthesis and glutathione synthesis. In this regard, the recent identification of cysteine-response elements in 5'-upstream regions of several yeast genes that are involved in sulfur amino acid metabolism is noteworthy (45
).
Association of hepatic glutathione and taurine concentrations with hepatic CDO, CSD and GCS levels.
Hepatic glutathione concentration was higher in liver of rats fed the protein- or sulfur amino acid-supplemented diets (3.43 ± 0.09 to 4.36 ± 0.04 µmol/g) than in liver of rats fed the LP diet (1.65 ± 0.010 µmol/g). As mentioned above, even though GCS activity was lower in rats fed the supplemented diets, the large increase in availability of cysteine for synthesis of glutathione apparently resulted in higher glutathione concentrations in liver. The decreases in GCS activity may have restricted the synthesis of glutathione so that concentrations did not increase in an unrestricted fashion as sulfur amino acid availability increased. Hepatic taurine concentrations seemed to reflect both the increase in cysteine availability and the increase in CDO activity in liver of rats fed protein- or sulfur amino acid-supplemented diets. Hepatic taurine concentration increased in a step-wise fashion with the increases in dietary protein or sulfur amino acid level. The down-regulation of CSD activity in response to supplementation of the diet with protein or sulfur amino acids may restrict the rate of taurine (vs. pyruvate plus sulfate) formation from cysteinesulfinate, but the increased availability of cysteine and the increased activity of CDO clearly prevailed to yield increases in rates of taurine production that were reflected in increased hepatic taurine concentrations.
Cysteine, glutathione and taurine concentrations in nonhepatic tissues.
Renal cysteine concentrations were notably high compared with other tissues (0.8 to 1.1 µmol/g or > 10-times hepatic concentrations), and changes in the renal cysteine level were not associated with changes in CSD activity. Renal glutathione levels increased by 25% with increases in dietary sulfur amino acid level. Renal taurine levels increased in a step-wise fashion in response to increases in the dietary protein or sulfur amino acid level. These increases in renal taurine may be related to increased production of taurine in the liver because neither renal CDO activity nor renal cysteine concentration were increased and renal CSD activity was actually decreased.
Cysteine concentrations in lung ranged from 0.06 to 0.08 µmol/g tissue and those in brain ranged from 0.02 to 0.03 µmol/g tissue. Although cysteine concentrations and enzyme activities did not vary in lung, the highest concentration of CDO was in lung of rats fed the cystine-supplemented diet; lungs of these rats also had the highest cysteine concentration (Tables 3
and 6)
. Additionally, although there were no differences in enzyme activities or tissue cysteine, glutathione or taurine concentrations in brain, levels of immunodetectable CDO were greater in the brain of rats fed the LP + C diet.
The higher levels of CDO protein in kidney, lung and brain of rats fed the LP + C diet, and to a lesser extent in these tissues of rats fed the LP + M diet, suggest that it is possible to increase sulfur amino acid concentrations in these nonhepatic tissues and that elevated cysteine levels in these tissues may up-regulate CDO levels. These relationships are worth further exploration. They suggest that the mechanisms involved in the regulation of hepatic CDO may be active in other tissues that express CDO should those tissues be exposed to higher concentrations of cysteine. This hypothesis is supported by studies of Beetsch and Olson (11
) with cultured rat astrocytes in which cysteine concentration was strongly associated with CDO activity.
Nevertheless, regulation of body cysteine, glutathione and taurine levels in response to dietary changes appears to be primarily by the liver, not by the nonhepatic tissues. This study clearly demonstrates that there is little difference in cysteine-metabolizing enzyme concentrations or in the levels of cysteine and cysteine metabolites in nonhepatic tissues of rats adapted to diets that contain varied levels of protein or sulfur amino acids.
|
FOOTNOTES |
|---|
3 Abbreviations used: CDO, cysteine dioxygenase; CSD, cysteinesulfinate decarboxylase; GCS, -glutamylcysteine synthetase; GCS-HS, -glutamylcysteine synthetase heavy (catalytic) subunit.
Manuscript received 9 July 2002. Initial review completed 17 July 2002. Revision accepted 11 August 2002.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Karlsen, R. L., Grofova, I., Malthe-Sorenssen, D. & Fonnum, F. (1981) Morphological changes in rat brain induced by L-cysteine injection in newborn animals. Brain Res. 208:167-180.[Medline]
2. Mathisen, G. A., Fonnum, F. & Paulsen, R. E. (1996) Contributing mechanisms for cysteine excitotoxicity in cultured cerebellar granule cells. Neurochem. Res. 21:293-298.[Medline]
3. Pedersen, O. O. & Karlsen, R. L. (1980) The toxic effect of L-cysteine on the rat retina. A morphological and biochemical study. Invest. Ophthalmol. Vis. Sci. 19:886-892.
4. Sandberg, M., Orwar, O. & Hehmann, A. (1991) L-Cysteine toxicity: effects on non-sulfur amino acids, sulfur-containing excitatory amino acids and -glutamyl peptides in the immature brain. J. Neurochem. 57:S152.
5. Bella, D. L., Hahn, C. & Stipanuk, M. H. (1999) Effects of nonsulfur and sulfur amino acids on the regulation of hepatic enzymes of cysteine metabolism. Am. J. Physiol. 277:E144-E153.
6. Bella, D. L., Hirschberger, L. L., Hosokawa, Y. & Stipanuk, M. H. (1999) Mechanisms involved in the regulation of key enzymes of cysteine metabolism in rat liver in vivo. Am. J. Physiol. 276:E326-E335.
7. Kwon, Y. H. & Stipanuk, M. H. (2001) Cysteine regulates expression of cysteine dioxygenase and -glutamylcysteine synthetase in cultured rat hepatocytes. Am. J. Physiol. Endocrinol. Metab. 280:E804-E815.
8. Garcia, R. A. & Stipanuk, M. H. (1992) The splanchnic organs, liver and kidney have unique roles in the metabolism of sulfur amino acids and their metabolites in rats. J. Nutr. 122:1693-1701.
9. Qusti, S., Parsons, R. B., Abouglila, K. D., Waring, R. H., Williams, A. C. & Ramsden, D. B. (2000) Development of an in vitro model for cysteine dioxygenase expression in the brain. Cell Biol. Toxicol. 16:243-255.[Medline]
10. Parsons, R. B., Barber, P. C., Waring, R. H., Williams, A. C. & Ramsden, D. B. (1998) Cysteine dioxygenase: regional expression of activity in rat brain. Neurosci. Lett. 248:101-104.[Medline]
11. Beetsch, J. W. & Olson, J. E. (1998) Taurine synthesis and cysteine metabolism in cultured rat astrocytes: effects of hyperosmotic exposure. Am. J. Physiol. 274:C866-C874.
12. Reeves, P. G., Nielsen, F. H. & Fahey, G. C., Jr. (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.
13. National Research Council (1978) Nutrient Requirements of Laboratory Animals 1978:13-16 National Academy of Sciences Washington, DC .
14. Bella, D. L. & Stipanuk, M. H. (1995) Effects of protein, methionine, or chloride on acid-base balance and on cysteine catabolism. Am. J. Physiol. 269:E910-E917.
15. Yan, C. C. & Huxtable, R. J. (1996) Effects of monocrotaline, a pyrrolizidine alkaloid, on glutathione metabolism in the rat. Biochem. Pharmacol. 51:375-379.[Medline]
16. Fariss, M. W. & Reed, D. J. (1987) High-performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives. Methods Enzymol. 143:101-109.[Medline]
17. Stipanuk, M. H., Bagley, P. J., Coloso, R. M. & Banks, M. F. (1992) Metabolism of cysteine to taurine by rat hepatocytes. Adv. Exp. Med. Biol. 315:413-421.[Medline]
18. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. & Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76-85.[Medline]
19. Hosokawa, Y., Matsumoto, A., Oka, J., Itakura, H. & Yamaguchi, K. (1990) Isolation and characterization of a cDNA for rat liver cysteine dioxygenase. Biochem. Biophys. Res. Commun. 168:473-478.[Medline]
20. Weinstein, C. L. & Griffith, O. W. (1987) Multiple forms of rat liver cysteinesulfinate decarboxylase. J. Biol. Chem. 262:7254-7263.
21. Shi, M. M., Kugelman, A., Iwamoto, T., Tian, L. & Forman, H. J. (1994) Quinone-induced oxidative stress elevates glutathione and induces -glutamylcysteine synthetase activity in rat lung epithelial L2 cells. J. Biol. Chem. 269:26512-26517.
22. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227:680-685.[Medline]
23. Kaisaki, P. J., Jerkins, A. A., Goodspeed, D. C., Steele, R. D. & Kaisakia, P. J. (1995) Cloning and characterization of rat cysteine sulfinic acid decarboxylase. Biochim. Biophys. Acta 1262:79-82.[Medline]
24. Yan, N. & Meister, A. (1990) Amino acid sequence of rat kidney -glutamylcysteine synthetase. J. Biol. Chem. 265:1588-1593.
25. Brown, C. (1993) Analysis of RNA by Northern and slot blot hybridization. Ausubel, F. M. Brent, R. Kingston, R. E. Moore, D. D. Seidman, J. G. Smith, J. A. Struul, K. eds. Current Protocols in Molecular Biology 1993:4.9.1-4.9.14 Wiley-Interscience New York .
26. Bagley, P. J., Hirschberger, L. L. & Stipanuk, M. H. (1995) Evaluation and modification of an assay procedure for cysteine dioxygenase activity: high-performance liquid chromatography method for measurement of cysteine sulfinate and demonstration of physiological relevance of cysteine dioxygenase activity in cysteine catabolism. Anal. Biochem. 227:40-48.[Medline]
27. Bagley, P. J. & Stipanuk, M. H. (1994) The activities of rat hepatic cysteine dioxygenase and cysteine sulfinate decarboxylase are regulated in a reciprocal manner in response to dietary casein level 1,2,3. J. Nutr. 124:2410-2421.
28. Bagley, P. J. & Stipanuk, M. H. (1995) Rats fed a low protein diet supplemented with sulfur amino acids have increased cysteine dioxygenase activity and increased taurine production in hepatocytes. J. Nutr. 125:933-940.
29. Sullivan, D. M. & Hoffman, J. L. (1983) Fractionation and kinetic properties of rat liver and kidney methionine adenosyltransferase isozymes. Biochemistry 22:1641-1645.[Medline]
30. Shimada, M., Koide, T., Kuroda, E., Tsuboyama, N., Hosokawa, Y. & Watanabe, M. (1998) Expression and localization of cysteine dioxygenase mRNA in the liver, lung, and kidney of the rat. Amino Acids (Vienna) 15:143-150.[Medline]
31. Hirschberger, L. L., Daval, S., Stover, P. J. & Stipanuk, M. H. (2001) Murine cysteine dioxygenase gene: structural organization, tissue-specific expression and promoter identification. Gene (Amst.) 277:153-161.[Medline]
32. Tsuboyama-Kasaoka, N., Hosokawa, Y., Kodama, H., Matsumoto, A., Oka, J. & Totani, M. (1999) Human cysteine dioxygenase gene: structural organization, tissue-specific expression and downregulation by phorbol 12-myristate 13-acetate. Biosci. Biotechnol. Biochem. 63:1017-1024.[Medline]
33. Jerkins, A. A. & Steele, R. D. (1991) Cysteine sulfinic acid decarboxylase activity in response to thyroid hormone administration in rats. Arch. Biochem. Biophys. 286:428-432.[Medline]
34. Parsons, R. B., Sampson, D., Huggins, C. C., Waring, R. H., Williams, A. C. & Ramsden, D. B. (2001) Renal localisation of rat cysteine dioxygenase. Nephron 88:340-346.[Medline]
35. Parsons, R. B., Waring, R. H., Williams, A. C. & Ramsden, D. B. (2001) Cysteine dioxygenase: regional localisation of protein and mRNA in rat brain. J. Neurosci. Res. 65:78-84.[Medline]
36. Reymond, I., Almarghini, K. & Tappaz, M. (1996) Immunocytochemical localization of cysteine sulfinate decarboxylase in astrocytes in the cerebellum and hippocampus: a quantitative double immunofluorescence study with glial fibrillary acidic protein and S-100 protein. Neuroscience 75:619-633.[Medline]
37. Reymond, I., Bitoun, M., Levillain, O. & Tappaz, M. (2000) Regional expression and histological localization of cysteine sulfinate decarboxylase mRNA in the rat kidney. J. Histochem. Cytochem. 48:1461-1468.
38. Li, S., Thompson, S. A., Kavanagh, T. J. & Woods, J. S. (1996) Localization by in situ hybridization of -glutamylcysteine synthetase mRNA expression in rat kidney following acute methylmercury treatment. Toxicol. Appl. Pharmacol. 141:59-67.[Medline]
39. Kang, Y., Oiao, X., Jurma, O., Knusel, B. & Andersen, J. K. (1997) Cloning/brain localization of mouse glutamylcysteine synthetase heavy chain mRNA. Neuroreport 8:2053-2060.[Medline]
40. Guder, W. G. & Ross, B. D. (1984) Enzyme distribution along the nephron. Kidney Int. 26:101-111.[Medline]
41. Jerkins, A. A., Jones, D. D. & Kohlhepp, E. A. (1998) Cysteine sulfinic acid decarboxylase mRNA abundance decreases in rats fed a high-protein diet. J. Nutr. 128:1890-1895.
42. Tang, X. W., Hsu, C. C., Schloss, J. V., Faiman, M. D., Wu, E., Yang, C. Y. & Wu, J. Y. (1997) Protein phosphorylation and taurine biosynthesis in vivo and in vitro. J. Neurosci. 17:6947-6951.
43. Soltaninassab, S. R., Sekhar, K. R., Meredith, M. J. & Freeman, M. L. (2000) Multi-faceted regulation of -glutamylcysteine synthetase. J. Cell Physiol. 182:163-170.[Medline]
44. Wild, A. C. & Mulcahy, R. T. (2000) Regulation of -glutamylcysteine synthetase subunit gene expression: insights into transcriptional control of antioxidant defenses. Free Radic. Res. 32:281-301.[Medline]
45. Miyake, T., Kanayama, M., Sammoto, H. & Ono, B. (2002) A novel cis-acting cysteine-responsive regulatory element of the gene for the high-affinity glutathione transporter of Saccharomyces cerevisiae. Mol. Genet. Genomics 266:1004-1011.[Medline]
This article has been cited by other articles:
|
|
 |
|
  G. Courtney-Martin, M. Rafii, L. J. Wykes, R. O. Ball, and P. B. Pencharz Methionine-Adequate Cysteine-Free Diet Does Not Limit Erythrocyte Glutathione Synthesis in Young Healthy Adult Men J. Nutr., November 1, 2008; 138(11): 2172 - 2178. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Furne, A. Saeed, and M. D. Levitt Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2008; 295(5): R1479 - R1485. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. N. Dilger and D. H. Baker Excess Dietary L-Cysteine Causes Lethal Metabolic Acidosis in Chicks J. Nutr., September 1, 2008; 138(9): 1628 - 1633. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-I. Lee, J. E. Dominy Jr., A. K. Sikalidis, L. L. Hirschberger, W. Wang, and M. H. Stipanuk HepG2/C3A cells respond to cysteine deprivation by induction of the amino acid deprivation/integrated stress response pathway Physiol Genomics, April 1, 2008; 33(2): 218 - 229. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Ueki and M. H. Stipanuk Enzymes of the Taurine Biosynthetic Pathway Are Expressed in Rat Mammary Gland J. Nutr., August 1, 2007; 137(8): 1887 - 1894. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ye, X. Wu, L. Wei, D. Tang, P. Sun, M. Bartlam, and Z. Rao An Insight into the Mechanism of Human Cysteine Dioxygenase: KEY ROLES OF THE THIOETHER-BONDED TYROSINE-CYSTEINE COFACTOR J. Biol. Chem., February 2, 2007; 282(5): 3391 - 3402. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. N. Dilger, S. Toue, T. Kimura, R. Sakai, and D. H. Baker Excess Dietary L-Cysteine, but Not L-Cystine, Is Lethal for Chicks but Not for Rats or Pigs J. Nutr., February 1, 2007; 137(2): 331 - 338. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
