Rat Liver Serine Dehydratase Research Articles

Down Regulation of Asparagine Synthetase and 3-Phosphoglycerate Dehydrogenase, and the Up-Regulation of Serine Dehydratase in Rat Liver from Intake of Excess Amount of Leucine Are Not Related to Leucine-Caused Amino Acid Imbalance.

Asparagine synthetase (ASNS), 3-phosphoglycerate dehydrogenase (PHGDH) and serine dehydratase (SDS) in rat liver are expressed in response to protein and amino acid intake. In the present study, we examined the expression of these enzymes in relation to amino acid imbalance caused by leucine. Rats were subjected to leucine administration in the diet or orally between meals. Consumption of more than 2% leucine in a 6% casein diet suppressed food intake and caused growth retardation in a dose-dependent manner, but this was not seen in a 12% or 40% casein diet. ASNS and PHGDH expression in the liver was significantly induced by the 6% casein diet and was suppressed by leucine in a dose-dependent manner, whereas the SDS expression was induced. These effects were leucine specific and not seen with ingestion of isoleucine or valine. However, leucine orally administered between meals did not change the food intake or growth of rats fed a 6% casein die, though it similarly affected the expression of ASNS, PHGDH and SDS in the liver. These results suggest that the growth retardation caused by leucine imbalance was mainly because of the suppression of food intake, and demonstrated that there are no causal relationships between ASNS, PHGDH and SDS expression and amino acid imbalance caused by leucine.

Excess Leucine Intake Induces Serine Dehydratase in Rat Liver

We have hypothesized that rat liver serine dehydratase (SDH) is induced in response to the amount of surplus amino acids from dietary protein. In the present study, we found that excess leucine intake strongly induced SDH activity in the liver but not in the kidney of rats. The increase in activity was accompanied by increases in the levels of SDH mRNA. On the other hand, isoleucine and valine had little effect on SDH induction. These results support our hypothesis and suggest that leucine is a signal for SDH induction.

Some biochemical and histochemical properties of human liver serine dehydratase

In rat, serine dehydratase (SDH) is abundant in the liver and known to be a gluconeogenic enzyme, while there is little information about the biochemical property of human liver serine dehydratase because of its low content and difficulty in obtaining fresh materials. To circumvent these problems, we purified recombinant enzyme from Escherichia coli, and compared some properties between human and rat liver serine dehydratases. Edman degradation showed that the N-terminal sequence of about 75% of human serine dehydratase starts from Met START-Met 2-Ser 3- and the rest from Ser 3-, whereas the N-terminus of rat enzyme begins from the second codon of Met START-Ala 2-. The heterogeneity of the purified preparation was totally confirmed by mass spectrometry. Accordingly, this observation in part fails to follow the general rule that the first Met is not removed when the side chain of the penultimate amino acid is bulky such as Met, Arg, Lys, etc. There existed the obvious differences in the local structures between the two enzymes as revealed by limited-proteolysis experiments using trypsin and Staphylococcus aureus V8 protease. The most prominent difference was found histochemically: expression of rat liver serine dehydratase is confined to the periportal region in which many enzymes involved in gluconeogenesis and urea cycle are known to coexist, whereas human liver serine dehydratase resides predominantly in the perivenous region. These findings provide an additional support to the previous notion suggested by physiological experiments that contribution of serine dehydratase to gluconeogenesis is negligible or little in human liver.

Inverse correlation between the nitrogen balance and induction of rat liver serine dehydratase (SDH) by dietary protein.

Rats of different ages (3 to 15-wk-old) were fed on a 25% casein diet for one week, and the nitrogen balance and liver serine dehydratase (SDH, EC 4.2.1.13) activity were then determined. The value for nitrogen balance decreased with the age of the rats, while the liver SDH activity increased. A statistical analysis showed clear inverse correlation between the two factors (R(2) = 0.7372, p < 0.01). This result suggests that SDH was induced by response to the amount of surplus amino acids from dietary protein taken beyond the body's requirement. The increase in SDH activity was accompanied by an increase in the level of SDH mRNA. Since the half-life of this mRNA did not change significantly, the induction was mainly controlled at the level of transcription. In addition, the induction seems not to be related to gluconeogenesis, since the mRNA levels of tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), other gluconeogenic enzymes, were not changed under these experimental conditions.

Response of the induction of rat liver serine dehydratase to changes in the dietary protein requirement.

Growing and mature rats were examined for the effect of a change in dietary protein requirements on the induction of liver serine dehydratase (SDH). The rats were fed on diets varying in casein content, and the weight change and nitrogen balance was determined. SDH activity and its gene expression were induced in both growing and mature rats when their protein intake exceeded their nutritional requirements.

Evidence for a dimeric structure of rat liver serine dehydratase

Rat liver serine dehydratase (SDH) is known to be involved in gluconeogenesis. It has long been believed to be a dimeric protein with the subunit molecular weight ( M r) of 34,000. Recently, sheep liver SDH was reported to be a monomer with a M r of 38,000. The native M r of rat SDH was only determined by the ultracentrifugation method more than three decades ago, and that of sheep SDH was done by the method of gel chromatography. The primary to quaternary structures of a given enzyme in a specific mammalian organ are usually conserved among various species. The aim of the present investigation is to clarify the structural differences between rat and sheep SDHs. First, we found that the amino acid composition reported for sheep SDH was statistically similar to that of rat SDH. Second, immunoblot analysis using anti-rat SDH IgG as the probe showed the size of sheep SDH to be a M r of 30,500, whereas that of SDH was about M r of 35,000. On the other hand, the native size of rat SDH was assessed by two methods: (1) the laser light scattering method demonstrated that rat SDH had a M r of 66,800, consistent with the previous value ( M r=64,000); (2) cross-linking experiments of the purified rat SDH with dimethyl suberimidate revealed the existence of a dimeric form by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present results clearly confirm that rat SDH is a dimer, and suggest that sheep SDH is similar to rat SDH immunologically, but with a molecular weight 7500 smaller than reported previously.

Rat Liver Serine Dehydratase: BACTERIAL EXPRESSION AND TWO FOLDING DOMAINS AS REVEALED BY LIMITED PROTEOLYSIS

A pCW vector harboring rat liver serine dehydratase cDNA was expressed in Escherichia coli. The expressed level was about 5-fold higher in E. coli BL21 than in JM109 cell extract; the former lacked two kinds of proteases. Immunoblot analysis revealed the occurrence of a derivative other than serine dehydratase in the JM109 cell extract. The recombinant enzyme was purified to homogeneity. Staphylococcus aureus V8 protease and trypsin cleaved the enzyme at Glu-206 and Lys-220, respectively, with a concomitant loss of enzyme activity. Spectrophotometrically, the nicked enzyme showed a approximately 50% reduced capacity for binding of the coenzyme pyridoxal phosphate and no spectral change of circular dichroism in the region at 300-480 nm, whereas circular dichroism spectra of both enzymes in the far-UV region were similar, suggesting that proteolysis impairs the coenzyme binding without an accompanying gross change of the secondary structure. Whereas the nicked enzyme behaved like the intact enzyme on Sephadex G-75 column chromatography, it was dissociated into two fragments on the column containing 6 M urea. Upon the removal of urea, both fragments spontaneously refolded. These results suggest that serine dehydratase consists of two folding domains connected by a region that is very susceptible to proteases.

Non-sympathetic Nerve Regulation of Daily Rhythms of Serine Dehydratase, D Site-binding Protein, and HMG-CoA Reductase mRNA Levels in Rat Liver

Rat liver serine dehydratase, D site-binding protein, HMG-CoA reductase mRNA levels are known to exhibit daily rhythms which are affected by lesions of the suprachiasmatic nuclei (Ogawa and Ansai (1995) Arch. Biochem. Biophys., 316: 844–850) but not by adrenalectomy (Ansai, Y. and Ogawa, H. (1996) Biol. Rhythm Res., 27: 175–184). Autonomic nerves have been reported to be important in some metabolic processes in the liver, but little is known about the role of innervation in the generation of these rhythms. To this end, rat sympathetic hepatic nerves were destroyed by surgical and chemical procedures, and after four days the animals were killed at 6-hour intervals. Concentrations of noradrenaline, a major catecholamine in the liver, decreased to below 5% of control levels, indicating the completeness of denervation. However, the above three mRNA levels at each time point were not substantially influenced by this treatment. These findings suggest that the daily rhythms of serine dehydratase, D site-binding protein, and HMG-CoA reductase mRNA levels are not under sympathetic nervous control.

Diurnal Rhythms of Rat Liver Serine Dehydratase, D-Site Binding Protein, and 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase mRNA Levels Are Altered by Destruction of the Suprachiasmatic Nucleus of the Hypothalamus

It is generally accepted that a clock generating biological rhythms exists in the suprachiasmatic nuclei (SCN) of the hypothalamus in rodents. We have examined if diurnal variations of rat liver serine dehydratase (SDH), D-site binding protein (DBP), and 3-hydrogy-3-methylglutaryl coenzyme A reductase (HMGR) mRNA levels, each of which exhibits low values during the daytime (06:00-18:00) and high values during the dark period, are controlled by the SCN, The SCN were destructed electrolytically, and, 2 weeks later, rats were sacrificed at 06:00, 12:00, 18:00, and 24:00. Completeness of SCN lesions was assessed by the alteration of spontaneous locomotor activity and histological examination of the brains. The SDH and DBP mRNA levels in SCN-lesioned rats were very low at 06:00 and almost eliminated at 18:00, whereas the HMGR mRNA level at 06:00 was low at 06:00 and slightly increased at 18:00 compared to the control values. When determined at 12:00 and 24:00, the SDH, DBP, and HMGR mRNA levels in sham-operated rats were higher at 24:00 than at 12:00, whereas those in SCN-lesioned rats were higher at 12:00 than at 24:00. The hepatic β-actin and glycine N-methyltransferase mRNA levels showed no significant diurnal variation and were not affected by SCN lesions. These results indicate that the SCN also play a pivotal role in determining rhythmic gene expressions in liver.

Periportal expression of the serine dehydratase gene in rat liver

The mRNA for rat liver serine dehydratase, a gluconeogenic enzyme, exhibits a circadian rhythm with a maximum at the onset of darkness marking the end of the fasting period and a minimum at the onset of light that marks the end of the feeding period, when rats have free access to food and water. In situ hybridization with an antisense cRNA probe revealed that serine dehydratase mRNA was localized in the periportal area of rat liver parenchyma in the evening, whereas it was scarce in the liver in the morning. The predominant localization of serine dehydratase mRNA in the periportal area also occurred in livers of rats that underwent laparotomy, glucagon and dexamethasone administration, and streptozotocin-induced diabetes mellitus, all of which are known to induce serine dehydratase mRNA levels remarkably. Immunostaining revealed that the localization of serine dehydratase protein agreed with that of succinate dehydrogenase, another enzyme known to be predominant in the periportal zone. Thus, the periportal serine dehydratase gene expression strongly supports the idea of metabolic zonation that gluconeogenesis from amino acids occurs preferentially in the periportal parenchyma of rat liver.

Laparotomy Causes a Transient Induction of Rat Liver Serine Dehydratase mRNA

Rat liver serine dehydratase mRNA shows rhythmicity with a high level at the onset of dark (19:00) and a low level at the onset of light (07:00). We have examined the effect of stress (laparotomy) on the rhythm, Upon laparotomy at 09:00 or 17:00, a marked induction of serine dehydratase mRNA occurred 2 h after operation. The elevated mRNA level then decreased and the original mRNA rhythm resumed 2 days later. By contrast, the transcription activator protein DBP mRNA level which shows a similar oscillation was not affected by this treatment. The induction was also seen in adrenalectomized rats that had been treated with hydrocortisone but not with saline or noradrenaline, indicating that glucocorticoids are absolutely necessary for the induction. Pretreatment of rats with phenoxybenzamine, and prazosin prevented the effect of laparotomy, but propranolol and yohimbine had no effect, indicating the necessity of the α1-adrenergic stimulation for the induction. These results suggest that laparotomy releases glucocorticoids and neural noradrenaline that stimulates α1-adrenergic receptors, thereby leading to the serine dehydratase gene expression.

Serine and threonine catabolism in Saccharomyces cerevisiae: the CHA1 polypeptide is homologous with other serine and threonine dehydratases.

The catabolic L-serine (L-threonine) dehydratase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source. Previously we have cloned the CHA1 gene by complementation of a mutant, cha1, lacking the dehydratase activity. Here we present the DNA sequence of a 1,766-bp fragment of the CHA1 region encompassing an open reading frame of 1080 bp. Comparison of the predicted amino acid sequence of the CHA1 polypeptide with that of other serine/threonine dehydratases revealed several blocks of sequence homology. Thus, the amino acid sequence of rat liver serine dehydratase (SDH2) and the CHA1 polypeptide are 44% homologous allowing for conservative substitutions, while 36% similarity is found between the catabolic threonine dehydratase (tdcB) of Escherichia coli and the CHA1 protein. This strongly suggests that CHA1 is the structural gene for the yeast catabolic serine (threonine) dehydratase. S1-nuclease mapping of the CHA1 mRNA ends showed a major transcription initiation site corresponding to an untranslated leader of about 19 nucleotides, while a major polyadenylation site was located about 86 nucleotides downstream from the open reading frame. Furthermore, we have mapped the chromosomal position of the CHA1 gene to less than 0.5 kb centromere proximal to HML on the left arm of chromosome III.

Identification of glucocorticoid- and cyclic AMP-responsive elements of the rat serine dehydratase gene: Difference in responses of the transfected and chromosomal genes

Transcription of the gene coding for serine dehydratase (SDH, EC 4.2.1.13) in rat liver is induced 3–4 fold by glucocorticoids plus glucagon, but not by either hormone alone. For identification of the DNA elements mediating the glucocorticoid- and cyclic AMP-regulated expression of the SDH gene, primary cultures of adult rat hepatocytes were transfected with a fusion gene consisting of the 2.15 kb 5′-flanking sequence of the SDH gene linked to the coding sequence of the gene for chloramphenicol acetyltransferase (CAT). CAT assay demonstrated that transient expression of the SDH-CAT fusion gene was inducible by either dexamethasone or dibutyryl cyclic AMP, but that the effects of these inducers were not additive or synergistic. These results suggest that some structural organization of the DNA influences the hormonal actions in regulation of gene expression.

Rat serine dehydratase gene codes for two species of mRNA of which only one is translated into serine dehydratase.

With the previously obtained rat liver serine dehydratase cDNA (SDH2; Ogawa, H., Miller, D.A., Dunn, T., Su, Y., Burcham, J. M., Peraino, C., Fujioka M., Babcock, K., and Pitot, H. C. (1988) Proc. Natl. Acad. Sci. U.S. A. 85, 5809-5813) as a probe, we isolated a different species of cDNA (SDH3) from the same cDNA library from which SDH2 was obtained. Nucleotide sequence analysis has indicated that SDH3 has an open reading frame which encodes 327 amino acid residues and which is identical to that of the cDNA obtained by Noda et al. (Noda, C., Ito, K., Nakamura, T., and Ichihara, A., (1988) FEBS Lett. 234, 331-335). Primer extension analysis and RNase protection mapping clarified that the SDH3 mRNA was the major mRNA for serine dehydratase in the liver, and its transcription begins with a T residue located 23 nucleotides down-stream of a TATA-like box. In vitro transcription/translation experiment demonstrated that SDH3 encoded a polypeptide of 35 kDa, a size in agreement with that of the subunit of the purified protein, whereas SDH2, despite having a size larger than SDH3, produced a peptide of much smaller size that reacted with anti-serine dehydratase IgG. SDH2 was found to have a stop codon early in the sequence and is predicted to encode a polypeptide of 8.9 kDa. Also, SDH2 has a 5'-noncoding sequence different from that of SDH3. These results indicate that alternative transcription initiation and different modes of splicing of the primary transcripts of rat serine dehydratase gene result in the formation of two species of mRNA, of which only one is translated into the mature serine dehydratase protein.

Human Liver Serine Dehydratase: cDNA cloning and sequence homology with hydroxyamino acid dehydratases from other sources

Rat liver serine dehydratase cDNA was used to screen a human liver cDNA library in λgt11. One positive clone occurred in every 5,000 clones. Fifteen positive clones were plaque purified. The largest cDNA obtained contained an open reading frame of 987 base pairs, and 5′ and 3′ noncoding regions of 89 and 317 base pairs, respectively. The deduced amino acid sequence, with a calculated Mr of 34,615, was similar to that of rat liver serine dehydratase except for the absence of a segment consisting of 36 amino acid residues. In vitro transcription/translation with the cDNA resulted in the formation of a polypeptide with an Mrof ∼35,000, which cross-reacted with the anti-rat serine dehydratase antibody. These results suggest that the human serine dehydratase is structurally cognate with the rat enzyme. Moreover, portions of the sequence postulated to be essential for activity in microbial threonine dehydratases are found in the mammalian serine dehydratases, suggesting that hydroxyamino and dehydratases may have originated from a common ancestor.

The peptide sequences near the bound pyridoxal phosphate are conserved in serine dehydratase from rat liver, and threonine dehydratases from yeast and Escherichia coli.

The blocked amino-terminal residue of rat liver serine dehydratase was shown to be acetylalanine by analysis of an isolated amino-terminal peptide after digestion with acylamino acid-releasing enzyme. Digestion of the borohydride-reduced, carboxymethylated enzyme with lysyl endopeptidase yielded a single epsilon-N-pyridoxyllysine-containing peptide, whose sequence is Met-Asp-Ser-Ser-Gln-Pro-Ser-Gly-Ser-Phe-Lys(Pxy)-Ile-Arg-Gly- His-Leu-Cys(Cm)-Lys. This peptide comprises residues 30-49 of the cDNA-deduced amino acid sequence. The sequence of seven amino acids around the bound pyridoxal phosphate is highly conserved in serine dehydratase from rat liver, and threonine dehydratases from yeast and Escherichia coli.

Isolation and nucleotide sequence of the cDNA for rat liver serine dehydratase mRNA and structures of the 5' and 3' flanking regions of the serine dehydratase gene.

Rat serine dehydratase cDNA clones were isolated from a lambda gt11 cDNA library on the basis of their reactivity with monospecific immunoglobulin to the purified enzyme. Using the cDNA insert from a clone that encoded the serine dehydratase subunit as a probe, additional clones were isolated from the same library by plaque hybridization. Nucleotide sequence analysis of the largest clone obtained showed that it has 1444 base pairs with an open reading frame consisting of 1089 base pairs. The deduced amino acid sequence contained sequences of several portions of the serine dehydratase protein, as determined by Edman degradation. Rat liver serine dehydratase mRNA virtually disappeared from livers of rats fed a protein-free diet for 5 days. Several genomic clones were isolated from two libraries. Determinations of the transcription start site and the structure of the 3' flanking region of the gene indicated that the coded mRNA is 1504 nucleotides long. The 5' promoter region contained a variety of sequences similar to several consensus sequences believed to be important for the regulation of specific gene expression.

Primary structure of rat liver serine dehydratase deduced from the cDNA sequence

The nucleotide sequence of serine dehydratase mRNA of rat liver has been determined from a recombinant cDNA clone, previously cloned in this laboratory, and from a recombinant cDNA clone screened from a primer-extended cDNA library. The sequence of 1322 nucleotides includes the entire protein coding region and noncoding regions on the 3′- and 5′-sides. The deduced polypeptide consists of 327 amino acid residues with a calculated molecular mass of 34462 Da. Comparison of the amino acid sequences of the serine dehydratase polypeptide with those of biosynthetic threonine dehydratase of yeast and biodegradative threonine dehydratase of E. coli revealed various extents of homology. A heptapeptide sequence, Gly-Ser-Phe-Lys-Ile-Arg-Gly, which is the pyridoxal-binding site in the yeast and E. coli threonine dehydratases, was found as a highly conserved sequence.

Molecular cloning of DNA complementary to mRNA of rat liver serine dehydratase

A cDNA clone containing sequences complementary to the mRNA cording for rat hepatic serine dehydratase was isolated to study the multihormonal regulation of this enzyme. Serine dehydratase mRNA was partially purified (50-fold enrichment, 8.2% of the total mRNA activity) from the liver of rats fed high protein diet by polysome immunoadsorption followed by oligo(dT)-cellulose column chromatography. This preparation was used as template for synthesis of cDNA. Double-stranded cDNA sequences were inserted into the plasmid pBR322 and cloned in Esherichia coli DHl. Of 860 transformants screened, 6 clones containing DNA complementary to serine dehydratase mRNA were identified by differential colony hybridization and hybrid-selected translation. The length of serine dehydratase mRNA was estimated to be 1,500 bases by Northern blot analysis. One cloned cDNA comprised about 1,000 base pairs, or 65% of the length of the mRNA. The amount of the mRNA was greatly increased in the liver of rats given high protein diet.

Effects of feeding and lighting stimuli on the synthesis of ornithine aminotransferase and serine dehydratase in rat liver

In a previous study we showed that rats fed ad libitum and maintained on a 12-h light/ 12-h dark cycle demonstrated out-of-phase circadian oscillations in the rates of ornithine aminotransferase and serine dehydratase synthesis. As part of an investigation of the factors regulating both the generation of these cycles and their dissimilarity, this paper ompares the circadian fluctuations in the rates of ornithine aminotransferase and serine dehydratase synthesis measured immunochemically in rats given a single 2-h daily feeding in conjunction with exposure to constant light or a 12-h light/12-h dark cycle. When the 2-hr feeding was administered to rats under constant light, reciprocal circadian oscillations in ornithine aminotransferase and serine dehydratase synthesis were observed regardless of the temporal location of the feeding interval. Ornithine aminotransferase synthesis began to increase after the feeding interval and reached a maximum 12 h later while serine dehydratase showed the opposite response. In rats maintained on both the restricted feeding regimen and a 12-h light/12-h dark cycle, however, retention of synthesis oscillations depended on the temporal location of the restricted feeding interval within the light-dark cycle. Rats fed for 2 h at the beginning of the dark phase exhibited circadian oscillations in serine dehydratase synthesis and a high nonoscillating level of ornithine aminotransferase synthesis, whereas rats fed for 2 h at the beginning of the light phase exhibited circadian oscillations in ornithine aminotransferase synthesis and a low nonoscillating level of serine dehydratase synthesis. These responses suggest the existence of meal-responsive and light-responsive regulators of ornithine aminotransferase and serine dehydratase synthesis.