Developmental and growth-related regulation of expression of serine dehydratase mRNA in rat liver

In rat liver, serine dehydratase mRNA is undetectable in the late prenatal period, but its level increases rapidly after birth to a transient peak, and then after decrease gradually increases again to a maximum 2 weeks after birth that is slightly higher than that of adult liver. To determine whether mature quiescent hepatocytes proliferate without loss of differentiated functions, we measured the serine dehydratase mRNA contents in regenerating liver and primary cultured hepatocytes from adult rats. Partial hepatectomy resulted in a dramatic decrease in the mRNA content within 24 h and then its recovery within a week. In subconfluent cultures of adult rat hepatocytes that did not grow even in the presence of mitogens, serine dehydratase mRNA was maintained at a high level. However, when the hepatocytes were cultured at low cell density without added mitogens, their serine dehydratase mRNA content decreases to a quarter of that of subconfluent cultures. The possibility that the expression of serine dehydratase mRNA is regulated in G0/G1 transition before entry into the S phase and the relationship of the mRNA with growth are discussed.     

Periportal expression of the serine dehydratase gene in rat liver

Summary 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.     

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 Escherichia coli DH1. 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.     

Regulation of the expression of the serine dehydratase gene in the kidney and liver of the rat

Serine dehydratase was induced in the kidneys of normal rats by the administration of either glucagon or dexamethasone. The increase in enzyme activity was associated with an increase in both enzyme protein and its mRNA, which were determined respectively by Western blot and RNA blot analysis. No apparent differences were observed between kidney and liver in the molecular weights of serine dehydratase proteins and the sizes of their mRNAs. Although kidney serine dehydratase was dramatically induced by either glucagon or dexamethasone, the liver enzyme was induced by glucagon but not by dexamethasone alone in the intact rat. On the other hand, liver serine dehydratase was induced in starvation, diabetes mellitus, and a high-protein diet. The kidney enzyme could not be induced under any of these conditions.     

Studies on the mechanism of 5-fluoroorotic acid inhibition of serine dehydratase induction

Administration of glucagon to rats fed a protein-free diet caused a significant induction of the liver enzyme, serine dehydratase. This effect of glucagon is inhibited by the concomitant administration of fluoroorotic acid. This inhibition was enhanced by pretreatment with glucosamine or galactosamine, probably through depletion of the intracellular uridine pools. Although less than a doubling of enzyme activity was observed after glucagon plus fluoroorotic acid administration, the amount of protein precipitable by antisera specifically reactive against serine dehydratase increased 4.5 times. Ouchterlony double-diffusion analysis showed a completely cross-reacting single precipitin band from liver extracts of untreated animals and rats treated with the analog. Analysis of the antigen-antibody complex by Na dodecyl sulfate-gel electrophoresis indicated that a single protein was being immunochemically precipitated from both the glucagon- and glucagon plus fluoroorotic acid-treated rats. In the latter, the precipitated protein had a molecular weight similar to purified serine dehydratase. These results are consistent with the concept that the incorporation of fluoroorotic acid into mRNA results in the synthesis of a protein with characteristics similar to authentic serine dehydratase but without normal enzymatic activity. Other possible mechanisms to explain the production of this abnormal protein are discussed.     

Occurrence of D-serine in rice and characterization of rice serine racemase

Germinated, unpolished rice was found to contain a substantial amount of D-serine, with the ratio of the D-enantiomer to the L-enantiomer being higher for serine than for other amino acids. The relative amount of D-serine (D/(D + L)%) reached approximately 10% six days after germination. A putative serine racemase gene (serr, clone No. 001-110-B03) was found in chromosome 4 of the genomic DNA of Oryza sativa L. ssp. Japonica cv. Nipponbare. This was expressed as serr in Escherichia coli and its gene product (SerR) was purified to apparent homogeneity. SerR is a homodimer with a subunit molecular mass of 34.5 kDa, and is highly specific for serine. In addition to a serine racemase reaction, SerR catalyzes D- and L-serine dehydratase reactions, for which the specific activities were determined to be 2.73 and 1.42 nkatal/mg, respectively. The optimum temperature and pH were respectively determined for the racemase reaction (35 °C and pH 9.0) and for the dehydratase reaction (35 °C and pH 9.5). SerR was inhibited by PLP-enzyme inhibitors. ATP decreased the serine racemase activity of SerR but increased the serine dehydratase activity. Kinetic analysis showed that Mg²⁺ increases the catalytic efficiency of the serine racemase activity of SerR and decreases that of the serine dehydratase activity. Fluorescence-quenching analysis of the tryptophan residues in SerR indicated that the structure of SerR is distorted by the addition of Mg²⁺, and this structural change probably regulates the two enzymatic activities.     

Adaptational modification of serine and threonine metabolism in the liver to essential amino acid deficiency in rats

It is known that plasma serine and threonine concentrations are elevated in rats chronically fed an essential amino acid deficient diet, but the underlying mechanisms including related gene expressions or serine and threonine concentrations in liver remained to be elucidated. We fed rats lysine or valine deficient diet for 4 weeks and examined the mRNA expressions of serine synthesising (3-phosphoglycerate dehydrogenase, PHGDH) and serine/threonine degrading enzymes (serine dehydratase, SDS) in the liver. Dietary deficiency induced marked elevation of hepatic serine and threonine levels associated with enhancement of PHGDH mRNA expression and repression of SDS mRNA expression. Increases in plasma serine and threonine levels due to essential amino acid deficiency in diet were caused by marked increases in hepatic serine and threonine levels. Proteolytic responses to the amino acid deficiency may be lessened by storing amino radicals as serine and inducing anorexia through elevation of threonine.     

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 34,462 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.     

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 MetSTART-Met2-Ser3- and the rest from Ser3-, whereas the N-terminus of rat enzyme begins from the second codon of MetSTART-Ala2-. 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.     

Cloning,sequencing, and overexpression in Escherichia coli of a phenylserine dehydratase gene from Ralstonia pickettii PS22

The structural gene coding for phenylserine dehydratase from Ralstonia pickettii PS22 was cloned into Escherichia coli cells, and the nucleotide sequence was identified. The predicted amino acid sequence had high sequence similarity to biodegradative and biosynthetic threonine dehydratases from E. coli and serine dehydratase from human liver. Transformed E. coli cells overproduced phenylserine dehydratase, and the recombinant enzyme was purified to homogeneity with a high yield and characterized.     

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.     

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.     

Enzymes of serine metabolism in normal and neoplastic rat tissues

Enzymes involved in the pathway of de novo serine biosynthesis (L-phosphoserine aminotransferase) and in alternative pathways of serine utilization (L-serine hydroxymethyltransferase, L-serine dehydratase and L-serine aminotransferase) were assayed in normal adult and fetal rat tissues and in a range of transplantable rat tumors. Serine dehydratase and serine aminotransferase activities were essentially confined to normal adult liver and kidney, whereas phosphoserine aminotransferase and serine hydroxymethyltransferase activities showed a more ubiquitous tissue distribution. In particular, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were appreciable in neoplastic tissues, in the absence of the other enzymes of serine utilization. The pattern of enzyme distribution suggests that the synthesis of serine de novo is metabolically coupled to its utilization for nucleotide biosynthesis in tumors of differing tissue origins.     

Enzymes of serine metabolism in normal and neoplastic rat tissues

Enzymes involved in the pathway of de novo serine biosynthesis (L-phosphoserine aminotransferase) and in alternative pathways of serine utilization (L-serine hydroxymethyltransferase, L-serine dehydratase and L-serine aminotransferase) were assayed in normal adult and fetal rat tissues and in a range of transplantable sat tumors. Serine dehydratase and serine aminotransferase activities were essentially confined to normal adult liver and kidney, whereas phosphoserine aminotransferase and serine hydroxymethyltransferase activities showed a more ubiquitous tissue distribution. In particular, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were appreciable in neoplastic tissues, in the absence of the other enzymes of serine utilization. The pattern of enzyme distribution suggests that the synthesis of serine de novo is metabolically coupled to its utilization for nucleotide biosynthesis in tumors of differing tissue origins.