Immunochemical studies of serine dehydratase and ornithine aminotransferase regulation in rat liver in vivo.

Previous studies of serine dehydratase (EC 4.2.1.13) and ornithine aminotransferase (EC 2.6.1.13) adaptation in rat liver showed that in rats on a high protein diet, glucocorticoid administration increased serine dehydratase activity while simultaneously reducing the activity of ornithine aminotransferase. The present study examines the role of enzyme synthesis in the expression of these and other dissimilar adaptive characteristics of the two enzymes. Both enzymes were purified to crystallinity and used to prepare specific antibodies. Changes in the rate of synthesis of each enzyme during adaptation were then measured immunochemically. In rats fed ad libitum, the synthetic rates for both enzymes exhibited circadian rhythm, although enzyme levels remained relatively constant. The circadian cycle for ornithine aminotransferase synthesis was in phase with the cycles for body weight and relative liver weight (maxima at 9 a.m., minima at 9 p.m.) but was approximately 12 hours out of phase with the cycle for serine dehydratase synthesis. 9alpha-Fluoro-11beta, 21-dihydroxy-16alpha, 17alpha-isopted at 9 a.m., increased serine dehydratase synthesis and simultaneously decreased the synthesis of ornithine aminotransferase. When triamcinolone was injected at 9 p.m., however, serine dehydratase synthesis was not stimulated, although the reduction of ornithine aminotransferase synthesis was still produced. These results suggest that: (a) circadian cycling of synthesis may be a general phenomenon in enzyme regulation even though for enzymes with relatively long half-lives, such cycling may not be reflected as fluctuations in enzyme levels; (b) such circadian rhythmicity may also involve cyclic changes in the responsiveness of the enzyme-forming system to regulatory stimuli; (c) whereas the adaptive behavior of serine dehydratase typifies that of amino acid-catabolizing enzymes in general, the responses of ornithine aminotransferase denote a functional association of this enzyme with anabolic processes. On this basis, the possibility that ornithine aminotransferase plays a pivotal role in the regulation of urea cycle activity and nitrogen balance is discussed.     

Sequential changes in ornithine decarboxylase thymidine kinase, and other enzyme activities in regenerating liver in rats on controlled feeding schedules.

The changes in activity of five enzymes including ornithine decarboxylase (ODC), tyrosine aminotransferase (TAT), thymidine kinase (TK), ornithine aminotransferase (OAT) and serine dehydratase (SDH) in the early stage of the regenerating rat liver have been studied under a controlled feeding and lighting schedule. The first three enzyme activities were stimulated sequentially by partial hepatectomy. The earliest response was observed in ODC activity. A significant increase in this enzyme activity was observed at 2 hrs and the maximal level was at 4 hrs after the operation. TAT began to increase at 4 hrs and the maximal level was at 8 hrs. The TK activity was induced at about 24 hrs and the highest value was at 48 hrs after partial hepatectomy.A significant decrease in OAT activity was observed at 24 hrs after the operation and subsequently. Although a decrease in SDH activity was also observed this decrease did not seem to correlate directly with the regeneration process, since a lowered level of the enzyme activity was also found in the sham operated group.     

Liver enzymes of serine metabolism during neonatal development of the rat.

The developmental patterns of L-serine hydroxymethyltransferase, L-phosphoserine aminotransferase, L-serine aminotransferase and L-serine dehydratase were determined in rat liver. The results point to an increased capacity for serine biosynthesis de novo in the perinatal period. It is suggested that serine at this time, and also at weaning, may serve as a precursor, via the serine hydroxymethyltransferase reaction, for nucleotide biosynthesis to support the rapid phases of liver growth. The role of the alternative pathways of serine metabolism during neonatal development is discussed.     

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.     

The key enzymes of the alternative pathways of serine utilization in gluconeogenesis in chickens during individual development

It has been shown that in embryonic liver and kidney of chicks, serine is involved into gluconeogenesis almost exclusively via serine-pyruvate aminotransferase. This relationship stands true also for the liver of adult hens. On the contrary, in the kidney of adult hens, there is a significant increase in the activity of serine dehydratase, as compared to the level of the activity of this enzyme in embryogenesis and in the liver of adult hens.     

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.     

Effects of high dietary methionine on activities of selected enzymes in the liver of kittens (felis domesticus)

• 1.1. Growing male kittens were fed an 18% casein diet supplemented with 2, 3, or 4% l-methionine (MET) for 6 weeks.
• 2.2. Free MET concentration in liver increased 30-fold and cystathionine two- to three-fold; the activity of adenosyl-MET transferase and cystathionase also increased but remained lower than previously found in rats.
• 3.3. Taurine concentration in liver decreased in cats fed excess MET and appeared to depend on taurine intake.
• 4.4. Alanine aminotransferase activity was high in all groups while serine dehydratase activity was very low.
• 5.5. Pyruvate kinase and malic enzyme activities which are normally low in cat liver increased after excess MET. Also, glucose 6-phosphate and 6-phosphogluconate dehydrogenases increased.
• 6.6. Cat liver metabolism showed limited adaptation to an excess dietary intake of methionine compared to that found in rats.

     

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.     

Flux of the L-serine metabolism in rabbit, human, and dog livers. Substantial contributions of both mitochondrial and peroxisomal serine:pyruvate/alanine:glyoxylate aminotransferase.

L-Serine metabolism in rabbit, dog, and human livers was investigated, focusing on the relative contributions of the three pathways, one initiated by serine dehydratase, another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Under quasi-physiological in vitro conditions (1 mM L-serine and 0.25 mM pyruvate), flux through serine dehydratase accounted for only traces, and that through SPT/AGT substantially contributed no matter whether the enzyme was located in peroxisomes (rabbit and human) or largely in mitochondria (dog). As for flux through serine hydroxymethyltransferase and GCS, the conversion of serine to glycine occurred fairly rapidly, followed by GCS-mediated slow decarboxylation of the accumulated glycine. The flux through GCS was relatively high in the dog and low in the rabbit, and only in the dog was it comparable with that through SPT/AGT. An in vivo experiment with L-[3-3H,14C]serine as the substrate indicated that in rabbit liver, gluconeogenesis from L-serine proceeds mainly via hydroxypyruvate. Because an important role in the conversion of glyoxylate to glycine has been assigned to peroxisomal SPT/AGT from the studies on primary hyperoxaluria type 1, these results suggest that SPT/AGT in this organelle plays dual roles in the metabolism of glyoxylate and serine.     

Effect of adenosine 3',5'-monophosphate on serine metabolism in chick tissues.

The effect of cyclic 3',5'-AMP and supplemental dietary glycine upon de novo synthesis of serine metabolic enzymes in chick livers were examined. Chicks fed crystalline amino acid diets containing 2% glycine had approximately twofold the activity in liver for 3-phosphoglycerate dehydrogenase and phosphoserine phosphatase compared to liver tissue from chicks fed diets lacking in dietary glycine. Chicks subjected to daily intraperitoneal injections of cyclic 3',5'-AMP and fed diets containing no dietary glycine contained biosynthetic enzyme activity similar to glycine-fed chicks suggesting a correlation between glycine and cyclic AMP for serine enzyme induction. The elevated enzyme activity in liver of chicks fed dietary glycine or injected with cyclic AMP was inhibited when chicks were also injected with actinomycin D indicating de novo synthesis of 3-phosphoglycerate dehydrogenase and phosphoserine phosphatase. Dietary glycine or cyclic AMP, however, did not change serine dehydratase and glycerate dehydrogenase activities in chick liver.     

Regulation of hepatic l-serine dehydratase and l-serine-pyruvate aminotransferase in the developing neonatal rat

1. The activities of l-serine dehydratase and l-serine–pyruvate aminotransferase were determined in rat liver during foetal and neonatal development. 2. l-Serine–pyruvate aminotransferase activity begins to develop in late-foetal liver, increases rapidly at birth to a peak during suckling and then decreases at weaning to the adult value. 3. l-Serine dehydratase activity is very low prenatally, but increases rapidly after birth to a transient peak. After a second transient peak around the time weaning begins, activity gradually rises to the adult value. Both of these peaks have similar isoenzyme compositions. 4. In foetal liver both l-serine dehydratase and l-serine–pyruvate aminotransferase activities are increased after injection in utero of glucagon or dibutyryl cyclic AMP. Cycloheximide or actinomycin D inhibited the prenatal induction of both enzymes and actinomycin D blocked the natural increase of l-serine dehydratase immediately after birth. Glucose or insulin administration also blocked the perinatal increase of l-serine dehydratase. 5. After the first perinatal peak of l-serine dehydratase, activity is increased by cortisol and this is inhibited by actinomycin D. After the second postnatal peak, activity is increased by amino acids or cortisol and this is insensitive to actinomycin D inhibition. Glucose administration blocks the cortisol-stimulated increase in l-serine dehydratase and also partially lowers the second postnatal peak of activity. 6. The developmental patterns of the enzymes are discussed in relation to the pathways of gluconeogenesis from l-serine. The regulation of enzyme activity by hormonal and dietary factors is discussed with reference to the changes in stimuli that occur during neonatal development and to their possible mechanisms of action.     

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.     

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.     

Enzymological characterization of a putative canine analogue of primary hyperoxaluria type 1.

This paper concerns an enzymological investigation into a putative canine analogue of the human autosomal recessive disease primary hyperoxaluria type 1 (alanine:glyoxylate/serine:pyruvate aminotransferase deficiency). The liver and kidney activities of alanine:glyoxylate aminotransferase and serine:pyruvate aminotransferase in two Tibetan Spaniel pups with familial oxalate nephropathy were markedly reduced when compared with a variety of controls. There were no obvious deficiencies in a number of other enzymes including D-glycerate dehydrogenase/glyoxylate reductase which have been shown previously to be deficient in primary hyperoxaluria type 2. Immunoblotting of liver and kidney homogenates from oxalotic dogs also demonstrated a severe deficiency of immunoreactive alanine:glyoxylate aminotransferase. The developmental expression of alanine:glyoxylate/serine:pyruvate aminotransferase was studied in the livers and kidneys of control dogs. In the liver, enzyme activity and immunoreactive protein were virtually undetectable at 1 day old, but then increased to reach a plateau between 4 and 12 weeks. During this period the activity was similar to that found in normal human liver. The enzyme activities and the levels of immunoreactive protein in the kidneys were more erratic, but they appeared to increase up to 8 weeks and then decrease, so that by 36 weeks the levels were similar to those found at 1 day. The data presented in this paper suggest that these oxalotic dogs have a genetic condition that is analogous, at least enzymologically, to the human disease primary hyperoxaluria type 1.