Ornithine Metabolism Research Articles

Enzymatic synthesis and purification of l-pyrroline-5-carboxylic acid

Δ 1-Pyrroline-5-carboxylic acid (P5C) is an intermediate in the metabolism of proline, ornithine, and glutamic acid. It has been obtainable by chemical synthesis only as a mixture of the d- and l-stereoisomers. We report a method for the enzymatic synthesis of P5C on a preparative scale. The P5C is formed from l-ornithine by purified ornithine aminotransferase and then isolated by Dowex-50W cation-exchange resin chromatography. The purified compound exists entirely as the biologically active l-stereoisomer. With [ 14C]ornithine as precursor, high specific activity uniformly labeled [ 14C]P5C can be obtained.

Arginine catabolism in Neurospora: cycling of ornithine.

We measured the metabolism of ornithine in Neurospora during the transition from minimal medium to arginine-supplemented medium. Within an hour after arginine supplementation, the amount of intracellular ornithine (95% of which had been stored in vesicles) dropped by 65%, even though the catabolism of arginine produces as much ornithine as had been produced on minimal medium. The arginine level in the cell rose 10-fold. Ornithine flux through the catabolic enzyme ornithine aminotransferase increased fivefold, but flux through the mitochondrial enzyme ornithine transcarbamylase (leading to arginine synthesis) was only 20% of the rate seen on minimal medium. During this transition to arginine catabolism, the enzymes of the arginine pathway operate as an ornithine cycle, but at a restricted rate. We suggest the hypothesis that high levels of arginine may inhibit the movement of ornithine into the vesicles and into the mitochondria.

Some genetical aspects of ornithine metabolism in Aspergillus nidulans

A possible minor route of ornithine catabolism in Aspergillus nidulans might begin with the ornithine decarboxylase reaction and end with the succinic semialdehyde dehydrogenase reaction. It is therefore of interest that the putative structural genes for these two enzymes, puA and ssuA, respectively, are tightly linked group II. However, this linkage is unlikely to have regulatory significance because ileA, the structural gene for threonine dehydratase, separates them. The gene order in this region is ssuA-ileA-puA-mauB-anB. (mauB- mutations result in loss of monoamine oxidase whilst anB- mutations lead to aneurin auxotrophy.) 2. An auxotrophy for ornithine or putrescine in A. nidulans occurs in double mutants lacking arginase and blocked before ornithine in the arginine biosynthetic pathway. Some residual ornithine synthesis in such double mutants can be catalysed by ornithine delta-transaminase, especially if it is synthesised constitutively.

Studies on functional and metabolic role of urea cycle intermediates in brain.

Abstract— The distribution of argininosuccinate synthetase, argininosuccinase and arginase, and the synthesis of urea in cerebullum. cerebral cortex and brain stem have been studied. Cerebral cortex had high levels of argininosuccinate synthetase and argininosuccinase. and a high ability to synthesize urea from aspartic acid and citrulline. Of the three regions, cerebullum had the highest arginase activity. The activities of the enzymes transamidinase and ornithine aminotransferase in the metabolism of arginine and ornithine in pathways other than urea formation have been studied in the three regions of the rat brain. The activity of creatine phosphokinase in all regions was the same: carbamylphosphatase activity was highest in cerebullum. Cerebral cortex had a high activity of aspartic acid transcarba‐mylase. The brain stem, among the three regions, had the lowest activities of glutamine synthetase and glutaminase. The activities of these enzymes in the different regions are discussed in relation to urea production and the utilization of the urea cycle intermediates.Intraperitoneal injection of high amounts of citrulline brought about a rise in the glutamine synthetase activity of cerebellum and brain stem and a rise in ornithine aminotransferase in cerebral cortex and liver. These results are discussed in relation to the mechanism of action of citrulline in alleviating the toxicity in hyperammonaemic states.

Polyamine and ornithine metabolism during the germination of conidia of Aspergillus nidulans.

1. The activities of ornithine decarboxylase, S-adenosylmethionine decarboxylase and ornithine-2-oxoglutarate aminotransferase were studied during the first 24 h of conidial germination in Aspergillus nidulans. 2. Increases (over 100-fold) in the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase occurred during the emergence of the germ-tube and before the doubling of DNA and this was followed by a sharp fall in the activities of both enzymes by 16h. 3. The increase in ornithine decarboxylase could be largely suppressed if 0.6 mM-putrescine was added to the growth medium. 4. Low concentrations of cycloheximide, which delayed germination by 2h, caused a corresponding delay in the changes in ornithine decarboxylase activity. 5. Ornithine-2-oxoglutarate aminotransferase activity increased steadily during the first 24h of germination. 6. Ornithine or arginine in the growth medium induced higher activity of ornithine-2-oxoglutarate aminotransferase, but did not affect ornithine decarboxylase activity. 7. The significance of these enzyme changes during germination is discussed.

Amino acid metabolizing enzymes in rat submaxillary gland, normal or neoplastic, and in pancreas.

The activities of 12 enzymes, many related to ornithine metabolism, were measured in rat submaxillary gland, submaxillary gland tumors and pancreas. In submaxillary gland, the activities of arginase, ornithine aminotransferase, pyrroline-5-carboxylate reductase and glutamine synthetase were high, but no ornithine transcarbamylase or proline oxidase could be detected. In the fetal submaxillary gland, arginase was at almost adult levels while ornithine aminotransferase reached 50% of its adult value postnatally. Submaxillary tumors deviated from their cognate tissue by lower levels of amino acid metabolizing enzymes and by high concentrations of thymidine kinase. In pancreas, none of the pyrroline-5-carboxylate metabolizing enzymes were as high as in either liver or submaxillary gland. The outstanding activities were those of gamma-glutamyl transpeptidase and glutamate dehydrogenase. Although arginase activities in submaxillary gland and pancreas were quantitatively similar, they differed qualitatively: submaxillary gland contained the same variant as liver while the pancreatic isozymes resembled those of other nonhepatic tissues.

Metabolism of Arginine and Ornithine in the Cow and Rabbit Mammary Tissue

Amino acid uptake by the bovine mammary gland was determined by arteriovenous difference. Extraction of arginine from the plasma by the lactating bovine mammary gland was in excess of requirements for milk protein synthesis. Ornithine and citrulline also were extracted by the gland but are not in milk protein. Incubations of slices of lactating mammary tissue from cows and rabbits indicate that nonessential amino acids, especially proline and glutamate, are the major end products of arginine and ornithine metabolism in the lactating mammary gland.

Intracellular Localization of Enzymes of Arginine Metabolism in Neurospora

Abstract The physical basis for the confinement of carbamyl phosphate, ornithine, and arginine to specific anabolic fates has been investigated. Cell extracts of Neurospora grown in minimal medium were fractionated by differential centrifugation to determine the subcellular localization of the relevant enzymes. Carbamyl-P synthase A (arginine-specific; EC 2.7.2.5), ornithine acetyltransferase, and ornithine carbamyltransferase (EC 2.1.2.2) were found to be associated with a particulate (organellar) fraction. All three enzymes co-sediment with mitochondria on linear sorbitol gradients, and appear to be components of the mitochondrial matrix. The mitochondrial membrane limits the ability of exogenous substrates to be metabolized by these enzymes in vitro. Arginine synthesis from citrulline, putrescine synthesis from ornithine, and the catabolism of ornithine and arginine are carried out by cytoplasmic enzymes. The catabolic enzymes are found at significant levels even during growth in minimal medium, but little catabolism occurs in vivo. The results indicate that the anabolic and catabolic pathways of ornithine metabolism are, in part, separated by the mitochondrial membrane. However, the mechanisms responsible for confining carbamyl-P and ornithine to arginine synthesis and arginine to protein synthesis are more complex than the simple physical separation of the anabolic and catabolic enzymes.

Ordered and specific pattern of gene expression in neoplasia

The present paper outlined the evidence for the specificity of the alterations observed in the neoplastic liver in terms of their ability to provide an integrated system of quantitative and qualitative discriminants that can clearly distinguish the neoplastic liver from the rapidly growing fetal, newborn, adult, or regenerating liver. This examination of the specificity of the imbalance of enzymes and metabolic pathways in hepatomas is based also on studies on the ordered nature of the biochemical pattern in hepatomas of different growth rates that has been documented in earlier publications. The evidence leads to the conclusion that in the model system of hepatomas of different growth rates and in a control system of rapidly growing differentiating and regenerating liver there is a biochemical pattern that is ordered and specific to neoplasia. The results examined in this paper deal with key enzymes and metabolic pathways in carbohydrate, pyrimidine, DNA and ornithine metabolism. Similar considerations should apply to other biological systems and control mechanisms. The application of the conceptual and experimental approaches of the molecular correlation concept to the cyclic AMP system is reported elsewhere.

Ornithine metabolism in the developing sheep.

Ornithine metabolism in the developing sheep.

HYPERAMMONEMIA DUE TO A DEFECT IN HEPATIC ORNITHINE TRANSCARBAMYLASE

A 9-year-old girl with vomiting, changes in behavior, coma, and evidence of hepatic dysfunction at 3½ years of age was found to have hyperammonemia and decreased activity of liver ornithine transcarbamylase. When her dietary protein was reduced, she had improvement in her clinical condition and a return to normal of all hepatic function indices. Despite a defect in an enzyme of ornithine metabolism, she did not have hyperaminoacidemia, specifically hyperornithinemia, even when she had hyperammonemia. When she ingested a large amount of ornithine (300 mg/kg) she developed hyperornithinemia and hyperornithinuria. She also had orotic aciduria despite having normal activities of red cell orotidylic decarboxylase and pyrophosphorylase. Treatment with a low protein diet and citric acid supplements has been successful in preventing hyperammonemia and promoting growth and development.

Metabolic Imbalance in Carbohydrate, Pyrimidine and Ornithine Utilization

The behavior of opposing key enzymes and pathways involved in the metabolic imbalance in carbohydrate, pyrimidine and ornithine utilization was examined in nutritional and hormonal alterations and in normal and neoplastic proliferative conditions. 1. 1. In carbohydrate metabolism a marked imbalance emerged in the ratios of opposing key enzymes of gluconeogenesis and glycolysis, glucose-6-phosphatase/glucokinase, fructose-1,6-diphosphatase/phosphofructokinase, pyruvate carboxylase/pyruvate kinase in starvation, in diabetes and in insulin treatment. There were marked and progressive alterations in differentiation and in liver neoplasia but not in the regenerating liver. 2. 2. In pyrimidine and DNA metabolism, on the other hand, the antagonistic controls were revealed in major shifts in the balance of opposing pathways when gene expression was modulated by alterations in the replicative process in regenerating liver, in differentiation and in neoplasia. In starvation and in diabetes both synthetic and catabolic utilization of thymidine decreased and insulin restored pyrimidine metabolism to normal levels. 3. 3. In ornithine metabolism starvation caused a decrease, whereas diabetes resulted in an increase, in the activity of ornithine carbamyl transferase. Insulin administration in diabetic rats returned the enzyme activity to normal range. In differentiation ornithine carbamyl transferase activity increased, whereas in hepatomas it gradually decreased in parallel with an increase in tumor growth rate. 4. 4. The metabolic imbalance elucidated in the hepatomas confers a selective biological advantage on the cancer cells. 5. 5. The metabolic imbalance in nutritional, hormonal and normal and neoplastic proliferative conditions emerged as a result of ordered alterations in gene expression which operate, in part at least, through setting the amounts, activities and ratios of opposing key enzymes and metabolic pathways.

The Change in Arginine Levels and the Metabolism of Urea and Ornithine in Cucurbita moschata Seedlings

AbstractDuring germination a decrease in both soluble and protein argmine occurs in pumpkin cotyledons (Cucurbita moschata Poir.) and this decrease parallels the decrease in total nitrogen. Arginine is readily metabolized to urea and ornithine and the metabolism of the latter compounds were studied by allowing cotyledons of intact plants to absorb urea−14C and by incubating cotyledon discs with ornithine‐2−14C. Of the urea absorbed, 90 % of the label was metabolized to 14CO2 with little free urea remaining in the plant. Urease activity in the cotyledons reached a maximum at 4 to 6 days of germination. Ornithine was extensively metabolized with 37 % of the label being incorporated into protein by 3 h. Soluble and protein arginine accounted for 23 % of the label suggesting that Krebs‐Henseliet cycle enzymes were present.

1838. Inhibition of DNA synthesis by beryllium: Witschi, H. (1968). Inhibition of deoxyribonucleic acid synthesis in regenerating rat liver by beryllium. Lab. Invest.19, 67

Glutamine, the most abundant amino acid in blood and tissues, is degraded by the renal and splanchnic tissues, especially the small intestinal mucosa. Due to the activity of glutaminase, it may be broken down in these tissues and contribute to ammoniagenicity. Glutamine, either directly or through ammonia production, may act as a nitrogenous source for pyrimidine biosynthesis. We have evaluated the effect of glutamine on orotate metabolism in mice, by gavaging (ig) L-glutamine, 1.0 to 4.0 mmol/100 g of body wt/day, during 6 weeks of experimentation. Glutamine at doses of 2.5 to 4.0 mmol/100 g of body wt caused a significant increase in plasma ammonia and urinary orotate. The regulation of the orotic acid biosynthesis and excretion was studied by testing the effects of various inhibitors in mice force-fed with glutamine (4 mmol/100 g of body wt, ig). The orotic aciduria was insensitive to acivicin (1 and 5 mg/100 g of body wt, ip), a specific inhibitor of the cytoplasmic carbamyl phosphate synthetase-II, thus pointing toward the mitochondrion as the principal source of carbamyl phosphate. Cycloheximide (15 and 100 mg/kg of body wt, ip) caused a significant decrease in urinary orotate indicating that the induction of orotate synthesis by glutamine may be associated with the translation of a specific protein. However, orotate excretion was significantly decreased by N-(phosphonoacetyl)-L-aspartate (PALA) (5 mg/100 g of body wt, ip) due to its inhibitory effect on the aspartate transcarbamylase activity. There was a significant increase of urinary orotate following ingestion of adenine supplemented diets (0.1% and (1.2%), suggesting the blockage of the utilization of orotate for nucleotide biosynthesis by glutamine. Since orotate synthesis may also be influenced by ornithine metabolism, we evaluated the effect of glutamine administration on various ornithine-metabolizing enzymes. There was a decrease in hepatic ornithine decarboxylase activity with no change in hepatic ornithine aminotransferase activity following the administration of glutamine. This observation indicates that an increased metabolic utilization of ornithine is not responsible for the increase in orotate excretion, which may be caused principally through an effect of glutamine on mitochondrial carbamyl phosphate synthesis.

Regulation of arginine and ornithine metabolism at the enzymic level in rat liver and kidney

The effects on the activities of the enzymes which metabolize ornithine and arginine in liver and kidney have been studied by feeding rats diets containing 8% ornithine, arginine, or proline and 10 or 20% casein. In liver the enzymes studied were arginase, ornithine transcarbamylase, argininosuccinic acid lyase, and ornithine-δ-transaminase. The ratios of their activities were 3000:414::10:1, respectively. In kidney the enzymes studied were arginase, ornithine-δ-transaminase, and arginine-glycine transamidinase. The ratios of their activities were 24:2::7:1.0. Several of these enzymes were inducible to higher activities by feeding diets with the added amino acids. Of these, the most sensitive to substrate induction was liver ornithine-δ-transaminase. This enzyme, which had the lowest specific activity of those studied, showed an increase in specific activity of 250.6% when ornithine was added to the 20% casein diet. Arginine and proline were somewhat less effective as inducers. This difference in inducing ability was further accentuated by the 10% casein diet. This indicated that ornithine is probably the primary inducer of liver ornithine-δ-transaminase, and that arginine and proline probably act by conversion to ornithine. Kidney ornithine-δ-transaminase was also inducible, but to a lesser extent (28.7%) and only by ornithine added to the 20% casein diet. Liver ornithine transcarbamylase was induced maximally by ornithine on the 20% casein diet. The induction of this enzyme was relatively much less (31.2%) than that of liver ornithine-δ-transaminase, but in absolute amounts its change in activity was 50 times greater than the change in liver ornithine-δ-transaminase. On the 10% protein diet, arginine produced an inhibition of growth and a decrease in liver weight. This was accompanied by a 37.7% depression in the activity of liver arginase and a 23.5% depression in the activity of liver ornithine transcarbamylase, changes which probably reflect a general inhibition of protein synthesis.

The Metabolism of Arginine, Citrulline, Ornithine, Proline and delta-Aminovaleric Acid and Phenylalanine by Rumen Bacteria

L-arginine is fermented by concentrated suspensions of rumen bacteria with the production of more than i molecule of ammonia per molecule of arginine. Citrulline was shown to be the first intermediate in the hydrolytic desimidation of arginine. Citrulline is converted to ornithine, proline, and δ-aminovaleric acid. δ-Aminovaleric acid is reduced to ammonia and valeric acid. Phenylalanine is deaminated to phenylacetic acid, and pheuylpyruvic acid was shown to be an intermediate.

Metabolism of ornithine and other amino acids in the cerebro-oculo-renal syndrome

A family of six with three male children affected with the cerebro-oculo-renal syndrome has been studied for a specific biochemical abnormality in amino acid metabolism. Oral administration of the single amino acids l-lysine, l-hydroxyproline and l-glycine did not affect the urinary amino acids excreted by the oldest affected boy. In contrast, ingestion of a comparable amount of l-ornithine resulted in intensification of the abnormal urinary amino acid pattern. Feeding the parents and sister equimolar amounts of ornithine resulted only in the mother in an abnormal urinary pattern of amino acids which resembled that of the affected male children. The data are consistent with a sex-linked mode of inheritance in this family. The syndrome described herein probably represents a variant of the cerebro-oculo-renal syndrome as originally described by Lowe.