Two routes for asparagine metabolism in Pisum sativum L.

Asparagine, a major transport compound, is metabolized in Pisum sativum by two enzymes, asparaginase (EC 3.5.1.1) and asparagine-pyruvate aminotransferase. The relative amount of the two enzymes varies between tissues. In developing seeds, there are very high levels of asparaginase but only trace amounts of the aminotransferase. Asparaginase is high in young leaves but falls rapidly during leaf growth; the aminotransferase remains high throughout development. Inhibitor studies with aminooxyacetate and methionine sulfoximine confirm that the aminotransferase is the main enzyme involved in asparagine utilisation in the leaf. Root tissue has low levels of asparaginase and only trace amounts of the aminotransferase. The asparaginase is potassium dependent, but is also partially activated by ammonium ions. The leaf aminotransferase has a lower K _m for asparagine (4.5 mM) than the leaf asparaginase (8 mM). The seed asparaginase has a lower K _m for asparagine (3 mM) than the leaf asparaginase.     

Permeability of human erythrocytes to asparagine

Asparagine is able to penetrate into human erythrocytes from the external medium. The dependence of the asparagine transport rate on its concentration can be described by the Michaelis-Menten equation with parameters: Km = 2.50 mM, V = 0.24 mmol/l cells per hour. Loading of erythrocytes with asparaginase does not influence their permeability to asparagine. Aspartate is accumulated inside these erythrocytes during incubation with asparagine, thus reflecting rapid transformation of penetrating asparagine by entrapped asparaginase.     

Arabidopsis mutants lacking asparaginases develop normally but exhibit enhanced root inhibition by exogenous asparagine

Asparaginase catalyzes the degradation of L-asparagine to L-aspartic acid and ammonia, and is implicated in the catabolism of transported asparagine in sink tissues of higher plants. The Arabidopsis genome includes two genes, ASPGA1 and ASPGB1, belonging to distinct asparaginase subfamilies. Conditions of severe nitrogen limitation resulted in a slight decrease in seed size in wild-type Arabidopsis. However, this response was not observed in a homozygous T-DNA insertion mutant where ASPG genes had been inactivated. Under nitrogen-sufficient conditions, the ASPG mutant had elevated levels of free asparagine in mature seed. This phenotype was observed exclusively under conditions of low illumination, when a low ratio of carbon to nitrogen was translocated to the seed. Mutants deficient in one or both asparaginases were more sensitive than wild-type to inhibition of primary root elongation and root hair emergence by L-asparagine as a single nitrogen source. This enhanced inhibition was associated with increased accumulation of asparagine in the root of the double aspga1-1/-b1-1 mutant. This indicates that inhibition of root growth is likely elicited by asparagine itself or an asparagine-derived metabolite, other than the products of asparaginase, aspartic acid or ammonia. During germination, a fusion between the ASPGA1 promoter and beta-glucuronidase was expressed in endosperm cells starting at the micropylar end. Expression was initially high throughout the root and hypocotyl, but became restricted to the root tip after three days, which may indicate a transition to nitrogen-heterotrophic growth.     

Methotrexate stimulation of asparagine synthetase activity in rat liver

Methotrexate was found to stimulate asparagine synthetase activity in vivo by approximately six-fold in rat liver. The maximum effect of methotrexate on hepatic asparagine synthetase activity was observed sixteen hours after intraperitoneal injection of the drug. Cycloheximide, like methotrexate, is a protein synthesis inhibitor and was used to determine that asparagine synthetase activity was not preferentially stimulated under stress. As expected, hepatic asparagine synthetase activity falls markedly with the decreased protein synthesis caused by injection of cycloheximide. It is proposed that methotrexate inhibits serine-dependent glycine biosyn-thesis by decreasing the concentration of tetrahydrofolate for serine hydroxymethyltransferase. This leads to a stimulation of asparagine synthetase to provide nitrogen for asparagine-dependent glycine synthesis. This may provide an explanation of the observed chemotherapeutic synergism between asparaginase and methotrexate treatment.     

Control of protein synthesis by amino acid supply. The effect of asparagine deprivation on the translation of messenger RNA in reticulocyte lysates

The enzyme asparaginase, which hydrolyses asparagine to aspartic acid, inhibited cell-free protein synthesis by reticulocyte lysates. The inhibition was rapid and complete when sufficient enzyme was added but could be prevented or reversed by the addition of asparagine. The initial effect of asparaginase appears to be a block in polypeptide chain elongation due to asparagine deprivation, but there are some indications that prolonged incubation under these conditions may give rise to a secondary decrease in initiation of protein synthesis.     

Biochemical and pathophysiological characterization of Helicobacter pylori asparaginase

Asparaginase was purified from Helicobacter pylori 26695 and its pathophysiological role explored. The K(m) value of asparagine was 9.75 ± 1.81 μM at pH 7.0, and the optimum pH range was broad and around a neutral pH. H. pylori asparaginase converted extracellular asparagine to aspartate. H. pylori cells were unable to take up extracellular asparagine directly. Instead, aspartate produced by the action of the asparaginase was transported into H. pylori cells, where it was partially converted to β-alanine. Asparaginase exhibited striking cytotoxic activity against histiocytic lymphoma cell line U937 cells via asparagine deprivation. The cytotoxic activity of live H. pylori cells against U937 cells was significantly diminished by deletion of the asparaginase gene, indicating that asparaginase functions as a cytotoxic agent of the bacterium. The cytotoxic effect was negligible for gastric epithelial cell line AGS cells, suggesting that the effect differs across host cell types. An asparaginase-deficient mutant strain was significantly less capable of colonizing Mongolian gerbils. Since asparagine depletion by exogenous asparaginase has been shown to suppress lymphocyte proliferation in vivo, the present results suggest that H. pylori asparaginase may be involved in inhibition of normal lymphocyte function at the gastric niche, allowing H. pylori to evade the host immune system.     

Role of glutamine depletion in directing tissue-specific nutrient stress responses to L-asparaginase

L-asparaginase is important in the induction regimen for treating acute lymphoblastic leukemia. Cytotoxic complications are clinically significant problems lacking mechanistic insight. To reveal tissue-specific molecular responses to this drug, mice were administered asparaginase from either Escherichia coli (clinically used) or Wolinella succinogenes (novel, glutaminase-free form). Both enzymes abolished serum asparagine, but only the E. coli form reduced circulating glutamine. E. coli asparaginase reduced protein synthesis in liver and spleen but not pancreas via increased phosphorylation of the translation factor eIF2. In contrast, treatment with Wolinella caused no untoward changes in protein synthesis in any tissue examined. Treating mice deleted for the eIF2 kinase, GCN2, with the E. coli enzyme showed eIF2 phosphorylation to be GCN2-dependent, but only initially. Furthermore, although eIF2 phosphorylation was not increased in the pancreas or by Wolinella asparaginase, expression of the amino acid stress response genes, asparagine synthetase and CHOP/GADD153, increased as a result of both enzymes, even in tissues demonstrating no change in eIF2 phosphorylation. Finally, signaling downstream of the mammalian target of rapamycin kinase was repressed in liver and pancreas by E. coli but not Wolinella asparaginase. These data demonstrate that the nutrient stress response to asparaginase is tissue-specific and exacerbated by glutamine depletion. Importantly, increased expression of asparagine synthetase and CHOP does not require eIF2 phosphorylation, signifying alternate or auxiliary means of inducing gene expression under conditions of amino acid depletion in the whole animal.     

Simple and complex carbohydrate-rich diets and muscle glycogen content of marathon runners

The effects of simple-carbohydrate (CHO)- and complex-CHO-rich diets on skeletal muscle glycogen content were compared. Twenty male marathon runners were divided into four equal groups with reference to dietary consumption: depletion/simple, depletion/complex, nondepletion/simple, and nondepletion/complex. Subjects consumed either a low-CHO (15% energy [E] intake), or a mixed diet (50% CHO) for 3 days, immediately followed by a high-CHO diet (70% E intake) predominant in either simple-CHO or in complex-CHO (85% of total CHO intake) for another 3 days. Skeletal muscle biopsies and venous blood samples were obtained one day prior to the start of the low-CHO diet or mixed diet (PRE), and then again one day after the completion of the high-CHO diet (POST). The samples were analysed for skeletal muscle glycogen, serum free fatty acids (FFA), insulin, and lactate and blood glucose. Skeletal muscle glycogen content increased significantly (p less than 0.05) only in the nondepletion/simple group. When groups were combined, according to the type of CHO ingested and/or utilization of a depletion diet, significant increases were observed in glycogen content. Serum FFA decreased significantly (p less than 0.05) for the nondepletion/complex group only, while serum insulin, blood glucose, and serum lactate were not altered. It is concluded that significant increases in skeletal muscle glycogen content can be achieved with a diet high in simple-CHO or complex-CHO, with or without initial consumption of a low-CHO diet.     

Glutamine-dependent asparagine synthetase in fetal, adult and neoplastic rat tissues.

Three enzyme reactions related to asparagine synthesis were studied in rat tissues: formation of aspartylhydroxamate, either from aspartate or by transfer from asparagine, and actual synthesis of asparagine from aspartate. Actual asparagine synthesis occurred at one-thousandth the rate of the other two reactions. Optimal conditions for quantitative assay of asparagine synthesis were determined in fetal liver extract, which is a rich source of the enzyme. Demonstrable activity in liver fell 6 days after birth to 20% of the fetal value and decreased slowly thereafter to the low adult value. Adult pancreas was the most active tissue found. The asparagine synthetase of fetal liver extracts was significantly inhibited when combined with adult liver or tumor extracts. The inhibitor fractionated with ammonium sulfate in close association with the asparagine synthetase. Therefore, demonstrable activities of asparagine synthetase in tissue extracts, measured in the presence of this inhibitor, do not necessarily parallel the concentrations of the enzyme present.     

Lipid oxidation in plasma and tissues from estrogenized chicken hens

Four groups of 5-month-old chicken hens were given estradiol treatments and/or 5% dietary oil supplement for 14 days, after which blood plasma, liver, heart, and skeletal muscle were analyzed for lipid oxidation by TBA assay for malonaldehyde. Plasma from estradiol-treated birds had 8-fold higher levels of malonaldehyde compared to untreated birds. The bulk of this effect was due to a 5-fold increase in plasma lipid, but this lipid also contained a 70% higher concentration of malonaldehyde. Estradiol treatments produced significantly increased TBA numbers in liver, heart, and skeletal muscle. Corn oil supplementation significantly increased the malonaldehyde concentration in fat extracted from liver and heart, but not from plasma or skeletal muscle. It was concluded that estradiol treatment, in addition to generally increasing the deposition of fat in plasma and organs, also enhanced the concentration of malonaldehyde equivalents in plasma and organ fat.     

Trafficking of dietary oleic, linolenic, and stearic acids in fasted or fed lean rats

Increasing evidence supports the notion that there are significant differences in the health effects of diets enriched in saturated, as opposed to monounsaturated or polyunsaturated fat. However, the current understanding of how these types of fat differ in their handling by relevant tissues is incomplete. To examine the effects of fat type and nutritional status on the metabolic fate of dietary fat, we administered (14)C-labeled oleic, linolenic, or stearic acid with a small liquid meal to male Sprague-Dawley rats previously fasted for 15 h (fasted) or previously fed ad libitum (fed). (14)CO(2) production was measured for 8 h after tracer administration. The (14)C content of gastrointestinal tract, serum, liver, skeletal muscle (soleus, lateral, and medial gastrocnemius), and adipose tissue (omental, retroperitoneal, and epididymal) was measured at six time points (2, 4, 8, 24, and 48 h and 10 days) after tracer administration. Plasma levels of glucose, insulin, and triglyceride were also measured. Oxidation of stearic acid was significantly less than that of either linolenic or oleic acid in both the fed and fasted states. This reduction was in part explained by a greater retention of stearic acid within skeletal muscle and liver. Oxidation of oleate and stearate were significantly lower in the fed state than in the fasted state. In the fasted state, liver and skeletal muscle were quantitatively more important than adipose tissue in the uptake of dietary fat tracers during the immediate postprandial period. In contrast, adipose tissue was quantitatively more important than skeletal muscle or liver in the fed state. The movement of carbons derived from dietary fat between tissues is a complex time-dependent process, which varies in response to the type of fat ingested and the metabolic state of the organism.     

Influence of dietary fluoride restriction on regulation of plasma nd soft tissue fluoride contents.

The adjustments in total fluoride concentration in plasma, bones, liver, and muscle were examined when rats were given a diet of very low fluoride content following a dietary regimen of elevated fluoride intake. The animals received a diet containing 34 ppm of fluoride and water with 50 ppm added fluoride in the 28-day initial period and in the depletion period they were given a diet containing only 0.21 ppm of fluoride and distilled water. The findings indicated a 12-fold increase in the fluoride content of the humeri after 28 days of high-flurodie intake with a greater increment by the epiphyses than by the diaphyses. During 21 days of the depletion period the skeletal fluoride was reduced by only 7.7% indicating a marked retention of fluoride during processes of bone remodeling and growth. The plasma, muscle, and liver total fluoride contents were significantly increased at the end of the period of high-fluoride intake, but these concentrations were found to be restored to base-line levels in 3-7 days of the depletion period. By comparison of the distribution of total fluoride with injected radiofluoride between tissue and plasma waters, it was concluded that muscle and liver contain bound fluoride that does not exchange completely with ionic fluoride.     

Asparagine utilization in Escherichia coli

Asparagine-requiring auxotrophs of Escherichia coli K-12 that have an active cytoplasmic asparaginase do not conserve asparagine supplements for use in protein synthesis. Asparagine molecules entering the cell in excess of the pool required for use of this amino acid in protein synthesis are rapidly degraded rather than accumulated. Supplements are conserved when asparagine degradation is inhibited by the asparagine analogue 5-diazo-4-oxo-l-norvaline (DONV) or mutation to cytoplasmic asparaginase deficiency. A strain deficient in cytoplasmic asparaginase required approximately 260 mumol of asparagine for the synthesis of 1 g of cellular protein. The cytoplasmic asparaginase (asparaginase I) is required for growth of cells when asparagine is the nitrogen source. This enzyme has an apparent K(m) for l-asparagine of 3.5 mM, and asparaginase activity is competitively inhibited by DONV with an apparent K(i) of 2 mM. The analogue provides a time-dependent, irreversible inhibition of cytoplasmic asparaginase activity in the absence of asparagine.     

Catabolism of aspartate and asparagine byBacteroides intermedius andBacteroides gingivalis

Aspartate as asparagine catabolism was studied in representative strains ofBacteroides intermedius strain T588 andB. gingivalis strain W83. Cell suspensions of both species deamidated asparagine. The enzyme asparaginase was constitutive and was unaffected by the addition of ammonium ions to the culture medium. The enzyme aspartase was not detected, but since malate dehydrogenase was known to occur and succinate was present as a major end product of metabolism, aspartate catabolism was postulated to occur via oxaloacetate, malate, and fumarate to succinate. All enzymes of this pathway were present in cell-free extracts, and some of the major properties of these enzymes were examined. The electron carriers cytochrome b and menaquinone-9 were present inB. gingivalis, whereasB. intermedius possessed cytochrome c and menaquinone-11. The membrane-bound enzyme fumarate reductase utilized NADH as an electron donor, but the reaction was inhibited by short wave ultraviolet radiation and 2-n-heptyl-4-hydroxyquinoline-N-oxide.     

Alkaloid production during the cultivation with shaking of Claviceps sp: Effects of asparagine

Among the nitrogen sources tested, asparagine stimulated alkaloid production maximally. Ammonium salts supported alkaloid production poorly. During the cultivation with shaking of Claviceps sp. strain SD-58 in asparagine containing medium, the activity of asparagine increased during the exponential growth (up to 8 days) with the intracellular accumulation of ammonium ions. Among the ammonia-assimilating enzymes we studied, NADP⁺-glutamate dehydrogenase (GDH) had a higher activity in the growth phase (up to 6 days), while in the intensive alkaloid producing phase (after 6 days) the activity of glutamine synthetase was higher. The latter was associated with increases in the intracellular level of tryptophan and alkaloid production.The levels of NADP⁺- and NAD⁺-alanine dehydrogenases and glutamate synthase were negligible.     

Consumption of a moderately Zn-deficient and Zn-supplemented diet affects soluble protein expression in rat soleus muscle

Zinc deficiency negatively affects muscle function, but there are limited biochemical data identifying the cause of this reduction in function. The objective of the present study was to identify soluble proteins in rat soleus muscle that were responsive to different levels of dietary zinc. Rats (n=21) were fed diets containing three concentrations of zinc: 5, 30 and 200 ppm for 42 days. There was no difference in body weights of the rats consuming the 5-ppm zinc diet compared to the rats consuming the 30- or 200-ppm zinc diets; however, bone zinc levels were significantly decreased in the 5-ppm dietary zinc group. Individual soluble protein fractions were isolated from these muscles and the samples were prepared for two-dimensional polyacrylamide gel electrophoresis. The expression levels of four proteins were significantly depressed by dietary Zn depletion and supplementation, S-glutathiolated carbonic anhydrase, myosin light polypeptide 3, heat shock protein 20 and heart fatty acid binding protein. This is the first report that indicates that both Zn depletion and supplementation result in protein expression profiles that may negatively affect skeletal muscle function. These results indicate that there are specific signaling pathways that require proper Zn nutriture for maintaining optimal muscle function and suggest that the consumption of pharmacologic doses of Zn may be detrimental to muscle function.     

Utilization of the amide groups of asparagine and 2-hydroxysuccinamic Acid by young pea leaves

The fate of nitrogen originating from the amide group of asparagine in young pea leaves (Pisum sativum) has been studied by supplying [¹⁵N-amide]asparagine and its metabolic product, 2-hydroxysuccinamate (HSA) via the transpiration stream. Amide nitrogen from asparagine accumulated predominantly in the amide group of glutamine and HSA, and to a lesser extent in glutamate and a range of other amino acids. Treatment with 5-diazo,4-oxo-L-norvaline (DONV) a deamidase inhibitor, caused a decrease in transfer of label to glutamine-amide. Virtually no ¹⁵N was detected in HSA of leaves supplied with asparagine and the transaminase inhibitor aminooxyacetate. When [¹⁵N]HSA was supplied to pea leaves, most of the label was also found in the amide group of glutamine and this transfer was blocked by the addition of methionine sulfoximine, which caused a large increase in NH₃ accumulation. DONV was not specific for asparaginase, and inhibited the deamidation of HSA, causing a decrease in transfer of ¹⁵N into glutamine-amide, NH₃, and other amino acids. It is concluded from these results that use of the amide group of asparagine as a nitrogen source for young pea leaves involves deamidation of both asparagine and its transamination product HSA (possibly also oxosuccinamate). The amide group, released as ammonia, is then reassimilated via the glutamine synthetase/glutamate synthase system.     

The effect of 5 days of aspartate and asparagine supplementation on glucose transport activity in rat muscle

The consumption of protein supplements containing amino acids is increasing around the world. Aspartate (Asp) and asparagine (Asn) are amino acids metabolized by skeletal muscle. This metabolism involves biochemical pathways that are involved in increasing Krebs cycle activity via anaplerotic reactions, resulting in higher glutamine concentrations. A connection between amino acid supplementation, glycogen concentration, and glucose uptake has been previously demonstrated. The purpose of this study was to evaluate the effect of Asp and Asn supplementation on glucose uptake in rats using three different glycogen concentrations. The results indicate that Asp and Asn supplementation in rats with high glycogen concentrations (fed state) further increased the glycogen concentration in the muscle, and decreased in vitro 2‐deoxyglucose (a glucose analog) uptake by the muscle at maximal insulin concentrations. When animals had a medium glycogen concentration (consumed lard for 3 days), glucose uptake was higher in the supplemented group at sub‐maximal insulin concentrations. We conclude that supplementation of Asp and Asn reduced glucose transport in rat muscle only at higher levels of glycogen. The ingestion of lard for 3 days changed the responsiveness and sensitivity to insulin, and that group had higher levels of insulin sensitivity with Asp and Asn supplementation. Copyright © 2009 John Wiley & Sons, Ltd.     

Isolation and characterization of Rhizobium etli mutants altered in degradation of asparagine.

Rhizobium etli mutants unable to grow on asparagine as the nitrogen and carbon source were isolated. Two kinds of mutants were obtained: AHZ1, with very low levels of aspartase activity, and AHZ7, with low levels of asparaginase and very low levels of aspartase compared to the wild-type strain. R. etli had two asparaginases differentiated by their thermostabilities, electrophoretic mobilities, and modes of regulation. The AHZ mutants nodulated as did the wild-type strain and had nitrogenase levels similar to that of the wild-type strain.