A radiochemical assay for argininosuccinate synthetase with [U-14C]aspartate

A simple and sensitive radiochemical procedure to assay argininosuccinate synthetase activity in crude tissue homogenates and lysates of cultured cells is described. The new method depends on the location of 14C, uniformly, in the four carbons of aspartate. On incubation in the presence of excess of L-[U-14C]aspartate, L-citrulline, ATP, and an ATP-generating system, argininosuccinase and arginase, the [14C]fumarate formed is measured as the sum of malate and fumarate. After acidification the latter two acids are separated from [14C]aspartate on a small Dowex-50 column by elution with a few milliliters of water; the unutilized amino acid substrates remain on the column. With a specific radioactivity of 9 X 10(4) cpm, 1 to 2 nmol of product can be accurately measured under kinetically optimum conditions.     

A new radiochemical assay for argininosuccinase with purified [14C]argininosuccinate

A sensitive and simple radiochemical assay is described to measure argininosuccinase activity in crude tissue homogenates and cultured cells. The method depends on the use of argininosuccinate labeled uniformly with ¹⁴C in the six carbons of the arginine moiety. On incubation in the presence of excess arginase, the [U-¹⁴C]arginine formed is measured as the sum of radioactivity in [U-¹⁴C]ornithine and [¹⁴C]urea. Separation from the substrate is accomplished on a small Domex 1-acetate column eluted with 25 mm acetic acid; ornithine and urea emerge in the first few milliliters while unutilized substrate remains on the column. [¹⁴C]Argininosuccinate was synthesized enzymatically from l-[U-¹⁴C]arginine and fumarate and isolated and purified as the barium salt. Development of a new purification step has brought the amino acid to a purity of 97% as judged by chromatographic and barium analysis. With the present specific radioactivity, as little as 5 to 10 nmol of product can be accurately measured under kinetically optimum conditions.     

Evidence for urea cycle activity in Sporosarcina ureae

Sporosarcina ureae BS 860, a motile, sporeforming coccus, possesses the enzymes required for a functioning urea (ornithine) cycle. This is only the second known example of urea cycle activity in a prokaryote. Specific activities are reported for ornithine carbamoyltransferase, argininosuccinase, arginase, and urease. Although argininosuccinate synthetase activity could not be detected directly in crude cell extracts, indirect evidence from radiocarbon tracing data for arginine synthesis from the substrate, l-[1-¹⁴C]-ornithine, strongly suggest the presence of this or other similar enzyme activity. Furthermore, good growth in defined media containing either 1.0% glutamine, ornithine, or citrulline as sole carbon sources suggests argininosuccinate synthetase activity is necessary for arginine synthesis. The effect of varying pH on arginase and urease activities indicate that these two enzymes may function within the context of the urea cycle to generate ammonia for amino acid synthesis, as well as for raising the pH of the growth micro-environment.     

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.     

Identification of fumonisin B1 as an inhibitor of argininosuccinate synthetase using fumonisin affinity chromatography and in vitro kinetic studies

Fumonisin B1, a fungal mycotoxin that grows on corn and other agricultural products, alters sphingolipid metabolism by inhibiting ceramide synthase. The precise mechanism of fumonisin B1 toxicity has not been completely elucidated; however, a central feature in the cytotoxicity is alteration of sphingolipid metabolism through interruption of de novo ceramide synthesis. An affinity column consisting of fumonisin B1 covalently bound to an HPLC column matrix was used to isolate a rat liver protein that consistently bound to the column. The protein was identified as argininosuccinate synthetase by protein sequencing. The enzyme-catalyzed formation of argininosuccinic acid from citrulline and aspartate by recombinant human and rat liver argininosuccinate synthetase was inhibited by fumonisin B1. Fumonisin B1 showed mixed inhibition against citrulline, aspartate, and ATP to the enzyme. Fumonisin B1 had a Ki' of approximately 6 mM with the recombinant human argininosuccinate synthase and a Ki' of 35 mM with a crude preparation of enzyme prepared from rat liver. Neither tricarballylic acid nor hydrolyzed fumonisin B1 inhibited recombinant human argininosuccinate synthetase. This is the first demonstration of fumonisin B1 inhibition of argininosuccinate synthethase, a urea cycle enzyme, which adds to the list of enzymes that are inhibited in vitro by fumonisin B1 (ceramide synthase, protein serine/threonine phosphatase). The extent of the inhibition of argininosuccinate synthetase in cells, and the possible role of this enzyme inhibition in the cellular toxicity of FB1, remains to be established.     

The determination by radiochemical assay of argininosuccinase produced in an Escherichia coli system in vitro.

A highly sensitive radiochemical assay used to measure the synthesis and regulation of the product of the argH gene, argininosuccinase, in an Escherichia coli system in vitro is described. With L-[guanidino-14C]argininosuccinic acid as a substrate, and in the presence of excess arginase and urease, 14CO2 is collected in a simply designed micro-vessel. With this method less than 1 nmol of product can be measured in the presence of various concentrations of L-arginine.     

Inhibition of Arginyl-Transfer Ribonucleic Acid Synthetase Activity of Escherichia coli by Arginine Biosynthetic Precursors

The arginine biosynthetic precursors, ornithine, citrulline, and argininosuccinate, inhibit arginyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.13, arginine: soluble RNA ligase, adenosine monophosphate) activity in the in vitro attachment assay system. Ornithine is the most potent, argininosuccinate is next, and citrulline is least effective. The implications of these results are discussed in relation to arginyl-tRNA synthetase activity and the level of the arginine biosynthetic enzymes during conditions of restricted and unrestricted supply of arginine to cells.     

Molecular basis of enzyme abnormalities in urea cycle disorders. With special reference to citrullinemia and argininosuccinic aciduria

This paper deals with enzymological, immunochemical and molecular genetic analyses of citrullinemia and argininosuccinic aciduria. Citrullinemia has been classified by Saheki et al. [J. inher. Metab. Dis. 8: 155-156, 1985] into three types from the properties of the deficient argininosuccinate synthetase (ASS) of the patients. Analysis of hepatic mRNA coding for ASS revealed certain characteristics in type II and III citrullinemic patients whose hepatic ASS protein was low. A newly developed enzyme-linked immunosorbent assay (ELISA) of argininosuccinate lyase (ASL) protein showed that 8 out of ten cases of argininosuccinic aciduria had no detectable ASL protein in the liver, erythrocytes, cultured skin fibroblasts or cultured amniocytes.     

Control of the flux to arginine in Neurospora crassa: de-repression of the last three enzymes of the arginine pathway

The steady state concentrations of arginine and related intermediary metabolites of the arginine biosynthetic pathway in the eukaryote Neurospora crassa were varied and the concurrent de-repression of the enzymes ornithine transcarbamylase, argininosuccinate synthetase and argininosuccinase was measured. Pool variation was achieved endogenously by the introduction and combination of mutant enzymes with reduced specific activities. Measurements of activities of the mutationally unaltered enzymes showed various degrees of de-repression. The highest activity level for each of the three enzymes was about five times that found in the fully repressed wild-type strain. The variations observed in the pools were as follows: ornithine, 7-fold; citrulline, 700-fold; argininosuccinic acid, 400-fold; arginine, 300-fold.By this means a quantitative analysis of the process of repression is made possible. A strong correlation was found between the degree of de-repression of the three enzymes and the concentration of arginine. The de-repression follows a sigmoid curve with respect to arginine concentration. This is consistent with the interpretation that the pathway enzymes are subject to a repression system with arginine, or a simple derivative of it, acting as a co-repressor.     

A simplified procedure for synthesis of di-[14C]acyl-labeled lecithins.

A simplified procedure for synthesis of 1,2-di-[1'-14C]oleoyl-, 1,2-di-[1'-14C]linoleoyl-, and 1,2-di-[1'-14C]eicosatrienoyl-sn-glycero-3-phosphorylcholine is described. The method involves acylation of the CdCl2 complex of glycerophosphorylcholine with a 14C-labeled fatty acid in the presence of trifluoracetic anhydride and pyridine. The 14C-labeled lecithin is isolated in pure form by preparative thin-layer chromatography and alumina column chromatography in an overall yield of 12-24%. No isomerization or peroxidation of the unsaturated acids was detected.     

Effects of adenosine triphosphate and magnesium chloride on affinity elution of aminoacyl-transfer ribonucleic acid synthetases from phosphocellulose with transfer ribonucleic acids.

Aminoacyl-tRNA synthetases of bakers' yeast (Saccharomyces cerevisiae) were adsorbed to a phosphocellulose (P-cellulose) column, and those specific for tyrosine [EC 6.1.1.1], threonine [EC 6.1.1.3], valine [EC 6.1.1.9], and isoleucine [EC 6.1.1.5] were eluted with several specific tRNAs. Elutions of these synthetases were affected by ATP and/or MgCl2. The effects of ATP and MgCl2 differ with synthetases. Elutions of tyrosyl- and valyl-tRNA synthetases with their cognate tRNAs were more specific in the presence of MgCl2. Isoleucyl-tRNA synthetase was eluted with its cognate tRNA in the presence of both ATP and MgCl2. On the other hand, threonyl-tRNA synthetase was eluted in the absence of ATP and MgCl2 with unfractionated tRNA but not with some non-cognate tRNAs. This suggests that elution of threonyl-tRNA synthetase is highly specific. The present data on the effects of ATP or MgCl2 or both on this affinity elution will be useful for simple and rapid purification of the synthetases.     

Synthesis of [(14)C]pyrroloquinoline quinone (PQQ) in E. coli using genes for PQQ synthesis from K. pneumoniae

Radiochemical forms of pyrroloquinoline quinone (PQQ) are of utility in studies to determine the metabolic role and fate of PQQ in biological systems. Accordingly, we have synthesized [(14)C]PQQ using a tyrosine auxotrophic strain of Escherichia coli (AT2471). A construct containing the six genes required for PQQ synthesis from Klebsiella pneumoniae was used to transform the auxotrophic strain of E. coli. E. coli were then grown in minimal M9 medium containing 3.7x10(9) Bq/mmol [(14)C]tyrosine. At confluence, the medium was collected and applied to a DEAE A-25 anionic exchange column; [(14)C]PQQ was eluted using a KCl gradient (0-2 M in 0.1 M potassium phosphate buffer, pH 7.0). Radioactivity co-eluting as PQQ was next pooled, acidified and passed through a C-18 column; [(14)C]PQQ was eluted with a phosphate buffer (0.1 M, pH 7.0). Reverse phase HPLC (C-18) using either the ion-pairing agent trifluoroacetic acid (0. 1%) and an acetonitrile gradient or phosphoric acid and a methanol gradient were used to isolate [(14)C]PQQ. Fractions were collected and analyzed by liquid scintillation counting. (14)C-labelled compounds isolated from the medium eluted corresponding to the elution of various tyrosine-derived products or PQQ. The radioactive compound corresponding to PQQ was also reacted with acetone to form 5-acetonyl-PQQ, which co-eluted with a 5-acetonyl-PQQ standard, as a validation of [(14)C]PQQ synthesis. The specific activity of synthesized [(14)C]PQQ was 3.7x10(9) Bq/mmol [(14)C]PQQ, equal to that of [U-(14)C]tyrosine initially added to the medium.     

Interference with the citrulline-based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts

There are many reports of an arginine-dependent nitric oxide synthase activity in plants; however, the gene(s) or protein(s) responsible for this activity have yet to be convincingly identified. To measure nitric oxide synthase activity, many studies have relied on a citrulline-based assay that measures the formation of L-citrulline from L-arginine using ion exchange chromatography. In this article, we report that when such assays are used with protein extracts from Arabidopsis, an arginine-dependent activity was observed, but it produced a product other than citrulline. TLC analysis identified the product as argininosuccinate. The reaction was stimulated by fumarate (> 500 microM), implicating the urea cycle enzyme argininosuccinate lyase (EC 4.3.2.1), which reversibly converts arginine and fumarate to argininosuccinate. These results indicate that caution is needed when using standard citrulline-based assays to measure nitric oxide synthase activity in plant extracts, and highlight the importance of verifying the identity of the product as citrulline.     

Determination of argininosuccinate in normal blood serum and liver

Argininosuccinate has been determined in normal serum and liver extract of rats by chromatographic analysis on Dowex-1-acetate after a brief period of in vivo labeling with l-[U-¹⁴C] citrulline. The amounts found were within the range of other amino acid intermediates of the ornithine cycle, determined in the same samples. Both ¹⁴C and ninhydrin color were measured. Chromatography on Amberlite columns, subsequent to fractionation on Dowex, allowed determination of the other amino acids. Fractionation on Dowex-1-acetate provides a reliable and highly selective method for the analysis of minute amounts of argininosuccinate in biological samples containing other amino acids.     

Channeling of urea cycle intermediates in situ in permeabilized hepatocytes

Preferential use of endogenously generated intermediates by the enzymes of the urea cycle was observed using isolated rat hepatocytes made permeable to low molecular weight compounds with alpha-toxin. The permeabilized cells synthesized [14C]urea from added NH4Cl, [14C]HCO3-, ornithine, and aspartate, using succinate as a respiratory substrate; with all substrates saturating, about 4 nmol of urea were formed per min/mg dry weight of cells. Urea usually accounted for about 40-50% of the total (NH3 + ornithine)-dependent counts, arginine for less than 10%, and citrulline for about 30%. Very tight channeling of arginine between argininosuccinate lyase and arginase was shown by the fact that the addition of a 200-fold excess of unlabeled arginine to the incubations did not decrease the percentage of counts found in urea or increase that found in arginine, even though a substantial amount of the added arginine was hydrolyzed inside the cells. The channeling of argininosuccinate between its synthetase and lyase was demonstrated by similar observations; unlabeled argininosuccinate added in 200-fold excess decreased the percentage of counts in urea by only 25%. Channeling of citrulline from its site of synthesis by ornithine transcarbamylase in the mitochondrial matrix to argininosuccinate synthetase in the cytoplasmic space was also shown. These results strongly suggest that the three "soluble" cytoplasmic enzymes of the urea cycle are grouped around the mitochondria and are spatially organized within the cell in such a way that intermediates can be efficiently transferred between them.     

Argininosuccinic aciduria: prenatal studies in a family at risk.

We have monitored two successive pregnancies in a family which we found to be at risk for argininosuccinic aciduria. We measured argininosuccinic acid (ASA) concentrations in amniotic fluid and utilized an indirect assay of ASA lyase activity in cultured amniotic fluid cells. The assay procedure is based on the uptake of 14C from [14C]citrulline and of [3H]leucine into protein. ASA was easily measured in amniotic fluid from the first fetus at risk, whereas none was detectable in control fluids. Amniotic fluid cells cultured from this fetus had only 5.5% of control ASA lyase activity. The pregnancy was terminated, and hepatic ASA lyase activity in the fetus was shown to be about 1.3% of control values. In addition, eight fetal tissues were analyzed for ASA, and all had significant accumulation. ASA was not detected in amniotic fluid from the second fetus at risk, and ASA lyase activity in cultured cells was 80% of control activity. Enzymatic analysis of erythrocyte lysate confirmed the diagnosis of an unaffected child (ASA lyase = 46% of control) and indicated heterozygosity. Thus, we provide further evidence that argininosuccinic aciduria can be diagnosed successfully in utero by indirect assay of ASA lyase activity in cultured amniotic fluid cells. In addition, high amniotic fluid ASA concentrations provide strong adjunctive evidence for such a prenatal determination, and may prove to be sufficient for diagnosis.     

Separation of putrescine oxidase and spermidine oxidase in foetal bovine serum with the aid of a specific radioactive assay of spermidine oxidase.

1. A sensitive and specific assay for spermidine oxidase is described. The method involves the separation of [14C]spermidine (substrate) from [14C]putrescine (product) and other 14C-labelled products on a Dowex 50 cation-exchange column: 92% of the putrescine applied to the column was eluted by 2.3 M-HCl, but this treatment left 96% of the spermidine bound to the column. Unchanged spermidine could be removed from the column by elution with 6 M-HCl. 2. By means of this assay, foetal and adult bovine serum were each shown to contain spermidine oxidase activity, putrescine being a major product of the oxidation of spermidine by the serum enzymes. 3. In foetal bovine serum, spermidine oxidase activity is separable from putrescine oxidase activity by chromatography on a cadaverine-Sephadex column, by gel filtration and by ion-exchange column chromatography. Putrescine oxidase was purified 1900-fold and spermidine oxidase 130-fold by these procedures. The former oxidized putrescine but not spermidine, and spermidine oxidase exhibited no activity with putrescine as substrate.     

Citrulline metabolism in normal and citrullinemic human lymphocyte lines

Citrullinemia is one of the five aminoacidurias associated with the Krebs-Henseleit urea cycle. A long-term lymphocyte line (UM-21) derived from a patient with this disease and nine of ten clones of this line were found to have no activity for the enzyme argininosuccinate synthetase (AS), as demonstrated by their inability to grow in medium in which citrulline had been substituted for arginine, by their inability to incorporate arginine-C14 derived from citrulline-C14 into cellular protein, and by direct enzyme assay. One clone had normal or nearly normal argininosuccinate synthetase activity, as demonstrated by the same criteria. Nutritional "variants" able to grow logarithmically in medium containing citrulline were isolated from UM-21 and three clones. The apparent Kms of AS for citrulline in UM-21, the ten clones, the variant lines, and a normal line were measured and fell into three groups: AS in UM-21 and nine clones had no measurable apparent Km for citrulline; AS in the variant cells had apparent Kms for citrulline of approximately 20 mM; and AS in the normal cell line and one clone had apparent Kms for citrulline of 0.2 mM. The data suggest that the defect in the citrullinemic cell lines is due to a mutation in the structural gene coding for argininosuccinate synthetase.     

The metabolism of d-galactosamine and N-acetyl-d-galactosamine in rat liver

d-[1-¹⁴C]Galactosamine appears to be utilized mainly by the pathway of galactose metabolism in rat liver, as evidenced by the products isolated from the acid-soluble fraction of perfused rat liver. These products were eluted in the following order from a Dowex 1 (formate form) column and were characterized as galactosamine 1-phosphate, sialic acid, UDP-glucosamine, UDP-galactosamine, N-acetylgalactosamine 1-phosphate, N-acetylglucosamine 6-phosphate, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and an unidentified galactosamine-containing compound. In addition, [1-¹⁴C]glucosamine was found in the glycogen, an incorporation previously shown to result from the substitution of UDP-glucosamine for UDP-glucose in the glycogen synthetase reaction. Analysis of the [1-¹⁴C]glucosamine-containing disaccharides released from glycogen by β-amylase provided additional evidence that they consist of a mixture of glucose and glucosamine in a 1:1 ratio, but with glucose predominating on the reducing end. UDP-N-acetylgalactosamine was shown to result from the reaction of UTP with N-acetylgalactosamine 1-phosphate in the presence of a rat liver extract.