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

doi:10.1016/0003-2697(80)90129-3

Analytical Biochemistry

Volume 106, Issue 1, 15 July 1980, Pages 134-147

https://doi.org/10.1016/0003-2697(80)90129-3 Get rights and content

Abstract

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.

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Citation Excerpt :
Fibulin-5 gene (FBLN5) promoter contains palindromic sequence CCAATTGG at upstream of the first translation initiation codon, which functions as a potential CCAAT/enhancer binding element [41]. The CCAATTGG motif is present in BEN (BANP, E5R, and NAC1) domain promoter of drosophila insensitive (Insv) gene [42], rat argininosuccinate lyase (AL) promoter [43], Nuclear Factor-Y (NF-Y) [44]. Literature reveals that the CCAAT sequence is vital in many cellular processes that involve cell proliferation and cell integrity [45–48].
We report the interaction of resveratrol with an octamer DNA sequence d(CCAATTGG)₂, present in the promoter region of many oncogenes, using a combination of absorption, fluorescence, calorimetric and nuclear magnetic resonance techniques to probe the binding. Resveratrol binds to the duplex sequence with a binding constant 2.20 × 10⁶ M⁻¹ in absorption studies. A ligand-duplex stoichiometry of 2.2:1 was obtained with binding constant varying from 10³ to 10⁴ M⁻¹ in fluorescence titration measurements. Spectral changes indicated external binding of resveratrol to duplex DNA. Circular dichroism data displayed minimal variation suggesting external binding. Melting temperatures of DNA and its 1:1 complex showed a difference of approximately 2.25 °C, supporting the external binding. Nuclear magnetic resonance data showed resveratrol binds to the minor groove region near the AT base pair from the nuclear Overhauser effect spectroscopic cross peaks. Distance restrained molecular dynamics was employed in explicit solvent condition to obtain the lowest energy structure. The complex was stable and retained the B DNA conformation. Findings in this study identify resveratrol as a minor groove binder to the AT region of DNA and pave the way for exploring resveratrol and its analogues as promising anticancer/antibacterial drug.
Coinduction of nitric oxide synthase, argininosuccinate synthetase, and argininosuccinate lyase in lipopolysaccharide-treated rats: RNA blot, immunoblot, and immunohistochemical analyses
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Nitric oxide (NO) is synthesized from arginine by nitric oxide synthase (NOS), and citrulline which is generated can be recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). Rats were injected with bacterial lipopolysaccharide (LPS), and expression of the inducible isoform of NOS (iNOS), AS, and AL was analyzed. In RNA blot analysis, iNOS mRNA was undetectable before the LPS treatment but was induced by LPS in the lung, heart, liver, and spleen, and less strongly in the skeletal muscle and testis. AS mRNA was induced in the lung and spleen, and AL mRNA was weakly induced in these tissues. AS and AL mRNAs were abundant in the control liver and remained unchanged after the treatment. Kinetic studies showed that iNOS mRNA increased rapidly in both spleen and lung, reached a maximum 2-5 h after the treatment, and decreased thereafter. On the other hand, AS mRNA increased more slowly and reached a maximum in 6-12 h (by about 10-fold in the spleen and 2-fold in the lung). AL mRNA in the spleen and lung increased slowly and remained high up to 24 h. In immunoblot analysis, increase of iNOS protein was evident in the lung, liver, and spleen, and there was an increase of AS protein in the lung and spleen. In immunohistochemical analysis, macrophages in the spleen that were negative for iNOS and AS before LPS treatment were strongly positive for both iNOS and AS after this treatment. As iNOS, AS, and AL were coinduced in rat tissues and cells, citrulline-arginine recycling seems to be important in NO synthesis under the conditions of stimulation.
Staining of argininosuccinate lyase activity in polyacrylamide gel
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A procedure for the direct staining of argininosuccinate lyase activity in polyacrylamide gel is described. The method was based on coupling one of the enzymatic products fumarate with fumarase and malic enzyme catalyzed reactions. Fumarate was first converted to l-malate by fumarase. Malic enzyme then catalyzed the oxidative decarboxylation of l-malate to give CO₂ and pyruvate with concomitant reduction of NADP⁺ to NADPH. Finally the reducing power of NADPH was coupled to phenazine methosulfate and in turn to nitroblue tetrazolium yielding a deeply colored insoluble formazan which may be quantitized or semiquantitized by densitometer.
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The in vivo synthesis of arginine from glutamate in mammals requires seven enzymes to cooperate. Pyrroline-5-carboxylate synthase (PCS) is the first enzyme required. In order to establish the interorgan dependency of arginine synthesis, we quantitated PCS activity in as many as 32 rat tissues and found that the activity was concentrated only in the upper small intestine. Minor activity was found in pancreas, thymus, lymph node, and some other tissues: this was confirmed by the dependency on specific substrates, the loss of activity in the presence of an inhibitor, and identifying the reduced product as proline. No difference in activity was found between male and female rats on a milligram protein basis. The strict tissue localization of PCS and the localization of other enzymes of arginine synthesis previously reported clearly indicate that the upper small intestine is an indispensable tissue for the arginine synthesis from glutamate. Many of the tissues examined showed an activity to form an unknown product from glutamate. When assayed by the previously reported radiometric assay procedure using an AG1-X8 column (acetate), the product was not separated from PC and caused false-positive activities of PCS. An improved procedure was developed to overcome this technical difficulty. The new procedure enabled us to detect even 20 pmol PC without contamination by the adjoining unknown product. A preliminary characterization of the unknown product was achieved.
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^☆: Aided by Research Grant AM-03428 from the United States Public Health Service.

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

Abstract

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Anal. Biochem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Arch. Biochem. Biophys

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