Asparagine synthesis in the chick embryo liver

doi:10.1016/0304-4165(67)90068-2

Biochimica et Biophysica Acta (BBA) - General Subjects

Volume 136, Issue 2, 22 March 1967, Pages 233-244

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Abstract

1.
1. Chick embryo liver homogenates were shown to catalyze the conversion of [¹⁴C]aspartic acid to [¹⁴C]asparagine in the presence of l-glutamine.
2.
2. [¹⁴C]Asparagine synthesis was dependent upon both the mitochondrial and supernatant fractions. Heat treatment, freezing and thawing, dialysis and filtration on Sephadex gels indicated that both fractions contain enzymic factors.
3.
3. The activity of the chick embryo liver system was inhibited by anaerobiosis, cyanide, sulfide, rotenone and 2,4-dinitrophenol. Although these results suggested that asparagine synthesis is dependent upon an energy source, such a requirement could not be demonstrated directly with ATP and ATP-generating systems. Mg²⁺ was found to give a stimulation of about 2-fold at concentrations of 1 mM, but became inhibitory at higher levels.
4.
4. The level of activity of homogenates of liver from chick embryos was virtually the same from the 13th day after fertilization to the 17th day and then decreased to 10–15% of that level by the 19th day. Activity was not detectable in livers of newly hatched chicks or adult chickens.
5.
5. Investigation of the activity of mitochondrial and supernatant fractions of 14-day-old chick embryo livers and newly hatched chick livers in synthesizing asparagine showed that the mitochondrial fraction from the older livers was inactive but that the supernatant fraction remains active. Supernatant fractions obtained from chick embryo brain or from adult rat liver were inactive.

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Cited by (13)

Effects of supplemented nonessential amino acids and nonprotein nitrogen on growth and nitrogen excretion characteristics of broiler chickens fed diets with very low crude protein concentrations
2020, Poultry Science
Citation Excerpt :
The lower NUE at CP160+Asp compared with CP160+Asp+Asn indicated that part of additional Asp that could not be used by the birds to form Asn had to be excreted. The conversion of Asp to Asn may have been limited by Gln because Gln is necessary when Asp is metabolized to Asn by asparagine synthetase (EC 6.3.5.4) (Arfin, 1967). Limited conversion of Asp to Asn due to insufficient dietary Gln supply is indicated by the lower UA-N/(UA-N+NH3-N) ratio at CP160+Asp compared with CP160+Asp+Asn.
Reducing dietary CP for broiler chickens below a certain threshold results in decreased growth, even when the supply of essential amino acids and glycine equivalent (Gly_equi) is adequate, probably because other nonessential amino acids (neAA) are growth-limiting. Nonprotein nitrogen (NPN) might be used for the synthesis of neAA. Therefore, the effects of specific neAA and ammonium chloride (NH₄Cl) supplementation on the growth and N-excretion characteristics of broiler chickens were investigated. Nine male Ross 308 broiler chickens were kept in each of 81 metabolism units from day 7 to 21 and received 1 of 9 diets in 9 replicates in a one-factorial arrangement of treatments. Two diets with different neAA concentrations, except for Gly_equi, were mixed resulting in CP levels of 180 (CP180) and 160 (CP160) g/kg. In six other diets, CP160 was supplemented with either l-Ala, l-Pro, l-Asp, a mix of l-Asp and l-Asn·H₂O, l-Glu, or a mix of l-Glu and l-Gln to achieve concentrations of the respective neAA as formulated in CP180. In a further diet, NH₄Cl was added to CP160 to achieve the CP concentration of CP180. The ADG and gain:feed ratio (G:F) from day 7 to 21 were highest at CP180. Reduced neAA concentrations in CP160 decreased ADG and G:F. Supplementation of Asp+Asn, Glu, and Glu+Gln to CP160 increased ADG and G:F, but not to the level found for CP180. Compared with CP160, addition of Asp increased G:F but not ADG. Supplementation of Asp+Asn caused higher ADG and G:F than supplementation of Asp alone. The N-utilization efficiency was highest at CP160 and at CP160 supplemented with Ala, Pro, and Glu. Lower N-utilization efficiency was found at CP180 than at CP160, without and with supplemented neAA. The treatment containing NH₄Cl presented the lowest ADG, G:F, and N-utilization efficiency. These results showed that individual supplementation of Asp+Asn, Glu, and Glu+Gln partly compensates for the growth-reducing effects of very low CP diets. Supplementation of NH₄Cl as NPN source is not suitable for broiler chickens.
17. Asparagine Synthesis
1974, Enzymes
This chapter discusses asparagine synthesis. Asparagine is one of the common amino acid constituents of proteins and it is also found in the free-state in many animal and plant cells; it accumulates in considerable quantities in certain plants. Asparagine metabolism appears to be restricted to (1) utilization for protein synthesis, (2) conversion to aspartate and ammonia by enzymic hydrolysis, and (3) transamination with a-ketoacids to yield the corresponding amino acids and a-ketosuccinamic acid. Other metabolic pathways cannot be excluded; for example, it is possible that asparagine participates in amide nitrogen transfer reactions of the type observed with glutamine. Transamination of asparagine is reversible unless α-ketosuccinamic acid undergoes dimerization or deamidation. Thus, asparagine can be formed by transamination; however, no metabolic route to α-ketosuccinamic acid except transamination or oxidative deamination of asparagine is as yet known. The biosynthesis of asparagine in a number of mammalian tissues is catalyzed by glutamine-dependent asparagine synthetase. Asparagine synthesis also occurs when glutamine is replaced by ammonia. Glutamine-dependent asparagine synthetase is a glutamine amido transferase and its properties resemble those of such enzymes as glutamine-dependent carbamylphosphate synthetase, DPN synthetase, and formylglycinamide ribonucleotide amido transferase.
In vivo and in vitro studies on asparagine biosynthesis in soybean seedlings
1973, Archives of Biochemistry and Biophysics
The biosynthesis of asparagine in plants was investigated by feeding radioactive metabolites to soybean cotyledons and by extracting an asparagine synthetase from the same tissue. Soybean cotyledon slices were supplied with radioactive succinate, malate, or aspartate in the presence or absence of various unlabeled metabolites for periods of up to 80 min. Neither aspartate nor malate was rapidly converted to asparagine; labeled aspartate was converted largely to malate. Labeled succinate was rapidly converted to asparagine, and several lines of evidence suggested that fumarate, malate, and aspartate are intermediates. The results suggest that asparagine biosynthesis in plant cells is compartmentalized beginning with succinate. Although results were also consistent with asparagine formation via aspartate, metabolism of a mixture of [¹⁴C] plus [³H]succinate resulted in a lower $^{14} C^{3} H$ ratio in asparagine than aspartate, suggesting that some asparagine may be formed via another pathway. Demonstration of a glutamine-linked asparagine synthetase in soybean cotyledons supports the idea that asparagine is formed via aspartate. The enzyme requires aspartate (K_m = 2.2 mm), glutamine (K_m = 0.12 mm), ATP (K_m = 0.066 mm), magnesium ion, and sulfhydryl protection. It has a pH optimum of 7.7 and is not located in mitochrondria. A small amount of asparagine was formed when ammonium ion was substituted for glutamine, but the K_m of the enzyme for ammonium ion was about 25-fold greater than the K_m for glutamine suggesting that glutamine is the physiologically important substrate. Soybean cotyledons actively convert [¹⁴C]-cyanide to asparagine, apparently via β-cyanoalanine. However, malate was also rapidly labeled from [¹⁴C]cyanide and this result cannot be explained by known metabolic pathways.
A rapid radioactive assay for glutamine synthetase, glutaminase, asparagine synthetase, and asparaginase
1970, Analytical Biochemistry
A glutamine-dependent asparagine synthetase from yellow lupine seedlings
1970, FEBS Letters
Asparagine Synthetase (Tumor Cells)
1970, Methods in Enzymology
This chapter presents the assay, purification, and properties of asparagine synthetase from a strain of mouse leukemia. The enzyme preparation is incubated with L-aspartate-4-¹⁴C, L-glutamine, Mg²⁺, and adenosine triphosphate (ATP). After incubation and deproteinization, an aliquot of the reaction mixture is subjected to paper electrophoresis; the area of the dried paper containing L-asparagine is cut out, and the ¹⁴C present is determined by scintillation counting. A modified assay procedure may also be used in which the reaction mixture, after incubation, is treated with L-aspartate-β-decarboxylase or with ninhydrin. After decarboxylation of the remaining L-aspartate-4-¹⁴C, the residual ¹⁴C due to asparagine is determined. The enzyme catalyzes the formation of adenosine monophosphate (AMP), PP_i, and L-glutamate in amounts equimolar to that of L-asparagine synthesis. L-asparagine inhibits noncompetitively with respect to L-aspartate and competitively with respect to L-glutamine. The enzyme catalyzes L-aspartate dependent incorporation of ³²PP_i into ATP. This finding is in accord with the postulate that the reaction mechanism involves the formation of an enzyme bound β-aspartyladenylate intermediate.

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^☆: These studies are taken from a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University, New York, N.Y., U.S.A.

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Asparagine synthesis in the chick embryo liver☆

Abstract

J. Biol. Chem.

J. Biol. Chem.

Flora-Jena

Develop. Biol.

Anal. Biochem.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

Biochim. Biophys. Acta

J. Nutrition