The kinetics of inhibition of Vigna catjang cotyledon and buffalo liver arginase by L-proline and branched-chain amino acids

The effect of proline, isoleucine, leucine, valine, lysine and ornithine under standard physiological conditions, on purified Vigna catjang cotyledon and buffalo liver arginases was studied. The results showed that V. catjang cotyledon arginase is inhibited by proline at a lower concentration than buffalo liver arginase and the inhibition was found to be linear competitive for both enzymes. Buffalo liver arginase was more sensitive to inhibition by branched-chain amino acids than V. catjang cotyledon. Leucine, lysine, ornithine and valine are competitive inhibitors while isoleucine is a mixed type of inhibitor of liver arginase. We have also studied the effect of manganese concentration which acts as a cofactor and leads to activation of arginase. The optimum Mn2+ concentration for Vigna catjang cotyledon arginase is 0.6 mM and liver arginase is 2.0 mM. The preincubation period required for liver arginase is 20 min at 55 degrees C, the preincubation period and temperature required for activation of cotyledon arginase was found to be 8 min at 35 degrees C. The function of cotyledon arginase in polyamine biosynthesis and a possible role of branched chain amino acids in hydrolysis of arginine in liver are discussed.     

Subcellular localization and kinetic properties of arginase from the liver of Genypterus maculatus

1. From the liver of the teleost fish Genypterus maculatus, a partially purified preparation of arginase was obtained and characterized. 2. The Km value for arginine was found to be 9.1 mM at pH 7.5 and 11.5 mM at the optimum pH of 9.5. At both pH values, competitive inhibition was caused by ornithine and lysine, whereas proline, leucine, valine and isoleucine caused a non-competitive inhibitory effect. Branched chain amino acids were more inhibitory than proline. 3. The enzyme was found localized in the mitochondrial matrix of the liver of Genypterus maculatus. It is suggested that this localization would be of importance in the use of arginine as an energy source.     

Purification, properties and alternate substrate specificities of arginase from two different sources: Vigna catjang cotyledon and buffalo liver

Arginase was purified from Vigna catjang cotyledons and buffalo liver by chromatographic separations using Bio-Gel P-150, DEAE-cellulose and arginine AH Sepharose 4B affinity columns. The native molecular weight of an enzyme estimated on Bio-Gel P-300 column for Vigna catjang was 210 kDa and 120 kDa of buffalo liver, while SDS-PAGE showed a single band of molecular weight 52 kDa for cotyledon and 43 kDa for buffalo liver arginase. The kinetic properties determined for the purified cotyledon and liver arginase showed an optimum pH of 10.0 and pH 9.2 respectively. Optimal cofactor Mn++ ion concentration was found to be 0.6 mM for cotyledon and 2 mM for liver arginase. The Michaelis-Menten constant for cotyledon arginase and hepatic arginase were found to be 42 mM and 2 mM respectively. The activity of guanidino compounds as alternate substrates for Vigna catjang cotyledon and buffalo liver arginase is critically dependent on the length of the amino acid side chain and the number of carbon atoms. In addition to L-arginine cotyledon arginase showed substrate specificity towards agmatine and L-canavanine, whereas the liver arginase showed substrate specificity towards only L-canavanine.     

The inhibition of arginase from the hepatopancreas of a terrestrial snail by amino acids.

The effects of L-amino acids on arginase from the hepatopancreas of the snail Ariophanta (=Cryptozona) ligulata were studied. This enzyme was inhibited by ornithine, valine, lysine, leucine, isoleucine, proline and threonine. The other amino acids were without any significant effect. Only ornithine was a non-competitive inhibitor, where as all the other inhibitory amino acids were competitive.     

The sensitivity of the moor frog Rana arvalis Nilss. tadpoles to natural L-amino acids and its alteration in ontogeny

The Rana arvalis tadpoles at 27-40 stages responded to solution of some L-amino acids in concentration 10(-2) mol/l with feeding reactions. The range of perceptible amino acids increased during development from 10 to 16. The best feeding stimuli were aspartic and glutamic acids; amino-acids (proline and oxyproline), methionine, leucine and isoleucine did not evoke the significant reactions. The sensitivity to some amino acids (alanine, glutamine, lysine, ornithine, proline and valine) was 10(-5)-10(-4) mol/l at earlier stages and increased to 10(-6)-10(-5) mol/l during larval ontogenesis.     

Lysine Uptake in Cultured Trypanosoma cruzi: Interactions of Competitive Inhibitors*

SYNOPSIS Three sites are involved in lysine transport in Trypanosoma cruzi, as inferred from interactions of inhibitory neutral amino acids. Phenylalanine and tyrosine inhibit one site; proline and the straight-chain amino acids, alanine, methionine and cysteine inhibit another; and glycine and the branched-chain valine, leucine, and isoleucine inhibit a 3rd. The curved rather than straight line obtained with a Lineweaver-Burk plot of uptake rates presumably results from the functioning of qualitatively different transport sites. Blocking every site but one results in the linear double-reciprocal plot characteristic of adsorption kinetics. The partial competitiveness of most of the inhibitions appears to denote qualitatively different sites for lysine transport and the reaction of the inhibitor with more than one site. Since most of the inhibitions were partially competitive, the specificities of the 3 lysine transport sites must overlap considerably.     

Lysosomal pool of free-amino acids

Free (non-protein) amino acids were measured in whole rat liver and in unmodified lysosomes which were prepared from rat liver by the technique of free-flow electrophoresis. Significant intralysosomal pools of threonine, serine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine and arginine were found. No efflux occurred from rat liver lysosomes in isotonic buffered sucrose at 0°C, but all amino acids showed various degrees of efflux at 20⁰ and 37⁰.     

Die Aminosäurenzusammensetzung der Proteine von Sonnen- und Schattenblättern der Blutbuche (Fagus sylvatica L. cv. Atropunicea)

Summary The amino acid contents of sun and shade leaves of the copper beech are significantly different. On the basis of dry matter, the concentration of the majority of amino acids is higher in shade leaves. Only the concentrations of proline, valine, histidine and arginine are about 30% lower compared with those of the other amino acids. Calculated on the basis of crude protein, the concentrations of lysine, histidine, arginine, valine, isoleucine and proline are considerably lower in shade leaves than in sun leaves. On the other hand, shade leaves contain more tyrosine, phenylalanine, and methionine.A hypothesis interpreting the different morphogenesis of sun and shade leaves in connection with the high proline content of sun leaves is discussed.     

Polyamine and amino acid content, and activity of polyamine-synthesizing decarboxylases, in liver of streptozotocin-induced diabetic and insulin-treated diabetic rats

1. Concentrations of polyamines, amino acids, glycogen, nucleic acids and protein, and activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, were measured in livers from control, streptozotocin-diabetic and insulin-treated diabetic rats. 2. Total DNA per liver and protein per mg of DNA were unaffected by diabetes, whereas RNA per mg of DNA and glycogen per g of liver were decreased. Insulin treatment of diabetic rats induced both hypertrophy and hyperplasia, as indicated by an increase in all four of these constituents to or above control values. 3. Spermidine content was increased in the livers of diabetic rats, despite the decrease in RNA, but it was further increased by insulin treatment. Spermine content was decreased by diabetes, but was unchanged by insulin treatment. Thus the ratio spermidine/spermine in the adult diabetic rat was more typical of that seen in younger rats, whereas insulin treatment resulted in a ratio similar to that seen in rapidly growing tissues. 4. Ornithine decarboxylase activity was variable in the diabetic rat, showing a positive correlation with endogenous ornithine concentrations. This correlation was not seen in control or insulin-treated rats. Insulin caused a significant increase in ornithine decarboxylase activity relative to control or diabetic rats. 5. S-Adenosylmethionine decarboxylase activity was increased approx. 2-fold by diabetes and was not further affected by insulin. 6. Hepatic concentrations of the glucogenic amino acids, alanine, glutamine and glycine were decreased by diabetes. Their concentrations and that of glutamate were increased by injection of insulin. Concentrations of ornithine, proline, leucine, isoleucine and valine were increased in livers of diabetic rats and were decreased by insulin. Diabetes caused a decrease in hepatic concentration of serine, threonine, lysine and histidine. Insulin had no effect on serine, lysine and histidine, but caused a further fall in the concentration of threonine.     

Competitive interrelationships between lysine and arginine in rat liver under normal conditions and in experimental hyperammonemia

The effects of lysine administration on arginine and ornithine liver levels were studied in normal and urease-treated rats. L-Arginine injections produced a rise in liver arginine with a parallel increase in liver ornithine. Pretreatment with L-lysine resulted in an elevation in liver arginine. Administration of lysine to urease treated rats induced a significant increase in liver arginine content with a parallel drop in ornithine/arginine ratio. A similar decrease in ornithine/arginine ratio due to lysine administration was observed in animals, in which arginine and ornithine levels had been raised by loading with arginine. The mechanism of the lysine effect is most likely by inhibition of liver arginase activity $\text{in vivo}$.     

Mouse model for human arginase deficiency

Deficiency of liver arginase (AI) causes hyperargininemia (OMIM 207800), a disorder characterized by progressive mental impairment, growth retardation, and spasticity and punctuated by sometimes fatal episodes of hyperammonemia. We constructed a knockout mouse strain carrying a nonfunctional AI gene by homologous recombination. Arginase AI knockout mice completely lacked liver arginase (AI) activity, exhibited severe symptoms of hyperammonemia, and died between postnatal days 10 and 14. During hyperammonemic crisis, plasma ammonia levels of these mice increased >10-fold compared to those for normal animals. Livers of AI-deficient animals showed hepatocyte abnormalities, including cell swelling and inclusions. Plasma amino acid analysis showed the mean arginine level in knockouts to be approximately fourfold greater than that for the wild type and threefold greater than that for heterozygotes; the mean proline level was approximately one-third and the ornithine level was one-half of the proline and ornithine levels, respectively, for wild-type or heterozygote mice--understandable biochemical consequences of arginase deficiency. Glutamic acid, citrulline, and histidine levels were about 1.5-fold higher than those seen in the phenotypically normal animals. Concentrations of the branched-chain amino acids valine, isoleucine, and leucine were 0.4 to 0.5 times the concentrations seen in phenotypically normal animals. In summary, the AI-deficient mouse duplicates several pathobiological aspects of the human condition and should prove to be a useful model for further study of the disease mechanism(s) and to explore treatment options, such as pharmaceutical administration of sodium phenylbutyrate and/or ornithine and development of gene therapy protocols.     

Arginine uptake by isolated rat liver mitochondria

The question of arginine uptake by mitochondria is important in that arginine is an allosteric effector of N-acetylglutamate synthetase. Thus, changes in mitochondrial arginine concentration have the potential for acutely modifying levels of N-acetylglutamate, a compound necessary for maximal activity of carbamyl phosphate synthesis. Mitochondria were isolated from chow-fed rats, incubated with [guanido-¹⁴C]arginine and were centrifuged through silicon oil into perchloric acid for determination of intramitochondrial metabolites. Arginine was separated from urea by cation-exchange resin. Mitochondrial water space was determined by [¹⁴C]urea arising from arginase activity associated with the mitochondrial preparations. Extramatrix space was determined by parallel incubations with [inulin-¹⁴C]carboxylic acid or [¹⁴C]sucrose There was considerable degradation of arginine by arginase associated with the mitochondrial preparation. This was inhibited by 7 mM ornithine and 7 mM lysine. Arginine was concentrated intramitochondrially to 4-times the extramitochondrial levels. The concentration ratio was decreased in the presence of ornithine and lysine but not with citrulline, NH₄Cl, glutamate, glutamate or leucine. No uptake was observed when mitochondria were incubated at 0°C. Mitochondria did not concentrate citrulline.     

Dynamics of the amino acid composition of the medium during cultivation of isolated organs by the directed perfusion method]

The dynamics of the amino acid composition of the medium under conditions of adequate perfusion of the isolated organs of a dog (sternum, kidney and liver) was studied. It was found that after a 6-hour perfusion of the complex of organs the amount in the perfusion medium of such amino acids as histidine, lysine, alanine, considerably increased, whereas the amount of arginine, serine, aspartic acid, threonine with glutamine, isoleucine, proline, leucine and valine decreased as compared with their initial concentration. The dynamics of the amino acid medium composition during a 4-hour perfusion was studied in experiments with the isolated sternum. The concentration of alanine, lysine and histidine increased in the medium. At the same time there was seen a decrease in the concentration of serine, aspartic acid, isoleucine, tyrosine and phenyl-alanine.     

Control of a futile urea cycle by arginine feedback inhibition of ornithine carbamoyltransferase in Agrobacterium tumefaciens and Rhizobia

In Agrobacterium tumefaciens and Rhizobia arginine can be used as the sole nitrogenous nutrient via degradation by an inducible arginase. These microorganisms were found to exhibit arginine inhibition of ornithine carbamoyltransferase activity. This inhibition is competitive with respect to ornithine (Km for ornithine = 0.8 mM; Ki for arginine = 0.05 mM). This type of urea cycle regulation has not been observed among other microorganisms which degrade arginine via an arginase. The competitive pattern of this inhibition leads to its being inoperative in ornithine-grown cells, where the intracellular concentration of ornithine is high. In arginine-grown cells, however, the intracellular arginine and ornithine concentrations are compatible with inhibition and ornithine recycling appears to be effectively blocked in vivo.     

Selection and characterisation of mutants lacking arginase in Neurospora crassa

Summary A Neurospora mutant (aga) lacking arginase was selected by virtue of its inability to utilize arginine as a source of ornithine, using a strain in which ornithine was needed to satisfy a proline requirement. It mapped in linkage group VII (right arm), close to wc. The most important characteristic of the mutant was its extreme sensitivity to arginine. Inclusion of 1 mM arginine in the medium lead to a 40-fold increase in the arginine pool and a 90% inhibition of growth. This inhibition was relieved by the addition of ornithine or proline. The high arginine pool was associated with only a slight repression of two biosynthetic enzymes examined and with a five-fold induction of ornthine transaminase, the second enzyme of arginine catabolism. It is expected that the aga mutant will be of value in further work on the regulation of arginine biosynthesis in Neurospora.     

Quantitative analysis of free amino acids and carbohydrates in spring barley anthers at different stages of maturity

The content of the carbohydrates glucose, fructose and sucrose was determined in spring barley anthers at different stages of maturity. During maturation the sucrose content of the anthers increased markedly. The following 17 free amino acids were detected in anthers of different stages of maturity: aspartic acid, glutamic acid, serine, alanine, arginine, leucine, isoleucine, lysine, α-aminobutyric acid, glutamine, proline, tyrosine, phenylalanine, valine, threonine, cystine and glycine. Quantitative analysis was only carried out in amino acids present in higher concentrations in the analysed samples. These were: aspartic acid, glutamic acid, α-aminobutyric acid, proline, serine, valine and glutamine, and a mixture of amino acids (leucine, isoleucine, valine and phenylalanine). The total content of free amino acids increased with increasing maturity of the anthers. However, not all amino acids followed contributed to this increase, but only proline, glutamic acid, aspartic acid and glutamine. A small difference was found in the variety Gopal in which the aspartic acid content did not increase significantly, but the content of the mixture of amino acids and serine did. With the exception of green anthers of the variety Firlbecks Union, proline was present in the highest concentration in all samples analysed.     

Effect of cypermethrin on the free amino acids pool in an organophosphorus-insecticide-resistant and a susceptible strain of Tribolium castaneum

1. Proline was found to be the major component of CTC-12 (44%) and FSS II (45%) strain.2. The cypermethrin treatment resulted in an increase in most of the amino acids of sixth instar larvae and all amino acids of adult beetles of CTC 12 strain.3. In the susceptible strain (FSS II), however, the tyrosine, phenylalanine and arginine increased, whereas serine, proline, glycine, alanine, valine, isoleucine, leucine and lysine were decreased significantly in the sixth instar larvae.4. In the FSS II adult beetles, only aspartic acid increased, while other amino acids either decreased (threonine, proline, glycine, alanine, valine, methionine, isoleucine, tyrososine, lysine, arginine) or remained unaffected (serine, glutamic acid, leucine, phenylalanine, histidine).     

Purification and properties of liver arginase from teleostean fish Clarias batrachus (L.).

Liver arginase of Clarias batrachus has been purified to 56.3-fold employing ammonium sulphate fraction, DEAE-cellulose and CM-cellulose chromatography. Bidirectional polyacrylamide gel electrophoresis shows the presence of two isoenzymes of arginase. The enzyme has a molecular weight of about 87,000 and Km 15.38 mM for L-arginine, optimum pH 9.5 and temperature 37 degrees C. Ornithine and leucine as competitive whereas valine and isoleucine act as non-competitive inhibitors with respect to L-arginine as substrate.     

The differential transport of amino acids into the phloem of Ricinus communis L. seedlings as shown by the analysis of sieve-tube sap

The cotyledons of castor bean (Ricinus communis L.) act as absorption organs for amino acids, which are supplied to the medium. The analysis of the sieve-tube sap, which exudes from the cut hypocotyl, demonstrated the ability of the cotyledons to load particular amino acids into the phloem and to reject the loading of others. The sieve-tube sap of cotyledons, which were embedded in the endosperm, contained 150 mM amino acids, with 50 mM glutamine as the major amino acid, and 10–15 mM each of valine, isoleucine, lysine and arginine. Removal of the endosperm led to a drastic decline in the amino-acid content of sieve-tube sap down to 16 mM. Addition of single amino acid species to the medium increased the amino acid concentration in the sieve-tube sap in specific manner: glutamine caused the largest increase (up to 140 mM in exudate), glutamate and alanine smaller increases (up to 60 mM), and arginine the smallest. In addition, the amino acid composition of the sieve-tube sap changed, for instance, glutamine or alanine readily appeared in the sieve-tube sap upon incubation in glutamine or alanine, respectively, whereas glutamate was hardly discernible even in the case of incubation with glutamate; arginine was loaded into the sieve tubes only reluctantly. In general, glutamine and alanine accumulated four- to tenfold in the sieve tubes. The uptake of amino acids and of sucrose into the sieve tubes was interdependent: the loading of sucrose strongly reduced the amino acid concentration in the sieve-tube exudate and loading of amino acids decreased the sucrose concentration. Comparison of the concentrations of various amino acids on their way from the endosperm via the cotyledon-endosperm interface, through the cotyledons and into the sieve tubes showed that glutamine, valine, isoleucine and lysine are accumulated on this pathway, whereas glutamate and arginine are more concentrated in the cotyledons than in the sieve tubes. Obviously the phloem-loading system has a transport specificity different from that of the amino acid uptake system of the cotyledon in general and it strongly discriminates between amino acids within the cotyledons.