A direct continuous pH-spectrophotometric assay for arginine kinase activity

A direct and continuous spectrophotometric method was developed for determining arginine kinase (phosphoarginine synthesis) activity. Protons are produced during the phosphoarginine synthesis course, so adding the complex acid-base indicator to this solution and monitoring the decrease of absorbance of the solution at 575 nm will follow the arginine kinase activity. For this condition, one activity unit of arginine kinase was defined as 1 micromol H+ produced in 1 minute in the enzymatic reaction catalyzed by arginine kinase.     

Inhibition of T4 polynucleotide kinase by the ATP analog, beta, gamma-imidoadenylyl 5'-triphosphate.

The inhibition of T4 polynucleotide kinase by beta,gamma-imidoadenylyl 5'-triphosphate has been investigated. It was found that the ATP analog was a competitive inhibitor with regard to ATP and a noncompetitive inhibitor with regard to DNA possessing a 5'-hydroxyl group. At pH 8.0, the Ki values were 3 and 11 mM, respectively. beta,gamma-imidoadenylyl 5'-triphosphate was not a substrate in the forward reaction, but would replace ADP and ATP in the reverse reaction. The reverse reaction was also used to make beta,gamma-imidoadenylyl 5'-tetraphosphate.     

Purification and characterization of arginine kinase from the American cockroach (Periplaneta americana)

The isolation and characterization of homogeneous arginine kinase from the cockroach is reported. The purification protocol produces 6.6 mg of pure enzyme from 6.8 g of whole cockroach. The purified enzyme cross-reacts with a heterologous antibody and monoclonal antibody against arginine kinase from the shrimp. Both antibody preparations also cross-react with extracts from several species known to contain monomeric arginine kinase, but fail to react with extracts from organisms containing dimeric arginine kinase. Cockroach arginine kinase has a molecular mass of approximately 43,000 determined from measurements by gel filtration and gel electrophoresis. Compared with other arginine kinases, the enzyme from the cockroach is relatively thermostable (50% activity retained at 50 degrees C for 10 min) and has a pH optima of 8.5 and 6.5-7.5, for the forward and reverse reactions, respectively. Treatment with 5,5'dithiobis[2-nitrobenzoic acid] indicates that arginine kinase has a single reactive sulfhydryl group and, interestingly, the reaction is biphasic. The Michaelis constants for the phosphagen substrates, arginine: 0.49 mM, phosphoarginine: 0.94 mM, and nucleotide substrates MgATP: 0.14 mM, MgADP: 0.09 mM, are in the range reported for other arginine kinases. A 1% solution of pure enzyme has an absorbance of 7.0 at 280 nm. Calculations based on circular dichroic spectra indicate that arginine kinase from the cockroach has 12% alpha-helical structure. The intrinsic protein fluorescence emission maximum at 340 nm suggests that tryptophan residues are below the surface of the protein and not exposed to solvent. Arginine kinase from the cockroach and shrimp are known to be deleterious immunogens towards humans. The availability of pure protein, its characterization and potential regulation of activity, will be useful in developing agents to control the cockroach population and its destructive role in agriculture and human health.     

31P-NMR saturation transfer study of the in vivo kinetics of arginine kinase in Carcinus crab leg muscle

The kinetics of the reaction catalyzed by arginine kinase have been determined at 9.5 and 23°C for in vivo leg muscle of Carcinus maenas (the common shore crab) using the noninvasive technique of ³¹P-NMR spectroscopy. Concentrations of mobile phosphorus metabolites were the same at both temperatures: 78.7 mM for arginine phosphate, 9.0 mM for adenosine triphosphate (ATP), and 2.6 mM for inorganic phosphate (P_i), as estimated from NMR resonance intensities and literature values for ATP concentration as assayed by traditional biochemical methods. Apparent unidirectional rate constants for formation of ATP from arginine phosphate and ADP were 0.09 s^?1 at 9.5°C and 0.27 s^?1 at 23°C. Pseudo-first-order rate constants for arginine phosphate generation from Arg and ATP were 0.38 and 1.10 s^?1 at 9.5 and 23°C, respectively. In vivo Q₁₀ for the arginine kinase reaction between 9.5 and 23°C was thus 2.2 for both directions. When the kinetic data are analyzed using the Arrhenius equation, activation energies of 126 kJ/mol for ATP formation and 105 kJ/mol for arginine phosphate formation are found. The measured chemical fluxes through arginine kinase in the forward reaction (arginine phosphate hydrolysis) were twice those in the reverse reaction, consistent with either compartmentation of substrates or participation of substrates in alternative metabolic pathways.     

Properties and reaction mechanism of C4 leaf pyruvate,Pi dikinase

The properties and reaction mechanism of maize leaf pyruvate,Pi dikinase are described. Km values were determined for the forward reaction substrates, pyruvate, ATP, and Pi, at pH 7.4 and 8.0 and for reverse reaction substrates at pH 7.4. Enzyme activity was almost totally dependent on added monovalent cations in both directions. NH+4 was most effective, with Ka values of about 0.38 mM for the forward reaction and 2 mM for the reverse reaction. K+ also completely activated the enzyme in the forward direction (Ka = 8 mM) but only partially activated in the reverse direction. Na+ had little effect on either reaction. The pH optimum for the forward reaction was about 8.2; the reverse reaction optimum was about 6.9. Maximum activity for the reverse direction was about twice the maximum forward direction rate. From data on the requirements for the ATP-AMP exchange reaction, on the mechanism of inhibition of the forward reaction by PEP, AMP, and PPi, and from the kinetics of the interaction of varying certain substrate pairs, it was concluded that the maize leaf pyruvate,Pi dikinase reaction proceeded by the two-step Bi Bi Uni Uni mechanism. This differs from the mechanism of catalysis by the bacterial enzyme.     

Functional expression of phosphagen kinase systems confers resistance to transient stresses in Saccharomyces cerevisiae by buffering the ATP pool

Phosphagen kinase systems provide different advantages to tissues with high and fluctuating energy demands, in particular an efficient energy buffering system. In this study we show for the first time functional expression of two phosphagen kinase systems in Saccharomyces cerevisiae, which does not normally contain such systems. First, to establish the creatine kinase system, in addition to overexpressing creatine kinase isoenzymes, we had to install the biosynthesis pathway of creatine by co-overexpression of L-arginine:glycine amidinotransferase and guanidinoacetate methyltransferase. Although we could achieve considerable creatine kinase activity, together with more than 3 mM intracellular creatine, this was not sufficient to confer an obvious advantage to the yeast under the specific stress conditions examined here. Second, using arginine kinase, we successfully installed an intracellular phosphagen pool of about 5 mM phosphoarginine. Such arginine kinase-expressing yeast showed improved resistance under two stress challenges that drain cellular energy, which were transient pH reduction and starvation. Although transient starvation led to 50% reduced intracellular ATP concentrations in wild-type yeast, arginine kinase overexpression stabilized the ATP pool at the pre-stress level. Thus, our results demonstrate that temporal energy buffering is an intrinsic property of phosphagen kinases that can be transferred to phylogenetically very distant organisms.     

Adenylate kinase of plants. Properties of adenylate kinase of pea leaves]

The properties of adenylate kinase in 2 ADP in equilibrium ATP + AMP reaction have been studied. The dependence of the enzyme activity on medium pH, protein concentration, substrates, Mg++ ions, AMP, adenine and adenosine has been also investigated. pH optimum is found to be 8.5 for forward reaction and 8-9--for the reverse one. The Michaelis constants are as follows: for ADP--1.17-10(-4) M, for ATP--3.33-10(-4) M at 24 degrees C, in 50 mM tris-HCl pH 7.6. The optimal ratio, Mg++ ions/substrates (ADP, ATP + AMP), is 1:2. The chelates of adenine nucleotides with Mg++ ions are proved to be "true" reaction substrates. Unlike adenine and adenosine, the product of AMP reaction inhibits adenylate kinase activity. It is concluded that the properties of adenylate kinase in plants are similar to those of animals and humans (moikinase).     

Estimation of effective concentrations of ATP-regenerating enzymes in cilia of Paramecium caudatum

The phosphoarginine shuttle system effectively regenerates ATP in the cilia of Paramecium caudatum. To estimate the effective concentration of ATP‐regenerating enzymes, we attempted to reconstitute certain ATP‐regenerating systems within the cilia of intact cortical sheets using exogenous enzymes and high‐energy substances. The addition of phosphoenolpyruvate, which is one of the substrates in glycolysis, did not increase the ciliary beat frequency, whereas phosphocreatine together with exogenous creatine kinase, effectively increased the ciliary beat frequency. In the presence of 0.6 mg/ml creatine kinase and 0.4 mM phosphocreatine, the ciliary beat frequency was comparable to that produced by the addition of phosphoarginine. This result indicates that the reconstituted phosphocreatine shuttle system can work as an artificial ATP‐regenerating system for ciliary movements. The effective concentration of creatine kinase in the reconstituted phosphocreatine shuttle system was estimated to be about 7.4 μM based on the molecular mass of creatine kinase (MW 81,000). Therefore, the effective concentration of arginine kinase in the cilia of live Paramecium is approximately 10 μM. This estimated concentration of intraciliary arginine kinase is sufficient to maintain a high ATP concentration throughout the cilia of P. caudatum.     

Screening of substrate analogs as potential enzyme inhibitors for the arginine kinase of Trypanosoma cruzi

Arginine kinase catalyzes the transphosphorylation between phosphoarginine and ADP. Phosphoarginine is involved in temporal ATP buffering and inorganic phosphate regulation. Trypanosoma cruzi arginine kinase phosphorylates only L-arginine (specific activity 398.9 x mUE-min(-1) x mg(-1)), and is inhibited by the arginine analogs, agmatine, canavanine, nitroarginine, and homoarginine. Canavanine and homoarginine also produce a significant inhibition of the epimastigote culture growth (79.7% and 55.8%, respectively). Inhibition constants were calculated for canavanine and homoarginine (7.55 and 6.02 mM, respectively). In addition, two novel guanidino kinase activities were detected in the epimastigote soluble extract. The development of the arginine kinase inhibitors of T. cruzi could be an important feature because the phosphagens biosynthetic pathway in trypanosomatids is different from the one in their mammalian hosts.     

Reversible autophosphorylation of a cyclic 3':5'-AMP-dependent protein kinase from bovine cardiac muscle.

Purified cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase of bovine cardiac muscles catalyzes the incorporation of 2 mol of 32P from [gamma-32P]ATP to seryl residues in its cAMP-binding protein. The reaction appears to be catalyzed by the protein kinase itself rather than by a protein kinase kinase and is enhanced by cAMP and by the addition of polyarginine. Phosphorylation of the purified enzyme facilitates its dissociation by cAMP (Erlichman, J., Rosenfeld, R., and Rosen, O.M. (1974) J. Biol. Chem. 249, 5000-5003) but does not affect cAMP binding. At equilibrium, 2 mol of cAMP are bound to both the phospho- and dephospho-enzymes. Phosphorylation of protein kinase is reversible. Upon addition of ADP and Mg2+, phosphate is transferred from the protein to ADP, and ATP is formed. The reverse reaction is optimal at pH 5.5 unlike the forward reaction which has a broad, more alkaline pH activity optimum. It is activated by polyarginine and dependent upon the addition of cAMP to a much greater degree than the forward reaction. The data suggest that the catalytic subunit of protein kinase catalyzes the forward and reverse reactions but do not exclude the possibility that the holoenzyme may also be active. Autophosphorylation by protein kinase and dephosphorylation by phosphrprotein phosphatases of by reverals of the autophosphorylation reaction may regulate the sensitivity of certain protein kinases to activation by cAMP in vivo.     

Changes in Glycosyldiacylglycerols during Germination of Black Mung Beans (Matpe)

The physico-chemical characteristics of purified arginine kinases from prawn and swimming crab were examined. The molecular weights of prawn and swimming crab enzymes were 40,500 and 40,000, respectively. Amino acid analysis indicated that there were some differences in the contents of proline, glycine, methionine, and lysine. The other amino acid compositions of these enzymes resembled each other.

Both enzymes were stable up to 20°C when they were treated for 10 min at various temperature levels. The enzymes lost their activities at temperatures higher than 25°C. They were more stable at pH 8.0 than pH 7.0. The optimum temperature for the enzyme of prawn was about 42°C and that for swimming crab was about 40°C. The pH optima for the activity of arginine kinase of prawn in the forward and in the reverse reactions were found to be 9.0 and 6.1, respectively. For the swimming crab, the similar optimum pHs at 9.2 in the forward reaction and 5.8 in the reverse reaction were observed. Both enzymes were activated most strongly with Mg^{2 +} and Mn^{2 +} followed by Ca^{2 +}, Co^{2 +}, and Fe^{2 +}. The enzymes were not activated by Sr^{2 +}, Cu^{2 +}, or Zn^{2 +}.

The optimum molar ratio of Mg^{2 +}: ATP in the forward reaction of prawn and swimming crab was found to be 1:1, and the molar ratio of Mg^{2 +} : ADP in the reverse reaction was 4:1 in both cases. Kinetic studies indicated that dissociation constants were rather different. In the prawn, dissociation constants for arginine, ATP, AP, and ADP were 0.19,0.31, 0.67, and 0.29 mM, respectively, but in the swimming crab, they were 0.10, 0.18, 0.22, and 0.11 mM, respectively.     

Purification and properties of two molecular forms of arginine kinase from the adductor muscle of the scallop, Pecten maximus.

1. Two molecular forms of arginine kinase, AK1 and AK2 have been purified from the adductor muscle of the scallop, Pecten maximus. AK2 was retained on a DEAE-cellulose column at pH 7.5, but AK1 was not. 2. Both forms were monomeric (mol. wt. approximately 42,000) and showed the same pH optimum (7.5-8.0) in the direction of phosphoarginine synthesis. 3. AK1 had slower electrophoretic mobility at pH 8.3 towards the anode, higher lysine content, lower glutamate content, lower Km for L-arginine and higher Km for Mg(2+)-ATP than AK2. Unlike AK1, AK2 was strongly inhibited at high concentrations of Mg(2+)-ATP. 4. Both molecular forms cross-reacted with antisera raised against native as well as performic acid-oxidized lobster muscle arginine kinase. However, AK1 showed a greater affinity than AK2 to anti-lobster arginine kinase antibodies, particularly to those raised against the native enzyme.     

Metabolic responses and arginine kinase expression under hypoxic stress of the kuruma prawn Marsupenaeus japonicus

In response to hypoxia at PO(2) 1.3-1.7 mg/L for 6 h, the kuruma prawn Marsupenaeus (Penaeus) japonicus showed a dramatic decrease in phosphoarginine storage in muscle, with normal levels restored during 4-h post-hypoxic recovery. Large stores of muscle glycogen only decreased between 4 and 6 h during hypoxia, but greatly diminished during recovery. Muscle ATP levels and energy charge decreased only slightly under hypoxia. Lactate levels increased slightly during hypoxia and promptly returned to control levels during recovery. These data indicate that phosphoarginine works in muscle as an ATP buffer during hypoxia and glycogen is utilized as an energy source during recovery. Under hypoxia, up- and down-regulated proteins were identified after 2D electrophoresis and partial sequences were obtained after protease digestion. Fructose bisphosphate aldolase was down-regulated during hypoxia, suggesting the suppression of glycolysis under hypoxia. Several partial sequences from three protein spots up-regulated under hypoxia were all assigned to arginine kinase, suggesting the existence of several isoforms of arginine kinase in the muscle of M. japonicus. This arginine kinase up-regulation under hypoxia may indicate a provision for oxygen re-supply after anaerobiosis. This is consistent with the prompt replenishment of phosphoarginine stores during recovery from hypoxia.     

Overproduction, purification, and characterization of recombinant bifunctional threonine-sensitive aspartate kinase-homoserine dehydrogenase from Arabidopsis thaliana.

In plant, the first and the third steps of the synthesis of methionine and threonine are catalyzed by a bifunctional enzyme, aspartate kinase-homoserine dehydrogenase (AK-HSDH). In this study, we report the first purification and characterization of a highly active threonine-sensitive AK-HSDH from plants (Arabidopsis thaliana). The specific activities corresponding to the forward reaction of AK and reverse reaction of HSDH of AK-HSDH were 5.4 micromol of aspartyl phosphate produced min(-1) mg(-1) of protein and 18.8 micromol of NADPH formed min(-1) mg(-1) of protein, respectively. These values are 200-fold higher than those reported previously for partially purified plant enzymes. AK-HSDH exhibited hyperbolic kinetics for aspartate, ATP, homoserine, and NADP with K(M) values of 11.6 mM, 5.5 mM, 5.2 mM, and 166 microM, respectively. Threonine was found to inhibit both AK and HSDH activities by decreasing the affinity of the enzyme for its substrates and cofactors. In the absence of threonine, AK-HSDH behaved as an oligomer of 470 kDa. Addition of the effector converted the enzyme into a tetrameric form of 320 kDa.     

Regulatory properties of phosphorylase from chicken skeletal muscle

The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM, respectively. The activity ratio at pH 6.8/8.2 was 0.1-0.4 for different preparations of phosphorylase kinase. Similar to the rabbit enzyme, chicken phosphorylase kinase had an absolute requirement for Ca2+ as demonstrated by complete inhibition in the presence of EGTA. Half-maximal activation occurred at [Ca2+] = 0.4 microM at pH 7.0. In the presence of Ca2+, the chicken enzyme from white and red muscles was activated 2-4-fold by saturating concentrations of calmodulin and troponin C. The C0.5 value for calmodulin and troponin C at pH 6.8 was 2 and 100 nM, respectively. Similar to rabbit phosphorylase kinase, the chicken enzyme was stimulated about 3-6-fold by glycogen at pH 6.8 and 8.2 with half-maximal stimulation occurring at about 0.15% glycogen. Protamine caused 60% inhibition of chicken phosphorylase kinase at 0.8 mg/ml. ADP (3 mM) at 0.05 mM ATP caused 85% inhibition with Ki = 0.2 mM. Unlike rabbit phosphorylase kinase, no phosphorylation of the chicken enzyme occurred in the presence of the catalytic subunit of cAMP-dependent protein kinase. Incubation with trypsin caused 2-fold activation of the chicken enzyme.     

Human 3'-phosphoadenosine 5'-phosphosulfate synthetase (isoform 1, brain): kinetic properties of the adenosine triphosphate sulfurylase and adenosine 5'-phosphosulfate kinase domains

Recombinant human 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase, isoform 1 (brain), was purified to near-homogeneity from an Escherichia coli expression system and kinetically characterized. The native enzyme, a dimer with each 71 kDa subunit containing an adenosine triphosphate (ATP) sulfurylase and an adenosine 5'-phosphosulfate (APS) kinase domain, catalyzes the overall formation of PAPS from ATP and inorganic sulfate. The protein is active as isolated, but activity is enhanced by treatment with dithiothreitol. APS kinase activity displayed the characteristic substrate inhibition by APS (K(I) of 47.9 microM at saturating MgATP). The maximum attainable activity of 0.12 micromol min(-1) (mg of protein)(-1) was observed at an APS concentration ([APS](opt)) of 15 microM. The theoretical K(m) for APS (at saturating MgATP) and the K(m) for MgATP (at [APS](opt)) were 4.2 microM and 0.14 mM, respectively. At likely cellular levels of MgATP (2.5 mM) and sulfate (0.4 mM), the overall endogenous rate of PAPS formation under optimum assay conditions was 0.09 micromol min(-1) (mg of protein)(-1). Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 micromol min(-1) (mg of protein)(-1). The kinetic constants of the ATP sulfurylase domain were as follows: V(max,f) = 0.77 micromol min(-1) (mg of protein)(-1), K(mA(MgATP)) = 0.15 mM, K(ia(MgATP)) = 1 mM, K(mB(sulfate)) = 0.16 mM, V(max,r) = 18.7 micromol min(-1) (mg of protein)(-1), K(mQ(APS)) = 4.8 microM, K(iq(APS)) = 18 nM, and K(mP(PPi)) = 34.6 microM. The (a) imbalance between ATP sulfurylase and APS kinase activities, (b) accumulation of APS in solution during the overall reaction, (c) rate acceleration provided by exogenous APS kinase, and (d) availability of both active sites to exogenous APS all argue against APS channeling. Molybdate, selenate, chromate ("chromium VI"), arsenate, tungstate, chlorate, and perchlorate bind to the ATP sulfurylase domain, with the first five serving as alternative substrates that promote the decomposition of ATP to AMP and PP(i). Selenate, chromate, and arsenate produce transient APX intermediates that are sufficiently long-lived to be captured and 3'-phosphorylated by APS kinase. (The putative PAPX products decompose to adenosine 3',5'-diphosphate and the original oxyanion.) Chlorate and perchlorate form dead-end E.MgATP.oxyanion complexes. Phenylalanine, reported to be an inhibitor of brain ATP sulfurylase, was without effect on PAPS synthetase isoform 1.     

Purification and characterization of 3-phosphoglycerate kinase from Ehrlich ascites carcinoma cells

3-Phosphoglycerate kinase (3-PGK) has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. The enzyme has been partially characterized and compared with the characteristics of this enzyme of other normal and malignant cells. The EAC cell 3-PGK is composed of a single subunit of 47 kDa. It has a broad pH optimum (pH 6.0-7.5) for its enzymatic activity. The apparent Km values of 3-phosphoglycerate (3-PGA) and ATP for 3-PGK have been found out to be 0.25 mM and 0.1 mM respectively. Similar to 3-PGK of other cells, the EAC enzyme requires either Mg2+ or Mn2+ for full activity; the optimum concentrations of Mg2+ and Mn2+ are 0.8 mM and 0.5 mM respectively. When ATP and 3-PGA act as substrates, ADP, the reaction product of 3-PGK-catalyzed reaction has been found to inhibit this enzyme. Kinetic studies were made on the inhibition of ADP in presence of the substrates ATP and 3-PGA. Attempts to hybridize 3-PGK and glyceraldehyde-3-phosphate dehydrogenase of EAC cells by NAD or glutaraldehyde were unsuccessful.     

Kinetic properties of arginine phosphokinase from honeybees, Apis mellifera L. (Hymenoptera, Apidae)

The kinetic properties of honeybee arginine phosphokinase (APK), which catalyzes the reaction: Arginine phosphate + ADP + H⁺ ? arginine + ATP, have been studied.In the direction of ATP synthesis, the pH optimum was around pH 7.2 and the activation energy over the range 18–44 °C was about 10,500 cal/mole. The optimum ratio of Mg²⁺:ADP was about 4:1.In the direction of arginine phosphate (AP) synthesis, the enzyme had a pH optimum around pH 8.3. The energy of activation for the reaction over the range 22–39 °C was about 7500 cal/mole. The optimum ratio of Mg²⁺:ATP was about 1:1.The initial velocities of the reactions in the direction of ATP and AP synthesis were measured at varying concentrations of one substrate while the concentration of the other substrate was held constant at several levels. The double reciprocal plots of the data obtained yielded a series of intersecting lines, indicating that the enzyme has a sequential mechanism. Radioisotope exchange experiment showed that arginine phosphokinase did not catalyze ATP ? ADP exchange in the absence of arginine. Product inhibition studies showed that arginine was competitive with AP and noncompetitive with ADP; whereas ATP was competitive with ADP and noncompetitive with arginine. The results from initial velocity, radioisotope exchange, and product inhibition studies suggested that the enzyme has a rapid equilibrium, random mechanism.     

Cryoenzymologic studies on arginine kinase: solvent, temperature and pH effects on the overall reaction.

The overall reaction catalyzed by the phosphotransferase arginine kinase was studied at normal and subzero temperatures. Ethylene glycol was used as the antifreeze and its effects on the Km values of substances, kcat and pH profiles were investigated in detail. a) The Km values for the substrate (2 mM for ATP and 0.6 mM for arginine) were little affected by the solvent composition or temperature of the reaction mixture. b) At concentration of ethylene glycol higher than 40% there was a sharp drop of enzyme activity. c) Ethylene glycol induces a large shift in the enzymic pK D) At -5 degrees C in 40% of solvent there was a break in the Arrhenius plot suggesting a change of the rate-limiting step. The relevance of these results to the reaction pathway of arginine kinase is discussed. In addition, controlled perturbations induced by cosolvent and temperature appear as useful tools for further kinetic investigations.     

Arginine kinase overexpression improves Trypanosoma cruzi survival capability

Arginine kinase catalyzes the reversible transphosphorylation between adenosine diphosphate (ADP) and phosphoarginine, which is involved in temporal and spatial adenosine triphosphate (ATP) buffering. Here we demonstrate that the homologous overexpression of the Trypanosoma cruzi arginine kinase improves the ability of the transfectant cells to grow and resist nutritional and pH stress conditions. The stable transfected parasites showed an increased cell density since day 10 of culture, when the carbon sources became scarce, which resulted 2.5-fold higher than the control group on day 28. Additional stress conditions were also tested. We propose that arginine kinase is involved in the adaptation of the parasite to environmental changes.