精氨酸抗氧化作用机制

精氨酸是一种功能性氨基酸,在机体生理功能、新陈代谢和营养等方面发挥着重要作用。精氨酸具有抗氧化能力。目前的体外研究表明精氨酸具有较强的清除DPPH自由基、ABTS自由基、超氧自由基能力以及一定的还原力。作为一种带电子的碱性氨基酸,精氨酸可能通过胍基基团向自由基提供电子并与其作用,终止自由基链式反应,从而显示出还原能力与体外抗氧化能力。体内实验则表明精氨酸能有效地提高机体总抗氧化能力,降低体内自由基含量,抑制ROS生成与积累,促进谷胱甘肽(GSH)合成与积累,增强内源性抗氧化酶(CAT、SOD、GPx等)活性,抑制氧化应激的产生。精氨酸能够通过精氨酸——一氧化氮途径、GSH途径、Nrf2信号通路途径及其他途径发挥体内抗氧化作用。本文主要综述了目前精氨酸体外与体内抗氧化功能及其相关作用机制的研究进展,为精氨酸的实际应用提供理论指导意义。     

Effective elution of antibodies by arginine and arginine derivatives in affinity column chromatography

It has been shown that the recovery of monomeric antibodies from protein A affinity chromatography is enhanced significantly by using arginine as an eluent. To extend the applications of arginine to antibody purification and obtain an insight into the mechanism of arginine elution, we compared arginine with citrate, guanidine hydrochloride (GdnHCl), arginine derivatives, and other amino acids in protein A chromatography. We also applied arginine to elution of polyclonal antibodies (pAbs) in antigen affinity chromatography. As described previously, arginine was effective in eluting monoclonal antibodies IgG1 and IgG4. Two arginine derivatives, acetyl-arginine and agmatine, resulted in efficient elution at pH 4.0 or higher, and this was comparable to arginine. On the other hand, other amino acids, such as glycine, proline, lysine, and histidine, are much less effective than arginine under identical pH conditions. Whereas elution increased with arginine concentration, elution with citrate was insignificant in excess of 1 M at pH 4.3. Arginine was also effective in fractionation of pAbs using antigen-conjugated affinity columns. Although GdnHCl was also effective under similar conditions, the eluted material showed more aggregation than did the protein eluted by arginine.     

Convenient method for the determination of arginine and its related compounds in rumen fluid by reversed-phase high-performance liquid chromatography

In order to clarify arginine (Arg) metabolism by rumen microorganisms and by the tissues of ruminant animals, a convenient method for the simultaneous determination of Arg, citrulline (Cit), ornithine (Orn), proline (Pro) and 5-aminovaleric acid (5AV), and 4-aminobutyric acid (4AB) and lysine (Lys), incidentally, in goat rumen fluid was established by reversed-phase high-performance liquid chromatography (RP-HPLC). The separation was carried out by stepwise isocratic elution with two mobile phases (solvent A and solvent B) on a LiChrospher 100 RP-18 column (150×4.6 mm I.D., 5 μm particle size) equipped with a guard column (4.0×4 mm, 5 μm particle size). Solvent A is composed of acetonitrile–sodium citrate buffer (pH 7.2) (15:85, v/v) containing tetrahydrofuran (5 ml/100 ml), with solvent B comprising acetonitrile–sodium citrate buffer (pH 5.4) (40:60, v/v). Five compounds (Cit, Arg, Pro, 4AB and 5AV) were separated within 33 min in solvent A and the other two (Orn and Lys) in solvent B. Solvent A was automatically switched to solvent B with the help of a valve controller. Complete separation needs 62 min after sample injection in a single chromatogram. Samples were derivatized with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl) and detected on a fluorescence detector at excitation and emission wavelengths of 263 and 611 nm, respectively. The minimum detectable concentrations (μM) (signal-to-noise ratio, S/N 3:1) of these compounds were: 0.65 for Cit, 0.65 for Arg, 1.9 for Pro, 1.3 for 4AB, 1.9 for 5AV, 0.12 for Orn and 0.48 for Lys. When applied to rumen fluid from goats, recoveries of all compounds added to the rumen fluid were 96.6–100.6% for an intra-day study and 93.9–99.4% for inter-day (5 days) studies. The average contents of Orn, 5AV and Lys in the rumen fluid of three goats before morning feeding were 7.3, 13.5 and 3.6 μM, but Cit, Arg, Pro, and 4AB were not found, although all these four compounds were detected 1 h after feeding. Pro (390 μM) and 5AV (497.6 μM) were highest 1 h after feeding and then decreased. Orn levels before morning feeding were most similar to those after feeding.     

Characterization of arginine decarboxylase in rat brain and liver: distinction from ornithine decarboxylase

We compared the properties of mammalian arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) in rat liver and brain. Mammalian ADC is thermally unstable and associated with mitochondrial membranes. ADC decarboxylates both arginine (Km = 0.75 mM) and ornithine (Km = 0.25 mM), a reaction not inhibited by the specific ODC inhibitor, difluoromethylomithine. ADC activity is inhibited by Ca2+, Co2+, and polyamines, is present in many organs being highest in aorta and lowest in testis, and is not recognized by a specific monoclonal antibody to ODC. In contrast, ODC is thermally stable, cytosolic, and mitochondrial and is expressed at low levels in most organs except testis. Although ADC and ODC are expressed in cultured rat C6 glioma cells, the patterns of expression during growth and confluence are very different. We conclude that mammalian ADC differs from ADC isoforms expressed in plants, bacteria, or Caenorhabditis elegans and is distinct from ODC. ADC serves to synthesize agmatine in proximity to mitochondria, an organelle also harboring agmatine's degradative enzyme, agmatinase, and a class of imidazoline receptor (I2) to which agmatine binds with high affinity.     

Toxoplasma gondii lacks the enzymes required for de novo arginine biosynthesis and arginine starvation triggers cyst formation

Two separate carbamoyl phosphate synthetase activities are required for the de novo synthesis of pyrimidines and arginine in most eukaryotes. Toxoplasma gondii is novel in possessing a single carbamoyl phosphate synthetase II gene that corresponds to a glutamine-dependent form required for pyrimidine biosynthesis. We therefore examined arginine acquisition in T. gondii to determine whether the single carbamoyl phosphate synthetase II activity could provide both pyrimidine and arginine biosynthesis. We found that arginine deprivation efficiently blocks the replication of intracellular T. gondii, yet has little effect on long-term parasite viability. Addition of citrulline, but not ornithine, rescues the growth defect observed in the absence of exogenous arginine. This rescue with citrulline is ablated when parasites are cultured in a human citrullinemia fibroblast cell line that is deficient in argininosuccinate synthetase activity. These results reveal the absence of genes and activities of the arginine biosynthetic pathway and demonstrate that T. gondii is an arginine auxotroph. Arginine starvation was also found to efficiently trigger differentiation of replicative tachyzoites into bradyzoites contained within stable cyst-like structures. These same parasites expressing bradyzoite antigens can be efficiently switched back to rapidly proliferating tachyzoites several weeks after arginine starvation. We hypothesise that the absence of gene activities that are essential for the biosynthesis of arginine from carbamoyl phosphate confers a selective advantage by increasing bradyzoite switching during the host response to T. gondii infection. These findings are consistent with a model of host-parasite evolution that allowed host control of bradyzoite induction by trading off virulence for increased transmission.     

Enzymes of arginine utilization and their formation in Aeromonas formicans NCIB 9232

In Aeromonas formicans two inducible catabolic pathways of L-arginine have been characterized. The arginine decarboxylase is induced by arginine which also induces the three enzymes of the arginine deiminase pathway but only in stress conditions such as a shift from aerobic growth conditions to very low oxygen tension. Addition of glucose to medium containing arginine leads to repression of the enzymes involved in the arginine deiminase pathway while exogenous cAMP prevents that repression of enzyme synthesis by glucose. This suggests that the induction of arginine deiminase pathway is regulated by carbon catabolite repression and the energetic state of the cell.     

Effect of intranasal arginine vasopressin on human headache

Arginine vasopressin (AVP), a nonapeptide hormone of posterior pituitary, reaches the central nervous system from systemic blood circulation with a difficulty because of the blood–brain barrier (BBB). The interest has been expressed in the use of the nasal route for delivery of AVP to the brain directly, exploiting the olfactory pathway. Our previous study has demonstrated that AVP in the brain rather than the spinal cord and blood circulation plays an important role in rat pain modulation. For understanding the role of AVP on pain modulation in human, the communication tried to investigate the effect of intranasal AVP on human headache. The results showed that (1) AVP concentration in both plasma and cerebrospinal fluid (CSF) increased significantly in headache patients, who related with the headache level; (2) there was a positive relationship between plasma and CSF AVP concentration in headache patients; and (3) intranasal AVP could relieve the human headache in a dose-dependent manner. The data suggested that intranasal AVP, which was delivered to the brain through olfactory region, could treat human headache and AVP might be a potential drug of pain relief by intranasal administration.     

High-performance liquid chromatographic analysis of muscular interstitial arginine and norepinephrine kinetics: A microdialysis study in rats

Complex interactions between the -arginine/nitric oxide synthase (NOS) pathway and the sympathetic nervous system have been reported. Methods capable of measuring -arginine and norepinephrine (NE) have mainly been reported for plasma. We report the use of the microdialysis technique combined with high-performance liquid chromatography (HPLC) for measurement of both -arginine and NE within the same tissue microdialysis sample. The microdialysis probe consisted of linear flexible probes (membrane length: 10 mm, outside diameter: 290 μm, molecular weight cut-off 50 kDa). The method used for -arginine measurement was HPLC with fluorescence detection, giving a within-run and a between-day coefficient of variation of 2.9 and 12.8%, respectively. The detection limit was 0.5 pM/20 μl injected for -/ -arginine. The method used for NE measurement was HPLC with electrochemical detection. The coefficients of variation were 4% for within-assay precision and 7.5% for between-assay precision. The detection limit for NE was 1 fmol/20 μl injected. The microdialysis technique coupled with HPLC system was validated in vivo to measure muscular interstitial concentrations of both arginine and NE under baseline conditions and after intravenous infusion of 500 mg/kg of -arginine or -arginine. In conclusion, the microdialysis technique coupled to HPLC allows the simultaneous measurements of both -arginine and NE within the same tissue microenvironment and will enable the study of the complex interactions between the -arginine/NO pathway and sympathetic nervous system within the interstitial space of different organs.     

Defective arginine metabolism impairs mitochondrial homeostasis in Caenorhabditis elegans

Arginine catabolism involves enzyme-dependent reactions in both mitochondria and the cytosol,defects in which may lead to hyperargininemia,a devastating developmental disorder.It is largely unknown if defective arginine catabolism has any effects on mitochondria.Here we report that normal arginine catabolism is essential for mitochondrial homeostasis in Caenorhabditis elegans.Mutations of the arginase gene argn-1 lead to abnormal mitochondrial enlargement and reduced adenosine triphosphate(ATP) production in C elegans hypodermal cells.ARGN-1 localizes to mitochondria and its loss causes arginine accumulation,which disrupts mitochondrial dynamics.Heterologous expression of human ARGl or ARG2 rescued the mitochondrial defects of argn-1 mutants.Importantly,genetic inactivation of the mitochondrial basic amino acid transporter SLC-25A29 or the mitochondrial glutamate transporter SLC-25A18.1 fully suppressed the mitochondrial defects caused by argn-1 mutations.These findings suggest that mitochondrial damage probably contributes to the pathogenesis of hyperargininemia and provide clues for developing therapeutic treatments for hyperargininemia.     

Interaction of arginine oligomer with model membrane

Short oligomers of arginine (R8) have been shown to cross readily a variety of biological barriers. A hypothesis was put forward that inverted micelles form in biological membranes in the presence of arginine oligomer peptides, facilitating their transfer through the membranes. In order to define the role of peptide-lipid interaction in this mechanism, we prepared liposomes as the model membrane to study the ability of R8 inducing calcein release from liposomes, the fusion of liposomes, R8 binding to liposomes and membrane disturbing activity of the bound R8. The results show that R8 binding to liposome membrane depends on lipid compositions, negative surface charge density and interior water phase pH values of liposomes. R8 has no activity to induce the leakage of calcein from liposomes or improve liposome fusion. R8 does not permeabilize through the membrane spontaneously. These peptides delivering drugs through membranes may depend on receptors and energy.     

Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site

Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (ΔH°) was 12.38 kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK.     

The involvement of arginine vasotocin in the maturation of the kitten brain

In order to investigate the effects of the nonapeptide hormone arginine vasotocin (AVT) on the maturation of the brain, the following developmental data were tabulated between 5 and 21 days of postnatal life, in kittens, after the daily intraperitoneal administration of 10^?6 mg synthetic AVT: sleep, daily increase of body weight and locomotor, and investigative activities (LIA). Likewise, the day of the eye opening was noted and the brain weight as well as the total lipid levels within the brain in the day of sacrifice (21 days of age) were measured. The daily administration of AVT induced: (1) an increase of the total amount as well as of the intensity of active sleep (AS); (2) a decrease of the LIA; (3) a decrease of the total lipid levels within the brain and (4) a retardation of the eye opening. These effects appeared to be specific because neither arginine vasopressin, nor oxytocin, in the same doses (10^?6 mg), were able to reproduce the effects of AVT. The present results demonstrate that chronic administration of AVT is associated with a retardation of brain maturation. Whether AVT induces this effect by an unique mechanism or there are different mechanisms for the reported developmental data that were affected by AVT, is unknown. However, the present results suggest that the pineal gland, by its effector within the brain, AVT, is involved by an inhibitory pathway in the brain maturation and the hypothesis is advanced that the decrease of AVT content of fetal and neonatal brain could represent a hormonal signal for triggering the beginning of the brain maturation phenomena.     

In vitro potentiation of the activity of synthetic ovine corticotropin-releasing factor by arginine vasopressin

The ability of arginine vasopressin (AVP) to potentiate the actions of synthetic ovine corticotropin-releasing factor (CRF) was examined using anterior pituitary fragments. Marked potentiation of ACTH release was observed upon incubating the fragments with a combination of 2 nM AVP and 1 nM CRF. Potentiation of CRF-induced ACTH release was also observed when the fragments were incubated with a combination of 1 nM AVP and 0.5 nM CRF. These results suggest that AVP may play a role in the release of ACTH from the adenohypophysis.     

On the unidirectionality of arginine uptake in the yeast Saccharomyces cerevisiae

The reversibility of arginine accumulation was followed in exponentially growing cells of Saccharomyces cerevisiae and in the same cells transferred to non-growing energized conditions. Under non-growing conditions the accumulated arginine is retained in the cells while in exponentially growing cells the accumulated radioactivity is released after the addition of high external concentrations of arginine. There are indications that the process is saturable. The accumulated arginine is not exchanged for other related amino acids (l-citrulline, l-histidine). Only l-lysine (a low-affinity substrate of the specific arginine permease) provokes partial radioactivity efflux from the cells. The switch of the arginine-related radioactive label efflux to its complete retention in the cells after changing the growth conditions occurs within a few minutes and is tentatively attributed to two concomitantly occurring events: (1) the actual presence of radioactive arginine (not its metabolite(s)) in the cell and (2) a modification of the specific arginine permease. The specific exchange of arginine described in the present study contrasts with the currently widely accepted opinion of unidirectionality of amino acid fluxes in yeast. The reasons why this phenomenon has not been observed before are discussed.