Arginine Decarboxylase of oat seedlings

Arginine decarboxylase activity in the shoots of seedlings was high in oats, intermediate in barley and low in rice, maize, wheat and rye. After partial purification, the arginine decarboxylase from the shoots of potassium deficient oat seedlings was separated into two fractions, A (MW 195 000) and B (MW 118 000), by gel chromatography. On gel electrophoresis, the mobilities of these fractions were respectively 0.12 and 0.55 relative to bromophenol blue at pH 9.5. Fraction A was twice as active as fraction B in extracts of seedlings grown with both normal and potassium deficient nutrition, despite the greater activity ( × 5) of the potassium deficient plants. The properties of the two fractions were similar with respect to pH optimum (7–7.5), K_m (3 × 10 ^?5M) and the effect of inhibitors. Fraction A was purified to apparent homogeneity by DEAE-cellulose chromatography. The enzyme was specific for l-arginine and it was strongly inhibited by NSD 1055, d-arginine and canavanine. Mercaptoethanol and dithiothreitol stimulated the enzyme by ca 50% and p-chloromercuribenzoate was an inhibitor. Pyridoxal phosphate stimulated activity by ca 30% and EDTA stimulated activity by 30%. Ca²⁺ and Mg²⁺ inhibited the enzyme by 50% at ca 20 mM. Putrescine and the polyamines showed only moderate inhibition at 10 mM, but agmatine reduced activity to 30% at this concentration.     

Rapid Methods for Determining Decarboxylase Activity: Arginine Decarboxylase

A rapid biochemical method for the determination of arginine decarboxylase (EC 4.1.1.19) activity has been developed for use in the routine clinical microbiology laboratory and correlated with similar procedures for ornithine and lysine decarboxylase (EC 4.1.1.18) systems. It is based on the detection of agmatine, the amine end product formed during growth on a synthetic medium containing arginine as the key amino acid. A modified diacetyl reagent is used to detect this amine after a differential butanol extraction of the cultures. This procedure can be used to detect this amine after a 1- to 4-hr incubation period (with the use of an initial concentrated inoculum) or with an overnight culture. Thus, both an indirect measurement based on the alkalinization of the medium and a lengthy incubation period were avoided. Parameters for optimal enzyme activity and the pertinent enzyme systems involved in arginine and agmatine catabolism are discussed in detail.     

Some Properties of the Arginine Decarboxylase in Vicia faba Leaves

Growth of Vicia faba seedlings is accompanied by a rapid increasein arginine decarboxylase (EC 4.1.1.19 [EC] ) in the leaves and epicotyl.Increased enzyme activity was observed under saline conditionsin the presence of NaCl and with osmotic stress by mannitol.The partially purified enzyme (about 86-fold) readily decarboxylatedL-arginine, while D-arginine, L-homoarginine, L-ornithine andL-lysine were decarboxylated very slowly, and L-citrulline andL-glutamic acid were not decarboxylated. The K_m value was 5.8?10^–4M for L-arginine. The optimal pH and temperature for activitywere 8.5 and 45?C, respectively. p-Chloromercuribenzoate andN-ethylmaleimide were effective inhibitors of the enzyme. Inhibitionby spermidine, putrescine and agmatine suggested a possiblefeed-back mechanism in the pathway of polyamine biosynthesis. (Received October 11, 1983; Accepted February 24, 1984)     

Uncovering the Protein Lysine and Arginine Methylation Network in Arabidopsis Chloroplasts

Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology.     

Purification and Synthesis under Anaerobic Conditions of Rice Arginine Decarboxylase

Arginine decarboxylase (ADC; EC 4.1.1.19 [EC] ) which catalyzes thesynthesis of putrescine, is involved in the responses of plantsto stress. The enzyme was purified 1,561-fold from rice coleoptilesby steps that included ammonium sulfate fractionation, gel filtration,ion-exchange chromatography and chromatofocusing. The purifiedenzyme had a pI of 5.3, a molecular mass of 176 kDa and appearedto be composed of three subunits of 63 kDa. A polyclonal antibodywas raised in a rabbit and the IgG fraction was purified fromserum. On Western blots the antibody recognized the ADC fromboth rice and E. coli. Immunoprecipitation with the ADC-specificantibodies allowed detection of radiolabelled ADC in extractsfrom aerobically and anaerobically grown rice seedlings thathad been supplied with a mixture of ¹⁴C-amino acids. This resultis discussed in relation to the role of ADC under anaerobicconditions. (Received April 28, 1994; Accepted September 27, 1994)     

Purification and Partial Characterization of Arginine Decarboxylase from Brassica campestris

Arginine decarboxylase (EC 4.1.1.19) has been purified and characterized from Brassica campestris cv B-9. The enzyme was purified 1120 fold and the recovery was 9%. The mol wt of the enzyme determined by gel filtration was 240 kD with identical subunits of 60 kD. The pH and temperature optima for the enzyme were 8.0 and 30°C respectively. The Km was 0.31mM. Polyamines inhibited the enzyme activity significantly. Immunodiffusion with ADC-specific antibodies showed cross reactivity against purified ADC from Brassica.     

Polyamine Biosynthetic Enzymes in Chlorella: Characterization of Ornithine and Arginine Decarboxylase

In a Chlorella culture grown asynchronously under autotrophicconditions, two biosynthetic enzymes of putrescine—ornithinedecarboxylase (ODC) and arginine decarboxylase (ADC)—weredetected. Both enzymes require pyridoxal phosphate and dithiothreitolfor their activity but differ in their optimal pH, the ionicstrength of their buffer, temperature of inactivation, and Km.In addition, L-canaline was found to inhibit the activity ofODC but not that of ADC. During the logarithmic phase of growth,ODC activity increased sharply, then decreased before the onsetof the stationary phase. ADC activity changed only slightlyduring growth. ³The work was performed in partial fulfillment of the requirementsfor the Ph.D. Thesis of E.C. (Received September 25, 1982; Accepted June 1, 1983)     

Agrobacterium-mediated Transfer of Arginine Decarboxylase and Ornithine Decarboxylase Genes to Datura innoxia Enhances Shoot Regeneration and Hyoscyamine Biosynthesis

The genes (adc and odc) for two enzymes, arginine decarboxylase and ornithine decarboxylase involved in polyamine biosynthesis, were introduced into anther-derived calli of Datura innoxia through Agrobacterium tumefaciens. The transformed calli exhibited increased regeneration frequency as compared to control. Transgenic lines showed higher polyamine levels, mainly in the putrescine titre, and such lines also yielded a high level of the alkaloid, hyoscyamine. The results suggest that polyamines can modulate in vitro morphogenesis and polyamine biosynthetic pathway can be exploited for enhancement of polyamine-derived alkaloids of pharmaceutical importance.     

Purification and Characterization of Arginine Decarboxylase from Soybean (Glycine max) Hypocotyls

Arginine decarboxylase (EC 4.1.1.19 [EC] ) was purified from soybean,Glycine max, hypocotyls by a procedure which includes ammoniumsulfate fractionation, acetone precipitation, gel filtrationchromatography, and affinity chromatography. Using this procedure,ADC was purified to one band in non-denaturing PAGE. The purifiedADC has an M_r of 240 kDa based on gel filtration chromatographyand is a trimer of identical subunits which has an estimatedM_r of 74 kDa based on SDS-PAGE. ADC is active between 30 and50°C and has a K_m value of 46.1 µM. ADC is very sensitiveto agmatine or putrescine but not to spermidine or spermine.In the presence of 0.5 mM agmatine (or putrescine), the enzymeactivity was inhibited by 70%. However, at the same concentrationof spermidine (or spermine), the enzyme activity was inhibitedby only 10–20%. (Received April 2, 1997; Accepted August 18, 1997)     

Polyamine Metabolism in Embryogenic Cells of Daucus carota: II. Changes in Arginine Decarboxylase Activity

Embryogenic cultured cells of Daucus carota have been shown to synthesize putrescine from exogenously supplied [¹⁴C]arginine at twice the rate of control nonembryogenic cells. In the present paper, the activity of arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19), an important enzyme in the synthesis of putrescine, was assayed and also found to be elevated by as much as 2-fold in embryogenic cells. This difference between embryogenic and nonembryogenic cells was observed as early as 6 hours after the induction of embryogenesis and appeared not to result from the presence of a diffusible inhibitor or activator. It seemed to be dependent upon concomitant RNA and protein synthesis, as judged using 6-methyl-purine and cycloheximide. After cycloheximide addition to the culture medium, arginine decarboxylase activity declined with a half-time of about 30 minutes in both embryogenic and nonembryogenic cells. It is suggested that elevated arginine decarboxylase activity is involved in the mechanism leading to elevated putrescine levels in these cells and hence may play a role in the embryogenic process.     

Seasonal Changes of Polyamine Concentrations and Arginine Decarboxylase Activities in Leaves of 4 Ecotyes of Reeds

The seasonal changes of polyamine concentrations and arginine decarboxylase (ADC, EC 4.1.1. 1. 9)activities were investigated in the leaves of 4 ecotypes of reeds (Phragamites comrnunis Trinius)distributed over Hexi Corridor of Gansu province. The leaves of all ecotypes of reeds contained the same kind of polyamines and showed the same trend of decrement in total amuonts of potyamines with change of seasons. From May to September, the reeds which grow in arid and saline habitat maintained higher level of spermidine (Spd)and spermine (Spm)with no accumulation of putrescine (Put), resulting in low ratios of Put to other polyamine (Spd and Spm), whereas opposite results were observed in swamp reeds. These results indicate that the adaption of reeds to drought and salt stresses may correlate with Put synthesis via ADC pathway and the quick transformation of Put into Spd and Spm.     

The Arginine Decarboxylase Pathways of Host and Pathogen Interact to Impact Inflammatory Pathways in the Lung

The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa. We determined agmatine is at the center of a competing metabolism in the human lung during airways infections and is influenced by the metabolic phenotypes of the infecting pathogens. Ultra performance liquid chromatography with mass spectrometry detection was used to measure agmatine in human sputum samples from patients with cystic fibrosis, spent supernatant from clinical sputum isolates, and from bronchoalvelolar lavage fluid from mice infected with P. aeruginosa agmatine mutants. Agmatine in human sputum peaks during illness, decreased with treatment and is positively correlated with inflammatory cytokines. Analysis of the agmatine metabolic phenotype in clinical sputum isolates revealed most deplete agmatine when grown in its presence; however a minority appeared to generate large amounts of agmatine presumably driving sputum agmatine to high levels. Agmatine exposure to inflammatory cells and in mice demonstrated its role as a direct immune activator with effects on TNF-α production, likely through NF-κB activation. P. aeruginosa mutants for agmatine detection and metabolism were constructed and show the real-time evolution of host-derived agmatine in the airways during acute lung infection. These experiments also demonstrated pathogen agmatine production can upregulate the inflammatory response. As some clinical isolates have adapted to hypersecrete agmatine, these combined data would suggest agmatine is a novel target for immune modulation in the host-pathogen dynamic.     

Simultaneous Phytochrome-controlled Promotion and Inhibition of Arginine Decarboxylase Activity in Buds and Epicotyls of Etiolated Peas

The specific activity of arginine decarboxylase (ADC; l-arginine carboxylase; EC 4.1.1.19) rises steadily over an 8 hour experimental period in the growing buds and subapical epicotyl internodes of 6-day-old totally etiolated pea seedlings. Treatment with red light (R) completely annuls this rise in epicotyls but increases it in buds, thus paralleling the opposite effects of R on the growth of these two organs. Far red light (FR) reverses both effects of R on ADC and is, in turn, reversed by R, indicating phytochrome control. Effects in both organs are clearly seen within 2 hours. By 6 hours after R, the post-irradiation rise in ADC specific activity in buds is 3 times greater than that of the dark controls. Over the same period, ADC specific activity in epicotyls is inhibited by 56% relative to dark controls, reflecting zero net change after R and a continued rise in the dark. Cycloheximide inhibits the rise in ADC activity in both rapidly growing organs (epicotyls in dark and buds after R) but is without effect in both slower growing organs. Actinomycin D inhibits only in dark grown epicotyls, whereas chloramphenicol produces no inhibition in any system tested.ADC is the first enzyme to show a two-way, organ-specific response to phytochrome conversion from Pr to Pfr. This finding is discussed in relation to the growing evidence that polyamines formed from arginine may be important growth regulators in plants, as well as in microbial and animal cells.     

Low-pH Rescue of Acid-Sensitive Salmonella enterica Serovar Typhi Strains by a Rhamnose-Regulated Arginine Decarboxylase System

For Salmonella, transient exposure to gastric pH prepares invading bacteria for the stresses of host-cell interactions. To resist the effects of low pH, wild-type Salmonella enterica uses the acid tolerance response and the arginine decarboxylase acid resistance system. However, arginine decarboxylase is typically repressed under routine culture conditions, and for many live attenuated Salmonella vaccine strains, the acid tolerance response is unable to provide the necessary protection. The objective of this study was to enhance survival of Salmonella enterica serovar Typhi vaccine strains at pHs 3.0 and 2.5 to compensate for the defects in the acid tolerance response imposed by mutations in rpoS, phoPQ, and fur. We placed the arginine decarboxylase system (adiA and adiC) under the control of the P_araBAD or P_rhaBAD promoter to provide inducible acid resistance when cells are grown under routine culture conditions. The rhamnose-regulated promoter P_rhaBAD was less sensitive to the presence of its cognate sugar than the arabinose-regulated promoter P_araBAD and provided tighter control over adiA expression. Increased survival at low pH was only observed when adiA and adiC were coregulated by rhamnose and depended on the presence of rhamnose in the culture medium and arginine in the challenge medium. Rhamnose-regulated acid resistance significantly improved the survival of ΔaroD and ΔphoPQ mutants at pHs 3 and 2.5 but only modestly improved the survival of a fur mutant. The construction of the rhamnose-regulated arginine decarboxylase system allowed us to render S. Typhi acid resistant (to pH 2.5) on demand, with survival levels approximately equivalent to that of the native arginine decarboxylase system.     

Diurnal Modulation of Arginine Decarboxylase Activity of Chenopodium rubrum L. by Temperature and Light

Arginine decarboxylase (ADC), one of the key enzymes of polyaminemetabolism in plants, was investigated in Chenopodium rubrumL. seedlings under constant and alternating temperature andlighting conditions. With seedlings grown at constant temperature,ADC activity of the whole seedling increased rapidly betweenthe second and the third day after sowing. This effect was alwayshigher under continuous light than in continuous darkness. Fromthe third to the seventh day after sowing, there was a markeddecrease in ADC activity of the whole seedling almost down tothe level of the second day. Under "normal" lighting and temperatureconditions (32.5?C/10?C, light/dark) there was a marked increasein ADC activity when plants were transferred to 10?C and a rapiddecrease when they were transferred to 32.5?C. The same timecourse was observed when an "inverse" light-temperature program(32.5?C/10?C; dark/light) was applied. This means that the timecourse of ADC activity in the seedlings is slightly light-dependent,but strongly temperature-dependent. The data are discussed withrespect to the chronopathological effects of the "inverse" light-temperatureprogram. (Received March 5, 1985; Accepted October 2, 1985)     

Arginase,Arginine Decarboxylase,Ornithine Decarboxylase,and Polyamines in Tomato Ovaries (Changes in Unpollinated Ovaries and Parthenocarpic Fruits Induced by Auxin or Gibberellin)

Arginase (EC 3.5.3.1) activity has been found in the ovaries and Young fruits of tomato (Lycopersicon esculentum Mill. cv Rutgers).Changes in arginase, arginine decarboxylase (EC 4.1.1.19), and ornithine decarboxylase activity (EC 4.1.1.17) and levels of free and conjugated putrescine, spermidine, and spermine were determined in unpollinated ovaries and in parthenocarpic fruits during the early stages of development induced by 2,4-dichlorophenoxyacetic acid (2,4-D) or gibberellic acid (GA3). Levels of arginase, free spermine, and conjugates of the three polyamines were constant in unpollinated ovaries and characteristic of a presenescent step. A marked decrease in arginase activity, free spermine, and polyamine conjugates was associated with the initiation of fruit growth due to cell division, and when cell expansion was initiated, the absence of arginase indicated a redirection of nitrogen metabolism to the synthesis of arginine. A transient increase in arginine decarboxylase and ornithine decarboxylase was also observed in 2,4-D-induced fruits. In general, 2,4-D treatments produced faster changes than GA3, and without treatment, unpollinated ovaries developed only slightly and senescence was hardly visible. Sensitivity to 2,4-D and GA3 treatment remained for at least 2 weeks postanthesis.     

Putrescine and Acid Stress : Induction of Arginine Decarboxylase Activity and Putrescine Accumulation by Low pH

Incubation of peeled oat Avena sativa L. var Victory leaf segments on media of pH 5.0 or below leads to a rapid and massive increase in the titer of putrescine while incubation at pH values above 5.0 causes little or no change. The low pH effect is independent of the buffer system employed. Putrescine levels rise within 3 hours and reach their peak 8 to 9 hours after acidification. At this time, putrescine titer is eight times greater at pH 3.5 than at 6.0. None of the other polyamines shows a response to changes in external pH. The increase in putrescine is blocked by the addition of cycloheximide or dl-alpha-difluoromethylarginine, a specific inhibitor of the putrescine biosynthetic enzyme, arginine decarboxylase. In one experiment, arginine decarboxylase activity was 110% greater at pH 4.0 than at 6.0 after a 4-hour incubation, although the average increase over many experiments was 47%. The activity of the other possible putrescine biosynthetic enzyme, ornithine decarboxylase, falls throughout the incubation period and is virtually equal at pH 4.0 and 6.0.     

Arginine Methylation of Vasa Protein Is Conserved across Phyla

Recent studies have uncovered an unexpected relationship between factors that are essential for germline development in Drosophila melanogaster: the arginine protein methyltransferase 5 (dPRMT5/Csul/Dart5) and its cofactor Valois, methylate the Piwi family protein Aub, enabling it to bind Tudor. The RNA helicase Vasa is another essential protein in germline development. Here, we report that mouse (mouse Vasa homolog), Xenopus laevis, and D. melanogaster Vasa proteins contain both symmetrical and asymmetrical dimethylarginines. We find that dPRMT5 is required for the production of sDMAs of Vasa in vivo. Furthermore, we find that the mouse Vasa homolog associates with Tudor domain-containing proteins, Tdrd1 and Tdrd6, as well as the Piwi proteins, Mili and Miwi. Arginine methylation is thus emerging as a conserved and pivotal post-translational modification of proteins that is essential for germline development.     

S-Adenosylmethionine Decarboxylase Activity Is Decreased in the Rat Cortex After Traumatic Brain Injury

Abstract: S -Adenosyl- l -methionine decarboxylase (SAMdc) and l -ornithine decarboxylase (ODC) are major enzymes regulating polyamine synthesis. Following ischemia, putrescine content increases as a result of post-traumatic activation of ODC and inhibition of SAMdc. These alterations are thought to mediate edema and cell death. The purpose of this study was to quantify SAMdc activity and edema in the brain following controlled cortical impact injury. Anesthetized adult male rats underwent a right parietal craniectomy and were subjected to cortical impact injury. Tissues were obtained from three bilateral regions: parietal cortex, motor area (CPm); parietal cortex, somatosensory area (CPs); and the pyriform cortex (CPF). SAMdc activity was determined in the postmitochondrial fraction from homogenates of fresh, unfrozen tissues by measuring the decarboxylation of S -adenosyl- l -[ carboxyl -¹⁴C]methionine. Basal SAMdc activity was determined in unoperated rats, and regional differences were noted: Activity was lower in the CPF than in the CPm and CPs. SAMdc activity decreased to the greatest extent in the ipsilateral CPm (impact site) from 1 to 72 h following traumatic brain injury. Significant edema was found in the ipsilateral CPm 1, 8, 16, 24, and 48 h after injury. Decreased SAMdc activity impairs the conversion of putrescine to polyamines and may contribute to delayed pathological changes in the brain after traumatic injury.