Control of arginine utilization in Neurospora.

The response of Neurospora to changes in the availibility of exogenous arginine was investigated. Upon addition of arginine to the growth medium, catabolism is initiated within minutes. This occurs prior to expansion of the arginine pool or augmentation of catabolic enzyme levels. (Basal levels are approximately 25% of those found during growth in arginine-supplemented medium.) Catabolism of arginine is independent of protein synthesis, indicating that the catabolic enzymes are active but that arginine is not available for catabolism unless present in the medium. Upon exhaustion of the supply of exogenous arginine, catabolism ceases abruptly, despite an expanded arginine pool and induced levels of the catabolic enzymes. The arginine pool supports protein synthesis until the cells regain their normal capacity for endogenous arginine synthesis. These observations, combined with the known small level of induction of arginine catabolic enzymes, non-repressibility of most biosynthetic enzymes, and vesicular localization of the bulk of the arginine pool, suggest that compartmentation plays a significant role in controlling arginine metabolism in Neurospora.     

Thermodynamics of the arginine kinase reaction.

The effect of temperature, pH, free [Mg(2+)], and ionic strength on the apparent equilibrium constant of arginine kinase (EC 2.7.3.3) was determined. At equilibrium, the apparent K' was defined as [see text] where each reactant represents the sum of all the ionic and metal complex species. The K' at pH 7.0, 1.0 mM free [Mg(2+)], and 0. 25 M ionic strength was 29.91 +/- 0.59, 33.44 +/- 0.46, 35.44 +/- 0. 71, 39.64 +/- 0.74, and 45.19 +/- 0.65 (n = 8) at 40, 33, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy (DeltaH degrees') is -8.19 kJ mol(-1), and the corresponding standard apparent entropy of the reaction (DeltaS degrees') is + 2. 2 J K(-1)mol(-1) in the direction of ATP formation at pH 7.0, free [Mg(2+)] =1.0 mM, ionic strength (I) =0.25 M at 25 degrees C. We further show that the magnitude of transformed Gibbs energy (DeltaG degrees ') of -8.89 kJ mol(-1) is mostly comprised of the enthalpy of the reaction, with 7.4% coming from the entropy TDeltaS degrees' term (+0.66 kJ mol(-1)). Our results are discussed in relation to the thermodynamic properties of its evolutionary successor, creatine kinase.     

Utilization of arginine by Klebsiella aerogenes.

Klebsiella aerogenes utilized arginine as the sole source of carbon or nitrogen for growth. Arginine was degraded to 2-ketoglutarate and not to succinate, since a citrate synthaseless mutant grows on arginine as the only nitrogen source. When glucose was the energy source, all four nitrogen atoms of arginine were utilized. Three of them apparently did not pass through ammonia but were transferred by transamination, since a mutant unable to produce glutamate by glutamate synthase or glutamate dehydrogenase utilized three of four nitrogen atoms of arginine. Urea was not involved as intermediate, since a unreaseless mutant did not accumulate urea and grew on arginine as efficiently as the wild-type strain. Ornithine appeared to be an intermediate, because cells grown either on glucose and arginine or arginine alone could convert arginine in the presence of hydroxylamine to ornithine. This indicates that an amidinotransferase is the initiating enzyme of arginine breakdown. In addition, the cells contained a transaminase specific for ornithine. In contrast to the hydroxylamine-dependent reaction, this activity could be demonstrated in extracts. The arginine-utilizing system (aut) is apparently controlled like the enzymes responsible for the degradation of histidine (hut) through induction, catabolite repression, and activation by glutamine synthetase.     

The arginine repressor of Escherichia coli.

This review tells the story of the arginine repressor of Escherichia coli from the time of its discovery in the 1950s until the present. It describes how the research progressed through physiological, genetic, and biochemical phases and how the nature of the repressor and its interaction with its target sites were unraveled. The studies of the repression of arginine biosynthesis revealed unique features at every level of the investigations. In the early phase of the work they showed that the genes controlled by the arginine repressor were scattered over the linkage map and were not united, as in other cases, in a single operon. This led to the concept of the regulon as a physiological unit of regulation. It was also shown that different alleles of the arginine repressor could result in either inhibition of enzyme formation, as in E. coli K-12, or in stimulation of enzyme formation, as in E. coli B. Later it was shown that the arginine repressor is a hexamer, whereas other repressors of biosynthetic pathways are dimers. As a consequence the arginine repressor binds to two palindromic sites rather than to one. It was found that the arginine repressor not only acts in the repression of enzyme synthesis but also is required for the resolution of plasmid multimers to monomers, a completely unrelated function. Finally, the arginine repressor does not possess characteristic structural features seen in other prokaryotic repressors, such as a helix-turn-helix motif or an antiparallel beta-sheet motif. The unique features have sustained continuous interest in the arginine repressor and have made it a challenging subject of investigation.     

Control of the flux in the arginine pathway of Neurospora crassa. The flux from citrulline to arginine.

The arginine pathway is a complex one, having many branch points and effector interactions. In order to assess the quantitative role of the various mechanisms that influence the flux in the pathway, the system was divided experimentally into two moieties by the introduction of a genetic block abolishing ornithine carbamoyltransferase activity. This normally produces citrulline from ornithine within the mitochondria. The endogenous citrulline supply was replaced by citrulline in the growth medium, and control of the influx rate was achieved by using glycine or histidine as uptake inhibitors. By modulating the influx rate over a large range of values, the importance of such factors as reversibility, saturation, inhibition and induction in affecting the flux and the sizes of intermediate pools between citrulline and arginine was assessed. The role of expansion fluxes as important controls in the exponentially growing system was established.     

Radioimmunoassay of arginine vasopressin in human plasma.

Antibodies for the radioimmunoassay of arginine vasopressin (AVP) described here were produced in rabbits using synthetic AVP coupled to rabbit gamma-globulin with carbodiimide. In three out of six rabbits, significant antibody titres were obtained. Using the best antisera produced, 40% of labeled AVP was bound at a final dilution of 1:50.000. After iodination of synthetic AVP with 125I using the chloramin-T method, a gel filtration on Sephadex G-25 was performed to purify the iodinated AVP. For separation of antibody bound and free hormone, a second antibody precipitation was used. There was no crossreactivity with oxytocin. AVP was extracted from plasma after ammoniumsulfate precipitation of the proteins by adsorption to Florisil. The recovery of AVP added to plasma in amounts between 5-25 pg/ml was 60 +/- 15% (n equals 6). The minimum amount of AVP detectable was 1 pg per ml plasma. The plasma level in normal adults under standard conditions was 3.4 +/- 2.2 pg/ml. This is in agreement with data recently published by other researchers. The applicability and reproducibility was further tested in measurements of samples taken hourly during the entire day under water diuresis and after hormonal stimulation of AVP.     

Identification of two novel arginine binding DNAs.

RNA tertiary structure is known to play critical roles in RNA-protein recognition and RNA function. To examine how DNA tertiary structure might relate to RNA structure, we performed in vitro selection experiments to identify single-stranded DNAs that specifically bind arginine, and compared the results with analogous experiments performed with RNA. In the case of RNA, a motif related to the arginine binding site in human immunodeficiency virus TAR RNA was commonly found, whereas in the case of DNA, two novel motifs and no TAR-like structures were found. One DNA motif, found in approximately 40% of the cloned sequences, forms of hairpin structure with a highly conserved 10 nucleotide loop, whereas the second motif is especially rich in G residues. Chemical interference and mutagenesis experiments identified nucleotides in both motifs that form specific arginine binding sites, and dimethylsulfate footprinting experiments identified single guanine residues in both that are protected from methylation in the presence of arginine, suggesting possible sites of arginine contact or conformational changes in the DNAs. Circular dichroism experiments indicated that both DNAs undergo conformational changes upon arginine binding and that the arginine guanidinium group alone is responsible for binding. A model for the G-rich motif is proposed in which mixed guanine and adenine quartets may form a novel DNA structure. Arginine binding DNAs and RNAs should provide useful model systems for studying nucleic acid tertiary structure.     

A reactive arginine in adenylate kinase.

Adenylate kinase (ATP:AMP phosphotransferase, EV 2.7.4.3) from pig heart is inactivated by the specific arginyl reagent phenylglyoxal. During inactivation two molecules of phenyglyoxal are incorporated into the protein indicating the modification of one of the 11 arginine residues. The modification of other amino acids is ruled out. Chemical modification of this essential residue is prevented by high concentrations of the substrates AMP, ADP and MgATP2-. The protection of the substrates is explained by the formation of a ternary abortive enzyme-substrate complex ESS. The dissociation constants KD = [ES] - [S]/[ESS] are determined from the kinetic data of inactivation and protection.     

Central arginine vasopressin and endogenous antipyresis.

Arginine vasopressin (AVP) is a centrally synthesized nonapeptide that exerts classical endocrine effects as well as a host of centrally mediated actions. A strong case can be argued in support of a neurotransmitter-neuromodulator role for AVP. Acting within the central nervous system (CNS), AVP has been demonstrated to be involved in the modulation of febrile body temperature. Because AVP acts to reduce pyrogen-induced fevers, but not normal body temperature, its actions are deemed to be antipyretic. However, to demonstrate an endogenous antipyretic function, AVP must be shown to be active during conditions where fever is naturally suppressed. This review will focus on five such conditions where the absence of pyrogen-induced fever can be linked to the endogenous activity of AVP within the brain. In the neonatal rat pup, the use of specific antagonists to the AVP receptor has revealed a role for CNS AVP in the absence of fever following peripheral injections of bacterial endotoxin. These results may help to explain a similar lack of fever in other newborn species. In parturient animals a reduced or absent febrile response has been linked to the increased presence of AVP within the septal area of the brain. The combined use of AVP receptor antagonism as well as immunohistochemistry has shown enhanced AVP activity within the ventral septal area of the rat and guinea pig brain during tolerance to intravenous pyrogens. These results suggest that the mechanism of fever suppression following repeated systemic injections of bacterial pyrogen includes centrally acting AVP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functionally important arginine residues of aspartate transcarbamylase.

The reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity (Kantrowitz, E. R., and Lipscomb, W. N. (1976) J. Biol. Chem. 251, 2688-2695). If N-(phosphonacetyl)-L-aspartate is used to protect the active site, we find that phenylglyoxal causes destruction of the enzyme's susceptibility to activation by ATP and inhibition by CTP. Furthermore, CTP only minimally protects the regulatory site from reaction with this reagent. The modified enzyme still binds CTP although with reduced affinity. After reaction with phenylglyoxal, the native enzyme shows reduced cooperativity. The hybrid with modified regulatory subunits and native catalytic subunits exhibits slight heterotropic or homotropic properties, while the reverse hybrid, with modified catalytic subunits and native regulatory subunits, shows much reduced homotropic properties but practically normal heterotropic interactions. The decrease in the ability of CTP to inhibit the enzyme correlates with the loss of 2 arginine residues/regulatory chain (Mr = 17,000). Under these reaction conditions, 1 arginine residue is also modified on each catalytic chain (Mr = 33,000). Reaction rate studies of p-hydroxymercuribenzoate, with the liganded and unliganded modified enzyme suggest that the reaction with phenylglyoxal locks the enzyme into the liganded conformation. The conformational state of the regulatory subunit is implicated as having a critical role in the expression of the enzyme's heterotropic and homotropic properties.     

Characterization of arginine decarboxylase from Dianthus caryophyllus.

Arginine decarboxylase (ADC, EC 4.1.1.9) is a key enzyme in the biosynthesis of polyamines in higher plants, whereas ornithine decarboxylase represents the sole pathway of polyamine biosynthesis in animals. Previously, we characterized a genomic clone from Dianthus caryophyllus, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 78 kDa. In the present study, the ADC gene was subcloned into the pGEX4T1 expression vector in combination with glutathione S-transferase (GST). The fusion protein GST-ADC was water-soluble and thus was purified by sequential GSTrap-arginine affinity chromatography. A thrombin-mediated on-column cleavage reaction was employed to release free ADC from GST. Hiload superdex gel filtration FPLC was then used to obtain a highly purified ADC. The identity of the ADC was confirmed by immunoblot analysis, and its specific activity with respect to (14)C-arginine decarboxylation reaction was determined to be 0.9 CO(2) pkat mg(-1) protein. K(m) and V(max) of the reaction between ADC and the substrate were 0.077 +/- 0.001 mM and 6.0 +/- 0.6 pkat mg(-1) protein, respectively. ADC activity was reduced by 70% in the presence of 0.1 mM Cu(2+) or CO(2+), but was only marginally affected by Mg(2+), or Ca(2+) at the same concentration. Moreover, spermine at 1 mM significantly reduced its activity by 30%.     

Prolactin-releasing activity of arginine vasotocin in vitro.

A significant elevation in both luteinizing hormone (LH) and prolactin release was observed in the culture medium of hemipituitaries from castrated estrogen-progesterone (EP) primed female rats incubated for 5 h with arginine vasotocin (AVT) and luteinizing hormone-releasing hormone (LRH) compared to corresponding halves incubated with LRH alone. However, AVT alone did not significantly alter the discharge of LH or prolactin from hemipituitaries of EP-treated rats in vitro. Arginine vasopressin, a natural analogue of AVT, inhibited prolactin release using this same model system. Normal male rat hemipituitaries incubated with AVT released significantly more LH and prolactin into the culture medium than did their corresponding halves. A log-dose response curve indicated that any dose from 100 ng to 10 mug AVT significantly promoted prolactin release. However, the terminal tripeptide of AVT, Pro-Arg-Gly(NH2), failed to modify the discharge of LH or prolactin into the culture medium.     

Chemical modification of arginine residues in alpha-bungarotoxin.

The reaction of alpha-bungarotoxin (alpha-BuTX) with 1,2-cyclohexanedione resulted in the modification of only Arg-72 but arginine at position 36 or 72, as well as both were modified by reaction of the toxin with p-hydroxyphenylglyoxal. No derivative modified at Arg-25 was obtained, indicating that this residue may be located in the interior region of alpha-BuTX molecule. Monoderivative at Arg-72 showed about 50% of the lethal toxicity and binding activity of alpha-BuTX to nicotinic acetylcholine receptor (AChR), while the activity was decreased to one-third when the invariant Arg-36 was modified, indicating that the latter residue is more closely related to the interaction of the toxin with AChR. Approx. 13% of the residual activity was observed when both arginine residues at 36 and 72 were modified. The antigenicity of alpha-BuTX was still retained essentially intact after Arg-36 or -72 was modified, whereas it decreased to 50% when both these arginine residues were modified. The present study indicates that Arg-36 and -72 in alpha-BuTX may be involved in the multipoint contact between the toxin and AChR, but neither is absolutely essential for the binding.     

Studies on the mechanism of repression of arginine biosynthesis in Escherichia coli. IV. Further studies on the role of arginine transfer RNA repression of the enzymes of arginine biosynthesis

Two column Chromatographic procedures have been utilized to purify and fractionate arginyl-transfer RNA: DEAE-Sephadex and reverse-phase column chromatography. Five iso-accepting species of tRNA^Arg were found and they recognize the six arginine codons.     

Isolation and characterization of Pseudomonas putida mutants affected in arginine, ornithine and citrulline catabolism: function of the arginine oxidase and arginine succinyltransferase pathways.

Pseudomonas putida mutants impaired in the utilization of arginine are affected in either the arginine succinyltransferase pathway, the arginine oxidase route, or both. However, mutants affected in one of the pathways still grow on arginine as sole carbon source. Analysis of the products excreted by both wild-type and mutant strains suggests that arginine is mainly channelled by the oxidase route. Proline non-utilizing mutants are also affected in ornithine utilization, confirming the role of proline as an intermediate in ornithine catabolism. Mutants affected in ornithine cyclodeaminase activity still grow on proline and become unable to use ornithine. Both proline non-utilizing mutants and ornithine-cyclodeaminase-minus mutants are unable to use citrulline. These results, together with induction of ornithine cyclodeaminase when wild-type P. putida is grown on citrulline, indicate that utilization of citrulline as a carbon source proceeds via proline with ornithine as an intermediate. Thus in P. putida, the aerobic catabolism of arginine on the one hand and citrulline and ornithine on the other proceed by quite different metabolic segments.     

Structure of the arginine repressor from Bacillus stearothermophilus.

The arginine repressor (ArgR) is a hexameric DNA-binding protein that plays a multifunctional role in the bacterial cell. Here, we present the 2.5 A structure of apo-ArgR from Bacillus stearothermophilus and the 2.2 A structure of the hexameric ArgR oligomerization domain with bound arginine. This first view of intact ArgR reveals an approximately 32-symmetric hexamer of identical subunits, with six DNA-binding domains surrounding a central oligomeric core. The difference in quaternary organization of subunits in the arginine-bound and apo forms provides a possible explanation for poor operator binding by apo-ArgR and for high affinity binding in the presence of arginine.     

精氨酸脱亚胺酶在大肠杆菌中的分泌型表达

将变形假单胞菌的精氨酸脱亚胺酶(ADI)编码基因arc A克隆至具有阿拉伯糖启动子的分泌型表达载体pBAD/gⅢB中,经鉴定得到重组质粒pBAD-ADI。将重组质粒转化大肠杆菌TOP10F'后进行诱导表达,分别考察了不同诱导物L-arabinose浓度、诱导温度、诱导时间对重组蛋白表达的影响,最适诱导条件为L-arabinose浓度0.002%(w/v),25℃下诱导5 h,全细胞的酶活为68 mU/mL(指单位发酵液体积,下同)。采用Osmotic Shock法使ADI从胞周质释放出来,经检测分泌到胞周质的重组蛋白活性为53 mU/mL,细胞内的酶活为34 mU/mL。SDS-PAGE分析显示,重组蛋白大小约为46 kD。