Cellular distribution of ornithine in Neurospora: anabolic and catabolic steady states.

During growth on minimal medium, cells of Neurospora contain three pools of ornithine. Over 95% of the ornithine is in a metabolically inactive pool in vesicles, about 1% is in the cytosol, and about 3% is in the mitochondria. By using a ureaseless strain, we measured the rapid flux of ornithine across the membrane boundaries of these pools. High levels of ornithine and the catabolic enzyme ornithine aminotransferase coexist during growth on minimal medium but, due to the compartmentation of the ornithine, only 11% was catabolized. Most of the ornithine was used for the synthesis of arginine. Upon the addition of arginine to the medium, ornithine was produced catabolically via the enzyme arginasn early enzyme of ornithine synthesis. The biosynthesis of arginine itself, from ornithine and carbamyl phosphate, was halted after about three generations of growth on arginine via the repression of carbamyl phosphate synthetase A. The catabolism of arginine produced ornithine at a greater rate than it had been produced biosynthetically, but this ornithine was not stored; rather it was catabolized in turn to yield intermediates of the proline pathway. Thus, compartmentation, feedback inhibition, and genetic repression all play a role to minimize the simultaneous operation of anabolic and catabolic pathways for ornithine and arginine.     

In Saccharomyces cerevisiae, expression of arginine catabolic genes CAR1 and CAR2 in response to exogenous nitrogen availability is mediated by the Ume6 (CargRI)-Sin3 (CargRII)-Rpd3 (CargRIII) complex

The products of three genes named CARGRI, CARGRII, and CARGRIII were shown to repress the expression of CAR1 and CAR2 genes, involved in arginine catabolism. CARGRI is identical to UME6 and encodes a regulator of early meiotic genes. In this work we identify CARGRII as SIN3 and CARGRIII as RPD3. The associated gene products are components of a high-molecular-weight complex with histone deacetylase activity and are recruited by Ume6 to promoters containing a URS1 sequence. Sap30, another component of this complex, is also required to repress CAR1 expression. This histone deacetylase complex prevents the synthesis of the two arginine catabolic enzymes, arginase (CAR1) and ornithine transaminase (CAR2), as long as exogenous nitrogen is available. Upon nitrogen depletion, repression at URS1 is released and Ume6 interacts with ArgRI and ArgRII, two proteins involved in arginine-dependent activation of CAR1 and CAR2, leading to high levels of the two catabolic enzymes despite a low cytosolic arginine pool. Our data also show that the deletion of the UME6 gene impairs cell growth more strongly than the deletion of the SIN3 or RPD3 gene, especially in the Sigma1278b background.     

Arginine catabolism in Neurospora: cycling of ornithine.

We measured the metabolism of ornithine in Neurospora during the transition from minimal medium to arginine-supplemented medium. Within an hour after arginine supplementation, the amount of intracellular ornithine (95% of which had been stored in vesicles) dropped by 65%, even though the catabolism of arginine produces as much ornithine as had been produced on minimal medium. The arginine level in the cell rose 10-fold. Ornithine flux through the catabolic enzyme ornithine aminotransferase increased fivefold, but flux through the mitochondrial enzyme ornithine transcarbamylase (leading to arginine synthesis) was only 20% of the rate seen on minimal medium. During this transition to arginine catabolism, the enzymes of the arginine pathway operate as an ornithine cycle, but at a restricted rate. We suggest the hypothesis that high levels of arginine may inhibit the movement of ornithine into the vesicles and into the mitochondria.     

Compartmentation and control of arginine metabolism in Neurospora.

The fate of [14-C]arginine derived from the medium or from biosynthesis has been examined in Neurospora growing in arginine-supplemented medium. In both cases the label enters the cytosol, where it is used efficiently for both protein synthesis and catabolism before mixing with the majority of the endogenous [12C]arginine pool. Both metabolic processes appear to use the same cytosolic arginine pool. It is calculated that the nonorganellar cytoplasm contains approximately 20% of the intracellular arginine pool when the cells are growing in arginine-supplemented medium. The results suggest that compartmentation of arginine is a significant factor in controlling arginine metabolism in Neurospora. The significance of these results for studies of amino acid metabolism in other eukaryotic systems is discussed.     

Use of External, Biosynthetic, and Organellar Arginine by Neurospora

The fate of very low amounts of (14)C-arginine derived from the medium or from biosynthesis was studied in Neurospora cells grown in minimal medium. In both cases, the label enters the cytoplasm, where it is very briefly used with high efficiency for protein synthesis without mixing with the bulk of the large, endogenous pool of (12)C-arginine. The soluble (14)C-arginine which is not used for protein synthesis is sequestered in a vesicle with the bulk of the endogenous arginine pool. After this time, it is selectively excluded from use in protein synthesis except by exchange with cytoplasmic arginine. The data suggest that in vivo, the non-organellar cytoplasm contains less than 5% of the soluble, cellular arginine. The cellular organization of Neurospora described here also prevents the catabolism of arginine. Our results are discussed in relation to previous work on amino acid pools of other eukaryotic systems.     

Regulation of Arginase Activity by Intermediates of the Arginine Biosynthetic Pathway in Neurospora crassa

It has been found that, in Neurospora crassa, arginine synthesized from exogenous citrulline was not as effectively hydrolyzed as exogenous arginine. This was explained by the observed inhibition of arginase in vitro and in vivo by citrulline. The high arginine pool formed from exogenous citrulline feedback inhibits the arginine pathway. These two factors allow exogenous citrulline to be used adventitiously and efficiently as an arginine source. Finally, it was found that ornithine was a strong inhibitor of arginase. This suggests that the characteristically high ornithine pool of minimal cultures of Neurospora may act to control a potentially wasteful catabolism of endogenous arginine by arginase.     

Control of arginine metabolism in Neurospora: flux through the biosynthetic pathway.

The flux into the arginine biosynthetic pathway of Neurospora crassa was investigated using a mutant strain lacking the ornithine-degrading enzyme ornithine aminotransferase (EC 2.6.1.13). Flux was measured by the increase in the sum of the radioactivity (derived from [14C]glutamic acid) in the ornithine pool, the arginine pool, and arginine incorporated into proteins. Complete cessation of flux occurred immediately upon the addition of arginine to the growth medium. This response occurred prior to expansion of the arginine pool. After short-term exposure to arginine (80 min), flux resumed quickly upon exhaustion of arginine from the medium. This took place despite the presence of an expanded arginine pool. Initiation of flux required approximately 80 min when the mycelia were grown in arginine-supplemented medium for several generations before exhaustion of the exogenous arginine. The arginine pool of such mycelia was similar to that found in mycelia exposed to exogenous arginine for only 80 min. The results are consistent with rapid onset and release of feedback inhibiton of arginine biosynthesis in response to brief exposure to exogenous arginine. The insensitivity of flux to the size of the arginine pool is consistent with a role for compartmentation in this regulatory process. The lag in initiation of flux after long-term growth in the presence of exogenous arginine suggests the existence of an additional regulatory mechanism(s). Several possibilities are discussed.     

Isolation and characterization of Neurospora crassa mutants impaired in feedback control of ornithine synthesis.

Thirty-two independent mutants were isolated which overcame the proline requirement of pro-3 mutations in Neurospora crassa. The mutations were not revertants, appeared to be allelic, were closely linked or allelic to arg-6, and in strains unable to degrade ornithine no longer suppressed the proline requirement. The suppressor mutations did not alter the levels of biosynthetic or catabolic enzymes, yet allowed accumulation of ornithine. Suppressed strains unable to degrade arginine still produced ornithine (as detected by growth) in arginine-supplemented medium. The results suggest that the suppressor mutants were impaired in the feedback inhibition of ornithine synthesis by arginine. The activity of the appropriate biosynthetic enzyme was less sensitive to inhibition by arginine. The potential usefulness of such mutations is discussed.     

Effect of Carbon Source on Enzymes and Metabolites of Arginine Metabolism in Neurospora

The levels of enzymes and metabolites of arginine metabolism were determined in exponential cultures of Neurospora crassa grown on various carbon sources. The carbon sources decreased in effectiveness (as determined by generation times) in the following order: sucrose, acetate, glycerol, and ethanol. The basal and induced levels of the catabolic enzymes, arginase (EC 3.5.3.1) and ornithine transaminase (EC 2.6.1.13), were lower in mycelia grown on poor carbon sources. Arginase was more sensitive to variations in carbon source than was ornithine transaminase. Induction of both enzymes was sensitive to nitrogen metabolite control, but this sensitivity was reduced in mycelia grown on glycerol or ethanol. The pools of arginine and ornithine were reduced in mycelia grown in unsupplemented medium containing poor carbon sources, but the biosynthetic enzyme ornithine transcarbamylase (EC 2.1.3.3) was not derepressed. The arginine pools were similar, regardless of carbon source, in mycelia grown in arginine-supplemented medium. The ornithine pool was reduced by growth on poor carbon sources. The rate of arginine degradation was proportional to the level of arginase in both sucrose- and glycerol-grown mycelia. The distribution of arginine between cytosol and vesicles was only slightly altered by growth on glycerol instead of sucrose. The slightly smaller cytosolic arginine concentration did not appear to be sufficient to account for the alterations in basal and induced enzyme levels. The results suggest a possible carbon metabolite effect on the expression or turnover of a variety of genes for enzymes of arginine metabolism in Neurospora.     

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.     

L-ornithine transaminase synthesis in Saccharomyces cerevisiae: Regulation by inducer exclusion

Summary The ornithine transaminase (EC.2.6.1.13) of Saccharomyces cerevisiae is induced by arginine, ornithine, and their analogs. Genetic regulatory elements which are involved in this induction process have been defined due to the isolation of specific mutants. Two classes of OTAse operator mutants have previously been described; three unlinked genes are presumed to code for a specific repressor, CARGR of both of the arginine catabolic enzymes, arginase, and ornithine transaminase. The level of transaminase of cells grown on ammonia plus arginine is much lower than it is when arginine is the sole nitrogen source. Ammonia thus seems to limit the amount of enzyme synthesized when arginine is present in the growth medium. Nevertheless, all attempts to disclose a nitrogen catabolite repression process in OTAse synthesis have failed; neither the action of mutations that release this regulation on arginase and other catabolic enzymes, nor the use of derepressing growth conditions, affect OTAse synthesis. A decrease of the cells' arginine pool when amonia or aminoacids (serine, glutamate) are added to arginine as a nitrogen nutrient results in a progressive reduction of transaminase synthesis. This suggests that arginine is the only physiological effector in those conditions: ammonia or some aminoacids would reduce the enzyme synthesis because of an inducer exclusion. The first stage of OTAse induction would then be operated by the CARGR repressor, and an additional regulatory element might take part in the full scale process. Preliminary data favoring the involvment of such an element are presented.     

Effect of the areA gene on regulation of arginine catabolism in Aspergillus nidulans.

The areA gene which is known to be involved in ammonium repression in Aspergillus nidulans was found to participate in regulation of arginine catabolism. Mutations in this gene are hypostatic to mutations in arcA, suDpro and suEpro genes which are responsible for regulation of synthesis of arginine catabolic enzymes.     

Metabolic regulation in Streptomyces parvulus during actinomycin D synthesis, studied with 13C- and 15N-labeled precursors by 13C and 15N nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry.

Recent studies have suggested that the onset of synthesis of actinomycin D in Streptomyces parvulus is due to a release from L-glutamate catabolic repression. In the present investigation we showed that S. parvulus has the capacity to maintain high levels of intracellular glutamate during the synthesis of actinomycin D. The results seem contradictory, since actinomycin D synthesis cannot start before a release from L-glutamate catabolic repression, but a relatively high intracellular pool of glutamate is needed for the synthesis of actinomycin D. Utilizing different labeled precursors, D-[U-13C]fructose and 13C- and 15N-labeled L-glutamate, and nuclear magnetic resonance techniques, we showed that carbon atoms of an intracellular glutamate pool of S. parvulus were not derived biosynthetically from the culture medium glutamate source but rather from D-fructose catabolism. A new intracellular pyrimidine derivative whose nitrogen and carbon skeletons were derived from exogenous L-glutamate was obtained as the main glutamate metabolite. Another new pyrimidine derivative that had a significantly reduced intracellular mobility and that was derived from D-fructose catabolism was identified in the cell extracts of S. parvulus during actinomycin D synthesis. These pyrimidine derivatives may serve as a nitrogen store for actinomycin D synthesis. In the present study, the N-trimethyl group of a choline derivative was observed by 13C nuclear magnetic resonance spectroscopy in growing S. parvulus cells. The choline group, as well as the N-methyl groups of sarcosine, N-methyl-valine, and the methyl groups of an actinomycin D chromophore, arose from D-fructose catabolism. The 13C enrichments found in the peptide moieties of actinomycin D were in accordance with a mechanism of actinomycin D synthesis from L-glutamate and D-fructose.     

Energetics of vacuolar compartmentation of arginine in Neurospora crassa

The energy requirements for the uptake and retention of arginine by vacuoles of Neurospora crassa have been studied. Exponentially growing mycelial cultures were treated with inhibitors of respiration or glycolysis or an uncoupler of respiration. Catabolism of arginine was monitored as urea production in urease-less strains. The rationale was that the rate and extent of such catabolism was indicative of the cytosolic arginine concentration. No catabolism was observed in cultures treated with an inhibitor or an uncoupler of respiration, but cultures treated with inhibitors of glycolysis rapidly degraded arginine. These differences could not be accounted for by alterations in the level or activity of arginase. Mycelia growing in arginine-supplemented medium and treated with an inhibitor or uncoupler of respiration degraded an amount of arginine equivalent to the cytosolic fraction of the arginine pool. The inhibitors and the uncoupler of respiration reduced the ATP pool and the energy charge. The inhibitors of glycolysis reduced the ATP pool but did not affect the energy charge. The results suggest that metabolic energy is required for the transport of arginine into the vacuoles but not for its retention. The latter is affected by inhibitors of glycolysis. The form of energy and the nature of the vacuolar transport mechanism(s) are discussed.     

Growth effects and metabolism of arginine in stationary and rapidly-dividing sugarcane cell suspensions

The effect of 300 μ M arginine on growth of sugarcane cell suspensions was studied. Cells transferred to defined media in the stationary growth stage showed a greater requirement for exogenous arginine than cells similarly transferred in the rapidly dividing stage. Cell arginine levels, rates of arginine synthesis, and enzymes of arginine synthesis all decreased in cells entering the stationary stage. It is concluded that stationary stage cells are deficient in their ability to synthesize arginine and are therefore dependent upon an exogenous supply to resume growth in fresh media.     

Oxygen and nitrate in utilization by Bacillus licheniformis of the arginase and arginine deiminase routes of arginine catabolism and other factors affecting their syntheses.

Bacillus licheniformis has two pathways of arginine catabolism. In well-aerated cultures, the arginase route is present, and levels of catabolic ornithine carbamoyltransferase were low. An arginase pathway-deficient mutant, BL196, failed to grow on arginine as a nitrogen source under these conditions. In anaerobiosis, the wild type contained very low levels of arginase and ornithine transaminase. BL196 grew normally on glucose plus arginine in anaerobiosis and, like the wild type, had appreciable levels of catabolic transferase. Nitrate, like oxygen, repressed ornithine carbamoyltransferase and stimulated arginase synthesis. In aerobic cultures, arginase was repressed by glutamine in the presence of glucose, but not when the carbon-energy source was poor. In anaerobic cultures, ammonia repressed catabolic ornithine carbamoyltransferase, but glutamate and glutamine stimulated its synthesis. A second mutant, derived from BL196, retained the low arginase and ornithine transaminase levels of BL196 but produced high levels of deiminase pathway enzymes in the presence of oxygen.     

Arginine Catabolism and the Arginine Succinyltransferase Pathway in Escherichia coli

Arginine catabolism produces ammonia without transferring nitrogen to another compound, yet the only known pathway of arginine catabolism in Escherichia coli (through arginine decarboxylase) does not produce ammonia. Our aims were to find the ammonia-producing pathway of arginine catabolism in E. coli and to examine its function. We showed that the only previously described pathway of arginine catabolism, which does not produce ammonia, accounted for only 3% of the arginine consumed. A search for another arginine catabolic pathway led to discovery of the ammonia-producing arginine succinyltransferase (AST) pathway in E. coli. Nitrogen limitation induced this pathway in both E. coli and Klebsiella aerogenes, but the mechanisms of activation clearly differed in these two organisms. We identified the E. coli gene for succinylornithine aminotransferase, the third enzyme of the AST pathway, which appears to be the first of an astCADBE operon. Its disruption prevented arginine catabolism, impaired ornithine utilization, and affected the synthesis of all the enzymes of the AST pathway. Disruption of astB eliminated succinylarginine dihydrolase activity and prevented arginine utilization but did not impair ornithine catabolism. Overproduction of AST enzymes resulted in faster growth with arginine and aspartate. We conclude that the AST pathway is necessary for aerobic arginine catabolism in E. coli and that at least one enzyme of this pathway contributes to ornithine catabolism.     

Depression of enzyme synthesis in response to arginine limitation in Neurospora crassa

Ornithine carbamoyltransferase and argininosuccinase, two enzymes involved in arginine synthesis, are regulated by cross-pathway amino acid control in Neurospora and show derepression in response to limitation of any one of a number of amino acids. The effects of varying the severity of arginine limitation upon the synthesis of these enzymes, in mycelial cultures of an arginine auxotrophic strain, are reported here. Depression occurred at arginine concentrations sufficient to allow normal rates of protein accumulation, leading to increases of not more than fourfold in the absolute rate of enzyme synthesis. On the other hand, differential rates of enzyme synthesis increased progressively up to 20-fold or more under extreme conditions of arginine limitation that also limit net protein synthesis. The major part of the derepression response thus occurred at arginine concentrations that allowed low net rates of protein synthesis. The physiological significance of this is not yet understood. Our evidence suggests that these responses were mediated entirely through the cross-pathway control system, and may not be untypical (allowing for variations in magnitude) of depression resulting through this mechanism in Neurospora.     

Caveolar localization of arginine regeneration enzymes, argininosuccinate synthase, and lyase, with endothelial nitric oxide synthase.

Although normal intracellular levels of arginine are well above the K(m), and should be sufficient to saturate nitric oxide synthase in vascular endothelial cells, nitric oxide production can, nonetheless, be stimulated by exogenous arginine. This phenomenon, termed the "arginine paradox," has suggested the existence of a separate pool of arginine directed to nitric oxide synthesis. In this study, we demonstrate that exogenous citrulline was as effective as exogenous arginine in stimulating nitric oxide production and that citrulline in the presence of excess intracellular and extracellular arginine further enhanced bradykinin stimulated endothelial nitric oxide production. The enhancement of nitric oxide production by exogenous citrulline could therefore be attributed to the capacity of vascular endothelial cells to efficiently regenerate arginine from citrulline. However, the regeneration of arginine did not affect the bulk intracellular arginine levels. This finding not only supports the proposal for a unique pool of arginine, but also suggested channeling of substrates that would require a functional association between nitric oxide production and arginine regeneration. To support this proposal, we showed that nitric oxide synthase, and the enzymes involved in arginine regeneration, argininosuccinate synthase and argininosuccinate lyase, cofractionated with plasmalemmal caveolae, a subcompartment of the plasma membrane. Overall, the results from this study strongly support the proposal for a separate pool of arginine for nitric oxide production that is defined by the cellular colocalization of enzymes involved in nitric oxide production and the regeneration of arginine.     

Pyruvate production and excretion by the luminous marine bacteria.

During aerobic growth on glucose, several species of luminous marine bacteria exhibited an imcomplete oxidative catabolism of substrate. Pyruvate, one of the products of glucose metabolism, was excreted into the medium during exponential growth and accounted for up to 50% of the substrate carbon metabolized. When glucose was depleted from the medium, the excreted pyruvate was promptly utilized, demonstrating that the cells are capable of pyruvate catabolism. Pyruvate excretion is not a general phenomenon of carbohydrate metabolism since it does not occur during the utilization of glycerol or maltose. When cells pregrown on glycerol were exposed to glucose, they began to excrete pyruvate, even if protein synthesis was blocked with chloramphenicol. Glucose thus appears to have an effect on the activity of preexisting catabolic enzymes.