Transcarbamoylase Activity Research Articles

The pyrimidine biosynthetic pathway and its regulation in Pseudomonas jessenii.

The control of the pyrimidine biosynthetic pathway by pyrimidine bases was examined in Pseudomonas jessenii ATCC 700870. The pyrimidine biosynthetic enzymes aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate (OMP) decarboxylase activities were found to be higher in the succinate-grown ATCC 700870 cells than the glucose-grown cells. All the enzyme activities were depressed in uracil-supplemented ATCC 700870 glucose-grown cells relative to the unsupplemented cells which was indicative of possible repression of enzyme synthesis by uracil. In the succinate-grown, ATCC 700870 cells, transcarbamoylase, dihydroorotase and dehydrogenase activities were decreased by uracil and orotate supplementation while decarboxylase activity was decreased following uracil addition. A pyrimidine auxotroph was isolated by conventional chemical mutagenesis and resistance to 5-fluoroorotic acid whose pyrimidine requirement was met by uracil or cytosine. The mutant strain was deficient for orotate phosphoribosyltransferase activity. Pyrimidine limitation of the mutant strain cells for 1 or 2h caused about a two-fold increase in aspartate transcarbamoylase or dihydroorotase activity independent of carbon source relative to excess uracil growth conditions. At the level of enzyme activity, aspartate transcarbamoylase activity in P. jessenii ATCC 700870 was inhibited strongly by pyrophosphate, ATP, UTP, GTP and UMP under saturating substrate concentrations.

Pyrimidine nucleotide synthesis in the emerging pathogen Pseudomonas monteilii.

Regulation of pyrimidine biosynthesis by pyrimidines in the emerging, opportunistic human pathogen Pseudomonas monteilii ATCC 700476 was evident. When wild-type cells were grown on succinate in the presence of uracil or orotic acid, the activities of all 5 pyrimidine biosynthetic enzymes were depressed while the activities of 3 of the enzymes decreased in glucose-grown cells supplemented with uracil or orotic acid compared with unsupplemented cells. Pyrimidine limitation of succinate- or glucose-grown pyrimidine auxotrophic cells lacking orotate phosphoribosyltransferase activity resulted in more than a doubling of the pyrimidine biosynthetic enzyme activities relative to their activities in uracil-grown cells. Independent of carbon source, pyrimidine-limited cells of the pyrimidine auxotrophic cells deficient for dihydroorotase activity generally resulted in a slight elevation or depression of the pyrimidine biosynthetic enzyme activities compared with their activities in cells grown under saturating uracil conditions. Aspartate transcarbamoylase activity in P. monteilii was regulated at the enzyme activity level, since the enzyme was strongly inhibited by CTP, UMP, GMP, GDP, ADP, and UTP. In summary, the regulation of pyrimidine biosynthesis in P. monteilii could be used to control its growth or to differentiate it biochemically from other related species of Pseudomonas.

Fermentative production of the diamine putrescine: system metabolic engineering of corynebacterium glutamicum.

Corynebacterium glutamicum shows great potential for the production of the glutamate-derived diamine putrescine, a monomeric compound of polyamides. A genome-scale stoichiometric model of a C. glutamicum strain with reduced ornithine transcarbamoylase activity, derepressed arginine biosynthesis, and an anabolic plasmid-addiction system for heterologous expression of E. coli ornithine decarboxylase gene speC was investigated by flux balance analysis with respect to its putrescine production potential. Based on these simulations, enhancing glycolysis and anaplerosis by plasmid-borne overexpression of the genes for glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase as well as reducing 2-oxoglutarate dehydrogenase activity were chosen as targets for metabolic engineering. Changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and changing threonine 15 of OdhI to alanine reduced 2-oxoglutarate dehydrogenase activity about five fold and improved putrescine titers by 28%. Additional engineering steps improved further putrescine production with the largest contributions from preventing the formation of the by-product N-acetylputrescine by deletion of spermi(di)ne N-acetyltransferase gene snaA and from overexpression of the gene for a feedback-resistant N-acetylglutamate kinase variant. The resulting C. glutamicum strain NA6 obtained by systems metabolic engineering accumulated two fold more putrescine than the base strain, i.e., 58.1 ± 0.2 mM, and showed a specific productivity of 0.045 g·g−1·h−1 and a yield on glucose of 0.26 g·g−1.

Pyrimidine nucleotide synthesis in Pseudomonas nitroreducens and the regulatory role of pyrimidines

Control of pyrimidine biosynthesis in the commercially important, hydrocarbon-utilizing bacterium Pseudomonas nitroreducens ATCC 33634 was investigated. When glucose-grown wild-type cells were supplemented with uracil or orotic acid, the pyrimidine biosynthetic activities were depressed. Pyrimidine limitation of glucose-grown cells of an orotate phosphoribosyltransferase mutant caused aspartate transcarbamoylase and dihydroorotase activities to increase by about 4-fold while the other enzyme activities about doubled. In succinate-grown phosphoribosyltransferase mutant cells subjected to pyrimidine limitation, transcarbamoylase and dehydrogenase activities rose by about 5-fold while dihydroorotase activity more than tripled. In an OMP decarboxylase mutant, pyrimidine limitation of glucose-grown cells increased transcarbamoylase, dihydroorotase, dehydrogenase and phosphoribosyltransferase activities by 4-, 10-, 6- and 3.8-fold, respectively. Pyrimidine limitation of the succinate-grown decarboxylase mutant cells increased aspartate transcarbamoylase or dihydroorotase by more than 4-fold and the other activities by about 2-fold. Pyrimidine biosynthetic enzyme synthesis appeared to be regulated by pyrimidines with the regulation being influenced by the carbon source present. Aspartate transcarbamoylase activity in Ps. nitroreducens was regulated at the level of enzyme activity since the enzyme was strongly inhibited by UDP, pyrophosphate, ATP and ADP. Overall, the regulation of pyrimidine biosynthesis in Ps. nitroreducens can be used to differentiate it from other taxonomically related species of Pseudomonas.

Improving putrescine production by Corynebacterium glutamicum by fine-tuning ornithine transcarbamoylase activity using a plasmid addiction system

Corynebacterium glutamicum shows a great potential for the production of the polyamide monomer putrescine (1,4-diaminobutane). Previously, we constructed the putrescine-producing strain PUT1 by deletion of argF, the gene for ornithine transcarbamoylase (OTC), and argR, encoding the L-arginine repressor, combined with heterologous expression of the Escherichia coli gene for L-ornithine decarboxylase SpeC. As a consequence of argF deletion, this strain requires supplementation of L-arginine and shows growth-decoupled putrescine production. To avoid costly supplementation with L-arginine and the strong feedback inhibition of the key enzyme N-acetylglutamate kinase (ArgB) by L-arginine, a plasmid addiction system for low-level argF expression was developed. By fine-tuning argF expression through modifications of the promoter, the translational start codon and/or the ribosome binding site, high productivity and titer could be obtained. OTC activity varied almost thousandfold between 960 and 1 mU mg⁻¹ resulting in putrescine yields on glucose from less than 0.001 up to 0.26 g g⁻¹, the highest yield in bacteria reported to date. The most promising strain, designated PUT21, was characterized comprehensively. PUT21 strain grew with a rate of 0.19 h⁻¹ in mineral salt medium without the need for L-arginine supplementation and produced putrescine with a yield of 0.16 g g⁻¹ glucose at a volumetric productivity of 0.57 g L⁻¹ h⁻¹ and a specific productivity of 0.042 g g⁻¹ h⁻¹. The carbon balance suggested that no major unidentified by-product was produced. Compared to the first-generation strain PUT1, the putrescine yield observed with PUT21 was increased by 60%. In fed-batch cultivation with C. glutamicum PUT21, a putrescine titer of 19 g L⁻¹ at a volumetric productivity of 0.55 g L⁻¹ h⁻¹ and a yield of 0.16 g g⁻¹ glucose could be achieved. Moreover, while plasmid segregation of the initial strain required antibiotic selection, plasmid segregation in C. glutamicum PUT21 was fully stable for more than 60 generations without antibiotic selection even in the presence of L-arginine. The ornithine decarboxylase gene speC was expressed from this argF addiction plasmid ensuring stable putrescine production by the engineered C. glutamicum strain.

Pyrimidine biosynthesis in Pseudomonas veronii and its regulation by pyrimidines

Pyrimidine biosynthesis in the nutritionally versatile bacterium Pseudomonas veronii ATCC 700474 appeared to be controlled by pyrimidines. When wild type cells were grown on glucose in the presence of uracil, four enzyme activities were depressed while all five enzyme activities increased in succinate-grown cells supplemented with uracil. Independent of carbon source, orotic acid-grown cells elevated aspartate transcarbamoylase, dihydroorotase, orotate phosphoribosyltransferase or OMP decarboxylase activity. Pyrimidine limitation of glucose-grown pyrimidine auxotrophic cells lacking OMP decarboxylase activity resulted in at least a doubling of the enzyme activities relative to their activities in uracil-grown cells. Less derepression of the enzyme activities was observed after pyrimidine limitation of succinate-grown mutant cells possibly due to catabolite repression. Aspartate transcarbamoylase activity in Ps. veronii was regulated at the level of enzyme activity since the enzyme was strongly inhibited by pyrophosphate, UDP, UTP, ADP, ATP and GTP. Overall, the regulation of pyrimidine biosynthesis in Ps. veronii could be used to differentiate it from other taxonomically related species of Pseudomonas.

Alterations in DNA metabolism in Elliptio complanata mussels after exposure to municipal effluents

This study sought to examine the genotoxic potential in Elliptio complanata freshwater mussels exposed to a physically and chemically treated municipal effluent before and after ozone treatment. Mussels were continuously exposed to increasing concentrations of the effluents for 14days. Genotoxicity was determined by tracking changes in key enzymes for purine and pyrimidine synthesis (dehydrofolate reductase and aspartate transcarbamoylase), catabolism of purines (xanthine oxido-reductase) and DNA strand-break levels as determined by the alkaline precipitation assay. Other biomarkers related to xenobiotic biotransformation (cytochrome P4503A and glutathione S-transferase activities), metal metabolism (labile zinc and redox state of metathioneins) and oxidative stress (superoxide dismutase activity) were also determined in the mussels. The data revealed that dehydrofolate reductase activity was reduced by the initial effluent and increased by the ozonated effluent. Aspartate transcarbamoylase activity was significantly induced only with the ozonated effluent. The levels of DNA strand breaks responded in a biphasic manner in mussels exposed to the physically and chemically treated effluent where an initial decrease was observed at a low effluent concentration (3% v/v) followed by an increase in DNA strand breaks at a higher effluent concentration (20%). This response pattern was lost in the ozonated effluent, where only a decrease in DNA breaks was found. Xanthine oxidoreductase activity was not significantly affected but did correlate significantly with dehydrofolate reductase activity. Multivariate factorial and canonical analyses revealed that oxidative stress and metal/xenobiotic metabolism markers were strongly correlated with DNA strand breaks in mussels, suggesting that the presence of metals (zinc) and planar hydroxylated hydrocarbons present in these effluents were strong contributors to the observed response. We conclude that municipal effluents contain a complex mixture of pollutants that could modulate DNA synthesis and repair mechanisms in mussels.

Control of pyrimidine nucleotide formation in Pseudomonas fulva

Control of pyrimidine formation was examined in Pseudomonas fulva ATCC 31418. Pyrimidine supplementation lowered pyrimidine biosynthetic pathway enzyme activities in cells grown on glucose or succinate as a carbon source indicating possible repression of enzyme synthesis. Pyrimidine limitation experiments were conducted using an orotidine 50-monophosphate decarboxylase mutant strain isolated in this study. Compared to uracil-supplemented, glucose-grown mutant cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities to increase about 6-, 13-, 3-, 15-fold, respectively, which confirmed regulation of enzyme synthesis by pyrimidines. At the level of enzyme activity, transcarbamoylase activity in Ps. fulva was strongly inhibited by pyrophosphate, CTP, GTP and GDP under saturating substrate concentrations.

Reduction of Alkali-Labile Phosphates in Mussels Exposed to Primary-Treated Wastewaters Undergoing Ozone and Ultraviolet Disinfection: A Pilot Study

Abstract The purpose of this study was to examine the estrogenic potential of a primary municipal effluent, undergoing ozone or ultraviolet disinfection, in Elliptio complanata mussels. Mussels were exposed for seven weeks, using a continuous flowthrough system, to a primary effluent from a major city before and after disinfection. Results showed that the effluents, regardless of the disinfection procedure, readily affected gametogenesis in mussels as determined by gonado-somatic index, and by DNA synthesis as determined by the activity of aspartate transcarbamoylase activity, a rate-limiting enzyme for pyrimidine synthesis. The estrogenic potential of the effluents was observed by the increased levels of alkali-labile phosphate, a generic measure for vitellogenin-like proteins, where the disinfection procedures did not completely remove the estrogenic effects of the primary effluent. Thus, the introduction of an ozone or ultraviolet disinfection step to a primary-treated effluent will likely diminish the estrogenic potential, but not entirely.

Regulation of pyrimidine formation inPseudomonas lundensis

The regulation of pyrimidine formation in the food spoilage agent Pseudomonas lundensis ATCC 49968 by pyrimidines was examined. In P. lundensis cells grown on glucose as a carbon source, the enzymes aspartate transcarbamoylase, dihydroorotase, and orotidine 5'-monophosphate decarboxylase were induced by orotic acid. Pyrimidine auxotrophs containing reduced transcarbamoylase or orotate phosphoribosyltransferase activity were isolated using chemical mutagenesis and selection procedures. Independent of carbon source, the maximum derepression of enzyme activity was observed for orotidine 5'-monophosphate decarboxylase after pyrimidine limitation of either auxotroph. In the glucose-grown cells of the transcarbamoylase mutant strain, orotic acid induced dihydroorotase and decarboxylase activities. Aspartate transcarbamoylase activity in succinate-grown P. lundensis cells was highly regulated by pyrophosphate as well as by pyrimidine and purine ribonucleotides. It was concluded that pyrimidine formation in P. lundensis was controlled both at the level of de novo pyrimidine biosynthetic enzyme synthesis and at the level of transcarbamoylase activity.

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha ATCC 9890 was investigated and the pyrimidine biosynthetic pathway enzyme activities were affected by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. In pyrimidine-grown ATCC 9890 cells, the activities of four de novo enzymes could be depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the succinate-grown mutant strain cells, pyrimidine limitation of this strain caused dihydroorotase activity to increase about 3-fold while dihydroorotate dehydrogenase and orotidine 5'-monophosphate decarboxylase activities rose about 2-fold. Regulation of de novo pathway enzyme synthesis by pyrimidines appeared to be occurring. At the level of enzyme activity, aspartate transcarbamoylase activity in P. synxantha ATCC 9890 was strongly inhibited in vitro by pyrophosphate, UTP, ADP, ATP, CTP and GTP under saturating substrate concentrations.

Neuroendocrine disruption in Mya arenaria clams during gametogenesis at sites under pollution stress

This study examined the neuroendocrine status of clams on intertidal mud flats in the St. Lawrence Estuary and Saguenay Fjord areas during late gametogenesis. The impact of pollution was determined by a test battery of early stress markers (metallothioneins, heme levels, glutathione S-transferase activity), tissue damage (lipid peroxidation and DNA damage) and morphologic characteristics (age, soft-tissue weight ratio and growth index). Neuroendocrinal status was examined by tracking serotonin and dopamine metabolism, monoamine adenylate cyclase activity in synaptosomes, monoamine oxidase and arachidonate cyclooxygenase activities in relation to gametogenetic activity: pyrimidine synthesis, (aspartate transcarbamoylase activity or ATC), vitellogenin-like proteins and gonado-somatic index. The results show that clam soft tissue weights were reduced at sites close to harbours and higher at sites near domestic wastewater outfalls. The age-to-length ratio of clams was generally higher at impacted sites, suggesting reduced growth. The biomarkers of stress or damage confirmed that oxidative stress, DNA damage, metallothioneins and glutathione S-transferase activity were significantly increased at varying degrees, at the polluted sites. Vitellogenin-like proteins and gametogenetic activity were significantly increased at sites influenced by domestic wastewaters. Furthermore, the clams were still in active gametogenesis and not ready for spawning, as indicated by the concordance of the serotonin/dopamine ratio with vitellogenin-like proteins and pyrimidine synthesis. However, gonadal cyclooxygenase activity was increased at polluted sites and significantly correlated with most of the stress biomarkers, suggesting that the clams were in a state of inflammation rather than at the spawning stage. Finally, a multivariate analysis revealed that the sites were readily discriminated with high efficiency (>71%) and that both neuroendocrine physiological markers and stress responses were identified as the major components, thus explaining the global physiological response of the clams. We conclude that the effects of pollution compromise the clams' health status and that the initiation of gametogenesis in environments contaminated by municipal wastewaters or harbours contributes to the toxic effects of pollution.

Regulation of the Pyrimidine Biosynthetic Pathway in Pseudomonas mucidolens

Control of pyrimidine biosynthesis was examined in Pseudomonas mucidolens ATCC 4685 and the five de novo pyrimidine biosynthetic enzyme activities unique to this pathway were influenced by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. When uracil was supplemented to glucose-grown ATCC 4685 cells, activities of four de novo enzymes were depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the glucose-grown mutant strain cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase activities to increase by more than 3-fold while OMP decarboxylase activity increased by 2.7-fold. The syntheses of the de novo enzymes appeared to be regulated by pyrimidines. At the level of enzyme activity, aspartate transcarbamoylase activity in P. mucidolens ATCC 4685 was subject to inhibition at saturating substrate concentrations. Transcarbamoylase activity was strongly inhibited by UTP, ADP, ATP, GTP and pyrophosphate.

Regulation of pyrimidine synthesis in Pseudomonas resinovorans

To investigate the regulation of de novo pyrimidine biosynthesis in the bacterium Pseudomonas resinovorans ATCC 14235. The pyrimidine biosynthetic pathway enzymes were measured in cell extracts from P. resinovorans ATCC 14235 and from an auxotroph lacking orotate phosphoribosyltransferase activity. Pyrimidine biosynthetic pathway enzyme activities in ATCC 14235 were affected by the addition of pyrimidine bases to the culture medium. The de novo enzyme activities of the phosphoribosyltransferase mutant strain increased after pyrimidine starvation indicating possible repression of the pathway by a pyrimidine-related compound. Aspartate transcarbamoylase activity in ATCC 14235 was inhibited in vitro by ATP, UTP and pyrophosphate. Pyrimidine biosynthesis in P. resinovorans was regulated at the level of enzyme synthesis and at the level of activity for aspartate transcarbamoylase. Its regulation of enzyme synthesis seemed to be similar to what has been observed in the taxonomically related species Pseudomonas oleovorans. This study found that pyrimidine biosynthesis is regulated in P. resinovorans. This could prove helpful to future studies investigating polyhydroxyalkanoate production by the bacterium.

Pyrimidine nucleotide synthesis inPseudomonascitronellolis

Pyrimidine biosynthesis was active in Pseudomonas citronellolis ATCC 13674 and appeared to be regulated by pyrimidines. When wild-type cells were grown on succinate in the presence of uracil, the de novo enzyme activities were depressed while only four enzyme activities were depressed in the glucose-grown cells. On either carbon source, orotic acid-grown cells had diminished aspartate transcarbamoylase, dihydroorotase or OMP decarboxylase activity. Pyrimidine limitation of glucose-grown pyrimidine auxotrophic cells resulted in de novo enzyme activities, except for transcarbamoyolase activity, that were elevated by more than 5-fold compared to their activities in uracil-grown cells. Since pyrimidine limitation of succinate-grown mutant cells produced less enzyme derepression, catabolite repression appeared to be a factor. At the level of enzyme activity, aspartate transcarbamoylase activity in P. citronellolis was strongly inhibited by all effectors tested. Compared to the regulation of pyrimidine biosynthesis in taxonomically-related species, pyrimidine biosynthesis in P. citronellolis appeared more highly regulated.

Regulation of pyrimidine nucleotide formation in Pseudomonas taetrolens ATCC 4683

The regulation of the de novo pyrimidine biosynthetic enzymes in the food spoilage agent Pseudomonas taetrolens ATCC 4683 was investigated. The de novo pyrimidine biosynthetic enzyme activities were determined in P. taetrolens ATCC 4683 cells and in cells from an auxotroph deficient for orotidine 5′-monophosphate decarboxylase activity. Pyrimidine supplementation to the culture medium affected the biosynthetic enzyme activities in ATCC 4683 cells. Transcriptional regulation of the biosynthetic pathway by pyrimidines was indicated after the auxotroph was subjected to pyrimidine limitation. At the level of enzyme activity, aspartate transcarbamoylase activity was strongly inhibited by pyrophosphate, ADP, ATP, UDP, UTP and GTP. Transcriptional regulation of pyrimidine synthesis in P. taetrolens was not as highly controlled as in the taxonomically-related species Pseudomonas fragi although both species contained transcarbamoylase activities subject to significant nucleotide inhibition.

Regulation of pyrimidine nucleotide formation in Pseudomonas reptilivora.

To study the regulation of de novo pyrimidine biosynthesis in the pathogenic bacterium Pseudomonas reptilivora ATCC 14836. The pyrimidine biosynthetic pathway enzymes were assayed in extracts of Ps. reptilivora ATCC 14836 cells and of cells from an auxotroph lacking aspartate transcarbamoylase activity. Pyrimidine biosynthetic pathway enzyme activities in ATCC 14836 were influenced by the addition of pyrimidine bases to the culture medium with orotic acid addition inducing dihydroorotase activity. Pyrimidine starvation of the transcarbamoylase mutant strain increased its de novo enzyme activities suggesting that the de novo pathway was also subject to repression by a pyrimidine-related compound. Aspartate transcarbamoylase activity in ATCC 14836 was inhibited in vitro by pyrophosphate and ATP. Regulation of pyrimidine biosynthesis in Ps. reptilivora was observed at the level of enzyme synthesis and at the level of activity for aspartate transcarbamoylase. Its regulation of enzyme synthesis seemed to be more highly controlled than what was observed in the related species Ps. fluorescens. This investigation found that pyrimidine biosynthesis is controlled in Ps. reptilivora. This could prove helpful to future studies exploring its pathogenicity.

A Fluorescent Probe-labeled Escherichia coli Aspartate Transcarbamoylase That Monitors the Allosteric Conformational State

A new system has been developed capable of monitoring conformational changes of the 240s loop of aspartate transcarbamoylase, which are tightly correlated with the quaternary structural transition, with high sensitivity in solution. Pyrene, a fluorescent probe, was conjugated to residue 241 in the 240s loop of aspartate transcarbamoylase to monitor changes in conformation by fluorescence spectroscopy. Pyrene maleimide was conjugated to a cysteine residue on the 240s loop of a previously constructed double catalytic chain mutant version of the enzyme, C47A/A241C. The pyrene-labeled enzyme undergoes the normal T to R structural transition, as demonstrated by small-angle x-ray scattering. Like the wild-type enzyme, the pyrene-labeled enzyme exhibits cooperativity toward aspartate, and is activated by ATP and inhibited by CTP at subsaturating concentrations of aspartate. The binding of the bisubstrate analogue N-(phosphonoacetyl)-l-aspartate (PALA), or the aspartate analogue succinate, in the presence of saturating carbamoyl phosphate, to the pyrenelabeled enzyme caused a sigmoidal change in the fluorescence emission. Saturation with ATP and CTP (in the presence of either subsaturating amounts of PALA or succinate and carbamoyl phosphate) caused a hyperbolic increase and decrease, respectively, in the fluorescence emission. The half-saturation values from the fluorescence saturation curves and kinetic saturation curves were, within error, identical. Fluorescence and small-angle x-ray scattering stopped-flow experiments, using aspartate and carbamoyl phosphate, confirm that the change in excimer fluorescence and the quaternary structure change correlate. These results in conjunction with previous studies suggest that the allosteric transition involves both global and local conformational changes and that the heterotropic effect of the nucleotides may be exerted through local conformational changes in the active site by directly influencing the conformation of the 240s loop.

Influence of carbon source on pyrimidine synthesis in Pseudomonas mendocina.

The effect of carbon source on the regulation of the de novo pyrimidine biosynthetic enzymes in the bacterium Pseudomonas mendocina was studied. When glucose was the carbon source, orotic acid supplementation of P. mendocina cells produced the greatest depression of aspartate transcarbamoylase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities while P. mendocina cells grown in the presence of uracil caused the maximal decrease in dihydroorotase and OMP decarboxylase activities. After the pyrimidine starvation of an orotate phosphoribosyltransferase mutant strain of P. mendocina grown on glucose, the pyrimidine biosynthetic pathway enzyme activities were generally diminished. With respect to pyrimidine starvation studies, the carbon source glucose appeared to lessen regulation at the level of enzyme synthesis compared to what has been observed when succinate served as the carbon source. The regulation of the pyrimidine biosynthetic pathway by carbon source in P. mendocina appeared to differ from how carbon source influenced the control of pyrimidine biosynthesis in the closely-related species Pseudomonas stutzeri.

Excretory nitrogen metabolism in the Chinese fire-belly newt Cynops orientalis in water, on land, or in high concentrations of environmental ammonia.

The Chinese fire-belly newt Cynops orientalis reverts to an aquatic mode of living when sexually mature. Despite living in water, sexually mature C. orientalis maintained high capacity for hepatic urea synthesis. However, it had a lower rate of urea production than other terrestrial amphibians because endogenous ammonia could diffuse out to the external medium as NH3. This conserves cellular energy because urea synthesis is energetically expensive. Simultaneously, C. orientalis also reduced the rate of urea excretion, and excreted 33% of the total nitrogenous waste as ammonia. Upon exposure to land, C. orientalis increased the rate of urea synthesis from accumulating endogenous ammonia. The increased rate of urea synthesis was within the inherent capacity of the hepatic ornithine-urea cycle; there was no induction of hepatic carbamoyl phosphate synthetase or ornithine transcarbamoylase activities and there was no reduction in ammonia production. When exposed to water containing 75 mmol.l(-1) NH4Cl, the rates of both urea synthesis and urea excretion increased. Under such experimental conditions, the ornithine-urea cycle may be operating close to its limit; glutamine began to accumulate in the body, and endogenous ammonia production via amino acid catabolism was reduced.