Transport of diamines by Enterococcus faecalis is mediated by an agmatine-putrescine antiporter.

Enterococcus faecalis ATCC 11700 is able to use arginine and the diamine agmatine as a sole energy source. Via the highly homologous deiminase pathways, arginine and agmatine are converted into CO2, NH3, and the end products ornithine and putrescine, respectively. In the arginine deiminase pathway, uptake of arginine and excretion of ornithine are mediated by an arginine-ornithine antiport system. The translocation of agmatine was studied in whole cells grown in the presence of arginine, agmatine, or glucose. Rapid uncoupler-insensitive uptake of agmatine was observed only in agmatine-grown cells. A high intracellular putrescine pool was maintained by these cells, and this pool was rapidly released by external putrescine or agmatine but not by arginine or ornithine. Kinetic analysis revealed competitive inhibition for uptake between putrescine and agmatine. Agmatine uptake by membrane vesicles was observed only when the membrane vesicles were preloaded with putrescine. Uptake of agmatine was driven by the outwardly directed putrescine concentration gradient, which is continuously sustained by the metabolic process. Uptake of agmatine and extrusion of putrescine by agmatine-grown cells of E. faecalis appeared to be catalyzed by an agmatine-putrescine antiporter. This transport system functionally resembled the previously described arginine-ornithine antiport, which was exclusively induced when the cells were grown in the presence of arginine.     

Modulation of serotonin uptake kinetics by ions and ion gradients in human placental brush-border membrane vesicles

The modulation of serotonin uptake kinetics by Na+, Cl-, H+, and K+ was investigated in brush-border membrane vesicles prepared from normal human term placentas. The presence of Na+ and Cl- in the external medium was mandatory for the function of the serotonin transporter. In both cases, the initial uptake rate of serotonin was a hyperbolic function of the ion concentration, indicating involvement of one Na+ and one Cl- per transport of one serotonin molecule. The apparent dissociation constant for Na+ and Cl- was 145 and 79 mM, respectively. The external Na+ increased the Vmax of the transporter and also increased the affinity of the transporter for serotonin. The external Cl- also showed similar effects on the Vmax and the Kt, but its effect on the Kt was small compared to that of Na+. The presence of an inside-acidic pH, with or without a transmembrane pH gradient, stimulated the NaCl-dependent serotonin uptake. The effect of internal [H+] on the transport function was to increase the Vmax and decrease the affinity of the transporter for serotonin. The presence of K+ inside the vesicles also greatly stimulated the initial rates of serotonin uptake, and the stimulation was greater at pH 7.5 than at pH 6.5. This stimulation was a hyperbolic function of the internal K+ concentration at both pH values, indicating involvement of one K+ per transport of one serotonin molecule. The apparent dissociation constant for K+ was 5.6 mM at pH 6.5 and 4.0 mM at pH 7.5. The effects of internal [K+] on the uptake kinetics were similar to those of internal [H+].(ABSTRACT TRUNCATED AT 250 WORDS)     

Compartmental behavior of ornithine in Neurospora crassa.

In Neurospora cells grown on minimal medium, most of the large ornithine pool is found in osmotically sensitive organelles, the "vesicles." In this paper kinetic studies on the compartmental behavior of ornithine and its derivatives are reported. Analysis of the metabolism of a 10(-7) M pulse of uniformly labeled L-[14C] ornithine supports the following conclusions: (a) Over 98% of the cellular ornithine is in the vesicles. (b) The amount of ornithine normally in the cytosol is about 0.3% of the cellular ornithine, as shown by the kinetics of incorporation of 14C into putrescine via the cytosolic enzyme, ornithine decarboxylase (EC 4.1.1.17). (c) Mitochondria, the site of ornithine synthesis, contain about 1% of the cellular ornithine, as demonstrated by the kinetics of incorporation of 14C into citrulline via the mitochondrial enzyme, ornithine transcarbamylase (EC 2.1.3.3). (d) Considerable ornithine exchange, and a net efflux of ornithine, takes place across the mitochondrial membrane. (e) Ornithine aminotransferase (EC 2.6.1.13), a catabolic enzyme, may have a special relation to the cell membrane in cells grown in minimal medium. This enzyme uses ornithine efficiently while it enters from the medium, but very poorly after all the [14C] ornithine is within the cell. (f) Citrulline and proline are not compartmented with respect to the enzymes using them. (g) In contrast, arginine is distributed such that over 99% is in vesicles. We suggest that the vesicles; with their ability to sequester ornithine and arginine, are potentially significant in regulation.     

Transport of basic amino acids by membrane vesicles of Lactococcus lactis.

The uptake of the basic amino acids arginine, ornithine, and lysine was studied in membrane vesicles derived from cells of Lactococcus lactis which were fused with liposomes in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (PMF)-generating system. In the presence of ascorbate N,N,N'N'-tetramethylphenylenediamine-cytochrome c as the electron donor, these fused membranes accumulated lysine but not ornithine or arginine under aerobic conditions. The mechanism of energy coupling to lysine transport was examined in membrane vesicles of L. lactis subsp. cremoris upon imposition of an artificial electrical potential (delta psi) or pH gradient or both and in fused membranes of these vesicles with cytochrome c oxidase liposomes in which the delta psi and delta pH were manipulated with ionophores. Lysine uptake was shown to be coupled to the PMF and especially to the delta psi, suggesting a proton symport mechanism. The lysine carrier appeared to be specific for L and D isomers of amino acids with a guanidine or NH2 group at the C6 position of the side chain. Uptake of lysine was blocked by p-chloromercuribenzene sulfonic acid but not by maleimides. Counterflow of lysine could not be detected in L. lactis subsp. cremoris, but in the arginine-ornithine antiporter-containing L. lactis subsp. lactis, rapid counterflow occurred. Homologous exchange of lysine and heterologous exchange of arginine and lysine were mediated by this antiporter. PMF-driven lysine transport in these membranes was noncompetitively inhibited by arginine, whereas the uptake of arginine was enhanced by lysine. These observations are compatible with a model in which circulation of lysine via the lysine carrier and the arginine-ornithine antiporter leads to accumulation of arginine.     

Basic and neutral amino acid transport in Aspergillus nidulans.

Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids. The Km value for arginine is 1 X 10(-5) M, and Vmax is 2-8 nmol/mg dry wt/min; Km for lysine is 8 X 10(-6) M; Kt for lysine as inhibitor of arginine uptake is 12 muM, and Ki for ornithine is mM. On minimal medium, methionine is transported with a Km of 0-I mM and Vmax about I nmol/mg dry wt/min; transport is inhibited by azide. Neutral amnio acids such as serine, phenylalanine and leucine are probably transported by the same system, as indicated by their inhibition of methionine uptake and the existence of a mutant specifically impaired in their transport. The recessive mutant nap3, unable to transport neutral amino acids, was isolated as resistant to selenomethionine and p-fluorophenylanine. This mutant has unchanged transport of methionine by general and specific sulphur-regulated permeases.     

Effects of halothane on transport of 5-hydroxytryptamine by platelet membranes.

Na+-dependent uptake of 5-HT (5-hydroxytryptamine) into plasma membrane vesicles derived from bovine blood platelets and ATP-dependent 5-HT uptake into storage vesicles in platelet lysates were measured. Na+-dependent uptake was temperature-dependent, inhibited by imipramine and exhibited Michaelis-Menten kinetics (apparent Km, 0.12 +/- 0.02 microM; Vmax. 559 +/- 54 pmol/min per mg of protein. Halothane had no effect on Na+-dependent transport of 5-HT in plasma-membrane vesicles. ATP-dependent 5-HT transport into storage granules also exhibited Michaelis-Menten kinetics (apparent Km 0.34 +/- 0.03 microM; Vmax. 34.3 +/- 1.7 pmol/min per mg of protein) and was inhibited by noradrenaline (norepinephrine), but not by imipramine. Exposure of the granules to halothane resulted in a progressive decrease in Vmax. The results demonstrate a possible site for disruption of platelet function by anaesthetics.     

arcD, the first gene of the arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa, encodes an arginine-ornithine exchanger.

In the absence of oxygen and nitrate, Pseudomonas aeruginosa metabolizes arginine via the arginine deiminase pathway, which allows slow growth on rich media. The conversion of arginine to ornithine, CO2, and NH3 is coupled to the production of ATP from ADP. The enzymes of the arginine deiminase pathway are organized in the arcDABC operon. The arcD gene encodes a hydrophobic polytopic membrane protein. Translocation of arginine and ornithine in membrane vesicles derived from an Escherichia coli strain harboring a recombinant plasmid carrying the arcD gene was studied. Arginine and ornithine uptake was coupled to the proton motive force with a bias toward the transmembrane electrical potential. Accumulated ornithine was readily exchangeable for external arginine or lysine. The exchange was several orders of magnitude faster than proton motive force-driven transport. The ArcD protein was reconstituted in proteoliposomes after detergent solubilization of membrane vesicles. These proteoliposomes mediate a stoichiometric exchange between arginine and ornithine. It is concluded that the ArcD protein is a transport system that catalyzes an electroneutral exchange between arginine and ornithine to allow high-efficiency energy conversion in the arginine deiminase pathway.     

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.     

Car: a cytoplasmic sensor responsible for arginine chemotaxis in the archaeon Halobacterium salinarum

A new metabolic signaling pathway for arginine, both a chemoeffector and a fermentative energy source, is described for Halobacterium salinarum. Systematic screening of 80+ potentially chemotactic compounds with two behavioral assays identified leucine, isoleucine, valine, methionine, cysteine, arginine and several peptides as strong chemoattractants. Deletion analysis of a number of potential halobacterial transducer genes led to the identification of Car, a specific cytoplasmic arginine transducer which lacks transmembrane helices and was biochemically shown to be localized in the cytoplasm. Flow assays were used to show specific adaptive responses to arginine and ornithine in wild-type but not Deltacar cells, demonstrating the role of Car in sensing arginine. The signaling pathway from external arginine to the flagellar motor of the cell involves an arginine:ornithine antiporter which was quantitatively characterized for its transport kinetics and inhibitors. By compiling the chemotactic behavior, the adaptive responses and the characteristics of the arginine:ornithine antiporter to arginine and its analogs, we now understand how the combination of arginine uptake and its metabolic conversion is required to build an effective sensing system. In both bacteria and the archaea this is the first chemoeffector molecule of a soluble methylatable transducer to be identified.     

Carrier-mediated transport system for cephalexin in human placental brush-border membrane vesicles

The uptake of cephalosporin antibiotics, cephalexin, was studied with brush-border microvillous plasma membrane vesicles prepared and purified from human full-term placental syncytiotrophoblasts. The uptake of cephalexin by the membrane vesicles was not stimulated in the presence of an Na+ gradient from the outside to the inside of the vesicles, whereas alpha-(methylamino)isobutyrate uptake into the vesicles of the same preparation was stimulated by an Na+ gradient. The equilibrium level of cephalexin uptake decreased with increasing osmolarity of the medium, which indicates that cephalexin is transported into the membrane vesicles. When cephalexin concentrations were varied, the initial rate of uptake obeyed Michaelis-Menten kinetics with Km and Vmax values of 2.29 mM and 2.98 nmol/mg of protein per 60 s, respectively. The uptake of cephalexin was inhibited by structural analogues and sulfhydryl modifying reagents. These results indicate the existence of a carrier-mediated transport system for cephalexin in the human placental brush-border membranes.     

Na+-H+ exchange in cardiac sarcolemmal vesicles

The transport of Na+ by a purified sarcolemmal vesicular preparation from canine ventricular tissue was studied as a function of both internal and external pH. The uptake of Na+ into sarcolemmal vesicles increased upon raising the extravesicular pH of the reaction medium. Half-maximal uptake of Na+ was observed at a pHo of about 8.1 and maximal uptake occurred at pH 8.6. The uptake of Na+ by sarcolemma was also dependent upon the intravesicular pH. Na+ uptake into sarcolemmal vesicles was greatly attenuated in the absence of a H+ gradient across the membrane. Transport of Na+ was potently inhibited by amiloride, a known blocker of Na+-H+ exchange. LiCl was also an effective inhibitor of Na+ transport. In the presence of optimal H+ gradients, Na+ uptake was linear for the first 5 seconds of the reaction and exhibited a Vmax of 290 nmol Na+/mg per min and a KNa of 3.5 mM. These experiments strongly indicate the presence of a Na+-H+ exchange system in cardiac sarcolemma. This activity appeared to be relatively specific for this membrane fraction. The identification of Na+-H+ exchange activity in a sarcolemmal vesicular fraction from the heart will permit extensive characterization of the regulation and kinetics of this antiporter in future investigations.     

Identification of the arginine/ornithine antiporter ArcD from Halobacterium salinarum

This paper identifies the first arginine/ornithine antiporter ArcD from the domain of archea. The functional role of ArcD is demonstrated by transport assays with radioactive labelled arginine, by its necessity to enable arginine fermentation under anaerobic growth conditions and by the consumption of arginine from the medium during growth. All three experimentally observables are severely disturbed when the deletion strain ΔArcD is used. The isolated protein is verified by mass spectrometry and reconstituted in vesicles. The proteoliposomes are attached to a membrane and capacitive currents are recorded which appear upon initiation of the transport process by change from arginine-free to arginine-containing buffer. This clearly demonstrates that the purified 34 kD protein is the functional unit.     

Kinetics Analysis of the Plasma Membrane Sucrose-H+ Symporter from Sugar Beet (Beta vulgaris L.) Leaves

The kinetics behavior of the H+-sucrose (Suc) symporter was investigated in plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves by analyzing the effect of external and internal pH (pHo and pHi, respectively) on Suc uptake. The apparent Km for Suc uptake increased 18-fold as the pHo increased from 5.5 to 7.5. Over this same pHo range, the apparent Vmax for Suc uptake remained constant. The effects of pHi in the presence or absence of internal Suc were exclusively restricted to changes in Vmax. Thus, proton concentration on the inside of the membrane vesicles ([H+]i) behaved as a noncompetitive inhibitor of Suc uptake. The Km for the proton concentration on the outside of the membrane vesicles was estimated to be pH 6.3, which would indicate that at physiological apoplastic pH Suc transport might be sensitive to changes in pHo. On the other hand, the [H+]i for half-maximal inhibition of Suc uptake was approximately pH 5.4, making regulation of Suc transport through changes in [H+]i unlikely. These results were interpreted in the framework of the kinetics models for co-transport systems developed by D. Sanders, U.-P. Hansen, D. Gradmann, and C. L. Slayman (J Membr Biol [1984] 77: 123-152). Based on their analysis, the behavior of the Suc symporter with respect to the [H+]i is interpreted as an ordered binding mechanism by which the binding of Suc on the apoplastic side of the membrane and its release on the symplastic side precedes that of H+ (i.e. a first-on, first-off model).     

Purification and characterization of Ca2+/H+ antiporter from Bacillus subtilis

Ca2+ was accumulated in inside-out membrane vesicles of Bacillus subtilis when NADH was used as an energy source. A delta pH (acid interior) could also drive Ca2+ accumulation in the membrane vesicles and the accumulation was inhibited by carbonylcyanide p-trifluoromethoxyphenylhydrazone and nigericin plus K+. These results indicate the presence of a Ca2+/H+ antiporter (exchanger) in this organism. The antiporter was isolated and purified to homogeneity from the membrane proteins by chromatography on hydroxyapatite, diethylaminoethyl(DEAE)-Toyopearl 650 M and butyl-Toyopearl 650 M. The purified antiporter has a molecular mass of about 45 000 daltons and an isoelectric point of 5.0. The fluorescence quenching of a cyanine dye (3,3'-dipropylthiodicarbocyanine iodide [diS-C3-(5)] during Ca2+ accumulation in proteoliposomes by the purified antiporter showed the generation of a membrane potential (interior negative) suggesting a H+/Ca2+ stoichiometry above 2 in the transport. This was also supported by the result that the K+-diffusion potential, interior positive, stimulated the Ca2+ uptake in the presence of a delta pH. The apparent Km for Ca2+ of the antiporter was about 40 microM and La3+ inhibited the transport. Amino acid analysis of the purified antiporter indicated the presence of large amounts of glutamic and aspartic acids and small amounts of histidine, lysine and arginine. This is consistent with the low isoelectric point (about 5.0) of the protein.     

Modification of the internal pH sensitivity of the Na+/H+ antiporter by parathyroid hormone in a cultured renal cell line

Sodium-proton antiporter activity can be modulated through changes Vmax and/or intracellular proton sensitivity of the antiporter. To characterize a parathyroid hormone (PTH)-induced decrease in antiporter activity in a continuous renal cell line (opossum kidney cells), the extracellular sodium and intracellular proton dependence of amiloride-inhibitable 22Na uptake was studied. The Km for extracellular sodium at intracellular pH 6.32 was 28 mM and was unaltered by PTH, whereas the Vmax was decreased by 26%. When intracellular pH was set over the range 5.87-7.57 by the potassium-nigericin method, antiporter activity increased as intracellular pH decreased. Hill analysis revealed Hill coefficients of 1.25 and 1.01 and half-maximal antiporter activity at intracellular pH values of 6.90 and 6.35 for control and PTH-treated cells, respectively. PTH decreased the apparent Vmax at low pH by 15% and the intracellular pH at which Na+/H+ exchange is half-maximal by 0.55 pH units.     

Ca2+ transport studied with arsenazo III in Tetrahymena microsomes. Effects of calcium ionophore A23187 and trifluoperazine

Transport of Ca2+ in microsomal membrane vesicles of the Tetrahymena has been investigated using arsenazo III as a Ca2+ indicator. The microsomes previously shown to carry a Mg2+-dependent, Ca2+-stimulated ATPase (Muto, Y. and Nozawa, Y. (1984) Biochim. Biophys. Acta 777, 67-74) accumulated calcium upon addition of ATP and Ca2+ sequestered into microsomal vesicles was rapidly discharged by the Ca2+ ionophore A23187. Kinetic studies indicated that the apparent Km for free Ca2+ and ATP are 0.4 and 59 microM, respectively. The Vmax was about 40 nmol/mg protein per min at 37 degrees C. The calcium accumulated during ATP-dependent uptake was released after depletion of ATP in the incubation medium. Furthermore, addition of trifluoperazine which inhibited both (Ca2+ + Mg2+)-ATPase and ATP-dependent Ca2+ uptake rapidly released the calcium accumulated in the microsomal vesicles. These observations suggest that Tetrahymena microsome contains both abilities to take up and to release calcium and may act as a Ca2+-regulating site in this organism.     

The high and low affinity transport systems for dipeptides in kidney brush border membrane respond differently to alterations in pH gradient and membrane potential

The principal aim of the present study was to investigate the effects of variation in proton gradient and membrane potential on the transport of glycyl-L-glutamine (Gly-Gln) by renal brush border membrane vesicles. Under our conditions of transport assay, Gly-Gln was taken up by brush border membrane vesicles almost entirely as intact dipeptide. This uptake was mediated by two transporters shared by other dipeptides and characterized as the high affinity (Kt = 44.1 +/- 11.2 microM)/low capacity (Vmax = 0.41 +/- 0.03 nmol/mg protein/5 s) and low affinity (Kt = 2.62 +/- 0.50 mM)/high capacity (Vmax 4.04 +/- 0.80 nmol/mg protein/5 s) transporters. In the absence of a pH gradient, only the low affinity system was operational, but with a reduced transport capacity. Imposing a pH gradient of 1.6 pH units increased the Vmax of both transporters. Kinetic analysis of the rates of Gly-Gln uptake as a function of external pH revealed Hill coefficients of close or equal to 1, indicating that transporters contain only one binding site for the interaction with external H+. The effects of membrane potential on Gly-Gln uptake were investigated with valinomycin-induced K+ diffusion potentials. The velocity of the high affinity system but not of the low affinity system increased linearly with increasing inside-negative K+ diffusion potentials (p less than 0.01). The Kt of neither system was affected by alterations in either pH gradient or membrane potential. We conclude that (a) the high affinity transporter is far more sensitive to changes in proton gradient and membrane potential than the low affinity transporter and (b) in the presence of a pH gradient, transport of each dipeptide molecule requires cotransport of one hydrogen ion to serve as the driving force.     

Kinetic studies of D-glucose transport in renal brush-border membrane vesicles of streptozotocin-induced diabetic rats

Renal brush-border membrane vesicles prepared from streptozotocin-induced 4-day-diabetic rats possessed a Na+-dependent D-glucose transport system that exhibited apparent Kt and Vmax values about 2-fold greater than normal. Apparently, hyperglycemia and probably other stimuli cause the induction and membrane incorporation of a low-affinity transporter in these membranes; this increased sugar-transport capacity is retained for at least 4 weeks so long as the animals maintained or increased their body weight. Membranes prepared from 28-day-diabetic, severely ill ketoacidotic animals lose this enhanced transport ability and the decrease in Vmax was found to correlate directly with the weight loss. Furthermore, the transporter in brush-border membranes prepared from these cachectic animals had an even lower affinity for glucose than those from the acute hyperglycemic animals. That these changes in the diabetic animals represent major alterations in renal brush-border membrane construction is further supported by our observation that the specific activity of the marker enzymes, alkaline phosphatase and neutral alpha-glucosidase, are profoundly increased and decreased, respectively, in this condition.     

Kinetics of the intestinal brush border proline (Imino) carrier

The kinetics of L-proline transport across intestinal brush borders via the Imino carrier were studied using membrane vesicles. The Imino carrier is defined as the agent responsible for L-alanine insensitive. Na+-dependent uptake of L-proline. Initial rate measurements were made under voltage clamped conditions (pD = 0) to investigate L-proline transport as a function of cis and trans Na+ and proline concentrations. Under zero-trans conditions, increasing cis Na+ activated proline uptake with a Hill coefficient of 1.7 and decreased the apparent Kt with no change in Jimax. The Jimax was approximately 60 pmol mg-1 s-1 and the apparent Kt ranged from 0.25 mM at cis Na = 100 to 1.0 mM at cis Na+ = 30 mM. Trans Na inhibited proline uptake via a reduction in Jimax. Trans proline had no significant effect in the absence of trans Na+, but it relieved the trans Na+ inhibition. Under equilibrium exchange conditions, the Jimax was twice that observed under zero-trans conditions. These kinetics of L-proline transport suggest a model in which uptake occurs by a rapid equilibrium iso-ordered ter ter system. Two Na+ ions bind first to the carrier on the cis face of the membrane to increase the affinity of the carrier for proline. The fully loaded complex then isomerizes to release the substrates to the trans side. The partially loaded Na+-only forms are unable to translocate across the membrane. A rate-limiting step appears to be the isomerization of unloaded carrier from the trans to the cis side of the membrane.     

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.