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Lactobacillus plantarum is often found in nutrient-rich habitats with elevated levels of inorganic carbon (IC), and IC-dependent growth is commonly encountered in natural isolates of this species. High CO(2)-requiring (HCR) prototrophs are unable to grow under conditions of low IC unless arginine and pyrimidines are provided. Prototrophy is restored under high IC conditions, that is in 4% CO(2)-enriched air or bicarbonate-supplemented medium. Bicarbonate is required for the synthesis of carbamoyl phosphate (CP), a precursor of both arginine and pyrimidine biosynthesis. We hypothesize that at low IC levels, intracellular CP pools limit growth through the limitation of arginine and nucleotide supplies. HCR mutants obtained in the laboratory can be classified into 3 functional groups: mutants with impaired CP synthesis, increased CP consumption or increased CP requirements relative to wild type. This classification provides a framework for investigating the origin of the HCR phenotype in natural environmental isolates of Lactobacillus species, and to investigate the hypothesis that a low level of carbamoyl phosphate is a major determinant of the CO(2)-dependent growth phenotype often observed in L. plantarum isolates.

The enzyme adenosine kinase (AK) plays a central role in regulating the intracellular and interstitial concentration of the purine nucleoside adenosine (Ado). In view of the beneficial effects of Ado in protecting tissues from ischemia and other stresses, there is much interest in developing AK inhibitors, which can regulate Ado concentration in a site- and event-specific manner. The catalytic activity of AK from different sources is dependent upon the presence of activators such as phosphate (Pi). In this work we describe several new phosphorylated compounds which either activate or inhibit AK. The compounds acetyl phosphate, carbamoyl phosphate, dihydroxyacetone phosphate and imidodiphosphate were found to stimulate AK activity in a dose-dependent manner comparable to that seen with Pi. In contrast, a number of phosphonate and bisphosphonate derivatives, which included clodronate and etidronate, were found to inhibit the activity of purified AK in the presence of Pi. These AK inhibitors (viz. clodronate, etidronate, phosphonoacetic acid, 2-carboxyethylphosphonic acid, N-(phosphonomethyl)-glycine and N-(phosphonomethyl)iminodiacetic acid), at concentrations at which they inhibited AK, were also shown to inhibit the uptake of (3)H-adenosine and its incorporation into macromolecules in cultured mammalian cells, indicating that they were also inhibiting AK in intact cells. The drug concentrations at which these effects were observed showed limited toxicity to the cultured cells, indicating that these effects are not caused by cellular toxicity. These results indicate that the enzyme AK provides an additional cellular target for the clinically widely used bisphosphonates and related compounds, which could possibly be exploited for a new therapeutic application. Our structure-activity studies on different AK activators and inhibitors also indicate that all of the AK activating compounds have a higher partial positive charge (delta(+)) on the central phosphorous atom in comparison to the inhibitors. This information should prove helpful in the design and synthesis of more potent inhibitors of AK.

In rats, sucrose increases dental caries and impairs odontoblastic function by reducing dentine apposition during primary dentinogenesis. A high-sucrose diet also affects negatively a pulp or dentine function that appears to regulate solute or fluid movement within rat dentinal tissue. In earlier work it was found that carbamyl phosphate could significantly reverse sucrose-induced cariogenesis and also stimulate sucrose-depressed movement of dentinal fluid through a mechanism involving parotid function(s). In the current study, the possibility that carbamyl phosphate could overcome the sucrose-induced reduction in dentine apposition was examined. Weanling rats were fed a high-sucrose diet supplemented or not with carbamyl phosphate for 5 weeks. Dentine apposition was measured planimetrically in sagittal sections of the molars. The effect of carbamyl phosphate was similarly tested in parotidectomized animals. Carbamyl phosphate significantly reduced the deleterious effect of sucrose on dentine apposition by 58% in the first molars. However, the reduction in dentine apposition that followed parotidectomy was not altered by carbamyl phosphate supplementation. The possibility that the beneficial effect of carbamyl phosphate on dentinogenesis involves a parotid function is entertained.

Human liver ornithine carbamoyltransferase undergoes absorbance changes in the UV region upon formation of the carbamoylphosphate-norvaline-enzyme ternary complex. The UV changes are similar in the presence of carbamoylphosphate alone, whilst they are lower in the presence of ornithine or norvaline alone. The extent of the UV changes correlates with the enzyme susceptibility to proteolytic degradation. The free native enzyme is completely and rapidly hydrolyzed by trypsin, whilst it is partially protected upon carbamoylphosphate binding. The extent of protection increases for the carbamoylphosphate-norvaline-enzyme ternary complex. These results strongly suggest that the binding of the first substrate, i.e. carbamoylphosphate, to human ornithine carbamoyltransferase induces a large protein isomerization, which regards the polar domain plus a part of equatorial domain of each subunit.

A revised and simplified purification scheme for aspartate transcarbamoylase (ATCase) from wheat-germ is reported, with an eightfold increase in scale (yielding approximately 10 mg of the pure protein from 4 kg of wheat-germ), and improved characteristics of stability and regulatory kinetics. The ATCase obtained is greater than 96% pure, as judged by polyacrylamide gel electrophoresis. The long-term stability (i.e. on a time-scale of several hours to weeks) of the activity of the purified enzyme, under various storage conditions, was investigated. At 4 degreesC and pH 7.5, stability was found to be strongly dependent on protein concentration (increased stability at high concentration), buffer concentration (decreased stability at high buffer concentration) and the inclusion of glycerol (increased stability with increasing glycerol concentration). The enzyme is routinely stored at 4 degreesC, in 0. 05 m Tris/HCl buffer containing 25% glycerol and at high protein concentration (approximately 1 mg.mL-1, or 10 microm in trimers). Under these conditions, the half-life of the enzyme activity is greater than 300 days. Over the time-scale of kinetic experiments (up to 20 min), the diluted activity (at around 1 nm of ATCase, in the presence of ligands) is completely stable. The specific activity remains constant in the range 0.1-10 nm, in the absence and presence of ligands, showing that dissociation of the trimeric enzyme into its subunits is negligible. Steady-state kinetics were examined using the enzyme at a concentration of 1.3 nm. Initial-rate curves for both allosteric ligands, carbamoylphosphate (CP) and uridine 5'-monophosphate (UMP), showed pronounced sigmoidicity, each in the presence of the other. In the absence of UMP, initial-rate curves for CP are hyperbolic. The initial rate data fit reasonably well to a trimeric Monod-Wyman-Changeux model, suggesting a two-state conformational mechanism, greatly favouring the active (R) state when both ligands are absent, in which the R-state binds CP exclusively (dissociation constant = 23.2 microm), and the T-state binds UMP exclusively (dissociation constant = 0.49 microm). This regulatory behaviour was found to be quite stable, and was indistinguishable from that of the enzyme in a freshly made crude extract, even after storage of the pure sample for 5 months. This enzyme preparation is therefore free of the anomalous allosteric kinetics produced by a previous purification scheme, in which the affinity for UMP was markedly reduced, CP rate curves showed no sigmoidicity, while UMP rate curves had sigmoidicity exaggerated by a low maximum.

The kinetic and regulatory properties of aspartate carbamoyltransferase (ACTase) of the human pathogen Helicobacter pylori were studied in situ in cell-free extracts. The presence of enzyme activity was established by identifying the end product as carbamoylaspartate using nuclear magnetic resonance spectroscopy. Activity was measured in all strains studied, including recent clinical isolates. Substrate saturation curves determined employing radioactive tracer analysis or a microtiter colorimetric assay were hyperbolic for both carbamoyl phosphate and aspartate, and there was no evidence for substrate inhibition at higher concentrations of either substrate. The apparent Km were 0.6 and 11.6 mm for carbamoyl phosphate and aspartate, respectively. Optimal pH and temperature were determined as 8.0 and 45 degrees C. Activity was observed with the l- but not the d-isomer of aspartate. Succinate and maleate inhibited enzyme activity competitively with respect to aspartate. The carbamoyl phosphate analogues acetyl phosphate and phosphonoacetic acid inhibited activity in a competitive manner with respect to carbamoyl phosphate. With limiting carbamoyl phosphate purine and pyrimidine nucleotides, tripolyphosphate, pyrophosphate, and orthophosphate inhibited competitively at millimolar concentrations. Ribose and ribose 5-phosphate at 10 mm concentration showed 20 and 35% inhibition of enzyme activity, respectively. N-Phosphonoacetyl-l-aspartate (PALA) was the most potent inhibitor studied, with 50% inhibition of enzyme activity observed at 0.1 microM concentration. Inhibition by PALA was competitive with carbamoyl phosphate (Ki = 0.245 microM) and noncompetitive with aspartate. The kinetic and regulatory data on the activity of the H. pylori enzyme suggest it is a Class A ACTase, but with some interesting characteristics distinct from this class., (Copyright 1997 Academic Press.)

Treatment of ornithine carbamoyltransferase from dolphin Stenella with pyridoxal phosphate, followed by reduction with NaBH4 resulted in complete loss of enzyme activity. The phosphate alone or the substrate analogue 2-aminovaleric acid moderately decreased the extent of inactivation, while carbamoyl phosphate plus 2-aminovaleric acid provided complete protection from inactivation. The partially inactivated enzyme showed K(m) values for substrates equivalent to those of native enzyme and lowered Kcat values. Two lysyl residues were substantially modified in the absence of ligands but only one of them was responsible for the inactivation of catalytic activity. Modification of a single subunit was sufficient to completely abolish the catalytic activity of the trimeric enzyme. The lysine involved has been identified as lysine 56 on the known primary structure of homologous human liver enzyme.

ArcA is one of several transcription factors required for optimal anaerobic induction of the pyruvate formatelyase (pfl) operon. To aid the study at the molecular level of the interaction of ArcA with the pfl promoter-regulatory region we developed a procedure for the isolation of ArcA. The purification of ArcA involved chromatography in heparin agarose, hydroxylapatite and Mono-Q matrices and delivered a protein that was > 95% pure. Gel retardation assays demonstrated that ArcA bound specifically to the pfl regulatory region. Three distinct ArcA-DNA complexes could be resolved depending on the ArcA concentration used. This finding suggested that either multiple ArcA-binding sites are present in the regulatory region or that ArcA can oligomerize at one or more sites. The DNA-binding activity of ArcA could be increased as estimated 10-fold by prior incubation of the protein with carbamoyl phosphate, suggesting that phosphorylation activates DNA binding or oligomerisation. DNase I footprint analyses identified four sites that were protected by ArcA from cleavage. Two of these sites spanned the transcription start site and -10 regions of promoters 6 and 7, while a third site partially overlapped the characterized binding site of integration host factor (IHF). ArcA exhibited the highest affinity for a stretch of DNA located between the IHF site and the transcription start site of promoter 7. These results are congruent with the hypothesis that a higher-order nucleoprotein complex comprising several proteins, including ArcA, is required to activate transcription from the multiple promoters of the pfl operon.

The quaternary structural change of Escherichia coli aspartate transcarbamylase (ATCase) was studied by time-resolved X-ray solution scattering following the binding of carbamoyl phosphate and of succinate, a competitive inhibitor of the natural substrate L-aspartate. Stopped-flow experiments at sub-zero temperatures in the presence of 30% ethylene glycol allowed us to monitor the evolution of the scattering pattern, including the characteristic scattering peak in an s (=2 sin theta/lambda) range of 0.01-0.06 A-1. The inhibitor binding promotes a quaternary structure change from the T state toward the R state, and as expected for a simple ligand binding process, ATCase remains in the R state, unlike the physiological enzyme reaction [Tsuruta, H., et al. (1990) FEBS Lett. 263, 66-68]. After equilibrium had been established, the final scattering pattern was recorded. When the succinate concentration was sufficiently high, this pattern was the same as that given by ATCase saturated with the bisubstrate analogue N-phosphonoacetyl-L-aspartate (PALA). This implies that, under cryogenic conditions, succinate and carbamoyl phosphate promote the same quaternary structure change as PALA, which is in good agreement with the crystallographic studies of Gouaux and Lipscomb [Gouaux, J.E., & Lipscomb, W.N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4205-4208]. Scattering patterns recorded during the course of the structural transition were satisfactorily reproduced by a linear combination of the initial and final patterns, suggesting that there is no significant concentration of quaternary structure intermediates between the T and R states. This is consistent with a concerted structural transition of ATCase.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined the pathway and energetics of urea-induced dissociation and unfolding of the catalytic trimer (c3) of aspartate transcarbamylase from Escherichia coli at low temperature in the absence and presence of carbamyl phosphate (CP; a substrate), N-(phosphonacetyl)-L-Asp (PALA; a bisubstrate analog), and 2 anionic inhibitors, Cl- and ATP, by analytical gel chromatography supplemented by activity assays and ultraviolet difference spectroscopy. In the absence of active-site ligands and in the presence of ATP, c3 dissociates below 2 M urea into swollen c chains that then gradually unfold from 2 to 6 M urea with little apparent cooperativity. Linear extrapolation to 0 M urea of free energies determined in 3 independent types of experiments yields estimates for delta Gdissociation at 7.5 degrees C of about 7-10 kcal m-1 per interface. delta Gunfolding of dissociated chains when modeled as a 2-state process is estimated to be very small, on the order of -2 kcal m-1. The data are also consistent with the possibility that the unfolding of the dissociated monomer is a 1-state swelling process. In the presence of the ligands CP and PALA, and in the presence of Cl-, c3 dissociates at much higher urea concentrations, and trimer dissociation and unfolding occur simultaneously and apparently cooperatively, at urea concentrations that increase with the affinity of the ligand.

We studied the effect of carbamoylphosphate (CP) on L-aspartate aminotransferase (GOT) and L-alanine aminotransferase (GPT), compared to its effect on L-threonine deaminase (TD). GPT and GOT were slightly inhibited by CP, while TD was strongly inhibited. GPT and TD, but not GOT, were inactivated when preincubated with CP. Only GOT was enhanced by pyridoxal 5'-phosphate (PLP), but not when the coenzyme was preincubated with CP. When the enzymes were resolved by p-chloromercuribenzoate (PCMB) treatment to apoenzymes, only GOT retained 47% of the original activity. Reconstitution of the apoenzymes with PLP also followed different course; activities of GPT and TD were completely restored while GOT remained partially inactivated. Treatment of apoenzymes with CP resulted in impairment of their reconstitution except GPT, activity of which could be completely restored. When PLP was pre-treated with CP before reconstitution, however, even GPT was only partially restored. The data indicated that CP affect activities of these enzymes at different levels, holoenzymes, PLP and probably apoenzymes. Under a concentration of PLP, activity of GOT would be most enhanced, followed by TD then GPT. In the presence of CP, this effect would be eliminated.

AlgR is a transcriptional regulator of mucoidy in Pseudomonas aeruginosa, a critical virulence factor expressed in cystic fibrosis. AlgR belongs to the superfamily of bacterial signal transduction systems, and has been shown to bind to the algD promoter, a critical point in the regulation of mucoidy. This protein, like other typical response regulators, contains highly conserved residues known to be critical for the phosphorylation and signal transduction processes. However, a typical second component interacting with AlgR has not been identified. Here we demonstrate that AlgR undergoes phosphorylation in vitro when interacting with the well-characterized histidine protein kinase CheA. These results indicate that AlgR is capable of undergoing phosphorylation typical of other two-component signal transduction systems. Moreover, the phosphotransfer reaction between CheA and AlgR was found to be affected by the presence of carbamoyl phosphate, acetyl phosphate, and salts of phosphoramidic acid, recently shown to act as small-molecular-weight phospho-donors in the process of phosphorylation of several response regulators. These findings suggest that AlgR may react with intermediary metabolites such as carbamoyl phosphate and acetyl phosphate, and that these processes may play a role in the control of mucoidy in P. aeruginosa.

Transcription of the Ntr regulon is controlled by the two-component system consisting of the response regulator NRI (NtrC) and the kinase/phosphatase NRII (NtrB), which both phosphorylates and dephosphorylates NRI. Even though in vitro transcription from nitrogen-regulated promoters requires phosphorylated NRI, NRII-independent activation of NRI also occurs in vivo. We show here that this activation likely involves acetyl phosphate; it is eliminated by mutations that reduce synthesis of acetyl phosphate and is elevated by a mutation expected to cause accumulation of acetyl phosphate. With purified components, we investigated the mechanism by which acetyl phosphate stimulates glutamine synthetase synthesis. Acetyl phosphate, carbamyl phosphate, and phosphoramidate but not ATP or phosphoenolpyruvate acted as substrates for the autophosphorylation of NRI in vitro. Phosphorylated NRI produced by this mechanism exhibited the properties associated with NRI phosphorylated by NRII, including the activated ATPase activity of the central domain of NRI and the ability to activate transcription from the nitrogen-regulated glutamine synthetase glnAp2 promoter.

The effects exerted by carbamoyl phosphate (CP) and cyanate (KCNO) on rat liver L-threonine deaminase have been studied. The two compounds showed that same effects, inhibiting through a competitive mechanism both the holoenzyme and the dialyzed enzyme; inhibition was more evident for the latter. Ki values, both for L-threonine and pyridoxal 5'-phosphate (PLP), were lower for the apoenzyme and the inhibitors also affected the Km of the apoenzyme for PLP. The effects of CP and KCNO are mainly due to an interference in the association reaction apoenzyme + PLP in equilibrium holoenzyme This was clearly demonstrated by the fact that, when PLP was incubated with CP or KCNO, it failed to enhance the activity of the holoenzyme nor did it reactivate the resolved apoenzyme. Such interference of CP and KCNO in the L-threonine deaminase activity was mainly due to a specific mechanism, the formation of a new derivative of PLP. The reaction of PLP with either CP or KCNO occurred readily, at low concentrations, under physiological conditions. The new compound was identified as 3,4-dihydro-2H-pyrido[3,4-e]1,3-oxazin-2-one derivative by ultraviolet-visible spectra, elemental analysis, infrared, NMR and MS spectra. In this paper we formulate the hypothesis that this compound is involved in the regulation of the CP and PLP intracellular content and in the control of PLP dependent enzymes.

The sigmoidal dependence of activity on substrate concentration exhibited by the regulatory enzyme aspartate transcarbamoylase (ATCase) of Escherichia coli is generally attributed to a ligand-promoted change in the quaternary structure of the enzyme. Although a global conformational change in ATCase upon the binding of ligands to some of the six active sites is well documented, a corresponding alteration in the structure of the wild-type enzyme upon the addition of the inhibitor, CTP, or the activator, ATP, has not been detected. Such evidence is essential for testing whether heterotropic, as well as homotropic, effects can be accounted for quantitatively in terms of coupled equilibria involving a conformational change in the enzyme and preferential binding of ligands to one conformation or the other. This evidence has now been obtained with a mutant form of ATCase in which Lys 143 in the regulatory chain was replaced by Ala, thereby perturbing interactions at the interface between the regulatory and catalytic chains in the enzyme and destabilizing the low-activity, compact (T) conformation relative to the high-activity, swollen (R) state. Difference sedimentation velocity experiments involving measurements of the changes caused by the binding of the bisubstrate analogue N-(phosphonacetyl)-L-aspartate demonstrated that the sedimentation coefficient of the mutant enzyme was intermediate between that observed for the T and R states of wild-type ATCase. We interpret the results as indicating that the [T]/[R] ratio in phosphate buffer at pH 7.0 is reduced from about 2 X 10(2) for the wild-type enzyme to 2.7 for r143Ala ATCase.(ABSTRACT TRUNCATED AT 250 WORDS)

We have adapted a chick embryo liver cell system for studying the synthesis of proteins secreted by hepatocytes. In primary liver cell cultures maintained for several days in arginine-deficient medium containing ornithine (0.7 mM) and carbamyl phosphate (1 mM), only hepatocytes demonstrated normal morphological and biosynthetic characteristics, indicating that they possessed a functional ornithine cycle as a source of arginine production. Non-parenchymal liver cells, such as fibroblasts, which lack the ornithine cycle were excluded. Hepatocytes in arginine-deficient or arginine-containing medium synthesized fibronectin (Fn) over several days at a constant rate of 3 micrograms +/- 1 microgram/mg cell protein per day, with fibronectin representing approximately 3% of the total secreted hepatocyte proteins during any culture period after the first 24 h. Pulse-chase experiments indicated that Fn synthesis and secretion was relatively rapid (t1/2 = 45 min) and represented approximately 95% of the intracellularly labelled Fn. This Fn is secreted predominantly as a 450 kD dimer with a subunit size that is indistinguishable from the plasma form as assessed by one-dimensional electrophoretic analysis. Continuous exposure of hepatocytes to insulin caused a moderate decrease (26%) in Fn synthesis, whereas there was no effect of short-term exposure. In contrast, dexamethasone stimulated Fn production 2-3-fold, consistent with its known ability to stimulate hepatocyte production of acute phase proteins. Under these conditions, electrophoretic analyses showed that an increased quantity of intact hepatocyte Fn was produced having the same molecular size of plasma Fn.

The regulation of the molybdate (MoO42-) transport activity of Clostridium pasteurianum has been studied by observing the effects of NH3, carbamyl phosphate, MoO42-, and chloramphenicol on the ability of cells to take up MoO42-. Compared with cells fixing N2, cells grown in the presence of 1 mM NH3 are greater than 95% repressed for MoO42- transport. Uptake activity begins to increase just before NH exhaustion (under Ar or N2) and continues to increase throughout the lag period as cells shift from NH3-growing to N2-fixing conditions. When cells are shifted from N2-fixing to NH3-growing conditions the transport activity per fixed number of cells decreases by increase of bells in absence of transport synthesis. Carbamyl phosphate (greater than or equal to 15 mM) but not NH3 inhibits 58% of the in vitro uptake activity. When 1 mM carbamyl phosphate is added just before the exhaustion of NH3, the transport activity, measured 2 h later, is 100% repressed. Cells grown in the presence of high MoO42- (1mM) are 80% repressed for MoO42- transport. Synthesis of the MoO42- transport system is also completely stopped when chloramphenicol (300 mug/ml) is added just before the exhaustion oNH 3 from the medium. These findings demonstrate that the ability of cells to transport MoO42- is dependent upon new protein synthesis and can be repressed by high levels of substrate. The regulation of MoO42- uptake by NH3 or carbamyl phosphate closely parallels the regulation of nitrogenase activity. Activity of neither nitrogenase component (Fe protein or MoFe protein) was detected even 3 h after the exhaustion of the NH3 if either MoO42- was absent or if WO42- was present in place of MoO42-. The duration of the diauxic lag increases with decreasing concentration of MoO42- in the medium. If no MoO42- is present the lag continues indefinitely. If MoO42- is added late in the lag period, growth under N2-fixing conditions resumes but only after a normal induction period.

Ornithine transcarbamoylase (OTC) is inactivated by liver lysosomes. Carbamoyl phosphate prevents the inactivation of OTC by lysosomes, while ATP, ADP, GTP, GDP 1,N6-ethenoadenosine 5'-triphosphate and particularly epsilon-ATP stimulate it. Both stimulation and protection occur at concentrations within the physiological range of ATP and carbamoyl phosphate. Inactivation of OTC is followed by extensive proteolysis. Since the inactivation is prevented by leupeptin, antipain and L-(tosylamido-2-phenyl)ethylchloromethyl ketone, the proteolytic susceptibility of OTC to lysosomes could be due to thiol endopeptidase(s). 1,N6-Ethenoadenosine 5'-triphosphate also markedly increases OTC susceptibility to trypsin and elastase. ATP analogs had no stimulatory effect on OTC inactivation by lysosomes; none of the inhibitors of ATPases tested inhibited the ATP effect. The ATP stimulation does not require Mg2+. These findings indicate a new role for ATP, GTP and related nucleotides in protein breakdown. The ATP, ADP, GTP, GDP stimulation, together with the carbamoyl protection of OTC, agree well with the molecular plasticity hypothesis model.

beta,gamma-Methylene adenosine triphosphate (AMPOPCP) has two effects on fragmented sarcoplasmic reticulum (FSR), i.e., inhibition of the rate of Ca uptake and the induction of Ca release from FSR filled with Ca. The Ca release brought about by AMPOPCP has many features in common with the mechanism of Ca-induced Ca release: i) it is inhibited by 10 mM procaine; ii) the amount of Ca release increases with increase in the extent of saturation of FSR with Ca; iii) increase of the Ca concentration in the extent of saturation of FSR with Ca; iii) increase of the Ca concentration in the medium facilitates the release of Ca. However, no facilitation of Ca release upon decrease of Mg concentration in the medium is observable. AMPOPCP and caffeine potentiate each other remarkably in their Ca-releasing action, irrespective of the kind of substrate. From the mode of action of AMPOPCP on the rate of Ca uptake, the amount of phosphorylated intermediate (EP), and the effect on Sr release, it is suggested that the state of the FSR-ATP complex is crucial for Ca-induced Ca release.

A sensitive radiometric enzyme-inhibition assay is described for the determination of N-phosphonacetyl-L aspartic acid (PALA, NSC-224,131) in plasma. The assay is based on the inhibition of PALA, at low concentrations of the competitive co-substrate carbamyl phosphate, of L-aspartate transcarbamylase prepared from mouse spleen. The assay responds with good reproducibility and in a proportional way to concentrations of PALA over the range of 2-20 nM; as such, it represents a 100-fold increase in sensitivity over existing methodologies. The applicability and pertinence of this assay to long-term pharmacokinetic studies with PALA is demonstrated by the one month persistence of nanomolar concentrations of PALA in the plasma of mice treated with a single dose of the drug.

Intra-arterial infusion of carbamyl phosphate or of carbamyl-DL-aspartic acid into rats on a cariogenic diet greatly stimulated the movement of fluid through the odontoblastic processes. The infusion of sodium cyanate also stimulated fluid movement. Guanidine HCL and L-asparagine were active at higher concentrations. Purifying the urea on a mixed-bed ion exchange resin virtually removed its stimulatory effect on dentinal fluid movement. The action of urea is apparently attributable to contamination with sodium cyanate.

Formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) from Clostridium cylindrosporum catalyzes phosphate transfer from carbamyl phosphate to ADP. This activity is lost when monovalent cations are removed and is recovered when K+ is added back. Carbamyl phosphate is an inhibitor of the formyltetrahydrolfolate synthetase forward reaction, and formate as well as phosphate inhibit the ATP synthesis reaction. Acetyl phosphate and phosphonoacetate are inhibitors of both reactions. The results of kinetic studies support the concept that carbamyl phosphate is an analog of the putative intermediate of the formyltetrahydrofolate synthetase reaction, formyl phosphate.

In eight patients with sickle cell anemia, weekly extracorporeal carbamylation of about 20% of the circulating red cell mass was carried out for 2 yr or longer. At each visit, a mean of 1.3+/-0.2 mol of cyanate were incorporated per mole of hemoglobin in the carbamylated erythrocytes. Within 3 mo, a stable level of about 35-50% of the circulating erythrocytes was carbamylated. This quantity and degree of hemoglobin carbamylation produced a decrease in mean whole blood P50 from 33 to 26 mm Hg. During the first 3 mo of carbamylation, the mean hemoglobin increased from 6.4 to 9.1 g/100 ml, while mean absolute reticulocytes decreased by 58% and circulating irreversibly sickled erythrocytes decreased by 65%. The mean red cell life span increased from 13 days before treatment to 21.6 days after 3 mo of carbamylation. Beyond the 3rd mo of carbamylation, blood P50, hemoglobin, and reticulocytes remained quite stable. No toxic effects of extracorporeal carbamylation of erythrocytes were noted. The capacity of blood to release oxygen at 30 mm Hg PO2 increased from 4.3 to 5.0 cm3/100 ml blood during carbamylation. The overall frequency of severe painful crises decreased by about 80% during carbamylation. Before carbamylation, 34% of the crises were induced by a concomitant illness, usually an infection. During carbamylation, the incidence of induced crises decreased 50% while spontaneous crises virtually disappeared. The marked improvements in hematologic parameters and the decreased frequency of severe painful crises observed during this study offer sufficient promise to warrant further exploration, hopefully using more efficient techniques, of the clinical efficacy of extracorporeal erythrocyte carbamylation in sickle cell anemia.

Although the allosteric enzyme aspartate transcarbamoylase from Escherichia coli has been the focus of numerous physical and enzymological studies for over 2 decades, the catalytic mechanism is still poorly understood. There has been much speculation regarding the role of a conserved histidine residue at position 134 which recent crystallographic studies have implicated in the catalytic mechanism as a general acid or as a general base. We have used a combination of site-directed mutagenesis, 13C-isotope incorporation, and high field NMR to probe the role of His134 in the catalytic mechanism of aspartate transcarbamoylase. By comparing the wild-type catalytic trimer with that from a partially active mutant in which His134 is replaced by alanine, we have assigned the 13C resonance for His134 in the wild-type enzyme. This residue is shown to have a pK less than 6, indicating that the imidazole ring is unprotonated at pH values optimal for enzymatic activity (pH 8.0). This result eliminates the possibility of His134 participating as a general acid in the carbamoyl transfer mechanism. Since the crystallographic studies indicate that His134 is close enough to hydrogen-bond to the carbonyl of the liganded bisubstrate analog N-(phosphonacetyl)-L-aspartate, the imidazole ring would be oriented as to make it unlikely that the N1 lone pair of electrons could participate in general base catalysis. Moreover, if His134 is implicated in base catalysis, we would have expected a much greater loss of activity upon its replacement by alanine. Perhaps the role of His134 is merely to help position the carbonyl group of carbamoyl phosphate for nucleophilic attack by the alpha-amino group of aspartate.

The release of Ca by quercetin from the sarcoplasmic reticulum has been claimed to be a result of the well-known inhibition of Ca2+-ATPase activity, or to be due to an intrinsic property of quercetin. To get a clearer understanding of the effect of quercetin, we examined it using fragmented sarcoplasmic reticulum (FSR) from bullfrog skeletal muscle. The rapid phase of Ca release (hereafter simply referred to as "Ca release") from loaded FSR was almost completed within 5 s after addition of quercetin in the presence of ATP. It cannot be ascribed to the inhibition of Ca2+-ATPase activity on the basis of following findings. First, when Ca uptake was driven by carbamylphosphate, no or little Ca release was observed in marked contrast to a stronger reduction in the rate of Ca uptake. Secondly, procaine reverses the Ca releasing action of quercetin, whereas it show a synergistic action in the inhibition of Ca2+-ATPase activity. Thirdly, HFSR released more Ca than LFSR, while the Ca2+-ATPase activities of both fractions were inhibited to a similar extent. The Ca release by quercetin is enhanced by ATP or beta, gamma-methylene adenosine triphosphate, and decreased by procaine or a high concentration of Mg2+. In the presence of 2.5 mM caffeine, the amount of Ca2+ released by quercetin was decreased, and the dose-effect relationship was shifted to higher doses of quercetin. This indicates that quercetin and caffeine probably overlap in the site(s) of the action, but that quercetin is dissimilar from halothane in the mode of its Ca-releasing action.

Ornithine transcarbamylase from Escherichia coli, strain W, exhibits negative cooperativity with respect to ornithine, and the enzymatic activity is further regulated by orotate. The effect of orotate on ornithine transcarbamylase is dependent not only upon the carbamylphosphate concentration, but also upon the concentration of ornithine. At high concentrations of carbamylphosphate (10 mM), a conversion from negative cooperativity to positive cooperativity is observed with 10 mM orotate. At 1 mM carbamylphosphate, however, 10 mM orotate activates the enzyme at low ornithine concentrations, but as the ornithine concentration is increased above 5 mM, inhibition is observed. Thus, a regulatory link has been established between the pathways of arginine biosynthesis and pyrimidine biosynthesis, each of which utilizes carbamylphosphate.

The interactions of Pi, PPi, and carbamyl-P with the hepatic glucose-6-phosphatase system were studied in intact and detergent-disrupted microsomes. Penetration of PPi and carbamyl-P into intact microsomes was evidenced by their reactions with the enzyme located exclusively on the luminal surface. Lack of effects of carbonyl cyanide m-chlorophenylhydrazone and valinomycin + KCl indicated that pH gradients and/or membrane potentials that could influence the kinetics of the system are not generated during metabolism of PPi and glucose-6-P by intact microsomes. With disrupted microsomes, only competitive interactions were seen among glucose-6-P, Pi, PPi, and carbamyl-P. With intact microsomes, Pi, PPi, and carbamyl-P were relatively weak, noncompetitive inhibitors of glucose-6-phosphatase, and PPi hydrolysis was inhibited competitively by Pi and carbamyl-P but noncompetitively by glucose-6-P. Analysis of the kinetic data in combination with findings from other studies that a variety of inhibitors of the glucose-6-P translocase (T1) does not affect PPi hydrolysis provide compelling evidence that permeability of microsomes to Pi, PPi, and carbamyl-P is mediated by a second translocase (T2). Some properties of the microsomal anion transporters are described. If the characteristics of the glucose-6-phosphatase system as presently defined in intact microsomes apply in vivo, glucose-6-P hydrolysis appears to be the predominant, if not the exclusive, physiologic function of the system. Both the "noncompetitive character" and the relative ineffectiveness of Pi as an inhibitor of glucose-6-phosphatase of intact microsomes result from the rate limitation imposed by T1 that prevents equilibration of glucose-6-P across the membrane. In microsomes from fed rats, where T1 is less rate restricting, about one-half as much Pi was required to give 50% inhibition compared with microsomes from fasted or diabetic rats. Thus, any treatment or agent that alters the kinetic relationship between transport and hydrolysis of glucose-6-P (e.g. endocrine or nutritional status) is an essential consideration in analyses of kinetic data for the glucose-6-phosphatase system.

No inhibition of nitrogenase activity by physiological levels of NH4+ or carbamyl phosphate was observed in extracts of Azotobacter vinelandii. All of the 15N2 reduced by cultures which received no NH4+ was found in the cells. By contrast, more than 95% of the 15N2 reduced by cultures which had been given NH4+ was found in the medium. Failure to examine the culture medium would lead to the erroneous conclusion that N2 fixation is inhibited by NH4+. Nitrogenase in a derepressed mutant strain of A. vinelandii was fully active in vivo in the presence of NH4+. The addition of NH4Cl to N2-fixing cultures resulted in no decrease in the N2-reducing activity of intact cells of Klebsiella pneumoniae or Clostridium pasteurianum and only a small (15%) decrease in A. vinelandii. Therefore, no significant inhibition of nitrogenase by NH4+ or metabolites derived from NH4+ exists in A. vinelandii, K. pneumoniae, or C. pasteurianum.

Equine muscle carbonic anhydrase (CA-III) behaves like ubiquitin in undergoing extensive acylation of N epsilon-lysine residues upon reacting with p-nitrophenyl esters. The enzyme undergoes extensive carbamoylation of lysine residues when reacted with carbamoyl phosphate. The modification of from 6 to 7 lysine residues results in the production of a series of more anodic electrophoretic components. The derivatization of the lysine residues leads to a marked decrease in the enzyme's ability to hydrate CO2. The equine CA-III possesses both acid and alkaline phosphatase activities in contrast to the rabbit which possesses only the former type.

Two carbamylating agents, carbamyl phosphate and sodium cyanate, are currently being evaluated as therapeutic drugs for the treatment of sickle cell anemia. Since the clinical usefulness of these drugs might be limited by toxicity, a comparative in vitro study of the relative inhibition of human sickle cell and rabbit reticulocyte protein synthesis was performed. Sodium cyanate was found to inhibit human sickle cell protein synthesis at concentrations one-eighth that of carbamyl phosphate. Carbamyl phosphate lowered reduced glutathione levels to a similar degree as sodium cyanate and was slightly more effective as an in vitro antisickling agent, which is evidence that carbamyl phosphate enters these cells. When rabbit reticulocyte protein synthesis was investigated, carbamyl phosphate was found to be noninhibitory at concentrations as high as 128 mol/mol of hemoglobin in the incubation medium, while cyanate was inhibitory at much lower concentrations. The effect of carbamyl phosphate on glutathione concentrations, however, was virtually identical to that of cyanate. These results show that the effect on protein synthesis does not result from the lowered glutathione levels. It is concluded that both the therapeutic and toxic effects of sodium cre, there might be differences in toxicity between animals and humans.

The effect of oral administration of dilithium carbamyl phosphate on adult beagle dogs is dose related. The levels of carbamylation as mol valine hydantoin/mol hemoglobin tetramer reaches a plateau of 0.3-0.38 with daily doses of 100 mg/kg body weight. Related changes in oxygen binding by whole blood and hemoglobin amount to a 5-15% left shift in oxygen isotherms. After discontinuing the administration of carbamyl phosphate, the disappearance of modified hemoglobin with a return to normal oxygen binding values follows the gradual replacement of old cells by new cells in the circulation. When the level of in vivo carbamylation in dog blood is greater than 0.16, it is similar to the levels of in vitro carbamylation of hemoglobin SS which result in the interference with sickling of erythrocytes at low levelsof oxygen.

The effects of several organic and inorganic nitrogen compounds on nitrogenase mRNA and enzyme activity levels were examined in anaerobic cultures of Anabaena variabilis 29413. Even low concentrations of exogenous ammonia (20 microM) prevented nitrogenase gene expression. Nitrate, in contrast, had little effect, even at very high concentrations. Neither compound had a significant direct effect on existing enzyme activity. The amino acids glutamine and glutamate did not repress nif gene expression. Methionine sulfoximine, but not 7-azatryptophan, was shown to eliminate the repressive effect of ammonia, and this action occurred at the mRNA level. Low concentrations of carbamyl phosphate caused a rapid decrease in nitrogenase mRNA levels. These results are consistent with the ideas that nif gene regulation in Anabaena spp. occurs primarily at the mRNA level and that ammonia, and possibly also glutamine and glutamate, is not the immediate effector of regulation.

Aspartate transcarbamoylase from Escherichia coli is composed of six catalytic (c) and six regulatory (r) polypeptides. We have studied the structure and function of this enzyme using chymotrypsin as a probe. The protease inactivates the isolated catalytic subunit (c3) but has not effects on the native enzyme (c6r6). Under identical conditions, the c3r6 complex is inactivated at a much slower rate than c3. The presence of the substrate analogue succinate together with carbamoyl phosphate reduces substantially the rate of inactivation. Extended exposure to chymotrypsin converts the catalytic subunit into a partially active derivative with a fourfold higher Michaelis constant. This derivative is indistinguishable from the unmodified catalytic subnit in gell electrophoresis under nondenaturing conditions. However, in the presence of sodium dodecyl sulfate, the major fragment in the electropherogram is smaller than that of the intact catalytic polypeptide. The results could be explained by postulating the presence of a chymotrypsin-sensitive peptide bond at or near the active site. Since X-ray crystallographic studies have indicated that the active sites are located in a central cavity, the resistance of the native enzyme towards inactivation may be due to the inability of chymotrypsin to enter this cavity.

Carbamoyl phosphate has been shown to inhibit carbonic anhydrase (CA) isozymes CA I, CA II and CA III. This physiologically important molecule is the most potent, naturally occurring inhibitor of carbonic anhydrase yet found. It is also unique, among carbonic anhydrase inhibitors discovered hitherto, in that it inhibits the 3 isozymes with equal effect, despite their strikingly different properties. The results imply the participation of carbonic anhydrase in the regulation of substrate availability for the urea cycle.

Carbamoyl phosphate inhibited acetylene reduction by whole cells and cell-free extracts of Anabaena cylindrica. Higher levels of both endogenous carbamoyl phosphate and carbamoyl phosphate synthase activity were present in NH4+-grown cells (in which acetylene reduction was absent) than in N2-grown cells (in which acetylene reduction was present). However, inhibition of acetylene reduction was observed also with cyanate, the main initial decomposition product under the conditions used. It is concluded that carbamoyl phosphate or one of its metabolites may act as a physiological regulator of both nitrogenase activity and synthesis, but caution must be used in interpreting effects observed several hours after the addition of carbamoyl phosphate, because the effects may be due to cyanate.

Ornithine transcarbamylase (OTC) is very stable in rat liver homogenates, or mitochondria. However, pure OTC from rat or beef liver is inactivated by broken lysosomes. ATP and a number of purine nucleotides, particularly epsilon-ATP, stimulate the inactivation of OTC by broken lysosomes. Inactivation of OTC by trypsin and elastase is also stimulated by epsilon-ATP. Carbamoyl phosphate protects OTC against proteolytic attack by elastase or by broken lysosomes. Thus, environmental factors, e.g., ATP and carbamoyl phosphate, appear to influence OTC stability to proteases.

The conformational changes upon anaerobic carbamyl phosphate addition to dilute hemoglobin solutions have been studied by means of circular dichroism. Freshly prepared carbamyl phosphate stabilizes the conformation of pure deoxyhemoglobin without detectable NH2-terminal carbamylation. Addition of preincubated (i.e. partially converted to cyanate) carbamyl phosphate, however, results in such carbamylation and in the formation of the conformationally different carbamyldeoxyhemoglobin which exhibits enhanced oxygen affinity. Fresh carbamyl phosphate carbamylates hemoglobin in deoxygenated hemolysates containing erythrocyte phosphatases. Therefore the reversal of SS erythrocyte sickling by carbamyl phosphate is attributable to carbamylation by the carbamyl phosphate hydrolysis product, cyanate.

Weanling rats fed a cariogenic diet supplemented with carbamyl phosphate, in combination with egg shell meal and trace elements, showed a striking reduction in the incidence of caries. The cariostatic efficacy of carbamyl phosphate is significant because it stimulates fluid movement within odontoblasts via the hypothalamic-parotid gland endocrine axis.