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Potassium iron hexacyanoferrate, or Prussian blue (PB), is investigated as a cathode material for nonaqueous divalent calcium ion batteries. PB is an attractive prospect due to its high specific capacity, nontoxicity, low cost, and simple synthesis. Charge/discharge performances are examined at current densities of 23 mAg−1, 45 mAg−1, 90 mAg−1, and 125 mAg−1 that produced reversible specific capacities ranging from 150 mAhg−1 (at 23 mAg−1 current density) to over 120 mAhg−1 (at 125 mAg−1 current density). These are the highest storage capacities to date for a divalent calcium ion cathode over extended period of charge/discharge cycling and are comparable in performance to monovalent intercalating ions.

A computationally simple yet predictive multi-scale simulation scheme is introduced to estimate zero-field charge mobilities for amorphous OSC materials. A percolating charge model is utilized to describe inhomogeneity of hopping trajectories of carriers in amorphous media. The prediction scheme is composed of the following stages: quantum chemical calculation of Marcus inner sphere reorganization energies, molecular dynamics simulations of the amorphous condensed phase bulk structure, automated quantum chemical calculations of the electronic coupling for dimer pairs in the amorphous solid, and calculation of the Marcus theory charge hopping rates and an estimated bulk mobility using the Einstein relation, corrected for the inhomogeneous hopping network of the solid. Comparisons with independent experimental measurements of hole mobility for ten OSC compounds show that this approach gives good correlation between predictions and measurements suitable for ranking systems, and useful quantitative agreement. This low-cost model with minimal complexity is well-suited for incorporation into a virtual materials discovery framework for advanced OSC solutions.

The influence of the precursors, namely potassium ferrocyanide and potassium ferricyanide on the particles sizes of Prussian Blue (PB) and Prussian Green (PG), under identical reaction conditions have been investigated. It was found that the particle sizes influence the gravimetric capacity utilization of these materials as cathodes for aqueous potassium (K + ) ion batteries. The PG particle sizes were on the order of 50–75 nm, whereas PB particles size were on the order of 2–10 microns. The PG cathodes demonstrated a reversible capacity of 121.4 mAhr/g, with a coulombic efficiency of 98.7% compared to PB cathodes which demonstrated 53.8 mAhr/g, with a coulombic efficiency of 100%. We interpret the increased capacity of PG batteries relative to PB batteries as being a result of the smaller particle size of PG, which results in greater accessibility of the cathode to K+ ions.

Potassium barium hexacyanoferrate (K2BaFe(CN)6) was investigated as a cathode material for reversible Ca2+ ion insertion/extraction type rechargeable battery using non-aqueous electrolytes. The electrochemical performance of K2BaFe(CN)6was evaluated using cyclic voltammetry and galvanic cycling at ambient temperature. It is shown that addition of water led to significant enhancement in intercalation and de-intercalation of Ca2+ ions, leading to improved charge/discharge capacity. The enhancement in performance is attributed to formation of solvation spheres around the intercalating Ca2+ ions which provide screening from the electrostatic charges of the BaFe(CN)6 lattice. A reversible capacity of 55.8 mA hr g−1 and a coulombic efficiency of 93.8% was demonstrated at the end of 30 charge/discharge cycles.

Chemical modification of graphene by covalently functionalizing its surface potentially allows a wider flexibility in engineering electronic structure, in particular the local density of states of the carbon atoms bound to the modifier that can result in opening of the band gap. Such binding can involve covalent hydrogenation of graphene to modify hybridization of carbon atoms from sp2 to sp3 geometry [1] , [2] , [3] . Methods have also been developed to functionalize graphene covalently with molecular species [4] , [5] , [6] , [7] , [8] . Among these, perfluorophenylazide (PFPA) functionalization of graphene is well-developed using a nitrene intermediate. Films of this molecule also act as adhesion layers that allow production of long ribbons of exfoliated graphene [7] , [8] , [9] . We have developed a theory to predict electrical properties of PFPA functionalized graphene and compared it to experimental results. Conductivity of these PFPA functionalized ribbons of exfoliated graphene show good agreement with our theory.

Dihydroorotases (EC 3.5.2.3) catalyze the reversible cyclization of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis. The X-ray structures of Aquifex aeolicus dihydroorotase in two space groups, C 222 1 and C 2, were determined at a resolution of 1.7 A. These are the first structures of a type I dihydroorotase, a class of molecules that includes the dihydroorotase domain of mammalian CAD. The type I enzymes are more ancient and larger, at 45 kDa, than the type II enzymes exemplified by the 38 kDa Escherichia coli dihydroorotase. Both dihydroorotases are members of the metallo-dependent hydrolase superfamily, whose members have a distorted “TIM barrel” domain containing the active site. However, A. aeolicus dihydroorotase has a second, composite domain, which the E. coli enzyme lacks and has only one of the two zinc atoms present in the E. coli enzyme. A. aeolicus dihydroorotase is unique in exhibiting significant activity only when complexed with aspartate transcarbamoylase, whereas the E. coli dihydroorotase and the CAD dihydroorotase domain are active as free proteins. The latency of A. aeolicus dihydroorotase can be related to two differences between its structure and that of E. coli dihydroorotase: (1) the monoclinic structure has a novel cysteine ligand to the zinc that blocks the active site and possibly functions as a “cysteine switch”; and (2) active site residues that bind the substrate in E. coli dihydroorotase are located in disordered loops in both crystal structures of A. aeolicus dihydroorotase and may function as a disorder-to-order “entropy switch”.

Dihydroorotase (DHOase) catalyzes the reversible condensation of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis. The enzyme from Aquifex aeolicus, a hyperthermophilic organism of ancient lineage, was cloned and expressed in Escherichia coli. The purified protein was found to be a 45-kDa monomer containing a single zinc ion. Although there is no other DHOase gene in the A. aeolicus genome, the recombinant protein completely lacked catalytic activity at any temperature tested. However, DHOase formed an active complex with aspartate transcarbamoylase (ATCase) from the same organism. Whereas the kcat of 13.8 ± 0.03 s–1 was close to the value observed for the mammalian enzyme, the K mfor dihydroorotate, 3.03 ± 0.05 mm was 433-fold higher. Gel filtration and chemical cross-linking showed that the complex exists as a 240-kDa hexamer (DHO3-ATC3) and a 480-kDa duodecamer (DHO6-ATC6) probably in rapid equilibrium. Complex formation protects both DHOase and ATCase against thermal degradation at temperatures near 100 °C where the organism grows optimally. These results lead to the reclassification of both enzymes: ATCase, previously considered a Class C homotrimer, now falls into Class A, whereas the DHOase is a Class 1B enzyme. CD spectroscopy indicated that association with ATCase does not involve a significant perturbation of the DHOase secondary structure, but the visible absorption spectrum of a Co2+-substituted DHOase is appreciably altered upon complex formation suggesting a change in the electronic environment of the active site. The association of DHOase with ATCase probably serves as a molecular switch that ensures that free, uncomplexed DHOase in the cell remains inactive. At pH 7.4, the equilibrium ratio of carbamoyl aspartate to dihydroorotate is 17 and complex formation may drive the reaction in the biosynthetic direction.

Aspartate transcarbamoylase from Pseudomonadaceae is a class A enzyme consisting of six copies of a 36-kDa catalytic chain and six copies of a 45-kDa polypeptide of unknown function. The 45-kDa polypeptide is homologous to dihydroorotase but lacks catalytic activity. Pseudomonas aeruginosa aspartate transcarbamoylase was overexpressed in Escherichia coli. The homogeneous His-tagged protein isolated in high yield, 30 mg/liter of culture, by affinity chromatography and crystallized. Attempts to dissociate the catalytic and pseudo-dihydroorotase (pDHO) subunits or to express catalytic subunits only were unsuccessful suggesting that the pDHO subunits are required for the proper folding and assembly of the complex. As reported previously, the enzyme was inhibited by micromolar concentrations of all nucleotide triphosphates. In the absence of effectors, the aspartate saturation curves were hyperbolic but became strongly sigmoidal in the presence of low concentrations of nucleotide triphosphates. The inhibition was unusual in that only free ATP, not MgATP, inhibits the enzyme. Moreover, kinetic and binding studies with a fluorescent ATP analog suggested that ATP induces a conformational change that interferes with the binding of carbamoyl phosphate but has little effect once carbamoyl phosphate is bound. The peculiar allosteric properties suggest that the enzyme may be a potential target for novel chemotherapeutic agents designed to combatPseudomonas infection.

CAD, a large multifunctional protein that carries carbamoyl phosphate synthetase (CPSase), aspartate transcarbamoylase, and dihydroorotase activities, catalyzes the first three steps of de novo pyrimidine biosynthesis in mammalian cells. The CPSase component, which catalyzes the initial, rate-limiting step, exhibits complex regulatory mechanisms involving allosteric effectors and phosphorylation that control the flux of metabolites through the pathway. Incubation of CAD with ATP in the absence of exogenous kinases resulted in the incorporation of 1 mol of P(i)/mol of CAD monomer. Mass spectrometry analysis of tryptic digests showed that Thr(1037) located within the CAD CPS.B subdomain was specifically modified. The reaction is specific for MgATP, ADP was a competitive inhibitor, and the native tertiary structure of the protein was required. Phosphorylation occurred after denaturation, further purification of CAD by SDS gel electrophoresis, and renaturation on a nitrocellulose membrane, strongly suggesting that phosphate incorporation resulted from an intrinsic kinase activity and was not the result of contaminating kinases. Chemical modification with the ATP analog, 5'-p-fluorosulfonylbenzoyladenosine, showed that one or both of the active sites that catalyze the ATP-dependent partial reactions are also involved in autophosphorylation. The rate of phosphorylation was dependent on the concentration of CAD, indicating that the reaction was, at least in part, intermolecular. Autophosphorylation resulted in a 2-fold increase in CPSase activity, an increased sensitivity to the feedback inhibitor UTP, and decreased allosteric activation by 5-phosphoribosyl-1-pyrophosphate, functional changes that were distinctly different from those resulting from phosphorylation by either the protein kinase A or mitogen-activated protein kinase cascades.

The carbamoyl phosphate synthetase domain of the multifunctional protein CAD catalyzes the initial, rate-limiting step in mammalian de novo pyrimidine biosynthesis. In addition to allosteric regulation by the inhibitor UTP and the activator PRPP, the carbamoyl phosphate synthetase activity is controlled by mitogen-activated protein kinase (MAPK)- and protein kinase A (PKA)-mediated phosphorylation. MAPK phosphorylation, both in vivo and in vitro, increases sensitivity to PRPP and decreases sensitivity to the inhibitor UTP, whereas PKA phosphorylation reduces the response to both allosteric effectors. To elucidate the factors responsible for growth state-dependent regulation of pyrimidine biosynthesis, the activity of the de novo pyrimidine pathway, the MAPK and PKA activities, the phosphorylation state, and the allosteric regulation of CAD were measured as a function of growth state. As cells entered the exponential growth phase, there was an 8-fold increase in pyrimidine biosynthesis that was accompanied by a 40-fold increase in MAPK activity and a 4-fold increase in CAD threonine phosphorylation. PRPP activation increased to 21-fold, and UTP became a modest activator. These changes were reversed when the cultures approach confluence and growth ceases. Moreover, CAD phosphoserine, a measure of PKA phosphorylation, increased 2-fold in confluent cells. These results are consistent with the activation of CAD by MAPK during periods of rapid growth and its down-regulation in confluent cells associated with decreased MAPK phosphorylation and a concomitant increase in PKA phosphorylation. A scheme is proposed that could account for growth-dependent regulation of pyrimidine biosynthesis based on the sequential action of MAPK and PKA on the carbamoyl phosphate synthetase activity of CAD.

To examine the reliability and validity of a new outcome measure, the Upper Body Musculoskeletal Assessment (UBMA).Twenty subjects physician-diagnosed as having work-related musculoskeletal disorders (WRMD) and ten healthy subjects were assessed using the UBMA on three separate occasions. All subjects with WRMD attributed their injury to equipment use on their job.The WRMD group had significantly higher UBMA scores on the side of equipment use than on the other side (p0.01), whereas the healthy group had similar scores on both sides (p0.05). UBMA scores for the WRMD group were significantly greater on both sides of the body than scores for the healthy group (p0.01). Only one test occasion was required to produce excellent reliability coefficients (ICCs0.88). Although group reliability was excellent, changes of 24% for patients with WRMD and 44% for healthy subjects would be required for confidence that UBMA scores for individual patients on the side of equipment use had changed from baseline.Although testing on one occasion produced reliable UBMA scores, healthy subjects could be distinguished from patients with WRMD, and the side of equipment use could be distinguished from the other side in patients with WRMD, prediction of individual UBMA scores was poor. In its present form, the UBMA is useful for making decisions about groups but not about individual patients. Modifications of the current UBMA are required to reduce measurement error.

Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis. Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and carbamoyl-phosphate synthetase domains of CAD. There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO3−, the other substrates needed for carbamoyl phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenuation subdomains. In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser44 in the GLN attenuation domain with alanine increases thekcat/Km for glutamine hydrolysis 680-fold. The formation of a functional hybrid between the mammalian Ser44 GLN domain and the Escherichia coli carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis. In contrast, ATP and HCO3− did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser44 → Ala mutant than that needed for carbamoyl phosphate synthesis. Ser44, the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and carbamoyl phosphate synthesis.

The kinetics of the coupled reactions between carbamoyl-phosphate synthetase (CPSase) and both aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase (OTCase) from the deep sea hyperthermophilic archaeon Pyrococcus abyssi demonstrate the existence of carbamoyl phosphate channeling in both the pyrimidine and arginine biosynthetic pathways. Isotopic dilution experiments and coupled reaction kinetics analyzed within the context of the formalism proposed by Ovádi et al. (Ovádi, J., Tompa, P., Vertessy, B., Orosz, F., Keleti, T., and Welch, G. R. (1989) Biochem. J. 257, 187-190) are consistent with a partial channeling of the intermediate at 37 degrees C, but channeling efficiency increases dramatically at elevated temperatures. There is no preferential partitioning of carbamoyl phosphate between the arginine and pyrimidine biosynthetic pathways. Gel filtration chromatography at high and low temperature and in the presence and absence of substrates did not reveal stable complexes between P. abyssi CPSase and either ATCase or OTCase. Thus, channeling must occur during the dynamic association of coupled enzymes pairs. The interaction of CPSase-ATCase was further demonstrated by the unexpectedly weak inhibition of the coupled reaction by the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA). The anomalous effect of PALA suggests that, in the coupled reaction, the effective concentration of carbamoyl phosphate in the vicinity of the ATCase active site is 96-fold higher than the concentration in the bulk phase. Channeling probably plays an essential role in protecting this very unstable intermediate of metabolic pathways performing at extreme temperatures.

The initial steps of pyrimidine biosynthesis in yeast and mammals are catalyzed by large multifunctional proteins of similar size, sequence and domain structure, but appreciable functional differences. The mammalian protein, CAD, has carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities. The yeast protein, ura2, catalyzes the first two reactions and has a domain, called pDHO, which is homologous to mammalian DHOase, but is inactive. In CAD, only CPSase is regulated, whereas both CPSase and ATCase in the yeast protein are inhibited by UTP. These functional differences were explored by constructing a series of mammalian yeast chimeras. The isolated ATCase domain is catalytically active, but is not regulated. The inclusion of the yeast sequences homologous to the mammalian regulatory domain (B3) and the intervening pDHO domain did not confer regulation. Chimeric proteins in which the homologous regions of the mammalian protein were replaced by the corresponding domains of ura2 exhibited full catalytic activity, as well regulation of the CPSase, but not the ATCase, activities. The yeast B3 subdomain confers UTP sensitivity on the mammalian CPSase, suggesting that it is the locus of CPSase regulation in ura2. Taken together, these results indicate that there are regulatory site(s) in ura2. Channeling is impaired in all the chimeric complexes and completely abolished in the chimera in which the pDHO domain of yeast is replaced by the mammalian DHO domain.

Carbamoyl-phosphate synthetase consists of an amidotransferase domain or subunit (GLN) that hydrolyzes glutamine and transfers the ammonia to the synthetase component (CPS) where the biosynthetic reaction occurs. The CPS domain is composed of two homologous subdomains, CPS.A and CPS.B, that catalyze different ATP-dependent reactions involved in carbamoyl phosphate synthesis. When the individual CPS.A and CPS.B subdomains were individually cloned and expressed in Escherichia coli (Guy, H. I., and Evans, D. R. (1996) J. Biol. Chem. 271, 13762-13769), they were found to be functionally equivalent and could each independently catalyze carbamoyl phosphate synthesis. The proposal was advanced that, although the monomers could catalyze the individual partial reactions, overall synthesis of carbamoyl phosphate required a homodimer of CPS.A or CPS.B. To test this hypothesis, the GLN-CPS.B dimer was reversibly dissociated at 1500 bar in a high pressure cell. Dissociation was accompanied by a loss of both glutamine- and ammonia-dependent CPSase activity. Activity was recovered once the protein was returned to atmospheric pressure. If the sample was cross-linked before exposure to high pressure, there was no dissociation and no loss of biosynthetic activity. In contrast, the bicarbonate-dependent ATPase and the carbamoyl phosphate-dependent ATP synthetase activities were largely unaffected by pressure-induced dissociation. These experiments confirmed the hypothesis that the synthesis of carbamoyl phosphate requires the concerted action of the two active sites within the homodimer.

Escherichia coli carbamoyl-phosphate synthetase (CPSase) is comprised of a 40-kDa glutaminase (GLN) and a 120-kDa synthetase (CPS) subunit. The CPS subunit consists of two homologous domains, CPS.A and CPS.B, which catalyze the two different ATP-dependent partial reactions involved in carbamoyl phosphate synthesis. Sequence similarities and controlled proteolysis experiments suggest that the CPS subdomains consist, in turn, of three subdomains, designated A1, A2, A3 and B1, B2, B3 for CPS.A and CPS.B, respectively. Previous studies of individually cloned CPS.A and CPS.B from E. coli and mammalian CPSase have shown that homologous dimers of either of these “half-molecules” could catalyze all three reactions involved in ammonia-dependent carbamoyl phosphate synthesis. Four smaller recombinant proteins were made for this study as follows: 1) A1-A2 in which the A3 subdomain was deleted from CPS.A, 2) B1-B2 lacking subdomain B3 of CPS.B, 3) the A2 subdomain of CPS.A, and 4) the B2 subdomain of CPS.B. When associated with the GLN subunit, A1-A2 and B1-B2 had both glutamine- and ammonia-dependent CPSase activities comparable to the wild-type protein. In contrast, the 27-kDa A2 and B2 recombinant proteins, which represent only 17% of the mass of the parent protein, were unable to use glutamine as a nitrogen donor, but the ammonia-dependent activity was enhanced 14–16-fold. The hyperactivity suggests that A2 and B2 are the catalytic subdomains and that A1 and B1 are attenuation domains which suppress the intrinsically high activity and are required for the physical association with the GLN subunit.

Escherichia coli carbamyl-phosphate synthetase consists of two subunits that act in concert to synthesize carbamyl phosphate. The 40-kDa subunit is an amidotransferase (GLN subunit) that hydrolyzes glutamine and transfers ammonia to the 120-kDa synthetase subunit (CPS subunit). The enzyme can also catalyze ammonia-dependent carbamyl phosphate synthesis if provided with exogenous ammonia. In mammalian cells, homologous amidotransferase and synthetase domains are carried on a single polypeptide chain called CAD. Deletion of the 29-residue linker that bridges the GLN and CPS domains of CAD stimulates glutamine-dependent carbamyl phosphate synthesis and abolishes the ammonia-dependent reaction (Guy, H. I., and Evans, D. R. (1997) J. Biol. Chem. 272, 19906-19912), suggesting that the deletion mutant is trapped in a closed high activity conformation. Since the catalytic mechanisms of the mammalian and bacterial proteins are the same, we anticipated that similar changes in the function of the E. coli protein could be produced by direct fusion of the GLN and CPS subunits. A construct was made in which the intergenic region between the contiguous carA and carB genes was deleted and the sequences encoding the carbamyl-phosphate synthetase subunits were fused in frame. The resulting fusion protein was activated 10-fold relative to the native protein, was unresponsive to the allosteric activator ornithine, and could no longer use ammonia as a nitrogen donor. Moreover, the functional linkage that coordinates the rate of glutamine hydrolysis with the activation of bicarbonate was abolished, suggesting that the protein was locked in an activated conformation similar to that induced by the simultaneous binding of all substrates.

The amidotransferase domain (GLNase) of mammalian carbamyl-phosphate synthetase II hydrolyzes glutamine and transfers ammonia to the synthetase domain where carbamyl phosphate is formed in a three-step reaction sequence. The synthetase domain consists of two homologous subdomains, CPS.A and CPS.B. Recent studies suggest that CPS.A catalyzes the initial ATP dependent-activation of bicarbonate, whereas CPS.B uses a second ATP to form carbamyl phosphate. To establish the function of these substructural elements, we have cloned and expressed the mammalian protein and its subdomains in Escherichia coli. Recombinant CPSase (GLNase-CPS.A-CPS.B) was found to be fully functional. Two other proteins were made; the first consisted of only GLNase and CPS.A, whereas the second lacked CPS.A and had the GLNase domain fused directly to CPS.B. Remarkably, both proteins catalyzed the entire series of reactions involved in glutamine-dependent carbamyl phosphate synthesis. The stoichiometry, like that of the native enzyme, was 2 mol of ATP utilized per mol of carbamyl phosphate formed. GLN-CPS.B is allosterically regulated, whereas GLN-CPS.A was insensitive to effectors, a result consistent with evidence showing that allosteric effectors bind to CPS.B. These properties are not peculiar to the mammalian protein, because the separately cloned CPS.A subdomain of the E. coli enzyme was also found to catalyze carbamyl phosphate synthesis. Gel filtration chromatography and chemical cross-linking studies showed that these molecules are dimers, a structural organization that may be a prerequisite for the overall reaction. Thus, the homologous CPS.A and CPS.B subdomains are functionally equivalent, although in the native enzyme they may have different functions resulting from their juxtaposition relative to the other components in the complex.

The amidotransferase or glutaminase (GLNase) domain of mammalian carbamyl phosphate synthetase (CPSase), part of the 243-kDa CAD polypeptide, consists of a carboxyl half that is homologous to all trpG-type amidotransferases and an amino half unique to the carbamyl phosphate synthetases. The two halves of the mammalian GLNase domain have been cloned separately, expressed in Escherichia coli, and purified. The 21-kDa carboxyl half, the catalytic subdomain, is extraordinarily active. The k is 347-fold higher and the K is 40-fold lower than the complete GLNase domain. Unlike the GLNase domain, the catalytic subdomain does not form a stable hybrid complex with the E. coli CPSase synthetase subunit. Nevertheless, titration of the synthetase subunit with the catalytic subdomain partially restores glutamine-dependent CPSase activity. The 19-kDa amino half, the interaction subdomain, binds tightly to the E. coli CPSase large subunit. Thus, the GLNase domain consists of two subdomains which can autonomously fold and function. The catalytic subdomain weakly interacts with the synthetase domain and has all of the residues necessary for catalysis. The interaction subdomain is required for complex formation and also attenuates the intrinsically high activity of the catalytic subdomain and, thus, may be a key element of the interdomain functional linkage.

Carbamyl-phosphate synthetases from different organisms have similar catalytic mechanisms and amino acid sequences, but their structural organization, sub-unit structure, and mode of regulation can be very different. Escherichia coli carbamyl-phosphate synthetase (CPSase), a monofunctional protein consisting of amido-transferase and synthetase subunits, is allosterically inhibited by UMP and activated by NH3, IMP, and ornithine. In contrast, mammalian CPSase II, part of the large multifunctional polypeptide, CAD, is inhibited by UTP and activated by 5-phosphoribosyl-1-pyrophosphate (PRPP). Previous photoaffinity labeling studies of E. coli CPSase showed that allosteric effectors bind near the carboxyl-terminal end of the synthetase subunit. This region of the molecule may be a regulatory subdomain common to all CPSases. An E. coli mammalian hybrid CPSase gene has been constructed and expressed in E. coli. The hybrid consists of the E. coli CPSase synthetase catalytic subdomains, residues 1-900 of the 1073 residue polypeptide, fused to the amino-terminal end of the putative 190-residue regulatory subdomain of the mammalian protein. The hybrid CPSase had normal activity, but was no longer regulated by the prokaryotic allosteric effectors. Instead, the glutamine- and ammonia-dependent CPSase activities and both ATP-dependent partial reactions were activated by PRPP and inhibited by UTP, indicating that the binding sites of both of these ligands are located in a regulatory region at the carboxyl-terminal end of the CPSase domain of CAD. The apparent ligand dissociation constants and extent of inhibition by UTP are similar in the hybrid and the wild type mammalian protein, but PRPP binds 4-fold more weakly to the hybrid. The allosteric ligands affected the steady state kinetic parameters of the hybrid differently, suggesting that while the linkage between the catalytic and regulatory subdomains has been preserved, there may be qualitative differences in interdomain signal transmission. Nevertheless, switching prokaryotic and eukaryotic allosteric controls argues for remarkable conservation of structure and regulatory mechanisms in this family of proteins.

The multifunctional protein CAD catalyzes the first three steps in de novo pyrimidine biosynthesis in mammalian cells. Glutamine-dependent carbamyl-phosphate synthetase (CPSase), aspartate transcarbamylase, and dihydroorotase activities are carried by a 243-kDa polypeptide chain that is organized into discrete functional domains connected by interdomain linkers. One of the connecting chain segments, the DA linker bridging the dihydroorotase and aspartate transcarbamylase domains, is unusually long (109 residues) and conserved in length in all eukaryotic species. A plasmid (pCK-CAD10) that encodes the entire 243-kDa polypeptide was constructed and expressed in Escherichia coli. The recombinant protein was purified to homogeneity by ion exchange and gel filtration chromatography. The purified protein had kinetic parameters that were close to those obtained for native CAD. Moreover, the CPSase activity was allosterically regulated. Gel filtration showed that the recombinant protein had the same molecular mass as native CAD. Thus, this complex mammalian protein is expressed and folds correctly in bacterial cells and, despite its extreme protease sensitivity, can be isolated intact. A deletion mutant that lacked the DA linker was then constructed. The kinetic parameters of the mutant protein were, for the most part, unaltered, showing that the DA linker is not essential for the proper folding or optimal functioning of the individual domains. However, a significant decrease in the thermal stability of the CPSase domain suggested that the linker helps to stabilize the complex. Moreover, the channeling of carbamyl phosphate, determined by measuring the extent to which the exogenously added intermediate could dilute the endogenous carbamyl phosphate pool, was appreciably reduced when the DA linker was removed. Thus, although the domains function autonomously, some of the linkers are important for interdomain interactions in CAD.

The trpG-type amidotransferases, a homologous but structurally diverse family of molecules, catalyze glutamine hydrolysis to supply ammonia for many biosynthetic reactions. The amidotransferase or glutaminase (GLNase) domain of mammalian carbamyl phosphate synthetase (CPSase), part of a 243-kDa polypeptide that initiates de novo pyrimidine biosynthesis, has been cloned and expressed in Escherichia coli. Complementation studies showed that a functional protein was produced in vivo which could provide ammonia for carbamyl phosphate synthesis by the host CPSase synthetase subunit. The recombinant 38-kDa protein was identified by immunoblotting, but when purified to homogeneity, had marginal glutaminase activity. Titration of the E. coli CPSase synthetase subunit with the mammalian GLNase domain resulted in the formation of a fully active 1:1 stoichiometric stable complex which catalyzed the glutamine-dependent overall reaction. The hybrid, isolated by gel filtration, had kinetic parameters (KGLNm = 102 microM, KATPm = 1.8 mM, kcat = 5.7 s-1) similar to those of the native E. coli CPSase. Thus, the amidotransferase activity of mammalian CPSase is carried by an autonomous domain which folds independently. However, optimal catalytic activity requires association of the glutaminase and synthetase domains. The conservation of this linkage in the mammalian E. coli hybrid suggests that the subunit interfaces must be nearly identical in the eukaryotic and prokaryotic proteins.

CAD is a multidomain protein that catalyzes the first three steps in mammalian de novo pyrimidine biosynthesis. The 243-kDa polypeptide consists of four functional domains; glutamine amidotransferase (GLNase), carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase), and dihydroorotase (DHOase). Controlled proteolysis of hamster CAD was found to cleave the molecule into 18 fragments which successively accumulate and disappear during the course of digestion. Each fragment was isolated and partially sequenced to determine its location in the polypeptide chain. Proteolysis was found to usually occur at the junctions between the domains and sub-domains identified by sequence homology. All proteases of low to moderate specificity cleaved the molecule in a similar fashion. The rate of proteolysis widely varied and the interdomain regions were not always accessible to proteases. Each of the major functional domains is postulated to consist of subdomains. The duplicated halves of the CPSase domain (116 kDa) have a homologous structure consisting of 11-, 25-26-, and 21-22-kDa subdomains. Prolonged digestion cleaved the DHOase domain (36.6 kDa) into two stable species suggesting that this region is comprised of 11.5- and 15.0-kDa subdomains. Similarly, proteolysis of the 21-kDa catalytic subdomain of the GLNase domain (40 kDa) indicated a bilobal structure consisting of 12.3- and 8.5-kDa chain segments. The connecting region between the two ATCase subdomains (16.4 and 18 kDa) was not cleaved. Copurification of many of the domains showed that they remain associated by noncovalent interactions even after the connecting segments have been cleaved. The chain segments, the linkers, which connect the domains and subdomains were conserved in length but not in sequence, were predicted to be relatively hydrophilic and flexible but did not show a tendency to assume a particular secondary structure. These studies provide a more detailed map of the structural organization of the CAD polypeptide.

CAD is a 243-kDa multidomain polypeptide which catalyzes the first three steps in mammalian de novo pyrimidine biosynthesis. The largest cDNA clone obtained thus far, pCAD142 (Shigesada, K., Stark, G.R., Maley, J. A., Niswander, L. A., and Davidson, J. N. (1985) Mol. Cell. Biol. 5, 1735), lacks the 5' end of the mRNA which encodes the amino terminus of CAD. To clone this missing segment, a synthetic oligonucleotide complementary to pCAD142 and poly(A)+ RNA template, isolated from a Syrian hamster cell line which overproduces the CAD mRNA, were used for cDNA synthesis. The resulting clone pKB11, which has a 1369-base pair (bp) cDNA insert, overlapping pCAD142 by 781 bp, was identified by hybridization methods and sequence analysis and found to contain the entire cDNA sequence for the amino end of the CAD polypeptide. The deduced amino acid sequence is homologous to seven carbamyl phosphate synthetases. Primer extension, oligonucleotide-directed RNase H digestion, and RNA sequencing indicated that pKB11 extends to within 68 bases of the 5' end of the CAD mRNA. This conclusion was confirmed by Northern blotting analysis of the 5'-flanking region of CAD gene. The probable 3' end of an unidentified gene which codes for a 1-kilobase (kb) transcript was identified immediately upstream of the CAD gene. Northern analysis using probes complementary to the region between the CAD and the 1-kb genes detected the presence of a small transcript of less than 300 nucleotides. The sequence revealed three potential translation initiation sites raising the possibility of more than one CAD translation product. The major translation start codon was identified as the first ATG in pKB11 by sequence homology, in vitro transcription and translation, and protein studies. Starting from this ATG within pKB11, the clone encodes a 143-residue domain of unknown function. This study completes the determination of the primary structure of the CAD polypeptide. The CAD mRNA is 7.5 kb in length and has 6675 bp of coding sequence and about 200 bp and 600 bp of untranslated sequence at the 5' and 3' ends, respectively.

The multifunctional protein CAD catalyzes the first three steps in pyrimidine biosynthesis in mammalian cells, including the synthesis of carbamyl phosphate from bicarbonate, MgATP and glutamine. The Syrian hamster CAD glutaminase (GLNase) domain, a trpG-type amidotransferase, catalyzes glutamine hydrolysis in the absence of MgATP and bicarbonate (Km = 95 microM and kcat = 0.14 s-1). Unlike E. coli carbamyl phosphate synthetase (Wellner, V.P., Anderson, P.M., and Meister, A. (1973) Biochemistry 12, 2061-2066), a stable thioester intermediate did not accumulate when the mammalian enzyme was incubated with glutamine. However, a covalent adduct could be isolated when the protein was denatured in acid. The steady state concentration of the intermediate increased with increasing glutamine concentration to nearly one mole per mole of enzyme with half saturation at 105 microM, close to the Km value for glutamine. The adduct formed at the active site of the glutaminase domain. The rate of breakdown of the intermediate (k4), determined directly, was 0.17 s-1 and the rate of formation (k3) was estimated as 0.52 s-1. In the absence of MgATP and bicarbonate, k4 = kcat indicating that the decomposition of the intermediate is the rate-limiting step. The intermediate was chemically and kinetically competent, and the glutamine dissociation constant (330 microM) and rate constants were consistent with steady state kinetics and accurately predicted the steady state concentration of the intermediate. These studies suggest a mechanism similar to the cysteine proteases such as recently proposed by Mei and Zalkin (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619) who identified a catalytic triad in glutamine phosphoribosyl-5'-pyrophosphate amidotransferase, a purF-type enzyme. MgATP and bicarbonate increased kcat of the glutaminase reaction 14-fold by accelerating both the rate of formation and the rate of breakdown of the intermediate, and prevented the accumulation of the intermediate; however, the Km value for glutamine was not significantly altered. The instability of the thioester intermediate leads to appreciable hydrolysis of glutamine in the absence of the other substrates. However, bicarbonate alone spares glutamine by increasing the Km and Ks of glutamine to 600 and 8960 microM, respectively, thus reducing kcat/Km 3-fold when MgATP is limiting. In the absence of MgATP and bicarbonate, ammonia decreased the rate of hydrolysis and the accumulation of the thioester intermediate indicating that ammonia had direct access to the thioester at the GLNase domain active site.(ABSTRACT TRUNCATED AT 400 WORDS)

Glutamine-dependent carbamoyl-phosphate synthetase (EC 6.3.5.5) catalyzes the first step in de novo pyrimidine biosynthesis. The mammalian enzyme is part of a 240-kDa multifunctional protein which also has the second (aspartate carbamoyltransferase, EC 2.1.3.2), and third (dihydroorotase, EC 3.5.2.3) activities of the pathway. Shigesada et al. (Shigesada, K., Stark, G.R., Maley, J.A., and Davidson, J.N. (1985) Mol. Cell Biol. 175, 1-7) produced a truncated cDNA clone from a Syrian hamster cell line that contained most of the coding region for this protein. We have completed sequencing this clone, known as pCAD142. The cDNA insert contained all of the coding region for the glutaminase (GLN) and carbamyl phosphate synthetase (CPS) domains but lacked a short amino-terminal segment. By comparing the primary structure of the mammalian chimera to monofunctional proteins we have identified the borders of the functional domains. The GLN domain is 21 kDa, close to the size of the functionally similar polypeptide products of the Escherichia coli pabA and hisH genes. The domain has the three regions of homology common to trpG-type glutamine amidotransferases, as well as a fourth region specific to the carbamyl phosphate synthetases. The CPSase domain is similar to other reported CPSases in size (120 kDa), primary structure (37-67% amino acid identity), and homology between its amino and carboxyl halves. Analysis of the nucleotide and amino acid sequence identities among the various carbamyl phosphate synthetases suggests that the gene fusion which joined the GLN and CPS domains was an early event in the evolution of eukaryotic organisms and that the Saccharomyces cerevisiae enzyme consisting of separate subunits arose by defusion from an ancestral multifunctional protein.

In situ magnetic field data from Jupiter and Saturn are used to interpret earth-based microwave observations for all areas except Branson's hot spot on Jupiter. It is found that Jupiter's field is strongly dipolar but has large high-order moments compared with the magnetic field of the earth. Decametric emissions of Jupiter have a complex rotational pattern which appears to have been stable since 1980. Microwave observations Saturn's radio emissions were strongly asymmetric along the rotational axis, indicating the presence of longitudinal variations in the magnetic fields a thousand kilometers from the cloud tops. The magnetic fields within a few thousand kilmeters of the cloud tops of both Jupiter and Saturn could not be identified.

Salsolinol, a substance that may participate in the development of alcoholism, has been identified in urine and other biological samples from alcoholics. Differentials have been observed between alcoholics and controls. Salsolinol forms when dopamine reacts with acetaldehyde, which may exist in higher concentrations in the blood of alcoholics after alcohol ingestion. Hence, it was postulated that there is a relationship between level of social drinking and the elaboration of salsolinol. Salsolinol is also found in certain food and beverage products. Eighty volunteers, balanced for gender, social drinking level, ethanol dose administered and experimental diet provided urine samples 90 minutes and three hours after ethanol was consumed. Salsolinol levels were analysed in urine using high performance liquid chromatography. A 24 hour carryover effect was observed. Diet, ethanol dose and social drinking level had main and interactive effects on excreted quantities of salsolinol. Gender, situational stress and cigarette smoking had minor if any influence on salsolinol excretion. While there was no evident difference in amounts of salsolinol excreted by light and heavy drinkers in the absence of external sources of salsolinol, heavy social drinkers excreted less salsolinol than did light drinkers after consuming a "salsolinol-enriched" diet, suggesting that they differ in some aspect of absorption, distribution, or metabolism of salsolinol after drinking ethanol. Accordingly, studies that attempt to determine whether salsolinol has any relationship to drinking behaviour in humans should be particularly concerned with salsolinol that occurs in exogenous sources.

The aspartate transcarbamylase activity of mammalian cells is carried by a large multifunctional protein, CAD, that catalyzes the first three steps in the de novo pyrimidine biosynthetic pathway. Controlled proteolysis of CAD cleaved the polypeptide chain into several separate structural domains which carried the individual activities of the complex. The aspartate transcarbamylase activity was associated with a 40,000-dalton proteolytic fragment, which kinetic studies showed was released in one of the first proteolytic cleavages. The species was purified to homogeneity by chromatography on carboxymethyl-Sephadex. The isolated species, designated the aspartate transcarbamylase domain, had a molecular weight under denaturing conditions of 40,000 +/- 1,500, a pI = 9.4, and a sedimentation coefficient (S20,w) of 6.2. The sedimentation coefficient suggested that the isolated domain was an oligomer consisting of two or three identical copies of the 40,000-dalton proteolytic fragment. The aspartate saturation curve obtained at a saturating concentration of carbamyl phosphate gave Km = 2.1 X 10(-2)M and Vmax = 119.5 mumol/min/mg, corresponding to a turnover number of 4,780 min-1. Like the aspartate transcarbamylase activity of CAD, the activity was strongly inhibited by high concentrations of aspartate. The corresponding parameters from the carbamyl phosphate saturation curve were Km = 2.07 X 10(-5)M, Vmax = 52.5 mumol/min/mg, and a turnover number of 2,153 min-1. The similarity of these parameters to those obtained from a steady state kinetic study of CAD indicated that the tertiary structure of this region of the polypeptide chain was largely preserved in the isolated species. In the absence of stabilizing agents, the half-life of aspartate transcarbamylase activity of CAD was 60.2 h, while that of the isolated domain was 10.6 h. This result suggested that there were interactions with other regions of the molecule which stabilized the structure of the aspartate transcarbamylase domain in the intact complex.

A steam distillation method for the determination of the TBA number in control and irradiated hake and tuna is presented. Chemical values are compared with sensoric evaluation results for the 30-day cold storage period. Close correlation has been observed between the organoleptic scores and the oxidative rancidity in tuna loins and hake fillets, whether control or irradiated.

CAD, is a multidomain polypeptide, with a molecular weight of over 200,000, that has glutamine-dependent carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase activity as well as regulatory sites that bind UTP and 5-phosphoribosyl 1-pyrophosphate. The protein thus catalyzes the first three steps of de novo pyrimidine biosynthesis and controls the activity of the pathway in higher eukaryotes. Controlled proteolysis of CAD isolated from Syrian hamster cells, cleaves the molecule into seven major proteolytic fragments that contain one or more of the functional domains. The two smallest fragments, which had molecular weights of 44,000 and 40,000, corresponded to the fully active dihydroorotase (DHO) and aspartate transcarbamylase (ATC) domains, respectively, but the larger fragments have not been previously characterized. In this study, enzymatic assays of partially fractionated digests and immunoblotting with antibodies specifically directed against the purified ATC domain, the purified dihydroorotase domain and an 80-kDa fragment of the putative carbamyl-phosphate synthetase domain established the precursor-product relationships among all of the major proteolytic fragments of CAD. These results indicate that 1) only the intact molecule had all of the functional domains, 2) a species with a molecular weight of 200,000 was produced in the first step of proteolysis which had glutamine-dependent carbamyl-phosphate synthetase and dihydroorotase activity, but neither aspartate transcarbamylase activity nor the antigenic determinants present on the isolated ATC domain, and 3) cleavage of the 200-kDa species produced a species, with a molecular mass of 150,000 which lacked both aspartate transcarbamylase and dihydroorotase domains. This 150-kDa species, containing the postulated carbamyl-phosphate synthetase, glutamine, and regulatory (UTP, 5-phosphoribosyl 1-pyrophosphate) domains, had two elastase-sensitive sites that divided this region of the polypeptide chain into 10-, 65-, and 80-kDa segments. The location of the functional sites on these segments has not yet been established. The immunochemical analysis also revealed the existence of possible precursors of the stable aspartate transcarbamylase and dihydroorotase domains, suggesting that the chain segments connecting the functional domains of CAD are extensive and that the overall size of the intact polypeptide chain has been underestimated. On the basis of these studies we have proposed a model of the domain structure of CAD.

A course designed to train graduate students in clinical psychology in a number of intervention techniques for adult clients is described, based on the behavioral systems and personalized instruction approaches to training. The objectives for the course are stated, and its features reviewed. The impact of the course on students' subsequent performance is briefly considered.

Dihydroorotase (DHOase) catalyzes the third step in eukaryotic de novo pyrimidine biosynthesis. In mammalian cells, this enzyme activity is carried by a large chimeric protein, CAD, that also catalyzes the first two steps in the pathway: glutamine-dependent carbamyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). Controlled elastase cleavage of CAD released a 44,000 +/- 2,000-dalton proteolytic fragment which catalyzed only the dihydroorotase reaction. We have devised a rapid and simple method for the isolation of the DHO domain from elastase digests. The domain, which was obtained in 36% yield, was found to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The domain was also characterized by amino acid analysis and analytical high pressure liquid chromatography peptide mapping. The amino terminus of both the DHO domain and intact CAD was blocked suggesting that this domain is located at the extreme amino terminus of the CAD polypeptide, a result consistent with the suspected juxtaposition of domains as DHO-CPS-ATC. The isoelectric point of the DHO domain was 5.1, while that of the ATC domain was 9.4, so that the ends of the CAD polypeptide are oppositely charged at physiological pH. Immunoblotting with DHO domain-specific antibodies showed that a 47-kDa species was generated in the early stages of controlled proteolysis of CAD. Thus there are two elastase cleavage sites within a 3-kDa connecting region that links the DHO and CPS domains. The domain was shown by atomic absorption spectrophotometry and by isolating a 65Zn-containing DHO domain from mammalian cells grown in the presence of the radionuclide to contain 1 g eq of tightly bound zinc in each polypeptide chain. Zinc was not found in any other CAD domain. Chelating agents inhibit dihydroorotase activity of the isolated domain supporting the conclusion, based on studies of intact CAD by others, that zinc participates in catalysis. At moderate protein concentrations the DHO domain was a 88,000 dimer with a Stokes radius of 37.6 A, a S20,w = 5.1 X 10(-13) s, a diffusion coefficient of 3.17 X 10(-7) cm2 s-1, and a frictional ratio of 1.26. On dilution the dimer dissociated and was in rapid concentration-dependent equilibrium with a 43,500 monomer. The hydrodynamic parameters of the monomer have also been estimated (Stokes radius of 29.8 A, D20,w = 4.11 X 10(-7) cm2 s-1, and f/f0 1.21).(ABSTRACT TRUNCATED AT 400 WORDS)

Ammonia-dependent carbamoyl-phosphate synthetase I (carbon-dioxide:ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16; formerly EC 2.7.2.5) isolated from hamster liver mitochondria is comprised of identical 160 kDa polypeptide chains. Controlled proteolysis by elastase sequentially cleaved this molecule into a small number of specific fragments. The first cleavage led to a complete loss of enzymatic activity and the formation of a 145 kDa species that was subsequently degraded into 83 kDa and 62 kDa fragments. Very different results were obtained when proteolysis was carried out in the presence of saturating ATP, MgCl 2 , NH 4 Cl, and the activator N- acetyl- l -glutamate . These ligands stabilized the molecule 8-fold against elastase digestion. Moreover, only small amounts of the 145 kDa species were generated. Instead, the molecule was initially cleaved into a fully active 120 kDa species anda 40 kDa proteolytic fragment. The same species were found in limit digests conducted in the presence and absence of ligands, indicating that only the sequence of elastase cleavages differed. Comparison of digests conducted in the presence of each ligand alone and in combination, showed that while NH 4 Cl and N- acetyl- l -glutamate were necessary for maximal stabilization of the molecule, the altered digestion pattern was produced specifically by MgATP. The MgATP-induced change in digestion pattern correlated well with the steady-state ATP saturation curve, suggesting that the production of the 120 kDa species resulted from ATP binding to the active site. The effect of MgATP on the proteolysis of carbamoyl-phosphate synthetase I was not the result of an alteration in oligomeric structure, but the protection of two elastase cleavage sites. The results were interpreted on the basis of the primary structure recently determined elsewhere.

The importance and also the lack of attention to assessment procedures in behaviour therapy has been stressed recently. The purpose of this study was to investigate whether differences exist in a number of subjective variables taken on two groups of exhibitionists, those successfully and those unsuccessfully treated by means of aversion therapy. Differences in age, education and marital status for the two groups were not significant. Significantly more of the subjects in the failure group were found to masturbate to deviant fantasy than in the success group. However, differences in the frequency of masturbation and sexual inter- course for the two groups were not significant. The failure group had been acting-out significantly longer and more frequently than had the success group. Further, the failure group took significantly more treatment trials to non-acting-out and no urges to act-out than did the success group. The importance of assessing cases for behaviour therapy on measures derived from learning experiments is stressed.