Spectroscopy of element 115 decay chains.

A high-resolution α, x-ray, and γ-ray coincidence spectroscopy experiment was conducted at the GSI Helmholtzzentrum für Schwerionenforschung. Thirty correlated α-decay chains were detected following the fusion-evaporation reaction 48Ca + 243Am. The observations are consistent with previous assignments of similar decay chains to originate from element Z=115. For the first time, precise spectroscopy allows the derivation of excitation schemes of isotopes along the decay chains starting with elements Z>112. Comprehensive Monte Carlo simulations accompany the data analysis. Nuclear structure models provide a first level interpretation.

The chemistry of the reaction determines the invariant amino acids during the evolution and divergence of orotidine 5'-monophosphate decarboxylase.

Orotidine 5'-phosphate (OMP) decarboxylase has the largest rate enhancement for any known enzyme. For an average protein of 270 amino acids from more than 80 species, only 8 amino acids are invariant, and 7 of these correspond to ligand-binding residues in the crystal structures of the enzyme from four species. It appears that the chemistry required for catalysis determines the invariant residues for this enzyme structure. A motif of three invariant amino acids at the catalytic site (DXKXXD) is also found in the enzyme hexulose-phosphate synthase. Although the core of OMP decarboxylase is conserved, it has undergone a variety of changes in subunit size or fusion to other protein domains, such as orotate phosphoribosyltransferase, during evolution in different kingdoms. The phylogeny of OMP decarboxylase shows a unique subgroup distinct from the three kingdoms of life. The enzyme subunit size almost doubles from Archaea (average mass of 24.5 kDa) to certain fungi (average mass of 41.7 kDa). These observed changes in subunit size are produced by insertions at 12 sites, largely in loops and on the exterior of the core protein. The consensus for all sequences has a minimal size of <20 kDa.

Nifedipine and nimodipine competitively inhibit uridine kinase and orotidine-phosphate decarboxylase: theoretical relevance to poor outcome in stroke.

Nifedipine and nimodipine, dihydropyridine calcium channel blockers, are commonly used as antihypertensive and antianginal agents in patients at risk for stroke. At least one stroke trial suggests that patients receiving calcium channel blockers at the time of an acute stroke have worse outcomes than those receiving other or no antihypertensive medications. We hypothesize that the poor outcome may not be related to blood pressure changes but instead may be mediated by competitive inhibition of important enzymes of pyrimidine synthesis whose products are needed to repair nerve cell membranes after an acute stroke. Both drugs acted as competitive inhibitors of the only enzymes that are known to synthesize the nucleotide uridine-5'-phosphate: uridine kinase and orotidine-5'-phosphate decarboxylase. Nifedipine produced Ki values of 28 microM for uridine kinase and 105 microM for orotidine-5'-phosphate decarboxylase. Nimodipine produced Ki values of 20 microM for uridine kinase and 18 microM for orotidine-5'-phosphate decarboxylase. For uridine kinase, these inhibitors bound more tightly than the physiologic substrates uridine or cytidine. For the decarboxylase, the inhibitors bound less tightly than the normal physiologic substrate orotidine-5'-phosphate. Additional experiments are needed to determine whether the concentrations of nifedipine or nimodipine, and of cytidine, uridine, and orotidine-5'-phosphate in human brain, are such that this inhibition would affect stroke outcome.

[Serum level-adjusted dosage of once-daily aminoglycoside therapy in critical illness: results of a prospective study]. / Serumspiegelorientierte Dosierung der einmal täglichen Aminoglykosidtherapie beim kritisch Kranken: Ergebnisse einer prospektiven Untersuchung.

OBJECTIVE: In critically ill patients, the adjustment of target peak and trough levels of tobramycin was investigated because aminoglycoside pharmacokinetics can be changed by multiple influences. Sufficient but not too high peak serum concentrations and low trough levels, however, should be achieved to ensure a therapeutic effect and to minimize toxicity. METHODS: 70 critically ill patients of 51 +/- 18 years were monitored daily during their aminoglycoside treatment on the intensive care unit targeting a peak of about 12 micrograms/ml 30 minutes after infusion and a trough level below 1 to 2 micrograms/ml. Dose recommendations were given daily, taking into consideration serum levels, dose predictions (Bayesian method, ABBOTTBASE), creatinine clearance and clinical findings. Creatinine clearance was estimated according to the Cockcroft-Gault-formula as well as directly by the urine collection method. RESULTS: The standardized initial dose of 400 mg tobramycin led to average peak serum levels of 14.2 +/- 3.9 micrograms/ml in the patients with an apparent distribution volume of 0.345 +/- 0.074 L/kg. In 95% of the patients, the initial peak was higher than 8.5 micrograms/ml; levels higher than 20 micrograms/ml were observed in 7%, extremely low concentrations (below 5 micrograms/ml) in 2%. With individually adjusted doses between 160 and 560 mg, a mean peak of 11.5 +/- 2.7 micrograms/ml was measured subsequently. The levels amounted to 96 +/- 23% of the predicted values, deviations greater than 50% occurred in 5%. The target trough level was achieved in 99%, in less than 3% the dosing interval was extended up to 72 hours. A tobramycin clearance below 80 ml/min/1.73 m2 was associated with average 80% and 33% higher creatinine clearance values according to the Cockcroft-method and the direct method, respectively. CONCLUSION: Target peak and trough aminoglycoside levels are adjustable even in critically ill patients. Reduced tobramycin clearance can be associated with normal creatinine clearance. Assuming an exact methodology, a reduced "direct" creatinine clearance, however, indicates a reduced drug clearance.

Uridine kinase: altered enzyme with decreased affinities for uridine and CTP.

Uridine kinase is the rate-limiting enzyme in the salvage pathway for uridine or cytidine of mammalian cells. Alignment of the uridine kinase sequence with other nucleoside and nucleotide kinases supports a common ancestor for all of these. Three polypeptide segments for the ATP site and three polypeptide segments for the acceptor nucleoside site have been identified. We report here the characterization of an altered form of the enzyme with a single amino acid change, Q146R, within or near the uridine-binding site. This single amino acid change leads to a 160-fold increase in Km for uridine (Km = 6.5 mM) and a decrease in kcat by more than 99%. This variant has normal affinity for ATP (Km = 130 microM), but shows substrate inhibition at ATP concentrations >3 mM. Mouse uridine kinase is normally an active tetramer that will dissociate to inactive monomers in response to CTP. In contrast, the altered protein is monomeric, but will associate to dimers and then to tetramers with increasing ATP. The Q146R enzyme has a 100-fold loss in affinity for the allosteric inhibitor CTP; this supports a model for CTP inhibition being caused by CTP binding backward at the catalytic site, as a bisubstrate analog.

Time-action profile of inhaled insulin.

We compared the pharmacodynamics of insulin after inhalation of 99 U microcrystalline solid insulin and subcutaneous injection of 10 U regular insulin and intravenous injection of 5 U regular insulin. The time-action profiles of the three insulin administrations were studied in 11 healthy volunteers using the euglycaemic glucose clamp technique. The insulins were administered to each volunteer on three separate occasions in random order. Onset of action, assessed as glucose infusion rate, after insulin inhalation was substantially more rapid than after subcutaneous injection and half-maximal action was reached earlier (31 +/- 17 vs 54 +/- 12 min; p < 0.001). Maximal metabolic response was reached earlier after insulin inhalation in comparison to subcutaneous injection (108 +/- 49 vs 147 +/- 53 min; p < 0.001). The maximal glucose infusion rate after inhalation of insulin was lower than after subcutaneous insulin injection (6.2 +/2- 2.4 vs 9.1 +/- 2.5 mg kg-1 min-1; p < 0.001). The glucose infusion rates in the first 60 min after inhalation were significantly greater than after insulin injection (area under the glucose infusion rate curve: 0.23 +/- 0.12 vs 0.13 +/- 0.08 g kg-1 60 min-1; p < 0.001). However, the total metabolic effect after inhalation was significantly lower than after insulin injection (1.44 +/- 0.68 vs 1.90 +/- 0.47 g kg-1 360 min-1; p < 0.001). Relative effectiveness of inhaled insulin calculated with regard to the data from the intravenous insulin application was 9.5 +/- 4.1% and of the subcutaneous insulin application was 7.6 +/- 2.9%. With its rapid onset of action, inhaled insulin might have potential for clinical use.

Cloning and expression of a cDNA encoding uridine kinase from mouse brain.

Uridine kinase is the rate-limiting enzyme in the pyrimidine salvage pathway of all mammalian cells. A cDNA for uridine kinase from mouse brain has been isolated, sequenced, and characterized. This is the first report of a complete nucleotide sequence for mammalian uridine kinase. The isolated cDNA is only 95% complete, missing the first 17 codons. The correct 5'-terminus sequence was obtained from high-stringency screening of a mouse liver genomic DNA library. The translated cDNA sequence encodes a protein of 277 amino acids (Mr 31,068). A truncated form of the cDNA was expressed in Escherichia coli. The expressed protein displayed uridine kinase activity and readily formed a tetramer, the most active form of the wild-type enzyme. Analysis of the amino acid sequence identified the three ATP-binding site consensus motifs. The predicted secondary structure for uridine kinase and the sequence comparison with three kinases having known crystal structures are consistent with uridine kinase having an alpha/beta core structure of the nucleotide-binding fold found in many kinases. We have also isolated and cloned a nonfunctional, processed pseudogene from mouse genomic DNA. This pseudogene sequence is 94% identical with the coding DNA.

Intrinsic activity and stability of bifunctional human UMP synthase and its two separate catalytic domains, orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase.

Human UMP synthase is a bifunctional protein containing two separate catalytic domains, orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (EC 4.1.1.23). These studies address the question of why the last two reactions in pyrimidine nucleotide synthesis are catalyzed by a bifunctional enzyme in mammalian cells, but by two separate enzymes in microorganisms. From existing data on subunit associations of the respective enzymes and calculations showing the molar concentration of enzyme to be far lower in mammalian cells than in microorganisms, we hypothesize that the covalent union in UMP synthase stabilizes the domains containing the respective catalytic centers. Evidence supporting this hypothesis comes from studies of stability of enzyme activity in vitro, at physiological concentrations, of UMP synthase, the two isolated catalytic domains prepared by site-directed mutagenesis of UMP synthase, and the yeast ODCase. The two engineered domains have activities very similar to the native UMP synthase, but unlike the bifunctional protein, the domains are quite unstable under conditions promoting the dissociated monomer.

Physiological concentrations of purines and pyrimidines.

The concentrations of bases, nucleosides, and nucleosides mono-, di- and tri-phosphate are compared for about 600 published values. The data are predominantly from mammalian cells and fluids. For the most important ribonucleotides, average concentrations +/- SD (microM) are: ATP, 3,152 +/- 1,698; GTP, 468 +/- 224; UTP, 567 +/- 460 and CTP, 278 +/- 242. For deoxynucleosides-triphosphate (dNTP), the concentrations in dividing cells are: dATP, 24 +/- 22; dGTP, 5.2 +/- 4.5; dCTP, 29 +/- 19 and dTTP 37 +/- 30. By comparison, dUTP is usually about 0.2 microM. For the 4 dNTPs, tumor cells have concentrations of 6-11 fold over normal cells, and for the 4 NTPs, tumor cells also have concentrations 1.2-5 fold over the normal cells. By comparison, the concentrations of NTPs are significantly lower in various types of blood cells. The average concentration of bases and nucleosides in plasma and other extracellular fluids is generally in the range of 0.4-6 microM; these values are usually lower than corresponding intracellular concentrations. For phosphate compounds, average cellular concentrations are: Pi, 4400; ribose-1-P, 55; ribose-5-P, 70 and P-ribose-PP, 9.0. The metal ion magnesium, important for coordinating phosphates in nucleotides, has values (mM) of: free Mg2+, 1.1; complexed-Mg, 8.0. Consideration of experiments on the intracellular compartmentation of nucleotides shows support for this process between the cytoplasm and mitochondria, but not between the cytoplasm and the nucleus.

The functions and consensus motifs of nine types of peptide segments that form different types of nucleotide-binding sites.

From an analysis of current data on 16 protein structures with defined nucleotide-binding sites consensus motifs were determined for the peptide segments that form such nucleotide-binding sites. This was done by using the actual residues shown to contact ligands in the different protein structures, plus an additional 50 sequences for various kinases. Three peptide segments are commonly required to form the binding site for ATP or GTP. Binding motif Kinase-1a is found in almost all sequences examined, and functions in binding the phosphates of the ligand. Variant versions, comparable to Kinase-1a, are found in a subset of proteins and appear to be related to unique functions of those enzymes. Motif Kinase-2 contains the conserved aspartate that coordinates the metal ion on Mg-ATP. Motif Kinase-3 occurs in at least four versions, and functions in binding the purine base or the pentose. Two protein structures show ATP-binding at a separate regulatory site, formed by the motifs Regulatory-1 and Regulatory-2. Structures for adenylate kinase and guanylate kinase show three different sequence motifs that form the binding site for a nucleoside monophosphate (NMP). NMP-1 and NMP-2 bind to the pentose and phosphate of the bound ligand. NMP-1 is found in almost all the kinases that phosphorylate AMP, CMP, GMP, dTMP, or UMP. NMP-3a is found in kinases for AMP, GMP, and UMP, while NMP-3b binds only GMP. For the binding of NTPs, three distinct types of nucleotide-binding fold structures have been described. Each structure is associated with a particular function (e.g. transfer of the gamma-phosphate, or of the adenylate to an acceptor) and also with a particular spatial arrangement of the three Kinase segments evident in the linear sequence for the protein.

Dissociation of enzyme oligomers: a mechanism for allosteric regulation.

Most enzymes exist as oligomers or polymers, and a significant subset of these (perhaps 15% of all enzymes) can reversibly dissociate and reassociate in response to an effector ligand. Such a change in subunit assembly usually is accompanied by a change in enzyme activity, providing a mechanism for regulation. Two models are described for a physical mechanism, leading to a change in activity: (1) catalytic activity depends on subunit conformation, which is modulated by subunit dissociation; and (2) catalytic or regulatory sites are located at subunit interfaces and are disrupted by subunit dissociation. Examples of such enzymes show that both catalytic sites and regulatory sites occur at the junction of 2 subunits. In addition, for 9 enzymes, kinetic studies supported the existence of a separate regulatory site with significantly different affinity for the binding of either a substrate or a product of that enzyme. Over 40 dissociating enzymes are described from 3 major metabolic areas: carbohydrate metabolism, nucleotide metabolism, and amino acid metabolism. Important variables that influence enzyme dissociation include: enzyme concentration, ligand concentration, other cellular proteins, pH, and temperature. All these variables can be readily manipulated in vitro, but normally only the first two are physiological variables. Seven of these enzymes are most active as the dissociated monomer, the others as oligomers, emphasizing the importance of a regulated equilibrium between 2 or more conformational states. Experiments to test whether enzyme dissociation occurs in vivo showed this to be the case in 6 out of 7 studies, with 4 different enzymes.

Cloning, sequencing, and expression of a cDNA encoding beta-alanine synthase from rat liver.

A cDNA for beta-alanine synthase from rat liver has been isolated, sequenced, and characterized. beta-Alanine synthase clones were isolated from rat liver cDNA libraries in lambda gt11, using affinity-purified polyclonal antibodies against beta-alanine synthase protein. beta-Alanine synthase protein was not expressed with equal efficiency by all clones. One of the expressed fusion proteins has normal specific enzyme activity, and a second has reduced specific activity. Both clones were completely sequenced and yielded identical DNA sequence, except that one clone contained an additional 36 bases of 5' sequence. The various clones of this cDNA code for an EcoRI insert of 1.5 +/- 0.1 kb, and the open reading frame corresponds to a protein of 393 amino acids (M(r) = 44,042), in good agreement with the M(r) of approximately 42,000 for the native enzyme on SDS-gel electrophoresis. An 11-amino acid sequence was obtained from a tryptic peptide of native beta-alanine synthase; 11 codons for these same amino acids were found at the expected site in the sequenced cDNA, and confirm the open reading frame of the beta-alanine synthase cDNA. Chemical analysis of the native enzyme shows 2 zinc atoms per subunit, and the sequence of beta-alanine synthase contains 2 putative zinc-binding site motifs. Comparison of amino acid sequence, deduced from the cDNA sequence, to sequences in the protein data base showed that it is a unique sequence and that it has about 20% identity to aspartate carbamoyltransferase, ornithine carbamoyltransferase, urease, and leucine aminopeptidase; enzymes that bind comparable ligands or have a similar mechanism.

beta-Alanine synthase: purification and allosteric properties.

beta-Alanine synthase has been purified greater than 1000-fold to homogeneity from rat liver. The enzyme has a subunit molecular weight of 42,000 and a native size of hexamer. The enzyme undergoes ligand-induced changes in polymerization: association in response to the substrate, N-carbamoyl-beta-alanine, and the inhibitor, propionate; and dissociation in response to the product, beta-alanine. The ability of the substrate to associate the pure native enzyme to a larger polymeric species was exploited in the final purification step. The purified enzyme had a pI of 6.7, a Km of 8 microM, and a kcat/Km of 7.9 x 10(4) M-1 s-1. Positive cooperativity was observed toward the substrate N-carbamoyl-beta-alanine, with nH = 1.9. Such cooperativity occurred at substrate concentrations below 12 nM, so that this activation most likely occurs at a regulatory site, with a significantly stronger affinity for N-carbamoyl-beta-alanine than that shown by the catalytic site. The enzyme was sensitive to denaturation, which could be minimized by avoiding heat steps during the purification and by the presence of reducing agents. Such denatured enzyme had little change in Vmax, but had much higher Km, and had also lost the ability to associate or dissociate in response to effectors. After purification, enzyme stability was achieved by the addition of glycerol and detergent.

Allosteric regulation of purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM).

Purine nucleoside phosphorylase. Allosteric regulation of a dissociating enzyme.

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is a trimeric enzyme that readily dissociates to the monomer. Dilution of enzyme from 20 to 0.02 microgram of protein/ml is accompanied by a greater than 50-fold increase in the specific activity (vtrimer = 0.23 nmol/min/microgram; vmonomer = 12.5 nmol/min/micrograms). Gel permeation chromatography in the presence of the substrate phosphate shows the enzyme to be predominantly trimeric at 50 mM Pi and predominantly monomeric at 100 mM Pi, when experiments are done at 24 degrees C. No significant dissociation was observed at 4 degrees C with Pi or at either temperature with the substrate inosine. As measured by dissociation, the L0.5 for Pi is 88 mM and thus significantly higher than the Km of 3.1 mM for Pi. Enzyme activity as a function of phosphate concentration showed negative cooperativity, but the conformational response measured by the change in native Mr during dissociation showed positive cooperatively toward Pi. These data support a model for two separate phosphate binding sites on the enzyme. The activity and stability of purine nucleoside phosphorylase are quite sensitive to the concentration of the enzyme as well as appropriate substrates. Although the monomer is interpreted as being a fully active form of the enzyme, the data in general are most consistent with the enzyme functioning in vivo as a regulated trimer.