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The formation of multiple ligand complexes with muscle pyruvate kinase was measured in terms of dissociation constants and the standard free energies of formation were calculated. The binding of Mn2+ to the enzyme (KA = 55 +/- 5 X 10(-6) M; deltaF degrees = -5.75 +/- 0.05 kcal/mol) and to the enzyme saturated with phosphoenolpyruvate (conditional free energy) KA' = 0.8 +/- 0.4 X 10(-6) M; deltaF degrees = -8.22 +/- 0.34 kcal/mol) has been measured under identical conditions giving a free energy of coupling, delta(deltaF degrees) = -2.47 +/- 0.34 kcal/mol. Such a large negative free energy of coupling is diagnostic of a strong positively cooperative effect in ligand binding. The binding of the substrate phosphoenolpyruvate to free enzyme and the enzyme-Mn2+ complex was, by necessity, measured by different methods. The free energy of phosphoenolpyruvate binding to free enzyme (KS = 1.58 +/- 0.10 X 10(-4)M; deltaF degrees = -5.13 +/- 0.04 kcal/mol) and to the enzyme-Mn2+ complex (K3 = 0.75 +/- 0.10 X 10(-6)M; deltaF degrees = -8.26 +/- 0.07 kcal/mol) also gives a large negative free energy of coupling, delta(deltaF degrees) = -3.16 +/- 0.08 kcal/mol. Such a large negative value confirms reciprocal binding effects between the divalent cation and the substrate phosphoenolpyruvate. The binding of Mn2+ to the enzyme-ADP complex was also investigated and a free energy of coupling, delta(deltaF degrees) = -0.08 +/- 0.08 kcal/mol, was measured, indicative of little or no cooperativity in binding. The free energy of coupling with Mn2+ and pyruvate was measured as -1.52 +/- 0.14 kcal/mol, showing a significant amount of cooperativity in ligand binding but a substantially smaller effect than that observed for phosphoenolpyruvate binding. The magnitude of the coupling free energy may be related to the role of the divalent cation in the formation of the enzyme-substrate complexes. In the absence of the activating monovalent cation, the coupling free energies for phosphoenolpyruvate and pyruvate binding decrease by 40-60% and 25%, respectively, substantiating a role for the monovalent cation in the formation of enzyme-substrate complexes with phosphoenolpyruvate and with pyruvate.

Formyltetrahydrofolate synthetase, a tetrameric protein, contains 23 half-cystine residues as determined by cysteic acid analysis and 24 free sulfhydryl groups as determined by four different methods thus indicating absence of disulfide bonds. All of the cysteine residues react with sulfhydryl reagents in the absence of denaturing agents. Titration of the enzyme with p-chloromercuribenzoate (CMB) caused a loss in activity which was a linear function of the amount of thiol groups reacted. A complete loss in activity occurred after 11 CMB molecules had reacted per mole of enzyme. The loss in activity was accompanied by the appearance of the monomeric form of the protein; however, the presence of tetramer which contained CMB was demonstrated by Sephadex G-200 chromatography of enzyme which had been partially inactivated with 14C-labeled CMB. The reaction of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was different from that with CMB. The relation between thiol groups reacted and loss in activity was not linear, and about 18 to 20 thiol groups had to react before a complete loss in activity occurred. Dissociation to the monomeric form did not occur until about 75% of the cysteine residues had reacted. Titration with HgCl2 showed that a linear decrease in activity occurred until complete inactivation was observed after 8 Hg2+ ions had been bound by the protein. Hg2+ could be bound to virtually all of the cysteine residues without causing dissociation of the enzyme. Reactivation by cysteine of enzyme inactivated by CMB or DTNB was variable and could be achieved only when the enzyme was still in the tetrameric form. Total reactivation of mercury enzyme was achieved with 2-mercaptoethanol or cysteine. The inactivation by CMB and DTNB was retarded by ATP and tetrahydrofolate and facilitated by MgATP. Formate and Mg2+ had no effect on the inactivation. It is concluded that the inactivation of formyltetrahydrofolate synthetase by sulfhydryl reagents results from conformational changes produced in the tetramer as well as from dissociation of the tetramer to the inactive monomer. The effects produced by the substrates probably result from conformational changes in the protein. There is no evidence which suggests that a sulfhydryl group or groups constitute part of the active site of the enzyme.

The optically pure d and l isomers of 2-phospholactate were synthesized by phosphorylation of the appropriate lactic acids. The interactions of these analogues and of phosphoglycolate with several enzymes that catalyze reactions of phosphoenolpyruvate were examined by kinetics and by direct binding studies measuring the longitudinal relaxation rate of water protons. With pyruvate kinase, the Ki of d-phospholactate (21 µm) and of phosphoglycolate (52 µm) agree with the Km of phosphoenolpyruvate (26 µm), while the Ki of l-phospholactate (380 µm) is 18-fold greater. This difference in affinity is confirmed in the direct binding studies. The enhancement of the ternary pyruvate kinase-Mn-inhibitor complex of d-phospholactate (et = 1.9) and of phosphoglycolate (et = 1.7) agree with that of phosphoenolpyruvate (et = 2.2), but differs from that of l-phospholactate (et = 3.8), indicating a structural difference in this latter metal bridge complex. With enolase, the inhibition was not stereospecific since the Ki values of both isomers of phospholactate and of phosphoglycolate were equal (370 µm) and larger than the Km of 2-phosphoglycerate (6.7 µm). Equal affinities for the analogues were confirmed by direct binding studies, but unequal ternary enhancements were observed for the substrate phosphoenolpyruvate (et = 3.7), d-phospholactate (et = 11.6), phosphoglycolate (et = 8.4), and l-phospholactate (et = 5.8) because of structural differences in their respective ternary complexes. Phosphoenolpyruvate carboxykinase, which catalyzes a reaction with a mechanism homologous with that of pyruvate kinase, shows the opposite inhibition pattern since it is preferentially inhibited by l-phospholactate (Ki = 28 µm) as compared with d-phospholactate (Ki = 292 µm). None of the analogues inhibited the reaction catalyzed by phosphoenolpyruvate synthetase. The patterns of inhibition by the phospholactates of pyruvate kinase (d >> l), enolase (d = l), and carboxykinase (l >> d) are explained in terms of the known stereochemistries of the reactions catalyzed by these enzymes and the structures at their respective active sites.

Pyruvate kinase, which has an absolute requirement for both a monovalent and a divalent cation for activity, can be activated by the monomethylammonium cation at a rate 0.5% that measured in the presence of K+. The kinetically determined dissociation constant for the monovalent activator in the presence of saturating substrates (K1 = 85 mm) agrees with that measured for K+ and the dissociation constant measured in the presence of saturating substrates and the divalent activator Mn2+, (K4 = 20.5 ± 3.3 mm) is somewhat larger than that for K+. The presence of carbonbound protons on the movovalent activator permits the investigation of the interaction of the monovalent activator with the enzyme-Mn2+ complexes by proton nuclear magnetic resonance techniques. The presence of the enzyme has no effect on the longitudinal (1/T1) and transverse (1/T2) relaxation rates of the carbon-bound protons. The formation of the enzyme-Mn2+ complex increases the relaxation rates due to the electron-nuclear dipolar interaction of the paramagnetic Mn2+ and the protons. In the presence of the substrate phosphoenolpyruvate, the relaxation rates are even further increased. A measure of the relaxation rates at two frequencies allows the evaluation of the correlation time for the electron-nuclear interaction, τc by several methods. No significant change in the correlation time is seen when the substrate complex is formed. The electron-nuclear distance can thus be evaluated from the correlation time, τc, and from the values measured for 1/T1. In the absence of substrate the protons of the methyl group are 8.3 ± 0.6 A from the enzyme-bound Mn2+. Upon formation of the pyruvate kinase-Mn2+-phosphoenolpyruvate complex, the protons move to 6.6 ± 0.2 A of the Mn2+. This result shows that the substrate affects the conformation of the active site by bringing the monovalent and divalent cation activators in closer proximity and that the monovalent activator can indeed bind in the active site of pyruvate kinase. The Mn2+-methyl proton distance measured is that predicted if the monovalent cation acts by binding P-enolpyruvate via the carboxyl group to form the active complex (Nowak, T., and Mildvan, A. S. (1972) Biochemistry 11, 2819). Monomethylammonium ion can thus serve as a useful probe in studying the interactions of monovalent cations with many biological systems with relative ease and should lead to a greater understanding of monovalent cation activation and function in biology.

Cell-associated flaviviruses contain the two membrane proteins V3 and NV2 besides the viral core protein V2 whereas extracellular viruses do contain V2 protein and the two membrane proteins V3 and V1. Since the V1 protein could not be detected in infected cells it has been suggested that V1 is generated from NV2 by proteolytic cleavage during the release of virus from cells ( D. Shapiro, W. E. Brandt, and P. K. Russell (1972) , Virology 50 , 906–911). We have isolated the viral structural proteins V1, V2, and NV2 from the flavivirus West Nile virus and determined their amino-terminal amino acid sequences and amino acid sequences of peptides derived from these proteins. We have also transcribed parts of the viral genome into cDNA and cloned and sequenced this cDNA. The analyses of the protein structure of V1, V2, and NV2 together with the determination of the amino-terminal sequence of V3 (data not shown) have allowed us to identify the nucleotide region coding for the structural proteins V2, NV2, and V1. The primary structure of this nucleotide sequence is presented in this report. The data show that the amino terminus of the viral core protein V2 is followed by the amino termini of the proteins NV2, V1, and V3, respectively. These data for the first time identify the exact order of all structural proteins of a flavivirus identified so far. Our data strongly support the above-mentioned hypothesis that V1 is derived from NV2 by proteolytic cleavage and furthermore indicate that V1 represents the nonglycosylated carboxy-terminal part of NV2 which contains those sequences which anchor NV2 in the viral membrane. A working hypothesis is presented in which two species of cellular enzymes, signalase(s) removing signal sequences and enzymes involved in cleaving polyproteins after a pair of basic amino acids, do generate the proteins V2, NV2, and V1 from the growing peptide chain synthesized during translation of the 42 S genome RNA which functions as mRNA for these proteins.

The use of implantable morphine pumps for the treatment of cancer pain is increasing. A sudden loss of previously adequate pain control should suggest pump malfunction. Pump revision should be considered, rather than automatically increasing oral analgesics. This case report details a rapid loss of pain control due to intraspinal catheter migration into the abdominal subcutaneous space

The interactions of nucleotide substrates with the enzyme phosphoenolpyruvate carboxykinase and its Mn2+ complex were investigated by several methods. Direct binding shows the formation of stoichiometric complexes. The presence of Mn2+ increases the affinity of the enzyme for nucleotide. A higher affinity for GTP (Kd less than 2 microM) than for GDP (Kd = 15 microM) was determined. Solvent proton relaxation rate studies indicate no substantial difference in titration curves for free nucleotide or for Mg-nucleotide to the enzyme-Mn complex. The effect of Mn2+ on the 31P relaxation rates of IDP and of ITP in the binary Mn-nucleotide complex indicates the formation of direct coordination complexes. The distances of the alpha- and beta-31P of IDP to Mn2+ are identical (3.5 A). The Mn2+ distance to the beta- and gamma-31P of ITP is also identical (3.7 A) and is 0.2 A further from the alpha-phosphorus. In the presence of P-enolpyruvate carboxykinase, the effect of Mn2+ on the 31P relaxation rates was measured at 40.5 MHz and at 121.5 MHz. The dipolar correlation time was calculated to be 0.6-5.4 ns, depending upon assumptions made. The Mn2+ to phosphorus distances indicate the nucleotide substrates form a second sphere complex to the bound Mn2+. From 1/T2 measurements, electron delocalization from Mn2+ to the phosphorus atoms is indicated; this effect occurs although direct coordination does not take place. The exchange rate of GTP from the enzyme-Mn complex (koff = 4 X 10(4) s-1) is rapid compared to kcat with a lower energy of activation (9.2 kcal/mol) than for catalytic turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction of fluorophosphate with muscle pyruvate kinase was investigated by 31P nuclear relaxation rate measurements. The fluorophosphate samples were highly purified and were first monitored by 19F and 31P relaxation rate measurements in the formation of the binary FPO3-Mn complex. The results of the binary complex demonstrated that FPO32− binds in the first coordination sphere of Mn2+ via the oxygen atoms but not via the fluorine. The enzyme experiments were designed under conditions where a significant fraction of the ligand is in the ternary enzyme-Mn-FPO3 complex. These studies demonstrate that the 31P relaxation rate of bound FPO3 ( 1 T 1 m = 1.58 ± 0.05 × 10 5 s −1 ) is consistent with the binding of this ligand in the first coordination sphere of enzyme-bound Mn2+ with an elongated Mn-O-P distance ( r Mn-P = 3.3 ± 0.2 A ). Such a structure is demonstrated in the ternary enzyme-Mn-FPO3 complex, in the complex containing HCO3−, and in the complex also containing HCO3− and ADP. The data further substantiate the binding of phosphoenolpyruvate analogs in the first coordination sphere of pyruvate kinase-bound Mn2+.

The genome RNA of the flavivirus West Nile (WN) virus has been transcribed into cDNA, the eDNA has been cloned, and the nucleotide sequences coding for the structural proteins have been determined ( Castle et al ., 1985 ; Wengler et al ., 1985 ). We have now determined the nucleotide sequence coding for all viral nonstructural proteins which comprises 7929 nucleotides. Together with our earlier sequence analyses these data show that a long open reading frame (ORF) containing 10,290 nucleotides is present on the genome of WN virus. The two largest nonstructural proteins which can be detected in flavivirus-infected cells are the proteins NV5 and NV4 which have an apparent molecular mass of 97,000 and 74,000 Da, respectively. Both proteins were isolated by preparative polyacrylamide gel electrophoresis, and partial amino acid sequences of peptides derived from these proteins were determined. These analyses allow us to localize the nucleotide regions which code for these proteins and show that the region coding for the NV5 protein is located at the 3′-terminus of the long ORF. Together with our earlier analyses these data show that the protein sequences of virus-specific proteins are present on the viral polyprotein translated from the long ORF in the order V2-NV2-V3-(nonstructural proteins of up to 75,000 Da)-NV4-(nonstructural proteins of up to 45,000 Da)-NV5. Our data indicate that virus-specific structural and nonstructural proteins which are synthesized from a single long ORF accumulate in large amounts in infected cells. A possible role of the presence of these molecules, which are associated to cellular membranes, in the accumulation of membrane vesicles which characteristically occurs in flavivirus-infected cells is discussed.

31P-NMR has been applied to the study of the metabolisms of the intact parasitic helminths Ascaris suum (the intestinal roundworm) and Fasciola hepatica (the liver fluke). After calibration of the chemical shift of Pi in muscle extracts the internal pH of adult Ascaris worms and the effect of the pH of the external medium on the organism's internal pH were measured. Assignments of nearly all of the observable 31P resonances could be made. A large resonance from glycerophosphorylcholine whose function is unclear was observed but no signals from energy storage compounds such as creatine phosphate were detected. The profiles of the phosphorus-containing metabolites in both organisms were monitored as a function of time. Changes in sugar phosphate distributions but not ATP/ADP were observed. Studies of the drug closantel on Fasciola hepatica were performed. Initial effects of the drug were a decrease in glucose 6-phosphate and an increase in Pi with no substantial change in ATP levels as observed by 31P-NMR. Studies involving treatment with closantel followed by rapid freezing, extraction, and analytical determination of glycolytic intermediates confirmed NMR observations. This NMR method can serve as a simple noninvasive procedure to study parasite metabolism and drug effects on metabolism.

Phosphoenolpyruvate carboxykinase [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] was purified from the liver of several species; the mitochondrial enzyme and the cytosolic enzyme were separated from each other. The species included guinea pig, monkey, rat, and chicken. Each enzyme was assayed for inhibition by the substrate analogues E-2-phosphoenolbutyrate and Z-2-phosphoenolbutyrate. Each enzyme tested displayed the same stereospecificity: the E diastereoisomer was the more potent inhibitor than the Z. These results suggest an active site homology for all carboxykinases. The absolute values for Ki measured show that in almost every case the mitochondrial enzyme is more susceptible to inhibition by these analogues than is the cytosolic enzyme. The Ki values are one-fifth those for the mitochondrial enzymes. These results imply subtle differences in ligand interactions at the active sites of these enzymes.

The binding of Mn 2+ to the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium was examined by electron paramagnetic resonance studies. Two types of binding sites were observed: one to two tight sites with a dissociation constant of 3–5 μ m and five to six weaker sites with a dissociation constant of 40–70 μ m . The activator constant for Mn 2+ was found to be 9 μ m for the glutamine-linked anthranilate synthetase activity and 4 μm for the phosphoribosyltransferase activity. These values are both in the range of the dissociation constant for the tight sites. Water proton relaxation rate measurements showed that the binary enhancement values for both classes of sites were equivalent, ϵ b = 10.7 ± 2.0. The addition of chorismate to the Mn 2+ -enzyme complexes when predominantly the tight Mn 2+ sites were occupied resulted in a large decrease in the observed enhancement ( ϵ T = 2.0). Addition of 5-phosphoribosyl-1-pyrophosphate to the enzyme-Mn 2+ complexes caused large decreases in the water proton relaxation rate ( ϵ T = 1.5) when tight or tight plus weaker Mn 2+ sites were occupied. No changes in the water proton relaxation rate were observed when glutamine, pyruvate, or anthranilate were added; a small decrease was observed when enzyme-Mn 2+ was titrated with tryptophan. Tryptophan significantly altered the effect of the binding of chorismate but not of 5-phosphoribosyl-1-pyrophosphate. The effect of tryptophan on the water proton relaxation rate of a Mn 2+ -enzyme-chorismate complex using a variant enzyme complex which is tryptophan hypersensitive ( P. D. Robison, and H. R. Levy, 1976 , Biochim. Biophys. Acta . 445 , 475–485) occurred at lower concentrations than for the normal enzyme complex. The uncomplexed anthranilate synthetase subunit was titrated with Mn 2+ and found to have one to two binding sites with a dissociation constant of 300 ± 100 μ m . This dissociation constant is much larger than the activator constant for Mn 2+ for uncomplexed anthranilate synthetase which was determined to be 4 μ m . These results indicate that the Mn 2+ -binding sites on anthranilate synthetase are altered when the enzyme complex is formed and that both chorismate and 5-phosphoribosyl-1-pyrophosphate interact closely with enzyme-bound Mn 2+ or cause a large effect upon its environment.

The proteolytic processes involved in the synthesis of the structural proteins of the West Nile (WN) flavivirus were analyzed: The carboxy-terminal sequences of the structural proteins were determined and the proteins translated in vitro in the presence of membranes from a mRNA coding for the structural polyprotein were analyzed. The results obtained indicate that the following proteolytic activities are involved in the synthesis and assembly of WN virus structural proteins: The growing peptide chain which contains the sequences of the structural proteins in the order C-pre-M-E is cleaved at three places by cellular signalase(s). This cleavage generates the primary amino acid sequence of the mature structural proteins pre-M and E (and the amino-terminus of the ensuing nonstructural protein NS 1). The amino-terminal part of the polyprotein containing the amino acid residues 1 to 123 is released as a molecule which migrates slightly slower than the mature viral core protein and which presumably is associated to the RER membranes via its carboxy-terminal sequence. This protein is called the anchored C protein. Priorto or during assembly of intracellular virus particles the anchored C protein is converted into mature C protein by removal of the carboxy-terminal hydrophobic segment containing the amino acid residues 106 to 123. Presumably a virus-coded protease which can cleave the polyprotein after two basic amino acid residues is responsible for this cleavage. The cell-associated WN virus particles are constructed from the proteins C, pre-M, and E which contain the amino residues 1–105, 124–290, and 291–787 of the polyprotein, respectively. Cleavage of the pre-M protein between amino acid residues 215 and 216, presumably by a cellular enzyme located in the Golgi vesicles, and loss of the amino-terminal fragment of this protein are associated with the release of virus from the cells.

Mobility in conventional ad-hoc networks is a challenge due to the constant invalidation end-to-end paths. We deal with mobile ad-hoc networks where humans are the main carriers of mobile devices. A good understanding of human mobility patterns aids the design of a realistic mobility model as a tool for evaluating network protocols. Conventional models for evaluating network protocols in early ad-hoc networks (e.g., random walks, random waypoints, random directions) fail to properly capture human mobility. In fact, recent studies have shown that human mobility is influenced by personal habits, social relationships, environmental features, and locations preferences. Therefore, a realistic model should be able to include these features. In this regard, we develop a heuristic to characterize human mobility based on the spatial, temporal, and connectivity features using real traces. Consequently, we uncover temporal dynamic movement clusters associated with individual users. We also study the distribution of the travel distance, pause time, angle of movement, contact duration, and inter-contact duration. Motivated by our findings, we proposed a new synthetic mobility model that mimics realistic features of human mobility. We validate the model by comparing its synthetic traces against real mobility measurements. Moreover, in a smart-campus environment, networks support applications for environmental monitoring and indoor/outdoor positioning, sometimes with a large deployment of sensors. Considering the limitation of sensors such as battery limitation, dynamicity, and low computing clock rate, sensor clocks need to have a common time to perform information fusion algorithms, implement energy management protocols, or real-time processing for safety applications. Given this, we proposed a pulse-coupled distributed clock synchronization algorithm for wireless sensor networks to reduce the clock skew due to the ambient conditions, mobility, or manufacturing defects. In our algorithm, sensors measure time differences by only exchanging zero-bit pulse instead of packets. Therefore our algorithm is lightweight and robust to the failure of the sensors in the network. The proposed algorithm is compared to previous work under static and mobile settings, and the results show that it can achieve reduced the clock skew, especially in a dynamic environment with high uncertainty in clock drift and unexpected topological changes like vehicular networks.; La mobilité dans les réseaux ad-hoc conventionnels est un défi en raison de l'invalidation constante des chemins de bout en bout. Nous traitons, dans cette thèse, spécifiquement des réseaux mobiles ad-hoc où les humains sont les principaux porteurs d'appareils mobiles communicants. Une bonne compréhension de la mobilité humaine permet la conception d'un modèle de mobilité réaliste en tant qu'outil d'évaluation des protocoles de réseau. Les modèles conventionnels d'évaluation des protocoles des premiers réseaux ad hoc (par exemple, random walks, random waypoints, random directions) ne parviennent pas à simuler correctement la mobilité humaine. Des études récentes ont montré que la mobilité humaine est influencée par les habitudes personnelles, les relations sociales, les caractéristiques environnementales et les préférences de localisation. Par conséquent, un modèle réaliste devrait inclure ces caractéristiques. À cet égard et à l'aide de traces réelles, nous avons développé une heuristique pour définir un modèle de mobilité humaine basée sur des caractéristiques spatiales, temporelles et de connectivité. Nous avons remarqué des clusters de mouvements dynamiques temporels associés à des utilisateurs individuels. Nous avons étudié la distribution de la distance parcourue, du temps de pause, de l'angle de déplacement, de la durée de contact et de la durée d'inter-contact. Motivés par nos résultats, nous avons proposé un nouveau modèle de mobilité qui imite de manière réaliste les caractéristiques de la mobilité humaine. Notre modèle a été validé en comparant ses traces synthétiques à des mesures de mobilité réelles. Dans un environnement de campus intelligent, les réseaux prennent en charge les applications de surveillance environnementale et de positionnement intérieur/extérieur, parfois avec un déploiement important de capteurs. Compte tenu des limitations des capteurs telles qu'autonomie énergétique, capacité de calcul limité, et la dynamique, les horloges des capteurs doivent être synchronisées pour exécuter des algorithmes de fusion de données, mettre en œuvre des protocoles de gestion de l'énergie ou un traitement temps réel des applications où la sécurité est importante. Compte tenu de cela, nous avons proposé un algorithme de synchronisation d'horloge distribuée à couplage d'impulsions pour des réseaux de capteurs sans fil. Notre algorithme permet de réduire les décalages d'horloge dus aux conditions ambiantes, à la mobilité ou aux défauts de fabrication. Pour ce faire, les capteurs mesurent les différences de temps en échangeant uniquement des impulsions au lieu de paquets. Par conséquent, notre algorithme est léger et robuste à la défaillance de capteurs du réseau. L'algorithme proposé est comparé aux travaux antérieurs avec des paramètres statiques et mobiles. Les résultats montrent qu'il peut réduire le décalage d'horloge, en particulier dans un environnement dynamique avec une dérive d'horloge importante et des changements topologiques inattendus comme ceux apparaissant dans les réseaux de véhicules.