Structural constraints on the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase from rotational-echo double-resonance NMR

The 46 kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, 15N labels were introduced selectively into the lysine, arginine and histidine residues of EPSP synthase and distances to a 13C label in Glp and to the 31P in S3P and Glp were measured by rotational-echo double-resonance NMR. Three lysine and four arginine residues are in the proximity of the phosphate group of S3P and the carboxyl and phosphonate groups of Glp. A single histidine residue is in the vicinity of the binding site (closer to Glp than to S3P) but is more distant than the lysine and arginine residues.     

Slowed enzymatic turnover allows characterization of intermediates by solid-state NMR

EPSP (5-enolpyruvylshikimate-3-phosphate) synthase catalyzes condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP, a precursor to the aromatic amino acids. S3P and [2-13C]POP were bound to mutant or wild type E. coli forms of the enzyme prior to lyophilization. CPMAS-echo and rotational-echo double-resonance (REDOR) NMR experiments, employing a slow catalytic EPSP synthase mutant and a long prelyophilization incubation interval, allowed our observation of the gradual formation of a strong 31P-13C coupling consistent with the well characterized tetrahedral intermediate. However, after shorter low temperature incubation intervals of substrates with mutant or wild-type enzymes, carbon CPMAS-echo NMR spectra showed the 13C label at 155 ppm, consistent with sp2 geometry of this carbon. REDOR revealed that the phosphorus of PEP was cleaved. However, phosphorus at a distance of 7.5 A was observed, due to the phosphate of a nearby bound S3P. Heating the sample allowed the reaction to progress, as shown by the diminution of the 155 ppm peak and growth of a peak at 108 ppm. The sp3 geometry implied by the 108 ppm peak strongly suggested formation of a S3P-PEP condensation product. REDOR indicated that phosphorus was still distant, but now only 6.1 (wild type) or 5.9 A (mutant) distant. We think that the early intermediates with peaks at 155 and 108 ppm are covalently bound to the enzyme. We also think that the tetrahedral intermediate that we observed was formed after product was generated.     

Analysis of fluoromethyl group chirality establishes a common stereochemical course for the enolpyruvyl transfers catalyzed by EPSP synthase and UDP-GlcNAc enolpyruvyl transferase

The stereochemistry of transient methyl group formation at C-3 of phosphoenolpyruvate (PEP) in the reaction catalyzed by 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase has been examined using the pseudosubstrates, (E)- and (Z)-3-fluorophosphoenolpyruvate (FPEP). Kinetically stable, chiral [1H, 2H]fluoromethyl analogs of the reaction tetrahedral intermediate were isolated and subjected to decomposition and stereochemical analysis. EPSP synthase was found to catalyze the 2-re face addition of solvent-derived hydrogen to C-3 of FPEP (corresponding to the 2-si face of PEP). Comparison of these data with prior analogous work on the MurA reaction [Kim, D.H., Lees, W.J., & Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381] suggests that the two enolpyruvyl transferases share a common stereochemical course, further strengthening the mechanistic, structural, and evolutionary relationship between the two enzymes.     

Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase

The site-directed mutagenesis of a number of proposed active site residues of 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is reported. Several of these mutations resulted in complete loss of enzyme activity indicating that these residues are probably involved with catalysis, notably K22R, K411R, D384A, R27A, R100A, and D242A. Of those, K22R, R27A, and D384A did not bind either the substrate shikimate-3-phosphate (S3P) or glyphosate (GLP). The K411R and D242A mutants bind S3P only in the presence of GLP. The kinetic characterization of mutants R100K, K340R, and E418A, which retain activity, is reported. Of those, R100K and K340R do not accumulate enzyme intermediate of enzyme-bound product under equilibrium conditions. These residues, while not essential for catalysis, are most likely important for substrate binding. All of the mutants are shown to be correctly folded by NMR spectroscopy.     

Reciprocal suppression of the AMPA and NMDA components of the excitatory postsynaptic potentials in the CA1 area of the rat hippocampus in vitro

The interaction between N-methyl-d-aspartate (NMDA)- and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-dependent components of excitatory postsynaptic potentials (EPSP) was studied in rat hippocampal slices. Responses evoked by stimulation of the collateral commissural fibers were recorded in the radial layer of the CA1 area. Contribution of the NMDA component was changed by application of solutions with different concentrations of magnesium. In solutions with low magnesium concentration, when both AMPA and NMDA components contribute significantly to EPSP, suppression of one of the components by application of selective antagonist resulted in increase in the area of another component. Thus, the sum of pharmacologically isolated AMPA and NMDA components was significantly higher than the control EPSP. For example, at 0.1 mM of magnesium in the extracellular solution the sum of the components was 340 +/- 120% of the control EPSP (p < 0.01, N = 6). The data imply that under the control conditions the EPSP components suppress each other. The mutual suppression of the AMPA and NMDA component of the EPSP can be an important factor which influences the conductivity and plastic properties of central glutamatergic synaptic pathways.     

Catalytic mechanism of Kdo8P synthase: transient kinetic studies and evaluation of a putative reaction intermediate

The mechanistic pathway for the reaction catalyzed by Kdo8P synthase has been investigated, and the cyclic bisphosphate 2 has been examined as a putative reaction intermediate. Two parallel approaches were used: (1) chemical synthesis of 2 and evaluation as an alternate substrate for the enzyme and (2) transient kinetic studies using rapid chemical quench methodology to provide direct observation and characterization of putative intermediate(s) during enzyme catalysis. The putative cyclic bisphosphate intermediate 2, possessing the stereochemistry of the beta-pyranose form, was synthesized and evaluated as a substrate and as an inhibitor of Kdo8P synthase. The substrate activity was examined by monitoring the release of anomeric phosphate over time using proton-decoupled 31P NMR spectroscopy. A very similar time course for the formation of inorganic phosphate was found in each experiment and the corresponding control experiment; i.e., no enzyme-catalyzed acceleration in the anomeric phosphate hydrolysis was detected. It was found however that 2 binds to the enzyme and is a competitive inhibitor with respect to phosphoenolpyruvate binding, having a Ki value of 35 microM. In a parallel study, we have performed single-turnover rapid chemical quench experiments to examine both the forward and reverse directions to identify a putative enzyme intermediate(s). Our results clearly demonstrate that the cyclic bisphosphate intermediate 2 does not accumulate under single-enzyme turnover conditions. This observation, coupled with the results obtained through the evaluation of synthetic 2 as a substrate, strongly suggests that the Kdo8P synthase catalytic pathway does not involve the formation of 2 as a reaction intermediate. Taken together, these combined results support the original hypothesis [Hedstrom, L., and Abeles, R. H. (1988) Biochem. Biophys. Res. Commun. 157, 816-820], which suggests a reaction pathway involving an acyclic bisphosphate intermediate 1.     

Metabolism and bioactivation of clozapine by human liver in vitro

The metabolism of clozapine by human liver has been investigated in vitro. Irreversible protein-binding and conjunction with model nucleophiles have been used as markers for bioactivation of clozapine, while stable metabolite formation has been assessed using radiometric HPLC. In all nine liver microsomal preparations investigated, clozapine was extensively metabolized to the stable products desmethylclozapine (range 19%-27.2%), N-oxide (1.5-20.5%) and three polar metabolites (0-20.8%), and was bioactivated to a protein-reactive metabolite (0.6-2.1%). The CYP2D6 genotype did not influence the capacity of the livers to form these metabolites. All metabolic pathways were inhibited by ketoconazole, indicating the involvement of the cytochrome P450 enzymes. Isozyme-selective inhibitor studies demonstrated that whereas demethylation was performed by CYP1A2, N-oxidation and chemically reactive metabolite formation were dependent upon multiple forms of P450. The N-oxide was readily reduced back to clozapine in the presence of NADPH, this conversion being inhibited by ascorbic acid. Glutathione (1 mM) decreased covalent binding by 70%. The amount of putative adduct formed in the presence of glutathione (13.4 +/- 0.9%) was much greater than the covalent binding (mean 1.1 +/- 0.2%). The bioactivation of clozapine was, like the N-oxidation of clozapine, a reversible process. In summary, our results indicate clozapine undergoes extensive metabolism by human liver to both stable and chemically reactive metabolites, the formation of which is catalyzed by the cytochrome P450 enzymes. The role of the reactive metabolite, which may be a free radical, in the pathogenesis of clozapine agranulocytosis and hepatotoxicity requires further study.     

Transient kinetics of formation and reaction of the uridylyl-enzyme form of galactose-1-P uridylyltransferase and its Q168R-variant: insight into the molecular basis of galactosemia

Galactose-1-phosphate uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate (Gal-1-P) to form UDP-galactose and glucose 1-phosphate (Glc-1-P) through a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP enzyme). Gln 168 in E. coli uridylyltransferase engages in hydrogen bonding with the phosphoryl oxygens of the UMP moiety, which is bonded to His 166 in the intermediate [Wedekind, J. E., Frey, P. A., and Rayment, I. (1996) Biochemistry 35, 11560-11569]. In humans, the point variant Q188R accounts for 60% of galactosemia cases. The corresponding E. coli variant Q168R has been overexpressed and purified. In preparation for kinetic correlation of Q168R and wild-type uridylyltransferases, we tested the kinetic competence of the wild-type UMP-enzyme. At 4 degreesC, the first-order rate constant for uridylylation by UDP-glucose is 281 +/- 18 s-1, and for deuridylylation it is 226 +/- 10 s-1 with Glc-1-P and 166 +/- 10 s-1 with Gal-1-P. Inasmuch as the overall turnover number at 4 degreesC is 62 s-1, the covalent intermediate is kinetically competent. The variant Q168R is uridylylated by UDP-glucose to the extent of about 65% of the potential active sites. Uridylylation reactions of Q168R with UDP-glucose proceed with maximum first-order rate constants of 2.2 x 10(-)4 s-1 and 4.2 x 10(-)4 s-1 at 4 and 27 degreesC, respectively. In experiments with uridylyl-Q168R and glucose-1-P, the mutant enzyme undergoes deuridylylation with maximum first-order rate constants of 4.8 x 10(-)4 s-1 and 1.68 x 10(-)3 s-1 at 4 and 27 degreesC, respectively. The value of Km for uridylylation of Q168R is slightly higher than for the wild-type enzyme, and for deuridylylation it is similar to the wild-type value. The wild-type enzyme undergoes uridylylation and deuridylyation about 10(6) times faster than Q168R. The wild-type activity in the overall reaction is 1.8 x 10(6) times that of Q168R. The wild-type enzyme contains 1.9 mol of Zn+Fe per mole of subunits, whereas the Q168R-variant contains 1.36 mol of Zn+Fe per mole of subunits. The mutation stabilizes the uridylyl-enzyme by 1.2 kcal mol-1 in comparison to the wild-type enzyme. These results show that the low activity of Q168R is not due to overstabilization of the intermediate or to the absence of structural metal ions. Instead, the main defect is very slow uridylylation and deuridylation.     

Transpeptidation by porcine pepsin catalyzed by a noncovalent intermediate unique to its iso-mechanism

Porcine pepsin proteolysis of the hexapeptide Leu-Ser-p-nitro-Phe-Nle-Ala-Leu-OMe (where OMe = methoxy and Nle = norleucine) in the presence of dipeptide Leu-Leu synthesizes a new hexapeptide Leu-Ser-p-nitro-Phe-Leu-Leu. Contrary to transpeptidation kinetics of other proteases, which depend upon an acyl-enzyme intermediate, the time course for pepsin-catalyzed transpeptidation displays a distinct lag before reaching a steady-state reaction velocity. Moreover, this lag is coupled to burst kinetics for the formation of proteolytic products, Leu-Ser-p-nitro-Phe and Nle-Ala-Leu-OMe. The lag requires that free Leu-Ser-p-nitro-Phe accumulate in the reaction medium during the lag phase and subsequently rebind for transpeptidation. Consistent with this dissociative kinetic mechanism are normal solvent isotope effects on formation of the proteolytic products Leu-Ser-p-nitro-Phe (vH/vD = 2.2 +/- 0.2) and Nle-Ala-Leu-OMe (vH/vD = 1.8 +/- 0.1) as opposed to an inverse effect on the formation of the transpeptidation product Leu-Ser-p-nitro-Phe-Leu-Leu (vH/vD = 0.40 +/- 0.09). Because proteolysis is slower in D2O but transpeptidation is faster, the isotopically sensitive step must occur after release of both products of proteolysis, which precludes putative acyl-enzyme covalent intermediates. Isotopically enhanced transpeptidation is a new type of isotope effect but one that is consistent with the Uni Bi iso-mechanism previously postulated on the basis of solvent isotope effects on Vmax but not on Vmax/Km (Rebholz, K. L., and Northrop, D. B. (1991) Biochem. Biophys Res. Commun. 179, 65-69) and confirmed by solvent isotope effects on the onset of inhibition by pepstatin (Cho, Y.-K., Rebholz, K. L., and Northrop, D. B. (1994) Biochemistry 33, 9637-9642). As a new biochemical mechanism for peptide bond synthesis that has a potential for applications in biotechnology, it is here proposed that the energy necessary to drive peptide synthesis from free peptides comes from the sizable free energy drop associated with rehydration of the active site of pepsin in 55 M water.     

Chloride and sodium transport across bovine ciliary body/epithelium (CBE)

PURPOSE: To study the chloride and sodium ion transports across the bovine ciliary body/epithelium (CBE) by a modified Ussing-Zerahn type chamber. METHODS: Isolated bovine CBE preparations were mounted in a modified Ussing-type chamber and the transepithelial electrical parameters were monitored. The inward (stroma to aqueous) and outward (aqueous to stroma) fluxes of 36[Cl] chloride and 22[Na] sodium ions across the CBE were measured under short-circuited conditions. The effect of 0.1 mM of furosemide and bumetanide on the chloride transport were studied. RESULTS: The potential difference (PD), the resistance and the short-circuit current (SCC) across the isolated bovine ciliary body were found to be -0.20+/-0.01 mV (aqueous negative), 75+/-1 omegacm2 and -2.70+/-0.17 microAcm(-2) (mean+/-SEM, n=50) respectively. A statistically significant net inward chloride ion flux of 1.12+/-0.41 microEq h(-1)cm(-2) (p < 0.01) was found (n=15). The net chloride transport was abolished when 0.1 mM furosemide (82% inhibition) and 0.1 mM bumetanide (100% inhibition) were applied bilateral. No significant net sodium ion flux was detected. CONCLUSIONS: Electrolyte and fluid transport across the bovine CBE may be via a bumetanide and furosemide-sensitive chloride transport mechanism. The Na-K-2Cl cotransporter plays a significant role in the trans-CBE chloride transport. The net chloride flux/current was about 12 times higher than the measured SCC, suggesting that the chloride ion transport may be coupled to other ion species.     

Involvement of calcitonin gene-related peptide (CGRP) receptors in insulin-induced vasodilatation in mesenteric resistance blood vessels of rats

1. The vascular effect of insulin in the mesenteric resistance blood vessel and the role of calcitonin generelated peptide (CGRP)-receptor in insulin-induced vascular responsiveness were investigated in rats. 2. The mesenteric vascular beds isolated from Wistar rats were perfused with Krebs solution, and perfusion pressure was measured with a pressure transducer. In preparations contracted by perfusion with Krebs solution containing methoxamine in the presence of guanethidine, the perfusion of insulin (from 0.1 to 3000 nM) caused a concentration-dependent decrease in perfusion pressure due to vasodilatation. The pD2 value and maximum relaxation (%) were 6.94+/-0.22 and 43.9+/-5.2, respectively. 3. This vasodilator response to insulin was unaffected by 100 nM propranolol (beta-adrenoceptor antagonist) plus 100 nM atropine (muscarinic cholinoceptor antagonist), 100 microM L-NG-nitroarginine (nitric oxide synthase inhibitor), 1 microM ouabain (Na+-K+ ATPase inhibitor), or 1 microM glibenclamide (ATP sensitive K+-channel inhibitor). 4. In preparations without endothelium, perfusion of insulin produced a marked vasodilatation. The pD2 value and maximum relaxation (%) were 7.62+/-0.21 and 81.0+/-4.6, respectively, significantly greater than in preparations with intact endothelium. 5. The vasodilator responses to insulin in the preparations without endothelium were significantly inhibited by CGRP[8 37], a CGRP receptor antagonist, whereas pretreatment with capsaisin, a toxin for CGRP-containing nerves, did not affect insulin-induced vasodilatation. 6. These results suggest that insulin induces non-adrenergic, non-cholinergic and endothelium-independent vasodilatation, which is partially mediated by CGRP receptors.     

Effects of cobalt or nickel on the sympathetically mediated contractile responses in rat-isolated vas deferens

Subchronic (30 days) exposure of rats to Co(NO3)2 or NiSO4 (20 mg.kg-1) in drinking water caused suppression of the isolated vas deferens contractile responses to exogenous adenosine 5'-triphosphate (ATP), noradrenaline, and l-phenylephrine, shifting the concentration-response curves to the relevant agonist to the right. Both metals facilitated the alpha 1-adrenoceptor antagonistic effects of prazosin, which resulted in increased pA2 values for the drug (9.68 +/- 0.13 in controls vs. 10.15 +/- 0.12 in Co(2+)-treated preparations and 12.60 +/- 0.67 in Ni(2+)-treated preparations). The inhibitory effect of clonidine on the contractions in response to low-frequency electrical field stimulation (EFS) in metal-treated preparations was decreased with pD2 values: 10.52 +/- 0.04 in controls, 9.56 +/- 0.13 in Co(2+)-treated preparations and 9.92 +/- 0.16 in Ni(2+)-treated preparations. The monophasic contractile responses to low-frequency EFS (0.1 Hz, 1 ms, 80 V) as well as the first phase of the biphasic contractions after high-frequency long-lasting EFS (300 pulses, 0.1 ms, 40 V, at 4, 8 or 20 Hz) were significantly increased in both groups of heavy metal-treated preparations. Therefore, subchronic exposure to Co2+ or Ni2+ leads to changes in pre- and postjunctional mechanisms underlying the sympathetically mediated contractile activity of isolated rat vas deferens.     

Carotid artery dissections presenting as isolated posterior cerebral artery infarctions

Our purpose was to delineate the course of the ureter in the female pelvis in relationship to several important surgical landmarks. Ten female cadavers with undissected pelves were used. The ureter was identified at the pelvic brim and traced inferiorly to the bladder. Sets of measurements (+/- 0.1 cm) that help define the location of the ureter were obtained at the three landmarks; the ischial spine, the obturator canal and the insertion of the arcus tendineus on the pubic bone. The mean distances from the ureter to the pelvic floor were ischial spine, 3.2 +/- 0.1 cm; obturator canal, 3.2 +/- 0.1 cm; and the insertion of the arcus tendineus on the pubic bone, 1.6 +/- 0.1 cm. The mean distances from the arcus tendineus to the pelvic floor were ischial spine, 1.9 +/- 0.1 cm; obturator canal, 2.8 +/- 0.1 cm; and the insertion of the arcus tendineus on the pubic bone, 3.2 +/- 0.1 cm. This study defines the relationship of the ureter to the pelvic floor through measurements taken at three landmarks. The data should be useful to pelvic surgeons and are important for the development of future surgical techniques.     

The role of glucose 6-phosphate in the control of glycogen synthase

Elevated blood glucose concentrations result in increased intracellular levels of glucose 6-phosphate in liver, skeletal muscle, and adipose tissue. In liver, blood glucose concentrations are the main factor in control of the synthesis of glycogen; insulin has only a potentiating effect. In skeletal muscle and adipocytes, glucose alone has little effect on the activity of glycogen synthase, the limiting enzyme in glycogen synthesis. However, insulin released as a result of elevated blood glucose stimulates the translocation of specific glucose transporters to the cell membrane, increases the uptake of glucose, and causes the covalent, dephosphorylation-mediated activation of glycogen synthase. We present evidence that elevated intracellular contents of glucose 6-phosphate provoke the activation of glycogen synthase in liver, muscle, and adipose tissue. In addition, glucose 6-phosphate may inhibit the phosphorylation of glycogen synthase by cyclic AMP-stimulated protein kinase. We show that the stimulated glucose uptake and phosphorylation appear to play a major role in the control by insulin of the enzymes involved in glycogen synthesis.     

Conformation of manganese(II)-nucleotide complexes bound to rabbit muscle creatine kinase: 13C NMR measurements using [2-13C]ATP and [2-13C]ADP

Conformations of cation-nucleotide complexes bound to rabbit muscle creatine kinase were investigated by measuring paramagnetic effects on 13C spin relaxation in E.Mn[2-13C]ATP and E.Mn[2-13C]ADP at three different frequencies, viz., 50, 75, and 125 MHz, and as a function of temperature in the range of 7-35 degrees C (at 75 MHz). Arrhenius plots of the temperature dependencies of relaxation rates show a positive slope with low activation energies of 1.3 +/- 0.2 kcal/mol and 2.0 +/- 0.2 kcal/mol for E.Mn ATP and E.MnADP, respectively. The relaxation rates of both complexes show strong frequency dependence, indicating that these rates are not exchange limited. Analysis of the data yields Mn(II)-2C distances of 10.0 +/- 0.5 A for E.MnATP and 8.6 +/- 0.5 A for E.MnADP. These data were interpreted, along with previously published information, on the location of the cation with respect to the phosphate chain [Jarori, G. K., Ray, B.D., & Nageswara Rao, B. D. (1985) Biochemistry 24, 3487-3494], and on the adenosine conformation [Murali, N., Jarori, G. K., & Nageswara Rao, B. D. (1993) Biochemistry 32, 12941-12948] in these complexes. The Mn(II)-2C distances depend on the orientation of the phosphate chain relative to the adenosine moiety. Conformational searches were performed by varying the two torsion angles, phi 1 (C4'-C5'-O5'-P alpha), and phi 2 (C5'-O5'-P alpha-O alpha beta), along with CHARMm energy computations, in order to determine acceptable conformations compatible with the distances determined. The significant difference in the Mn(II)-2C distances in E.MnATP and E.MnADP is indicative of the structural alterations occurring at the active site as the enzyme turns over.     

Patterns of occurrence of second primary non-mammary malignancies in breast cancer patients: results from 1382 consecutive autopsies

In a previous study, we demonstrated the existence of a 3.2 +/- 0.2 ppm peak in the 1H NMR spectrum at 60 MHz from human pancreatic adenocarcinomas (Capan-1 cell) heterotransplanted into nude mice. This peak, which is not present in normal human pancreas, was attributed to enhanced membrane fluidity and/or or an increase in phospholipid turnover. The present study was designed to identify this signal by comparing the 1H NMR spectra recorded in vivo at 100 MHz from Capan-1 tumors, after suppression of the tissular water proton peak, to those recorded from normal pancreatic tissue, and to those recorded at 300 MHz from lipid extracts. The 1H NMR spectra at 100 MHz of the Capan-1 tumors in vivo exhibited three main peaks in the 3.2 +/- 0.2 ppm region: 1. A peak at 2.8 +/- 0.1 ppm from CH2 protons of the acyl chains of unsaturated phospholipids; 2. A peak at 3.2 +/- 0.1 ppm from the protons of the N(CH3)3 group of choline; and 3 A peak at 3.5 +/- 0.1 ppm attributed to GPC. The NMR 1H 300 MHz spectrum of phospholipid extracts of Capan-1 tumors displayed 12 principal resonances, of which only the N(CH3)3 peak of PC had a similar chemical shift to that observed at low resolution (3.2 +/- 0.2 ppm). This peak had a higher intensity in the xenografts than in normal human pancreatic tissue. HPLC analysis of the same lipid extracts from Capan-1 cells in culture, of tumors derived from these cells and from normal pancreas showed: 1. Identical concentrations of the different phospholipids from cancerous human pancreatic cells in vivo and in culture; and 2. A significantly higher level of PC in the extracts of normal human pancreatic tissue. The increase in intensity of the N(CH3)3 peak of PC in the Capan-1 tumors was not thought to be caused by an increase in PC concentration, but to a difference in conformation or mobility of the PC protons in the xenografts. The increase in relaxation time in cancerous tissue (from 60 to 125 ms) was also taken to be evidence in favor of a high mobility of protons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies of 5-fluorodeoxyuridine 5'-monophosphate binding to carboxypeptidase A-inactivated thymidylate synthase from Lactobacillus casei

The binding of 5-fluorodeoxyuridylate (FdUMP) to carboxypeptidase-inactivated thymidylate synthase obtained from methotrexate-resistant Lactobacillus casei was investigated using [3H]FdUMP in a trichloroacetic acid precipitation assay and by 19F nuclear magnetic resonance spectroscopy. The cleavage of 1 valine residue from the carboxyl terminus of one of the identical subunits of the enzyme dimer correlates with complete loss of thymidylate synthesis (Aull, J. L., Loeble, R. B., and Dunlap, R. B. (1974) J. Biol. Chem. 249, 1167-1172). We have further investigated the phenomenon of carboxypeptidase A-dependent inactivation of thymidylate synthase by employing immobilized carboxypeptidase A in order to facilitate the isolation and characterization of the inactivated enzyme. The time course of carboxypeptidase treatment of thymidylate synthase has been profiled by the spectrophotometric assay, tritium release assay, trichloroacetic acid precipitation assay (covalent adduct analysis), 19F nuclear magnetic resonance spectroscopy, and amino acid analysis. The techniques utilized in this study yielded results which showed that the completely inactivated enzyme (failure to catalyze thymidylate formation) continued to catalyze both covalent FdUMP-enzyme interactions and the formation of the covalent inhibitory ternary complex with the cofactor, 5,1O-methylenetetrahydrofolate, although to a reduced extent, thus effectively uncoupling these processes from thymidylate synthesis activity.     

Calcium-mediated intracellular messengers modulate the serotonergic effects on axonal excitability

We carried out experiments to investigate the mechanisms of serotonin-induced axonal excitability changes using isolated dorsal columns from young (seven to 11-day-old) Long-Evan's hooded rats. Conducting action potentials were activated by submaximal (50%) and supramaximal constant current electrical stimuli and recorded with glass micropipette electrodes. In experiment 1, to study Ca(2+)-mediated mechanisms, we superfused the preparations with Ringer solutions containing varying Ca2+ concentrations. Following superfusion with Ca(2+)-free Ringer solution for 4 h, we tested initial responses to serotonin agonists. Studies then were repeated after preparations had been washed for 1 h with Ringer solution containing 1.5 mM Ca2+ and 1.5 mM Mg2+. After 4 h superfusion of Ca(2+)-free Ringer solution, quipazine (a serotonin2A agonist, 100 microM) did not induce significant axonal excitability changes (amplitude change of 1.4 +/- 1.3%, percentage of predrug control level, +/-S.D., n = 6). A 100 microM concentration of 8-hydroxy-dipropylaminotetralin (a serotonin1A agonist) reduced response amplitudes by 36.3 +/- 4.2% (+/-S.D., P < 0.0005, n = 7) and prolonged latencies by 22.3 +/- 4.3% (+/-S.D., P < 0.0005, n = 7). Application of serotonin (100 microM) decreased amplitudes by 6.6 +/- 5.0% (+/-S.D., P < 0.05, n = 6). Extracellular calcium concentration ([Ca2+]e) was measured at various depths in the dorsal column with ion-selective microelectrodes. Four hours' superfusion with Ca(2+)-free Ringer solution reduced [Ca2+]e to less than 0.1 mM in dorsal columns. In 1.5 mM Ca2+ Ringer solution, quipazine increased the amplitudes by 38.3 +/- 5.8% (P < 0.0005, n = 6). Likewise, serotonin increased the amplitudes by 13.8 +/- 4.9% (P < 0.005, n = 6). In contrast however, 8-hydroxy-dipropylaminotetralin still reduced amplitudes by 35.0 +/- 6.4% (P < 0.0005, n = 7) and prolonged latencies by 24.1 +/- 4.5% (P < 0.0005, n = 7). In experiment 2, we investigated calcium-dependent and cAMP-mediated protein kinase signalling pathways to evaluate their role as intracellular messengers for serotonin2A receptor activation. Two protein kinase inhibitors, 50 microM H7 (an inhibitor of protein kinase C and c-AMP dependent protein kinase) and 100 microM D-sphingosine (an inhibitor of protein kinase A and C) effectively eliminated the excitatory effects of the serotonin2A agonist. 100 microM cadmium (a Ca2+ channel blocker) also blocked the effects of quipazine. Neither these protein kinase inhibitors nor cadmium alone affected action potential amplitudes. These results suggest that replacing Ca2+ with Mg2+ blocks the excitatory effects of quipazine but does not prevent the inhibitory effects of 8-hydroxy-dipropylaminotetralin, and calcium-mediated protein kinase mechanisms modulate axonal excitability changes induced by serotonin and its agonist.     

Comparative enzymatic properties of GapB-encoded erythrose-4-phosphate dehydrogenase of Escherichia coli and phosphorylating glyceraldehyde-3-phosphate dehydrogenase

GapB-encoded protein of Escherichia coli and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) share more than 40% amino acid identity. Most of the amino acids involved in the binding of cofactor and substrates to GAPDH are conserved in GapB-encoded protein. This enzyme shows an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase activity (Zhao, G., Pease, A. J., Bharani, N., and Winkler, M. E. (1995) J. Bacteriol. 177, 2804-2812) but a low phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity, whereas GAPDH shows a high efficient phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity and a low phosphorylating erythrose-4-phosphate dehydrogenase activity. To identify the structural factors responsible for these differences, comparative kinetic and binding studies have been carried out on both GapB-encoded protein of Escherichia coli and GAPDH of Bacillus stearothermophilus. The KD constant of GapB-encoded protein for NAD is 800-fold higher than that of GAPDH. The chemical mechanism of erythrose 4-phosphate oxidation by GapB-encoded protein is shown to proceed through a two-step mechanism involving covalent intermediates with Cys-149, with rates associated to the acylation and deacylation processes of 280 s-1 and 20 s-1, respectively. No isotopic solvent effect is observed suggesting that the rate-limiting step is not hydrolysis. The rate of oxidation of glyceraldehyde 3-phosphate is 0.12 s-1 and is hydride transfer limiting, at least 2000-fold less efficient compared with that of erythrose 4-phosphate. Thus, it can be concluded that it is only the structure of the substrates that prevails in forming a ternary complex enzyme-NAD-thiohemiacetal productive (or not) for hydride transfer in the acylation step. This conclusion is reinforced by the fact that the rate of oxidation for erythrose 4-phosphate by GAPDH is 0.1 s-1 and is limited by the acylation step, whereas glyceraldehyde 3-phosphate acylation is efficient and is not rate-determining (>/=800 s-1). Substituting Asn for His-176 on GapB-encoded protein, a residue postulated to facilitate hydride transfer as a base catalyst, decreases 40-fold the kcat of glyceraldehyde 3-phosphate oxidation. This suggests that the non-efficient positioning of the C-1 atom of glyceraldehyde 3-phosphate relative to the pyridinium of the cofactor within the ternary complex is responsible for the low catalytic efficiency. No phosphorylating activity on erythrose 4-phosphate with GapB-encoded protein is observed although the Pi site is operative as proven by the oxidative phosphorylation of glyceraldehyde 3-phosphate. Thus the binding of inorganic phosphate to the Pi site likely is not productive for attacking efficiently the thioacyl intermediate formed with erythrose 4-phosphate, whereas a water molecule is an efficient nucleophile for the hydrolysis of the thioacyl intermediate. Compared with glyceraldehyde-3-phosphate dehydrogenase activity, this corresponds to an activation of the deacylation step by >/=4.5 kcal.mol-1. Altogether these results suggest subtle structural differences between the active sites of GAPDH and GapB-encoded protein that could be revealed and/or modulated by the structure of the substrate bound. This also indicates that a protein engineering approach could be used to convert a phosphorylating aldehyde dehydrogenase into an efficient non-phosphorylating one and vice versa.     

Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta

Synaptic efficacy at the rat Ia-motoneuron synapse has been reported to increase in vivo, within 3 d of sectioning a single muscle nerve (). We provide an indirect test of the hypothesis that this increase is caused by altered probability of transmitter release of axotomized afferents. Experiments consisted of in vivo recording of maximal composite group I EPSPs evoked in intact rat medial gastrocnemius (MG) motoneurons by stimulation of the lateral gastrocnemius-soleus nerve (LG-S). We compared the maximal LG-S EPSP amplitude and the response to high-frequency stimulation (modulation) recorded in untreated rats, with the same measures recorded in rats that had the LG-S nerve axotomized 3 d before data collection. In confirmation of previous work, the mean amplitude of LG-S EPSPs evoked by stimulation of axotomized afferents was significantly larger than that measured in untreated rats (3.9 +/- 0. 34 and 2.3 +/- 0.19 mV, respectively). The increase in EPSP amplitude was accompanied by significantly greater negative modulation (depression) of EPSP amplitude during high-frequency stimulation (-39 +/- 4% and -53 +/- 4%, untreated and treated, respectively). Modulation would not be expected to change if the increase in EPSP amplitude was attributable solely to a greater number of afferent connections (). Therefore, the present results are consistent with the hypothesis that the initial axotomy-induced increase in synaptic efficacy occurs because of an increase in the probability of transmitter release. Furthermore, these results suggest that the probability of transmitter release at this synapse is regulated by either afferent activity and/or trophic communication with the target muscle.