Role of protons in activation of cardiac sympathetic C-fibre afferents during ischaemia in cats. (65/5891)

1. Chest pain caused by myocardial ischaemia is mediated by cardiac sympathetic afferents. The mechanisms of activation of cardiac afferents during ischaemia remain poorly understood. Increased lactic acid production is associated closely with myocardial ischaemia. The present study examined the role of protons generated during ischaemia in activation of cardiac sympathetic C-fibre afferents. 2. Single-unit activity of cardiac afferents innervating both ventricles was recorded from the left sympathetic chain in anaesthetized cats. Epicardial tissue pH was measured within 1-1.5 mm of the surface by a pH-sensitive needle electrode. Responses of cardiac afferents to myocardial ischaemia, lactic acid, sodium lactate, acidic phosphate buffer and hypercapnia were determined. 3. Occlusion of the coronary artery for 5 min decreased epicardial tissue pH from 7.35 +/- 0.21 to 6.98 +/- 0.22 (P < 0.05). Epicardial placement of isotonic neutral phosphate buffer, but not saline, prevented the ischaemia-induced decrease in epicardial pH. This manoeuvre significantly attenuated the response of 16 afferents to 5 min of ischaemia (1.56 +/- 0.23 pre-treatment vs. 0.67 +/- 0.18 impulses s-1). Topical application of 10-100 microg ml-1 of lactic acid, but not sodium lactate, concentration-dependently stimulated 18 cardiac afferents. Inhalation with high-CO2 gas failed to activate 12 separate cardiac afferents. Furthermore, lactic acid stimulated cardiac afferents to a greater extent than acidic phosphate buffer solution, applied at a similar pH to the same afferents. 4. Collectively, this study provides important in vivo evidence that protons contribute to activation/sensitization of cardiac sympathetic C-fibre afferents during myocardial ischaemia.  (+info)

Connection between the taxonomic substates and protonation of histidines 64 and 97 in carbonmonoxy myoglobin. (66/5891)

Infrared spectra of heme-bound CO in sperm whale carbonmonoxy myoglobin and two mutants (H64L and H97F) were studied in the pH range from 4.2 to 9.5. Comparison of the native protein with the mutants shows that the observed pH effects can be traced to protonations of two histidine residues, H64 and H97, near the active site. Their imidazole sidechains experience simple, uncoupled Henderson-Hasselbalch type protonations, giving rise to four different protonation states. Because two of the protonation states are linked by a pH-independent equilibrium, the overall pH dependence of the spectra is described by a linear combination of three independent components. Global analysis, based on singular value decomposition and matrix least-squares algorithms enabled us to extract the pK values of the two histidines and the three basis spectra of the protonating species. The basis spectra were decomposed into the taxonomic substates A(0), A(1), and A(3), previously introduced in a heuristic way to analyze CO stretch spectra in heme proteins at fixed pH (see for instance, Biophys. J. 71:1563-1573). Moreover, an additional, weakly populated substate, called A(x), was identified. Protonation of H97 gives rise to a blue shift of the individual infrared lines by about 2 cm(-1), so that the A substates actually appear in pairs, such as A(0) and A(0)(+). The blue shift can be explained by reduced backbonding from the heme iron to the CO. Protonation of the distal histidine, H64, leads to a change of the infrared absorption from the A(1) or A(3) substate lines to A(0). This behavior can be explained by a conformational change upon protonation that moves the imidazole sidechain of H64 away from the CO into the high-dielectric solvent environment, which avoids the energetically unfavorable situation of an uncompensated electric charge in the apolar, low-dielectric protein interior. Our results suggest that protonation reactions serve as an important mechanism to create taxonomic substates in proteins.  (+info)

Proton nuclear magnetic resonance study of the binary complex of cytochrome P450cam and putidaredoxin: interaction and electron transfer rate analysis. (67/5891)

A 1H nuclear magnetic resonance study of the complex of cytochrome P450cam-putidaredoxin has been performed. Isocyanide is bound to cytochrome P450cam in order to increase the stability of the protein both in the reduced and the oxidized state. Diprotein complex formation was detected through variation of the heme methyl proton resonances which have been assigned in the two redox states. The electron transfer rate at equilibrium was determinated by magnetization transfer experiments. The observed rate of oxidation of reduced cytochrome P450 by the oxidized putidaredoxin is 27 (+/- 7) per s.  (+info)

Proton-nuclear magnetic resonance analyses of the substrate specificity of a beta-ketolase from Pseudomonas putida, acetopyruvate hydrolase. (68/5891)

A revised purification of acetopyruvate hydrolase from orcinol-grown Pseudomonas putida ORC is described. This carbon-carbon bond hydrolase, which is the last inducible enzyme of the orcinol catabolic pathway, is monomeric with a molecular size of approximately 38 kDa; it hydrolyzes acetopyruvate to equimolar quantities of acetate and pyruvate. We have previously described the aqueous-solution structures of acetopyruvate at pH 7.5 and several synthesized analogues by (1)H-nuclear magnetic resonance (NMR)-Fourier transform (FT) experiments. Three (1)H signals (2.2 to 2.4 ppm) of the methyl group are assigned unambiguously to the carboxylate anions of 2,4-diketo, 2-enol-4-keto, and 2-hydrate-4-keto forms (40:50:10). A (1)H-NMR assay for acetopyruvate hydrolase was used to study the kinetics and stoichiometries of reactions within a single reaction mixture (0.7 ml) by monitoring the three methyl-group signals of acetopyruvate and of the products acetate and pyruvate. Examination of 4-tert-butyl-2,4-diketobutanoate hydrolysis by the same method allowed the conclusion that it is the carboxylate 2-enol form(s) or carbanion(s) that is the actual substrate(s) of hydrolysis. Substrate analogues of 2,4-diketobutanoate with 4-phenyl or 4-benzyl groups are very poor substrates for the enzyme, whereas the 4-cyclohexyl analogue is readily hydrolyzed. In aqueous solution, the arene analogues do not form a stable 2-enol structure but exist principally as a delocalized pi-electron system in conjugation with the aromatic ring. The effects of several divalent metal ions on solution structures were studied, and a tentative conclusion that the enol forms are coordinated to Mg(2+) bound to the enzyme was made. (1)H-(2)H exchange reactions showed the complete, fast equilibration of (2)H into the C-3 of acetopyruvate chemically; this accounts for the appearance of (2)H in the product pyruvate. The C-3 of the product pyruvate was similarly labelled, but this exchange was only enzyme catalyzed; the methyl group of acetate did not undergo an exchange reaction. The unexpected preference for bulky 4-alkyl-group analogues is discussed in an evolutionary context for carbon-carbon bond hydrolases. Routine one-dimensional (1)H-NMR in normal (1)H(2)O is a new method for rapid, noninvasive assays of enzymic activities to obtain the kinetics and stoichiometries of reactions in single reaction mixtures. Assessments of the solution structures of both substrates and products are also shown.  (+info)

Functional characterization and expression analysis of the amino acid permease RcAAP3 from castor bean. (69/5891)

A polymerase chain reaction-based library screening procedure was used to isolate RcAAP3, an amino acid permease cDNA from castor bean (Ricinus communis). RcAAP3 is 1.7 kb in length, with an open reading frame that encodes a protein with a calculated molecular mass of 51 kD. Hydropathy analysis indicates that the RcAAP3 protein is highly hydrophobic in nature with nine to 11 putative transmembrane domains. RcAAP3-mediated uptake of citrulline in a yeast transport mutant showed saturable kinetics with a K(m) of 0.4 mM. Transport was higher at acidic pH and was inhibited by the protonophore carbonylcyanide-m-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. Citrulline uptake was strongly inhibited (72%) by the permeable sulfydryl reagent N-ethylmaleimide, but showed lower sensitivity (30% inhibition) to the nonpermeable reagent p-chloromercuribenzenesulfonic acid. Diethylpyrocarbonate, a histidine modifier, inhibited citrulline uptake by 80%. A range of amino acids inhibited citrulline uptake, suggesting that RcAAP3 may be a broad substrate permease that can transport neutral and basic amino acids with a lower affinity for acidic amino acids. Northern analysis indicated that RcAAP3 is widely expressed in source and sink tissues of castor bean, and that the pattern of expression is distinct from RcAAP1 and RcAAP2.  (+info)

Role of synaptic vesicle proton gradient and protein phosphorylation on ATP-mediated activation of membrane-associated brain glutamate decarboxylase. (70/5891)

Previously, we have shown that the soluble form of brain glutamic acid decarboxylase (GAD) is inhibited by ATP through protein phosphorylation and is activated by calcineurin-mediated protein dephosphorylation (Bao, J., Cheung, W. Y., and Wu, J. Y. (1995) J. Biol. Chem. 270, 6464-6467). Here we report that the membrane-associated form of GAD (MGAD) is greatly activated by ATP, whereas adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP), a non-hydrolyzable ATP analog, has no effect on MGAD activity. ATP activation of MGAD is abolished by conditions that disrupt the proton gradient of synaptic vesicles, e.g. the presence of vesicular proton pump inhibitor, bafilomycin A1, the protonophore carbonyl cyanide m-chorophenylhydrazone or the ionophore gramicidin, indicating that the synaptic vesicle proton gradient is essential in ATP activation of MGAD. Furthermore, direct incorporation of (32)P from [gamma-(32)P]ATP into MGAD has been demonstrated. In addition, MGAD (presumably GAD65, since it is recognized by specific monoclonal antibody, GAD6, as well as specific anti-GAD65) has been reported to be associated with synaptic vesicles. Based on these results, a model linking gamma-aminobutyric acid (GABA) synthesis by MGAD to GABA packaging into synaptic vesicles by proton gradient-mediated GABA transport is presented. Activation of MGAD by phosphorylation appears to be mediated by a vesicular protein kinase that is controlled by the vesicular proton gradient.  (+info)

Structural and functional roles of the N1- and N3-protons of psi at tRNA's position 39. (71/5891)

Pseudouridine at position 39 (Psi(39)) of tRNA's anticodon stem and loop domain (ASL) is highly conserved. To determine the physicochemical contributions of Psi(39)to the ASL and to relate these properties to tRNA function in translation, we synthesized the unmodified yeast tRNA(Phe)ASL and ASLs with various derivatives of U(39)and Psi(39). Psi(39)increased the thermal stability of the ASL (Delta T (m)= 1.3 +/- 0.5 degrees C), but did not significantly affect ribosomal binding ( K (d)= 229 +/- 29 nM) compared to that of the unmodified ASL (K (d)= 197 +/- 58 nM). The ASL-Psi(39)P-site fingerprint on the 30S ribosomal subunit was similar to that of the unmodified ASL. The stability, ribosome binding and fingerprint of the ASL with m(1)Psi(39)were comparable to that of the ASL with Psi(39). Thus, the contribution of Psi(39)to ASL stability is not related to N1-H hydrogen bonding, but probably is due to the nucleoside's ability to improve base stacking compared to U. In contrast, substitutions of m(3)Psi(39), the isosteric m(3)U(39)and m(1)m(3)Psi(39)destabilized the ASL by disrupting the A(31)-U(39)base pair in the stem, as confirmed by NMR. N3-methylations of both U and Psi dramatically decreased ribosomal binding ( K (d)= 1060 +/- 189 to 1283 +/- 258 nM). Thus, canonical base pairing of Psi(39)to A(31)through N3-H is important to structure, stability and ribosome binding, whereas the increased stability and the N1-proton afforded by modification of U(39)to Psi(39)may have biological roles other than tRNA's binding to the ribosomal P-site.  (+info)

Hydrogen bonding interaction of the amide group of Asn and Gln at distal E7 of bovine myoglobin with bound-ligand and its functional consequences. (72/5891)

Asn and Gln with an amide group at gamma- and delta-positions, respectively, were substituted for distal His-E7 of bovine myoglobin to establish a system where hydrogen bonding interaction between the distal residue and bound-ligand can be altered by changing donor-acceptor distance. Two mutant myoglobins showed nearly identical (1)H-NMR spectral pattern for resolved heme peripheral side-chain and amino acid proton signals and similar two-dimensional NMR connectivities irrespective of cyanide-bound and -unbound states, indicating that the heme electronic structure and the molecular structure of the active site are not affected by a difference in one methylene group at the E7 position. Chemical exchange rate of Asn-E7 N(delta)H proton in met-cyano myoglobin is larger than that of Gln-E7 N(epsilon)H proton by at least two orders of magnitude, suggesting a considerable difference in the strength of hydrogen bond between the E7 side-chain and bound-ligand, due to the differential donor-acceptor distance between the two mutants. Thus a comparative study between the two proteins provides an ideal system to delineate a relationship between the stabilization of bound-ligand by the hydrogen bond and myoglobin's ligand affinity. The Asn-mutant showed a faster dissociation of cyano ion from met-myoglobin than the Gln-mutant by over 30-fold. Similarly, oxygen dissociation is faster in the Asn-mutant than in the Gln-mutant by approximately 100-fold. Association of cyanide anion to the mutant met-myoglobin was accelerated by changing Gln to Asn by a 4-fold. Likewise, oxygen binding was accelerated by approximately 2-fold by the above substitution. The present findings confirm that hydrogen bonding with the distal residue is a dominant factor for determining the ligand dissociation rate, whereas steric hindrance exerted by the distal residue is a primary determinant for the ligand association.  (+info)