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PubMed1997

Fractional Ca2+ currents through capsaicin- and proton-activated ion channels in rat dorsal root ganglion neurones.

1. Capsaicin and protons cause excitation and sensitization of primary nociceptive afferents. In a subset of dorsal root ganglion (DRG) neurones, which probably represent nociceptive neurones, both capsaicin and protons induce slowly inactivating non-selective cation currents. Whole-cell as well as single channel currents activated by these two stimuli share many biophysical and physiological properties in these neurones. This has lead to the suggestion that protons and capsaicin might activate the same ion channels. 2. In this study we simultaneously measured fluorescence signals and whole-cell currents activated by capsaicin or protons in acutely isolated DRG neurones filled with a high concentration (1 mM) of the Ca2+ indicator dye fura-2. From these measurements the fractional contribution of Ca2+ (Pf; the portion of the whole-cell current carried by Ca2+) to capsaicin- and two types of proton-induced (fast and slowly inactivating) membrane currents was determined. 3. Capsaicin- and slowly inactivating proton-induced currents were accompanied by a change in fluorescence that was dependent on the presence of extracellular Ca2+. With 1.6 mM extracellular Ca2+ and at a holding potential of -80 mV Pf of capsaicin-induced currents (at pH 7.3) was 4.30 +/- 0.17% (mean +/- S.E.M.; no. of experiments, n = 16) and of slowly inactivating proton-induced currents (at pH 5.1) was 1.65 +/- 0.11% (n = 17). Pf of fast inactivating proton-induced currents was negligible. 4. Pf of capsaicin- and slowly inactivating proton-induced currents increased with increasing extracellular Ca2+ concentration (0.5-4.8 mM). 5. Pf of both current types decreased linearly with decreasing extracellular pH by about 0.7% per pH unit over the pH range investigated. When determined at the same extracellular pH Pf values were significantly different for the two current types at all pH values tested. 6. In summary, our results provide evidence that capsaicin and protons activate ion channels which are markedly permeable to Ca2+. The fractional contribution of Ca2+, however, was significantly different for capsaicin- and slowly inactivating proton-induced currents. This strongly suggests that the two stimuli activate different populations of ion channels and supports the possibility that Ca2+ influx through these channels may be important for Ca(2+)-dependent sensitization of primary nociceptive neurones.

Animals↗

PubMed2000

Evaluation of bone contusions with fat-saturated fast spin-echo proton-density magnetic resonance imaging.

OBJECTIVE: To evaluate the efficacy of fast spin-echo proton-density magnetic resonance imaging (MRI) with fat saturation sequences in the evaluation of bone contusions at the knee. METHODS: Analysis of 46 consecutive knee MRI examinations performed on patients referred from a sports medicine clinic after knee trauma. All examinations included coronal fast spin-echo proton-density fat saturation, fast spin-echo proton-density and fast spin-echo T2-weighted sequences. All 3 coronal sequences were blindly reviewed independently of each other by 3 experienced musculoskeletal radiologists to identify and grade bone contusions. RESULTS: Thirty-five bone contusions were identified in 24 patients. All bone contusions were identified on fast spin-echo proton-density fat saturation sequences, which was significantly greater than the percentage identified on either fast spin-echo T2-weighted sequences (21/35, 60%, p < 0.001) or fast spin-echo proton-density sequences (10/35, 29%, p < 0.001). Fourteen (40%) of the contusions were identified only on the fast spin-echo proton-density fat saturation sequences. The average grade of contusion for all 35 examinations was also significantly higher on the fast spin-echo proton-density fat saturation sequences than on the fast spin-echo proton-density and fast spin-echo T2-weighted sequences (p < 0.05). CONCLUSION: Fast spin-echo proton-density fat saturation sequences are more sensitive in the detection of bone contusions than fast spin-echo proton-density and fast spin-echo T2-weighted sequences. Assessment of other structures in the knee with fast spin-echo proton-density fat saturation MRI provides good spatial resolution and adequate T2-weighted information. It may have advantages over the more heavily T2-weighted fast spin-echo T2 fat saturation and inversion recovery sequences.

Adult↗

PubMed2000

Proton radiation therapy for chordomas and chondrosarcomas of the skull base.

Most patients with conventional radiotherapy after surgery die with local disease progression. The superior local tumor control and overall survival achieved with fractionated proton RT can be attributed to improved dose localization characteristics of protons, resulting in higher doses delivered. Patients with base of skull neoplasms are increasingly considered for stereotactic radiosurgery. Recently, Muthukumar et al reported for the University of Pittsburgh group on cobalt-60 Gamma Knife (Elekta Instruments, Atlanta, GA) therapy for 15 patients with chordomas or chondrosarcomas of the base of the skull. With tumor volumes ranging between 0.98 and 10.3 mL (mean, 4.6 mL), doses to the tumor margin varying from 12 to 20 Gy (median, 18 Gy) were delivered. Two patients were treated without histologic tumor confirmation. After a median follow-up time of 40 months, 2 patients had died of disease, 2 patients had succumbed to intercurrent disease, and 1 patient surviving at the time of analysis had developed tumor progression. Neither actuarial local control nor actuarial survival data were presented. In the LLUMC series, most tumors exceeded sizes reportedly suitable for radiosurgery or were of a highly irregular configuration. Nevertheless, in 11 patients, tumors less than 15 mL in size remained locally controlled as did tumors sized between 15 and 25 mL in 11 additional patients; these patients were thus potential candidates for stereotactic radiosurgery. At present, too few reports on radiosurgery contain sufficient patient numbers and statistical analyses to permit one to draw conclusions about the feasibility of radiosurgery for chordomas and chondrosarcomas of the base of the skull. A principal difference between proton RT and radiosurgery as currently practiced in most centers concerns target definition. In proton RT, the GTV is treated. In addition, a clinical volume is defined, which is distinctly different from the GTV in size and shape, to include the operative site and other areas of microscopic risk. In many instances, only the GTV is targeted in radiosurgery. Although it is certainly appropriate to explore the role that radiosurgical techniques may have in treating these tumors, results should be evaluated against the excellent outcome that can be achieved with fractionated proton RT, particularly in patients with tumors small enough and of favorable configuration and location to make them candidates for radiosurgery. The present problem of particle therapy is its limited availability. In the United States, only two proton centers can currently provide treatment for base of skull lesions. The HCL is soon to be replaced by a hospital-based facility at the MGH. Several other proton centers in the United States are currently under active consideration. Proton RT is an evolutionary process. Recent developments in proton RT include intensity modulated therapy and improvements in beam delivery systems, namely, the introduction of active beam scanning. These should further increase the degree of dose conformity. In addition, other heavy particles are also being investigated so as to combine the physical advantages of protons with the differential increased biologic effectiveness of particles in tumor as compared to normal tissues. A report from the Heavy Ion Research Facility in Darmstadt, Germany, has not revealed any increased acute toxicities in the first 13 patients with skull base chordomas or chondrosarcomas treated using carbon ions. Several important factors have emerged from recently published results: Patients with low-grade chondrosarcomas and male patients with chordomas have an excellent chance of durable tumor control and long-term survival after proton RT. Severe complications are within the acceptable range considering the high doses delivered and given the major morbidity associated with uncontrollable tumor growth in such patients. Female patients with chordomas experience increased early and late failures

Chondrosarcoma↗

PubMed1996

Evidence for vacuolar-type proton pumps in nonmitochondrial and inositol 1,4,5-trisphosphate-sensitive calcium stores of insulin-secreting cells.

This study examines whether acidic, vacuolar-type, proton-pump-carrying organelles of insulin-secreting cells (clonal endocrine pancreatic cell line INS-1) function as rapidly exchanging, inositol 1,4,5-trisphosphate-sensitive calcium stores. Calcium uptake into calcium stores will be modulated by the proton concentration within the stores, since calcium pumps in general appear to mediate a countertransport of calcium with protons. We therefore tested for sensitivity of calcium sequestration by nonmitochondrial stores (inhibition of mitochondrial calcium uptake by 2 microM ruthenium red) in saponin-permeabilized cells to proton-conducting ionophores and proton pump inhibition, using this as a marker for involvement of acidic organelles. Calcium sequestration was partially inhibited by the protonophores nigericin (10-50 microM) and carbonylcyanide m-chlorophenylhydrazone (CCCP; 20-50 microM), as well as by inclusion of 30 mM NH4Cl. Bafilomycin A1, a potent and selective inhibitor of vacuolar-type proton pumps, alone (1 - 500 nM) had no effect on calcium sequestration. however, it induced an inhibitory effect in the presence of nigericin or CCCP, even at low concentrations (5 microM) of these ionophores, lacking itself an inhibitory action on calcium sequestration. Bafilomycin A1 then was already maximally active at a concentration as low as 10 nM. Corres ponding to inhibition of total nonmitochondrial calcium sequestration, filling of inositol 1,4,5-trisphosphate-sensitive stores was decreased or even abolished by the protonophores alone or the protonophores combined with bafilomycin A1. We conclude that vacuolar-type proton pumps are present in at least a part of nonmitochondrial and inositol 1,4,5-trisphosphate-sensitive calcium stores in INS-1 cells. This assigns these stores to organelles such as secretory granules, the trans Golgi network, or endosomes. Luminal acidity of these stores will stimulate calcium sequestration by providing more protons for countertransport of calcium by calcium pumps. High concentrations of protonophores may be required for inhibitory effects because otherwise the proton pumps may be able to compensate sufficiently for ionophore-mediated proton loss. The lack of effect of bafilomycin A1 without protonophores may be due to a sufficient luminal buffering capacity or to preceding inhibition of the pump by an inside-positive transmembrane potential.

Ammonium Chloride↗

PubMed2005

Relative biologic effectiveness determination in mouse intestine for scanning proton beam at Paul Scherrer Institute, Switzerland. Influence of motion.

PURPOSE: To determine the relative biologic effectiveness (RBE) of the Paul Scherrer Institute (PSI) scanning proton beam in reference conditions and to evaluate the influence of intestine motion on the proton dose homogeneity. METHODS AND MATERIALS: First, RBE was determined for crypt regeneration in mice after irradiation in a single fraction. Irradiation was performed at the middle of a 7-cm spread out Bragg peak (SOBP; reference position), as well as in the proximal part of the plateau and at the distal end of the SOBP. Control gamma-irradiation was randomized with proton irradiation and performed simultaneously. Second, motion of mouse intestine was determined by radiographs after copper wire markers had been placed on the jejunum and intestinal wall. RESULTS: Proton RBE (reference (60)Co gamma) was equal to 1.16 for irradiation at the middle of the SOBP and to 1.11 and 1.21 for irradiation in the initial plateau and end of the SOBP, respectively. The confidence intervals for these RBE values were much larger than those obtained in the other proton beams we have tested so far. They exceeded +/-0.20 (compared with the usual value of +/-0.07), which resulted from the unusually large dispersion of the individual proton data. The instantaneous positions of the mice intestines varied by +/-2 mm in the course of irradiation. CONCLUSION: The results of this study have shown that the RBE of the PSI proton beam is in total accordance with the RBE obtained at the other centers. This experiment has corroborated that proton RBE at the middle of the SOBP is slightly larger than the generic value of 1.10 and that there is a slight tendency for the RBE to increase close to the end of the SOBP. Also, excessive dispersion of individual proton data may be considered to result from intestine motion, taking into account that irradiation at the PSI is delivered dynamically by scanning the target volume with a pencil proton beam ("spot scanning"). Because 2-mm movements resulted in significant variations in local dose depositions, this should be considered for moving targets. Strategies to reduce this effect for the spot scanning technique have been developed at the PSI for radiotherapy of humans.

Animals↗

PubMed2005

The protonation state of a heme propionate controls electron transfer in cytochrome c oxidase.

In cytochrome c oxidase (CcO), exergonic electron transfer reactions from cytochrome c to oxygen drive proton pumping across the membrane. Elucidation of the proton pumping mechanism requires identification of the molecular components involved in the proton transfer reactions and investigation of the coupling between internal electron and proton transfer reactions in CcO. While the proton-input trajectory in CcO is relatively well characterized, the components of the output pathway have not been identified in detail. In this study, we have investigated the pH dependence of electron transfer reactions that are linked to proton translocation in a structural variant of CcO in which Arg481, which interacts with the heme D-ring propionates in a proposed proton output pathway, was replaced with Lys (RK481 CcO). The results show that in RK481 CcO the midpoint potentials of hemes a and a(3) were lowered by approximately 40 and approximately 15 mV, respectively, which stabilizes the reduced state of Cu(A) during reaction of the reduced CcO with O(2). In addition, while the pH dependence of the F --> O rate in wild-type CcO is determined by the protonation state of two protonatable groups with pK(a) values of 6.3 and 9.4, only the high-pK(a) group influences this rate in RK481 CcO. The results indicate that the protonation state of the Arg481 heme a(3) D-ring propionate cluster having a pK(a) of approximately 6.3 modulates the rate of internal electron transfer and may act as an acceptor of pumped protons.

Animals↗

PubMed1996

GTP synthases. Proton pumping and phosphorylation in ligand-receptor-G alpha-protein complexes.

A structural model for a ligand-receptor-Gs alpha-protein complex to function as a GTP synthase is presented. The mechanism which is dependent on the movement and rotation of the G alpha-protein alpha 2-helix is seen to involve the delivery of, at least, one proton to the phosphorylation site in the rotation of this helix. The cycle is driven by a ligand-mediated proton pump through the alpha-helices of the receptor, attachment of the conserved Tyr-Arg-Tyr receptor proton shuttle being made to an aspartate group on the Gs alpha-protein terminal sidechain, which is itself linked to the Asn-Gln interaction known to control movement and rotation of the alpha 2-helix between .GDP and .GTP structures. The energetics of proton transfer through the shuttle mechanism and delivery of a proton to the aspartate group are shown to be sufficient to rupture this controlling interaction and its associated backbone bond. The complex leads to full spatial and energetic definition of the receptor proton shuttle mechanism, while there is a striking association of further Tyrosine and Arginine residues in the vicinity of the Gs alpha-protein Asn-Gln interaction. Calculations at the HF 6-31G** level confirm that a critical balance between ion pair and neutral forms of Tyr-Arg interactions under multiply hydrogen bonded conditions in a hydrophobic environment controls proton transfer and recovery mechanisms. The intrinsic preference of the neutral Tyr-Arg form over the ion-pair is 14.0 kcal/mol. Activation of the Tyrosine oxygen atom in the neutral form by single-NH or -OH groups reduces this difference by some 6.4-8.6 kcal/mol but the dominance of the neutral form is maintained. The expected slight overestimates are consistent with the maximum activation enthalpy of 11.0-12.0 kcal/ mol required to initiate proton transfer through the shuttle. The extended form of the shuttle with the Arginine acting competitively between the two Tyrosine residues allows interpretation of observed enthalpic differences in ligand binding with and without the presence of GTP. The uniqueness of Gs proteins among the G alpha-proteins is seen as their inability to transfer a proton directly through the alpha 2-helix switch Asn-Gln residues. A possible proton pathway to the mid-point of the Gs alpha-protein alpha 2 helix is outlined.

Amino Acid Sequence↗

PubMed2000

Importance of gas-phase proton affinities in determining the electrospray ionization response for analytes and solvents.

The effect of gas-phase proton transfer reactions on the mass spectral response of solvents and analytes with known gas-phase proton affinities was evaluated. Methanol, ethanol, propanol and water mixtures were employed to probe the effect of gas-phase proton transfer reactions on the abundance of protonated solvent ions. Ion-molecule reactions were carried out either in an atmospheric pressure electrospray ionization source or in the central quadrupole of a triple-quadrupole mass spectrometer. The introduction of solvent vapor with higher gas-phase proton affinity than the solvent being electrosprayed caused protons to transfer to the gas-phase solvent molecules. In mixed solvents, protonated solvent clusters of the solvent with higher gas-phase proton affinity dominated the resulting mass spectra. The effect of solvent gas-phase proton affinity on analyte response was also investigated, and the analyte response was suppressed or eliminated in solvents with gas-phase proton affinities higher than that of the analyte.

Atmospheric Pressure↗

PubMed2002

Structural and energetic aspects of the protonation of phenol, catechol, resorcinol, and hydroquinone.

The various protonated forms of phenol (1), catechol (2), resorcinol (3), and hydroquinone (4) were explored by ab initio quantum chemical calculations at the MP2/6-31G(d) and B3LYP/6-31G(d) levels. Proton affinities (PA) of 1-4 were calculated by the combined G2(MP2,SVP) method, and their gas-phase basicities were estimated after calculation of the change in entropy on protonation. These theoretical data were compared with the corresponding experimental values determined in a high-pressure mass spectrometer. This comparison confirmed that phenols are essentially carbon bases and that protonation generally occurs in a position para to the hydroxyl group. Resorcinol is the most effective base (PA = 856 kJ mol-1) due to the participation of both oxygen atoms in the stabilization of the protonated form. Since protonation is accompanied by a freezing of the two internal rotations, a significant decrease in entropy is observed. The basicity of catechol (PA = 823 kJ mol-1) is due to the existence of an intramolecular hydrogen bond, which is strengthened upon protonation. The lower basicity of hydroquinone (PA = 808 kJ mol-1) is a consequence of the fact that protonation necessarily occurs in a position ortho to the hydroxyl group. When the previously published data are reconsidered and a corrected protonation entropy is used, a proton affinity value of 820 kJ mol-1 is obtained for phenol.

Binding Sites↗

PubMed2007

Protonated heme.

The ions formally corresponding to protonated heme [Fe(II)-hemeH](+) have been obtained by collision-induced dissociation from the electrospray ionization of microperoxidase (MP11) and their gas-phase chemistry has been studied by FTICR mass spectrometry. H/D-exchange reactions, used as a tool to gain information on the protonation sites in polyfunctional molecules, show that labile hydrogens pertain to the propionyl substituents at the periphery of the protoporphyrin IX. Several conceivable isomers for protonated heme have been evaluated by density functional theory. The most stable among the species investigated is the one corresponding to protonation at the beta carbon atom of a vinyl group, yielding a proton affinity (PA) value for [Fe(II)-heme] of 1220 kJ mol(-1). This high PA is consistent with the inertness of the hydrogen atoms at the protonation site towards H/D exchange with ND(3) and CD(3)CO(2)D. Peculiar features of this [Fe(II)-hemeH](+) isomer emerge by analysis of its electronic structure, showing that the vinyl group undergoing formal protonation has gained significant radical character due to electron transfer from the metal center. As a consequence, the iron atom acquires partial iron(III) character and none of the two formal descriptions [Fe(II)-hemeH(+)] and [Fe(III)-hemeH(.)](+) alone may adequately illustrate the protonated heme ion. In agreement with this description, the reactivity of protonated heme presents dual facets, resembling iron(III) in some aspects and iron(II) in others. On the one hand, protonated heme behaves like [Fe(III)-heme](+) ions in H/D-exchange reactions. On the other, it shows markedly decreased reactivity towards the addition of ligands with the notable exception of NO, in line with the high affinity shown by iron(II) complexes towards this molecule, NO, of key biological role.

Computer Simulation↗

PubMed1993

Decoupling of photo- and proton cycle in the Asp85-->Glu mutant of bacteriorhodopsin.

Surface bound pH indicators were applied to study the proton transfer reactions in the mutant Asp85-->Glu of bacteriorhodopsin in the native membrane. The amino acid replacement induces a drastic acceleration of the overall rise of the M intermediate. Instead of following this acceleration, proton ejection to the extracellular membrane surface is not only two orders of magnitude slower than M formation, it is also delayed as compared with the wild-type. This demonstrates that Asp85 not only accepts the proton released by the Schiff's base but also regulates very efficiently proton transfer within the proton release chain. Furthermore, Asp85 might be the primary but is not the only proton acceptor/donor group in the release pathway. The Asp85-->Glu substitution also affects the proton reuptake reaction at the cytoplasmic side, although Asp85 is located in the proton release pathway. Proton uptake is slower in the mutant than in the wild-type and occurs during the lifetime of the O intermediate. This demonstrates a feed-back mechanism between Asp85 and the proton uptake pathway in bacteriorhodopsin.

Asparagine↗

PubMed2003

Fragmentation reactions of protonated peptides containing glutamine or glutamic acid.

A variety of protonated dipeptides and tripeptides containing glutamic acid or glutamine were prepared by electrospray ionization or by fast atom bombardment ionization and their fragmentation pathways elucidated using metastable ion studies, energy-resolved mass spectrometry and triple-stage mass spectrometry (MS(3)) experiments. Additional mechanistic information was obtained by exchanging the labile hydrogens for deuterium. Protonated H-Gln-Gly-OH fragments by loss of NH(3) and loss of H(2)O in metastable ion fragmentation; under collision-induced dissociation (CID) conditions loss of H-Gly-OH + CO from the [MH - NH(3)](+) ion forms the base peak C(4)H(6)NO(+) (m/z 84). Protonated dipeptides with an alpha-linkage, H-Glu-Xxx-OH, are characterized by elimination of H(2)O and by elimination of H-Xxx-OH plus CO to form the glutamic acid immonium ion of m/z 102. By contrast, protonated dipeptides with a gamma-linkage, H-Glu(Xxx-OH)-OH, do not show elimination of H(2)O or formation of m/z 102 but rather show elimination of NH(3), particularly in metastable ion fragmentation, and elimination of H-Xxx-OH to form m/z 130. Both the alpha- and gamma-dipeptides show formation of [H-Xxx-OH]H(+), with this reaction channel increasing in importance as the proton affinity (PA) of H-Xxx-OH increases. The characteristic loss of H(2)O and formation of m/z 102 are observed for the protonated alpha-tripeptide H-Glu-Gly-Phe-OH whereas the protonated gamma-tripeptide H-Glu(Gly-Gly-OH)-OH shows loss of NH(3) and formation of m/z 130 as observed for dipeptides with the gamma-linkage. Both tripeptides show abundant formation of the y(2)'' ion under CID conditions, presumably because a stable anhydride neutral structure can be formed. Under metastable ion conditions protonated dipeptides of structure H-Xxx-Glu-OH show abundant elimination of H(2)O whereas those of structure H-Xxx-Gln-OH show abundant elimination of NH(3). The importance of these reaction channels is much reduced under CID conditions, the major fragmentation mode being cleavage of the amide bond to form either the a(1) ion or the y(1)'' ion. Particularly when Xxx = Gly, under CID conditions the initial loss of NH(3) from the glutamine containing dipeptide is followed by elimination of a second NH(3) while the initial loss of H(2)O from the glutamic acid dipeptide is followed by elimination of NH(3). Isotopic labelling shows that predominantly labile hydrogens are lost in both steps. Although both [H-Gly-Glu-Gly-OH]H(+) and [H-Gly-Gln-Gly-OH]H(+) fragment mainly to form b(2) and a(2) ions, the latter also shows elimination of NH(3) plus a glycine residue and formation of protonated glycinamide. Isotopic labelling shows extensive mixing of labile and carbon-bonded hydrogens in the formation of protonated glycinamide.

Deuterium↗

PubMed2004

Modeling of the gas-phase ion chemistry of protonated arginine.

Arginine is often involved at the C-terminus of peptides obtained from tryptic digests of proteins. The very basic guanidine group of the side-chain of arginine has a large effect on the backbone fragmentation of protonated peptides. Furthermore, arginine exhibits specific fragmentation reactions involving its side-chain. Various tautomerization states, conformers and side-chain dissociation channels of protonated arginine were studied using theoretical methods. The guanidine loss of protonated arginine is proved to be an S(N)2 substitution on the delta-carbon of the side-chain, starting from species containing the N(epsilon)H-C(+)(N(eta)H(2))(N(eta')H(2)) or -N(epsilon) (+)H(2)-C(N(eta)H)(N(eta')H(2)) moieties and leads to formation to either protonated guanidine or protonated proline. In the corresponding transition structures the proline moiety is protonated. Under low-energy collision conditions the extra proton transfers to the guanidine moiety, leading to the formation of C(+)(NH(2))(3). On the other hand, the lifetime of the fragmenting species under high-energy collision conditions is shorter, resulting in enhanced formation of protonated proline and its dissociation products. The first step of ammonia loss is the leaving of a preformed NH(3) from tautomers containing the -N(epsilon)H-C(N(eta)H(3) (+))(N(eta')H) or -N(epsilon)-C(N(eta)H(3) (+))(N(eta')H(2)) moieties. The resulting protonated carbodiimide group can be stabilized by intramolecular nucleophilic attack, leading to ring formation. Overall, reactions involved in the ammonia loss from protonated arginine can be considered as an S(N)1 substitution on the central zeta-carbon of the guanidine group.

Ammonia↗

PubMed1996

Proton exchange rates from amino acid side chains--implications for image contrast.

The proton exchange rates between water and the hydroxyl protons of threonine, serine, tyrosine, the amino protons of lysine, and the guanidinium protons of arginine were measured in the pH range 0.5 to 8.5 and for the temperatures 4 degrees C, 10 degrees C, 20 degrees C, 30 degrees C, and 36 degrees C. The intrinsic exchange rates of the hydroxyl and amino protons at pH 7.0 degrees C and 36 degrees C were found to be in the range 700 to about 10,000 s-1. In addition, the exchange catalysis by phosphate, carbonate, carboxyl-, and amino-groups was investigated. The presence of these exchange catalysts at physiological concentrations increased the proton exchange rates from hydroxyl and amino groups several fold. The proton exchange rates are sufficiently fast that the total magnetization transfer between biomolecules and free bulk water is not rate limited by the proton exchange rate, but by the intramolecular cross-relaxation rates between the exchangeable and nonexchangeable protons of the biomolecules. Since the cross-relaxation rates between surface hydration water molecules and biomolecules are usually vanishingly small because of too rapid exchange with the free bulk water, it is proposed that the contrast in MR images is a fingerprint of the number of the exchangeable protons from OH and NH groups of the tissue, as far as the contrast depends on the magnetization transfer between biomolecules and water.

Amino Acids↗

PubMed1985

Transient proton inflows during illumination of anaerobic Halobacterium halobium cells.

In Halobacterium halobium strain R1 containing both bacteriorhodopsin (bR) and halorhodopsin (hR), the light-driven proton uptake has been experimentally resolved into three transient inflows which are superimposed on the larger proton outflow. Under anaerobic conditions the early proton uptake consists of two components: (i) an inflow which can be blocked using the ATPase inhibitor, Dio-9, and (ii) an inflow which can be abolished by low concentrations (less than 125 nM) of triphenyltin chloride (TPT) with no inhibition of ATP synthesis. At pH 6 these two inflows are approximately equal in magnitude and duration. Measurements of buffering capacity and internal pH indicate that Dio-9 does not alter the passive proton-hydroxyl permeability of the cell membrane and that TPT at these low concentrations slightly decreases it. At later times of illumination (iii) another transient light-driven proton inflow occurs. This inflow is most evident during the first illumination after cells have been stored for extended times in the dark. The internal potassium concentration is not changed by storage, but apparently sodium is taken up, and we attribute the third inflow to sodium extrusion in exchange for protons. These results demonstrate the existence of three distinct triggered secondary proton inflows through the cell membrane. The proton inflow, which can be inhibited by Dio-9, correlates with proton-dependent ATP synthesis. The second inflow, which disappears in the presence of low TPT concentrations, is a passive proton uptake through an otherwise unidentified channel in response to electrogenic chloride pumping by bacteriorhodopsin and/or halorhodopsin. The third system correlates with the Na+/H+ antiporter function that has been demonstrated in H. halobium cell envelope vesicles. In contrast to observations on hR-containing vesicles, which can develop substantial Cl- gradients, the electroneutral OH-/Cl- exchange function can be demonstrated in intact cells only at TPT concentrations greater than 500 nM.

Adenosine Triphosphate↗

PubMed1995

Proton release from HeLa cells and alkalization of cytoplasm induced by diferric transferrin or ferricyanide and its inhibition by the diarylsulfonylurea antitumor drug N-(4-methylphenylsulfonyl)-N'-(4-cholorophenyl) urea (LY181984).

Proton release from HeLa cells was stimulated by an external oxidant, potassium ferricyanide, or by the growth factor diferric transferrin. This stimulated proton release was inhibited by the antitumor sulfonylurea LY181984 [N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea] over the concentration range 10 nM to 1 microM. The antitumor-inactive sulfonylurea analog LY181985 [N-(4-methylphenylsulfonyl)-N'-(phenyl)urea] was without effect at 1 microM and required 10-100 microM concentrations to inhibit proton release. Diferric transferrin-induced alkalization of the cytoplasm estimated by BCECF [2',7'-bis(2-carboxyethyl)-5,(and 6)-carboxyfluorescein] fluorescence also was inhibited by 1 microM LY181984 but not by 1 microM LY181985. The inhibited component appeared to be amiloride resistant. The proton release induced by either ferricyanide or diferric transferrin was inhibited by about 35% at a near optimal amiloride concentration of 0.2 mM or at a dimethylamiloride concentration of 0.075 mM. However, the induced proton release was inhibited further by LY181984. Conversely, when proton release was inhibited fully by LY181984 at a near optimal concentration of 10 microM (50% inhibition), increasing concentrations of amiloride or dimethylamiloride resulted in additional inhibitions of 16 and 23%, respectively. However, the inhibitions by LY181984 and the amilorides were additive, suggesting that amiloride and the sulfonylureas may act independently. Evidence for an action of the sulfonylurea in inhibiting proton efflux differently from that of the amilorides came from measurements of sodium uptake either by fluorometry or by direct measurement with 22Na+. Sodium uptake was not inhibited by either LY181984 or LY181985 in HeLa cells at concentrations of LY181984 sufficient to inhibit proton efflux by 80% or more. The results show LY181984 to be a potent inhibitor of diferric transferrin- or ferricyanide-induced proton efflux and cytoplasmic alkalization in HeLa cells and that the inhibition may involve a component of proton transport that is resistant to amiloride.

Amiloride↗

PubMed1994

Supporting evidence for negative modulation by protons of an ion channel associated with the N-methyl-D-aspartate receptor complex in rat brain using ligand binding techniques.

The addition of L-glutamic acid (Glu) alone, both Glu and glycine (Gly) or Glu/Gly/spermidine (SPD) was effective in potentiating [3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine (MK-801) binding before equilibrium to an ion channel associated with the N-methyl-D-aspartate (NMDA) receptor complex in brain synaptic membranes extensively washed and treated with Triton X-100. The binding dependent on Glu almost linearly increased in proportion to decreasing proton concentrations at a pH range of 6.0 to 9.0 in external incubation medium, while a Gly-dependent portion of the binding increased with decreasing proton concentrations up to a pH of 7.5 with a plateau thereafter. In contrast, the SPD-dependent binding increased in proportion to decreasing proton concentrations up to a pH of 7.0 with a gradual decline thereafter. Similar profiles were also obtained with [3H]MK-801 binding at equilibrium, with an exception that significant binding of [3H]MK-801 was detected in the absence of any added agonists. The potency of SPD to potentiate [3H]MK-801 binding before equilibrium increased in proportion to decreasing proton concentrations, with those of both Glu and Gly being unchanged. In contrast, the ability of (+)MK-801 to displace [3H]MK-801 binding at equilibrium was not significantly affected by a decrement of external proton concentrations from pH 7.5 to pH 8.5 in the presence of Glu/Gly and Glu/Gly/SPD added. However, similar changes in external proton concentrations did not similarly affect binding of several radioligands for the NMDA and Gly domains on the receptor complex. Decreasing proton concentrations were effective in exponentially potentiating binding of [3H]SPD at a pH range of 6.0 to 9.0 without virtually altering [3H]D,L-alpha-amino-3- hydroxy-5-methyl-isoxazole-4-propionic acid binding. In addition, [3H]kainic acid binding markedly decreased with decreasing proton concentrations only in the presence of Ca2+ ions. These results suggest that protons negatively modulate neuronal responses mediated by the NMDA receptor ionophore complex through interference with opening mechanisms of the channel domain without disturbing association processes of the endogenous agonists with the respective recognition domains in rat brain. Moreover, possible modulation by protons of responses mediated by the kainate receptor in the presence of Ca2+ ions at concentrations that occur in vivo is also suggested.

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PubMed1986

The proton-pumping site of cytochrome c oxidase: a model of its structure and mechanism.

Cytochrome c oxidase is an electron-transfer driven proton pump. In this paper, we propose a complete chemical mechanism for the enzyme's proton-pumping site. The mechanism achieves pumping with chemical reaction steps localized at a redox center within the enzyme; no indirect coupling through protein conformational changes is required. The proposed mechanism is based on a novel redox-linked transition metal ligand substitution reaction. The use of this reaction leads in a straightforward manner to explicit mechanisms for achieving all of the processes previously determined (Blair, D.F., Gelles, J. and Chan, S.I. (1986) Biophys. J. 50, 713-733) to be needed to accomplish redox-linked proton pumping. These processes include: (1) modulation of the energetics of protonation/deprotonation reactions and modulation of the energetics of redox reactions by the structural state of the pumping site; (2) control of the rates of the pump's redox reactions with its electron-transfer partners during the turnover cycle (gating of electrons); and (3) regulation of the rates of the protonation/deprotonation reactions between the pumping site and the aqueous phases on the two sides of the membrane during the reaction cycle (gating of protons). The model is the first proposed for the cytochrome oxidase proton pump which is mechanistically complete and sufficiently specific that a realistic assessment can be made of how well the model pump would function as a redox-linked free-energy transducer. This assessment is accomplished via analyses of the thermodynamic properties and steady-state kinetics expected of the model. These analyses demonstrate that the model would function as an efficient pump and that its behavior would be very similar to that observed of cytochrome oxidase both in the mitochondrion and in purified preparations. The analysis presented here leads to the following important general conclusions regarding the mechanistic features of the oxidase proton pump. (1) A workable proton-pump mechanism does not require large protein conformational changes. (2) A redox-linked proton pump need not display a pH-dependent midpoint potential, as has frequently been assumed. (3) Mechanisms for redox-linked proton pumps that involve transition metal ligand exchange reactions are quite attractive because such reactions readily lend themselves to the linked gating processes necessary for proton pumping.

Animals↗