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PubMed2004

Dose conformation of intensity-modulated stereotactic photon beams, proton beams, and intensity-modulated proton beams for intracranial lesions.

PURPOSE: This study evaluates photon beam intensity-modulated stereotactic radiotherapy (IMSRT) based on dynamic leaf motion of a micromultileaf collimator (mMLC), proton beams, and intensity-modulated proton therapy (IMPT) with respect to target coverage and organs at risk. METHODS AND MATERIALS: Dose plans of 6 stereotactically treated patients were recalculated for IMSRT by use of the same field setup and an inverse planning algorithm. Proton and IMPT plans were calculated anew. Three different tumor shapes, multifocal, ovoid, and irregular, were analyzed, as well as dose to organs-at-risk (OAR) in the vicinity of the planning target volume (PTV). Dose distributions were calculated from beam-setup data for a manual mMLC for stereotactically guided conformal radiotherapy (SCRT), a dynamic mMLC for IMSRT, the spot-scanning technique for protons, and a modified spot-scanning technique for IMPT. SCRT was included for a part of the comparison. Criteria for assessment were PTV coverage, dose-volume histograms (DVH), volumes of specific isodoses, and the dose to OAR. RESULTS: Dose conformation to the PTV is equally good for all three techniques and tumor shapes considered. The volumes of the 90% and 80% isodose were comparable for all techniques. For the 50% isodose volume, a divergence between the two modes was seen. In 3 cases, this volume is smaller for IMSRT, and in the 3 other cases, it is smaller for IMPT. This difference was even more pronounced for the volumes of the 30% isodose; IMPT shows further improvement over conventional protons. OAR in concavities (e.g., the brainstem) were similarly well spared by protons and IMSRT. IMPT spares critical organs best. Fewer proton beams are required to achieve similar results. CONCLUSIONS: The addition of intensity modulation improves the conformality of mMLC-based SCRT. Conformation of dose to the PTV is comparable for IMSRT, protons, and IMPT. Concerning the sparing of OAR, IMSRT is equivalent to IMPT, and IMPT is superior to conventional protons. The advantage of protons lies in the lower integral dose.

Algorithms↗

PubMed2006

Proton-coupled electron transfer drives the proton pump of cytochrome c oxidase.

Electron transfer in cell respiration is coupled to proton translocation across mitochondrial and bacterial membranes, which is a primary event of biological energy transduction. The resulting electrochemical proton gradient is used to power energy-requiring reactions, such as ATP synthesis. Cytochrome c oxidase is a key component of the respiratory chain, which harnesses dioxygen as a sink for electrons and links O2 reduction to proton pumping. Electrons from cytochrome c are transferred sequentially to the O2 reduction site of cytochrome c oxidase via two other metal centres, Cu(A) and haem a, and this is coupled to vectorial proton transfer across the membrane by a hitherto unknown mechanism. On the basis of the kinetics of proton uptake and release on the two aqueous sides of the membrane, it was recently suggested that proton pumping by cytochrome c oxidase is not mechanistically coupled to internal electron transfer. Here we have monitored translocation of electrical charge equivalents as well as electron transfer within cytochrome c oxidase in real time. The results show that electron transfer from haem a to the O2 reduction site initiates the proton pump mechanism by being kinetically linked to an internal vectorial proton transfer. This reaction drives the proton pump and occurs before relaxation steps in which protons are taken up from the aqueous space on one side of the membrane and released on the other.

Adenosine Triphosphate↗

PubMed2000

Proton therapy for base of skull chordoma: a report for the Royal College of Radiologists. The Proton Therapy Working Party.

Chordomas of the base of the skull are rare. They are locally infiltrative and frequently arise close to radiosensitive structures, which limits the ability to deliver a high dose of radiotherapy. Complete surgical excision is not usually possible. Conventional postoperative radiotherapy can result in approximately 50% 5-year survival and effective palliation, but long-term local control and cure are rare. The well-defined Bragg peak of protons allows planning with a sharp cut-off outside the target volume. This permits a higher dose of radiotherapy to be delivered to the tumour while avoiding excessive irradiation to radiosensitive structures. Outcome after proton irradiation is superior to that reported for conventional photon irradiation. Radiotherapy schedules involving a mixed schedule of protons and photons have achieved an approximately 60% local control rate at 5 years. Some of this improvement may have resulted from better surgical techniques. Proton irradiation is also effective for base of skull chondrosarcomas. Protons of sufficient energy to treat base of skull tumours are not available in the UK. Patients have been referred to the proton facilities at the Harvard cyclotron, and at Orsay, France. They will continue to require referral abroad for proton therapy for base of skull tumours. Proton therapy has become standard treatment for ocular melanoma and this is available at the Douglas cyclotron at Clatterbridge Hospital. Proton therapy has the potential for improved dose distribution compared with conformal photon radiotherapy. This may be exploited effectively to irradiate target volumes close to radiosensitive structures such as the spinal cord. There is a need for further clinical research to evaluate proton therapy for tumours such as spinal and paraspinal sarcomas, and paediatric brain tumours.

Chordoma↗

PubMed1986

Proton pump activity and Mg-ATPase activity in rat kidney cortex brushborder membranes: effect of 'proton ATPase' inhibitors.

In order to further characterize the ATP driven proton pump present in the luminal membrane of the renal proximal tubule, brushborder membranes were isolated from rat kidney cortex and the effect of various proton ATPase inhibitors on intravesicular ATP hydrolysis in sealed brushborder membrane vesicles and on Mg-ATPase activity in permeabilized brushborder membranes was investigated. The protonophor induced intravesicular ATP hydrolysis (ATP driven proton pump) was inhibited by DCCD and filipin but not by diethylstilbestrol and duramycin. All four compounds decreased Mg-ATPase activity, the two former inhibited the ATPase activity with a lower potency than the proton pump. NEM--up to 10 mM--and orthovanadate did not affect intravesicular ATP hydrolysis nor Mg-ATPase activity. From the relative sensitivity of the proton pump and the Mg-ATPase activity to the inhibitors it is concluded that about 35% of the Mg-ATPase activity found in the brushborder membrane can be attributed to the ATP-driven proton pump. Furthermore, the results obtained with NEM and duramycin suggest that the brushborder membrane proton pump has different properties than the proton pump in clathrin-coated vesicles or endosomes. The results presented above raise the possibility that the brushborder membrane proton pump is predominantly involved in acid secretion by the proximal convoluted tubule whereas the proton pump in clathrin-coated vesicles may be predominantly involved in the endocytosis of larger peptides and proteins.

Animals↗

PubMed2003

A role for subunit III in proton uptake into the D pathway and a possible proton exit pathway in Rhodobacter sphaeroides cytochrome c oxidase.

Protons are transferred from the inner surface of cytochrome c oxidase to the active site by the D and K pathways, as well as from the D pathway to the outer surface by a largely undefined proton exit route. Alteration of the initial proton acceptor of the D pathway, D132, to alanine has previously been shown to greatly inhibit oxidase turnover and slow proton uptake into the D pathway. Here it is shown that the removal of subunit III restores a substantial rate of O(2) reduction to D132A. Presumably an alternative proton acceptor for the D pathway becomes active in the absence of subunit III and D132. Thus, in the absence of subunit III cytochrome oxidase shows greater flexibility in terms of proton entry into the D pathway. In the presence of DeltaPsi and DeltapH, turnover of the wild-type oxidase or D132A is slower in the absence of subunit III. Comparison of the turnover rates of subunit III-depleted wild-type oxidase to those of the zinc-inhibited wild-type oxidase containing subunit III, both reconstituted into vesicles, leads to the hypothesis that the absence of subunit III inhibits the ability of the normal proton exit pathway to take up protons from the outside in the presence of DeltaPsi and DeltapH. Thus, subunit III appears to affect the transfer of protons from both the inner and outer surfaces of cytochrome oxidase, perhaps accounting for the long-observed lower efficiency of proton pumping by the subunit III-depleted oxidase.

Amino Acid Substitution↗

PubMed2006

Dissociative protonation sites: reactive centers in protonated molecules leading to fragmentation in mass spectrometry.

It is often found in mass spectrometry that when a molecule is protonated at the thermodynamically most favorable site, no fragmentation occurs, but a major reaction is observed when the proton migrates to a different position. For benzophenones, acetophenones, and dibenzyl ether, which are all preferentially protonated at the oxygen, deacylation or dealkylation was observed in the collision-induced dissociation of the protonated molecules. For para-monosubstituted benzophenones, electron-withdrawing substituents favor the formation of RC6H4CO+ (R = substituent), whereas electron-releasing groups favor the competing reaction leading to C6H5CO+. The ln[(RC6H4CO+)/(C6H5CO+)] values are well-correlated with the sigmap+ substituent constants. In the fragmentation of protonated acetophenones, deacetylation proceeds to give an intermediate proton-bound dimeric complex of ketene and benzene. The distribution of the product ions was found to depend on the proton affinities of ketene and substituted benzenes, and the kinetic method was applied in identifying the reaction intermediate. Protonated dibenzyl ether loses formaldehyde upon dealkylation, via an ion-neutral complex of the benzyloxymethyl cation and neutral benzene. These gas-phase retro-Friedel-Crafts reactions occurred as a result of the attack of the proton at the carbon atom to which the carbonyl or the methylene group is attached on the aromatic ring, which is described as the dissociative protonation site.

Acetophenones↗

PubMed1986

Proton inhibition of chloride exchange: asynchrony of band 3 proton and anion transport sites?

The inhibition of chloride exchange at 0 degrees C by protons at the cytoplasmic and the extracellular surface of the band 3 protein of human erythrocytes was measured between pH 4.6 and 7.6. At constant external pH and chloride concentration, internal protons were a mixed inhibitor of chloride flux, with the apparent pK2 = 6.1 for protonation of the inward-facing empty transporter conformation and the apparent pK3 = 5.7 for protonation of the chloride-transporter complex. The activation of chloride exchange by external chloride was inhibited by internal protons, and internal protonation of the externally facing empty conformation had a pK1 = 6.1. External protons were also a mixed inhibitor of chloride exchange with the apparent pK1 = 5.0 for the empty outward-facing transporter conformation. Because of the pHo dependence of self-inhibition, the value of pK3 on the outside for chloride could not be accurately determined, but the apparent pK3 for protonation of the iodide-transporter complex on the extracellular surface was 4.9. The data support a mechanism with a single proton binding site that can alternatively have access to the cytoplasmic and extracellular solutions. It appears that this proton binding and transport site can be coupled to the single anion transport site for cotransport, but the two sites can be on opposite sides of the membrane at the same time and thus can be asynchronously transported by conformational changes of band 3.

Adult↗

PubMed2005

Transition in the temperature-dependence of GFP fluorescence: from proton wires to proton exit.

In green fluorescent protein, photo-excitation leads to excited-state proton transfer from its chromophore, leaving behind a strongly fluorescing anion, while the proton is commonly thought to migrate internally to Glu-222. X-ray data show that the protein contains more extended hydrogen-bonded networks that can support proton migration (i.e., proton wires). Here we study the temperature-dependence of the transient fluorescence from both the acid and anionic forms up to 15 ns. At low temperatures, we find that the (lifetime-corrected) fluorescence of the acidic form decays asymptotically as t(-1/2), following quantitatively the solution of a one-dimensional diffusion equation for reversible geminate recombination with quenching. This indicates proton migration along the internal proton wires. A small degree of geminate proton quenching is attributed to the formation of the zwitterion by proton migration on a side-branch of the proton wire. Above 230 K, the fluorescence kinetics undergo a transition, exhibiting an asymptotic t(-3/2) decay, and the quenching effect disappears. We interpret these findings as evidence for a conformational change enabling the rotation of Thr-203, which eventually allows the proton to escape to the exterior solution.

Animals↗

PubMed1984

Helix opening in deoxyribonucleic acid from a proton nuclear magnetic resonance study of imino and amino protons in d(CG)3.

All exchangeable protons in a short DNA helix, d(CG)3 sodium salt, have been studied by proton nuclear magnetic resonance. The cytidine and guanosine amino protons have been assigned for the first time. As a function of temperature the cytidine amino protons and the imino protons behave very similarly, their relaxation is dominated by exchange with solvent above 30 degrees C. The guanosine amino protons, however, show that helix opening can only be described by a multistate model. The most rapid process observed is probably a twist about the helix axis which lengthens or breaks the guanosine amino hydrogen bond and allows rotation of the amino group. The second fastest process is a scissor opening into the major groove which gives rise to solvent exchange with the imino and cytidine amino protons. The slowest process observed is the complete base pair opening in which the guanosine amino protons also exchange with solvent. For the ammonium salt of the oligonucleotide, a specific ammonium ion complex is observed which at low temperature may catalyze exchange of the guanosine amino protons with the protons of the ammonium ion, but retards exchange with solvent. The complex appears to be specific for the sequence d(CpG).

Amines↗

PubMed1999

Proton relay system in the active site of maltodextrinphosphorylase via hydrogen bonds with large proton polarizability: an FT-IR difference spectroscopy study.

Maltodextrinphosphorylase (MDP) was studied in the pH range 5.4-8.4 by Fourier transform infrared (FT-IR) spectroscopy. The pK(a) value of the cofactor pyridoxalphosphate (PLP) was found between 6.5 and 7.0, which closely resembles the second pK(a) of free PLP.FT-IR difference spectra of the binary complex of MDP + alpha-D-glucose-1-methylenephosphonate (Glc-1-MeP) minus native MDP were taken at pH 6.9. Following binary complex formation, two Lys residues, tentatively assigned to the active site residues Lys533 and Lys539, became deprotonated, and PLP as well as a carboxyl group, most likely of Glu637, protonated. A system of hydrogen bonds which shows large proton polarizability due to collective proton tunneling was observed connecting Lys533, PLP, and Glc-1-MeP. A comparison with model systems shows, furthermore, that this hydrogen bonded chain is highly sensitive to local electrical fields and specific interactions, respectively. In the binary complex the proton limiting structure with by far the highest probability is the one in which Glc-1-MeP is singly protonated. In a second hydrogen bonded chain the proton of Lys539 is shifted to Glu637. In the binary complex the proton remains located at Glu637. In the ternary complex composed of phosphorylase, glucose-1-phosphate (Glc-1-P), and the nonreducing end of a polysaccharide chain (primer), a second proton may be shifted to the phosphate group of Glc-1-P. In the doubly protonated phosphate group the loss of mesomeric stabilization of the phosphate ester makes the C1-O1 bond of Glc-1-P susceptible to bond cleavage. The arising glucosyl carbonium ion will be a substrate for nucleophilic attack by the nonreducing terminal glucose residue of the polysaccharide chain.

Biophysical Phenomena↗

PubMed1993

Light-induced proton uptake by photosynthetic reaction centers from Rhodobacter sphaeroides R-26.1. II. Protonation of the state DQAQB2-.

Proton uptake associated with the two-electron reduction of QB was investigated in reaction centers (RCs) from Rhodobacter sphaeroides R-26.1 using pH-sensitive dyes. An uptake of two protons was observed at pH < or = 7.5, consistent with the formation of the dihydroquinone QBH2. At higher pH, the proton uptake decreased with an apparent pKa of approx. 8.5, i.e., to 1.5 H+/2 e- at pH 8.5. A molecular model is presented in which the apparent pKa is due to the protonation of either the carbonyl oxygen on QB or of an amino acid residue near QB (e.g., His-L190). Experimental evidence in favor of the protonation of the oxygen is discussed. The kinetics of the electron transfer from QA-QB- to QAQB2- and the associated proton uptake were compared at several pH values and temperatures. At pH 8.5 (21.5 degrees C) the rate constants for the proton uptake and electron transfer are the same within the precision of the measurement. At lower pH, the proton uptake rate constant is smaller than that for electron transfer. The difference between the rate constants is temperature dependent, i.e., it varies from 12 +/- 4% at 21.5 degrees C (pH 7.5) to 28 +/- 4% at 4.0 degrees C (pH 7.5). We show that the kinetics can be explained by a previously proposed model (Paddock, M. L., McPherson, P. H., Feher, G. and Okamura, M. Y. (1990) Proc. Natl. Acad. Sci. USA 87, 6803-6807) in which the uptake of two protons by doubly reduced QB occurs sequentially, one concomitant with and the other after electron transfer.

Amino Acids↗

PubMed2001

Time-resolved Fourier transform infrared spectroscopy of the polarizable proton continua and the proton pump mechanism of bacteriorhodopsin.

Nanosecond-to-microsecond time-resolved Fourier transform infrared (FTIR) spectroscopy in the 3000-1000-cm(-1) region has been used to examine the polarizable proton continua observed in bacteriorhodopsin (bR) during its photocycle. The difference in the transient FTIR spectra in the time domain between 20 ns and 1 ms shows a broad absorption continuum band in the 2100-1800-cm(-1) region, a bleach continuum band in the 2500-2150-cm(-1) region, and a bleach continuum band above 2700 cm(-1). According to Zundel (G., J. Mol. Struct. 322:33-42), these continua appear in systems capable of forming polarizable hydrogen bonds. The formation of a bleach continuum suggests the presence of a polarizable proton in the ground state that changes during the photocycle. The appearance of a transient absorption continuum suggests a change in the polarizable proton or the appearance of new ones. It is found that each continuum has a rise time of less than 80 ns and a decay time component of approximately 300 micros. In addition, it is found that the absorption continuum in the 2100-1800-cm(-1) region has a slow rise component of 190 ns and a fast decay component of approximately 60 micros. Using these results and those of the recent x-ray structural studies of bR(570) and M(412) (H. Luecke, B. Schobert, H.T. Richter, J.-P. Cartailler, and J. K., Science 286:255-260), together with the already known spectroscopic properties of the different intermediates in the photocycle, the possible origins of the polarizable protons giving rise to these continua during the bR photocycle are proposed. Models of the proton pump are discussed in terms of the changes in these polarizable protons and the hydrogen-bonded chains and in terms of previously known results such as the simultaneous deprotonation of the protonated Schiff base (PSB) and Tyr185 and the disappearance of water molecules in the proton release channel during the proton pump process.

Bacteriorhodopsins↗

PubMed1985

Effect of buffer on kinetics of proton equilibration with a protonable group.

The laser-induced proton pulse generates a massive, brief, proton pulse capable of perturbing biochemical equilibria. The time resolution of the monitoring system can follow the diffusion-controlled protonation of specific sites on macromolecular bodies [Gutman, M. (1984) Methods Biochem. Anal. 30, 1-103]. In order to apply this method in enzymology, one must first evaluate how the buffer capacity of biochemical systems (substrates and proteins) will affect the observed dynamics. Unlike equilibrium measurements, where buffer is an inert component, in kinetic studies buffer modulates the observed dynamics. In this paper we analyze the effect of buffer on the dynamics of protonation in a model system. We describe the experimental technique and introduce the mathematical formalism that determines the various rate constants involved in the reaction. The analysis of the experiments indicates that in buffered solution proton flux is carried by two mechanisms: (A) proton dissociation followed by free proton diffusion; (B) collisional proton transfer between small diffusing solutes. We demonstrate how to evaluate the contribution of each pathway to the overall proton flux.

Arylsulfonates↗

PubMed2005

Slow proton transfer through the pathways for pumped protons in cytochrome c oxidase induces suicide inactivation of the enzyme.

In the absence of subunit III the aa(3)-type cytochrome c oxidase exhibits a shortened catalytic life span (total number of turnovers) due to an increased probability of undergoing irreversible inactivation during steady-state turnover. Inactivation results from structural alteration of the heme a(3)-Cu(B) active site in subunit I [Hosler (2004) Biochim. Biophys. Acta 1655, 332-339]. The absence of subunit III also dramatically slows proton uptake to the active site via the D proton pathway, as well as inhibiting the proton backflow/exit pathway that connects the active site/proton pump with the outer surface of the oxidase complex. Here we demonstrate that these phenomena are linked: slow proton delivery to the active site through these pathways induces suicide inactivation, thus shortening the catalytic life span of the enzyme. Mutations that inhibit the D pathway, but not the K pathway, increase the probability of suicide inactivation. Strong inhibition of the D pathway allows suicide inactivation to occur even in the presence of subunit III. Arachidonic acid, which stimulates proton uptake by the D pathway, retards suicide inactivation. Steady-state turnover in the presence of DeltaPsi and DeltapH, which inhibits proton uptake from the inner surface of the protein, enhances suicide inactivation. Simultaneous inhibition of proton uptake from both sides of the protein by a double mutation affecting the D pathway and the proton backflow/exit pathway greatly shortens the catalytic life span of the oxidase even in the presence of subunit III. Thus, maintenance of rapid proton transfer through the D pathway and the backflow/exit pathway is one mechanism by which subunit III normally functions to prevent suicide inactivation of cytochrome c oxidase. The experiments suggest that increased lifetimes of the heme a(3) oxoferryl intermediates as well as the anionic form of Glu286 of the D pathway cause suicide inactivation in the active site.

Animals↗

PubMed2000

Localized control of proton transfer through the D-pathway in cytochrome c oxidase: application of the proton-inventory technique.

In the reaction cycle of cytochrome c oxidase from Rhodobacter sphaeroides, one of the steps that are coupled to proton pumping, the oxo-ferryl-to-oxidized transition (F --> O), displays a large kinetic deuterium isotope effect of about 7. In this study we have investigated in detail the dependence of the kinetics of this reaction step ¿k(FO)(chi) on the fraction (chi) D(2)O in the enzyme solution (proton-inventory technique). According to a simplified version of the Gross-Butler equation, from the shape of the graph describing k(FO)(chi)/k(FO)(0), conclusions can be drawn concerning the number of protonatable sites involved in the rate-limiting proton-transfer reaction step. Even though the proton-transfer reaction during the F --> O transition takes place over a distance of at least 30 A and involves a large number of protonatable sites, the proton-inventory analysis displayed a linear dependence, which indicates that the entire deuterium isotope effect of 7 is associated with a single protonatable site. On the basis of experiments with site-directed mutants of cytochrome c oxidase, this localized proton-transfer rate control is proposed to be associated with glutamate (I-286) in the D-pathway. Consequently, the results indicate that proton transfer from the glutamate controls the rate of all events during the F --> O reaction step. The proton-inventory analysis of the overall enzyme turnover reveals a nonlinear plot characteristic of at least two protonatable sites involved in the rate-limiting step in the transition state, which indicates that this step does not involve proton transfer through the same pathway (or through the same mechanism) as during the F --> O transition.

Cytochrome c Group↗

PubMed1995

Rapid long-range proton diffusion along the surface of the purple membrane and delayed proton transfer into the bulk.

The pH-indicator dye fluorescein was covalently bound to the surface of the purple membrane at position 72 on the extracellular side of bacteriorhopsin and at positions 101, 105, 160, or 231 on the cytoplasmic side by reacting bromomethylfluorescein with the sulfhydryl groups of cysteines introduced by site-directed mutagenesis. At position 72, on the extracellular surface, the light-induced proton release was detected 71 +/- 4 microseconds after the flash (conditions: pH 7.3, 22 degrees C, and 150 mM KCl). On the cytoplasmic side with the dye at positions 101, 105, and 160, the corresponding values were 77, 76, and 74 +/- 5 microseconds, respectively. Under the same conditions, the proton release time in the bulk medium as detected by pyranine was around 880 microseconds--i.e., slower by a factor of more than 10. The fact that the proton that is released on the extracellular side is detected much faster on the cytoplasmic surface than in the aqueous bulk phase demonstrates that it is retained on the surface and migrates along the purple membrane to the other side. These findings have interesting implications for bioenergetics and support models of local proton coupling. From the small difference between the proton detection times by labels on opposite sides of the membrane, we estimate that at 22 degrees C the proton surface diffusion constant is greater than 3 x 10(-5) cm2/s. At 5 degrees C, the proton release detection time at position 72 equals the faster of the two main rise times of the M intermediate (deprotonation of the Schiff base). At higher temperatures this correlation is gradually lost, but the curved Arrhenius plot for the proton release time is tangential to the linear Arrhenius plot for the rise of M at low temperatures. These observations are compatible with kinetic coupling between Schiff base deprotonation and proton release.

Affinity Labels↗

PubMed2000

Identification of the proton pathway in bacterial reaction centers: replacement of Asp-M17 and Asp-L210 with asn reduces the proton transfer rate in the presence of Cd2+.

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the reduction and protonation of a bound quinone molecule Q(B) (the secondary quinone electron acceptor). We investigated the proton transfer pathway by measuring the proton-coupled electron transfer, k(AB)((2)) [Q(A)Q(B) + H(+) --> Q(A)(Q(B)H)(-)] in native and mutant RCs in the absence and presence of Cd(2+). Previous work has shown that the binding of Cd(2+) decreases k(AB)((2)) in native RCs approximately 100-fold. The preceding paper shows that bound Cd(2+) binds to Asp-H124, His-H126, and His-H128. This region represents the entry point for protons. In this work we investigated the proton transfer pathway connecting the entry point with Q(B) by searching for mutations that greatly affect k(AB)((2)) ( greater, similar10-fold) in the presence of Cd(2+), where k(AB)((2)) is limited by the proton transfer rate (k(H)). Upon mutation of Asp-L210 or Asp-M17 to Asn, k(H) decreased from approximately 60 s(-1) to approximately 7 s(-1), which shows the important role that Asp-L210 and Asp-M17 play in the proton transfer chain. By comparing the rate of proton transfer in the mutants (k(H) approximately 7 s(-1)) with that in native RCs in the absence of Cd(2+) (k(H) >/= 10(4) s(-1)), we conclude that alternate proton transfer pathways, which have been postulated, are at least 10(3)-fold less effective.

Aspartic Acid↗

PubMed1986

Intracellular pH regulation and proton transport by rabbit renal medullary collecting duct cells. Role of plasma membrane proton adenosine triphosphatase.

Proton secretion in the renal medullary collecting duct is thought to occur via a luminal proton-ATPase. In order to determine what mechanism(s) participate in proton transport across medullary collecting duct (MCD) cells membranes, intracellular pH (pHi) regulation and proton extrusion rates were measured in freshly prepared suspensions of rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pHi was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Proton extrusion rates were measured using a pH stat. The resting pHi of MCD cells was 7.19 +/- 0.05 (SE) in a nonbicarbonate medium of pH 7.30. When cells were acidified by exposure to acetate salts or by abrupt withdrawal of ammonium chloride, they exhibited pHi recovery to the resting pHi over a 5-min time-course. Depletion of greater than 95% of cellular ATP content by poisoning with KCN in the absence of glucose inhibited pHi recovery. ATP depletion inhibited proton extrusion from MCD cells. Treatment with N-ethylmaleimide also inhibited pHi recovery. In addition, cellular ATP content was dependent on transmembrane pH gradients, suggesting that proton extrusion stimulated ATP hydrolysis. Neither removal of extracellular sodium nor addition of amiloride inhibited pHi recovery. These results provide direct evidence that a plasma membrane proton-ATPase, but not a Na+/H+ exchanger, plays a role in proton transport and pHi regulation in rabbit MCD.

Acetates↗