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PubMed1994

The causes and functions of mitochondrial proton leak.

The non-linear relationship between respiration rate and protonmotive force in isolated mitochondria is explained entirely by delta p-dependent changes in the proton conductance of the mitochondrial inner membrane and is not caused by redox slip in the proton pumps. Mitochondrial proton leak occurs in intact cells and tissues: the futile cycle of proton pumping and proton leak accounts for 26% +/- 7% of the total oxygen consumption rate or 33% +/- 7% of the mitochondrial respiration rate of isolated hepatocytes (mean +/- S.D. for 43 rats); 52% of the oxygen consumption rate of resting perfused muscle and up to 38% of the basal metabolic rate of a rat, suggesting that heat production may be an important function in the proton leak in homeotherms. Together with non-mitochondrial oxygen consumption, it lowers the effective P/O ratio in cells from maximum possible values of 2.33 (palmitate oxidation) or 2.58 (glucose oxidation) to as low as 1.1 in liver or 0.8 in muscle. The effective P/O ratio increases in response to ATP demand; the ability to allow rapid switching of flux from leak to ATP turnover may be an even more important function of the leak reaction than heat production. The mitochondrial proton conductance in isolated mitochondria and in hepatocytes is greatly modulated by thyroid hormones, by phylogeny and by body mass. Usually the reactions of ATP turnover change in parallel so that the coupling ratio is not greatly affected. Changes in proton leak in tissues are brought about in the short term by changes in mitochondrial protonmotive force and in the longer term by changes in the surface area and proton permeability of the mitochondrial inner membrane. Permeability changes are probably caused by changes in the fatty acid composition of the membrane phospholipids.

Adenosine Triphosphate↗
PubMed2004

A cooperative model for proton pumping in cytochrome c oxidase.

In this paper, the mechanism of proton pumping in cytochrome c oxidase is examined. Data on cooperative linkage of vectorial proton translocation to oxido-reduction of Cu(A) and heme a in the CO-inhibited, liposome-reconstituted bovine cytochrome c oxidase are reviewed. Results on proton translocation associated to single-turnover oxido-reduction of the four metal centers in the unliganded, membrane-reconstituted oxidase are also presented. On the basis of these results, X-ray crystallographic structures and spectrometric data for a proton pumping model in cytochrome c oxidase is proposed. This model, which is specifically derived from data available for the bovine cytochrome c oxidase, is intended to illustrate the essential features of cooperative coupling of proton translocation at the low potential redox site. Variants will have to be introduced for those members of the heme copper oxidase family which differ in the redox components of the low potential site and in the amino acid network connected to this site. The model we present describes in detail steps of cooperative coupling of proton pumping at the low potential Cu(A)-heme a site in the bovine enzyme. It is then outlined how this cooperative proton transfer can be thermodynamically and kinetically coupled to the chemistry of oxygen reduction to water at the high potential Cu(B)-heme a(3) center, so as to result in proton pumping, in the turning-over enzyme, against a transmembrane electrochemical proton gradient of some 250 mV.

Animals↗
PubMed2005

Regeneration and inhibition of proton pumping activity of bacteriorhodopsin blue membrane by cationic amine anesthetics.

Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning alpha-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of "the cation" in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca2+, Mg2+ and Mn2+) and monovalent metal cations (Li+, Na+, K+ and Cs+), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation<monovalent amine anesthetic cation<divalent metal cation. We found that organic cations such as the amine anesthetics can also regenerate the proton pump in the bR protein. The inhibition of proton transport in the bR protein by the anesthetic cations was elucidated using the wild type, the E204Q and the D96N mutated bRs. The hydrophobic interaction of the amine anesthetics with the bR protein plays an important part in inhibiting the bR proton pump.

Amines↗
PubMed2005

Proton radiotherapy for orbital rhabdomyosarcoma: clinical outcome and a dosimetric comparison with photons.

BACKGROUND: Over 85% of pediatric orbital rhabdomyosarcoma (RMS) are cured with combined chemotherapy and radiation. However, the late effects of photon radiation compromise function and cosmetic outcome. Proton radiation can provide excellent tumor dose distributions while sparing normal tissues better than photon irradiation. METHODS AND MATERIALS: Conformal 3D photon and proton radiotherapy plans were generated for children treated with proton irradiation for orbital RMS at Massachusetts General Hospital. Dose-volume histograms (90%, 50%, 10%) were generated and compared for important orbital and central nervous system structures. Average percentages of total dose prescribed were calculated based on the 3 dose-volume histogram levels for normal orbital structures for both the proton and photon plans. The percent of normal tissue spared by using protons was calculated. RESULTS: Seven children were treated for orbital rhabdomyosarcoma with proton irradiation and standard chemotherapy. The median follow-up is 6.3 years (range, 3.5-9.7 years). Local and distant controls compare favorably to those in other published accounts. There was an advantage in limiting the dose to the brain, pituitary, hypothalamus, temporal lobes, and ipsilateral and contralateral orbital structures. Tumor size and location affect the degree of sparing of normal structures. CONCLUSIONS: Fractionated proton radiotherapy is superior to 3D conformal photon radiation in the treatment of orbital RMS. Proton therapy maintains excellent tumor coverage while reducing the radiation dose to adjacent normal structures. Proton radiation therapy minimizes long-term side effects.

Brain↗
PubMed2000

Common themes and problems of bioenergetics and voltage-gated proton channels.

The existence of a proton-selective pathway through a protein is a common feature of voltage-gated proton channels and a number of molecules that play pivotal roles in bioenergetics. Although the functions and structures of these molecules are quite diverse, the proton conducting pathways share a number of fundamental properties. Conceptual parallels include the translocation by hydrogen-bonded chain mechanisms, problems of supply and demand, equivalence of chemical and electrical proton gradients, proton wells, alternating access sites, pK(a) changes induced by protein conformational change, and heavy metal participation in proton transfer processes. An archetypal mechanism involves input and output proton pathways (hydrogen-bonded chains) joined by a regulatory site that switches the accessibility of the bound proton from one 'channel' to the other, by means of a pK(a) change, molecular movement, or both. Although little is known about the structure of voltage-gated proton channels, they appear to share many of these features. Evidently, nature has devised a limited number of mechanisms to accomplish various design strategies, and these fundamental mechanisms are repeated with minor variation in many superficially disparate molecules.

Cell Membrane↗
PubMed2004

Protons versus photons: a status assessment at the beginning of the 21st Century.

At present modern photon delivery techniques permit high dose isodose conformality similar to protons in many cases. However, a proton advantage appears to still be present for target volumes of higher degrees of complexity and concavity. Also, for selected histologies and sites, notably skull base tumors, protons have established a "gold standard" and photon data have yet to duplicate those proton results in clinical practice. Proton radiation therapy offers superior dose distribution by reduced low-dose integral irradiated volume. The avoidance of functional and cosmetic side effects in children by protons is at present acknowledged by the radiation oncology community and is expected to gain similar recognition in adult patients. This advantage of protons and disadvantage for photons constitutes an "inherent physical gap" that will likely be long lasting. Although the priorities of proton irradiation advantages have shifted over the decades, clinical advantages remain and are of sufficient significance and importance to justify further development and installation of proton radiation facilities worldwide.

Humans↗
PubMed2001

Potential role of intensity modulated proton beams in prostate cancer radiotherapy.

PURPOSE: The present study was undertaken to assess the potential benefit of intensity modulated (IM) proton beams in optimizing the dose distribution to safely escalate the tumor dose in prostate cancer radiotherapy. METHODS AND MATERIALS: Four treatment plans were compared in a prostate cancer patient aiming to deliver 81 Gy to the target: 1) conformal 18 MV X-rays, 6-fields; 2) 214 MeV protons, 2-fields; 3) IM 15 MV X-rays, 5-fields; and 4) 177-200 Mev IM protons, 5-fields as in Plan 3. In addition, IM methods were used to further escalate the tumor dose to 99 Gy. Dose-volume histograms (DVH) were used to physically compare the treatment plans. DVH data were also used to obtain normal tissue complication probabilities (NTCP) for the rectum, bladder, femoral heads, and tumor control probabilities. RESULTS: Although the planning target volume dose distribution was satisfactory with the four treatment plans, the homogeneity was slightly reduced in both X-ray plans (IM and standard) and the low-to-medium doses delivered to all organs at risk, and other normal tissues were significantly reduced by both proton plans. For a prescribed dose of 81 Gy, only the IM X-ray and IM proton plans both succeeded in predicting an acceptably low NTCP for the rectum (<5%, Grade 3). The integral nontarget dose was significantly reduced with IM proton beams (i.e., 3.1, 1.3, and 1.7 times less than Plans 1, 2, and 3, respectively). When escalating the dose to 99 Gy, no additional improvement between IM protons and IM X-ray beams was observed. CONCLUSION: Both IM X-ray and proton beams were able to optimize the dose distribution and comply with the goal of delivering the highest dose to the target while reducing the risk of severe morbidity to acceptable levels. The main advantage compared to IM X-rays was that IM protons succeeded in significantly reducing the low-to-medium dose to the nontarget tissues and achieved a small improvement in planning target volume (PTV) dose heterogeneity.

Femur Head↗
PubMed1997

Potential role of proton therapy in the treatment of pediatric medulloblastoma/primitive neuroectodermal tumors: reduction of the supratentorial target volume.

PURPOSE: One of the components of radiotherapy (RT) in medulloblastoma/primitive neuroectodermal tumors is the prophylactic irradiation of the whole brain (WBI). With the aim of reducing late neuropsychologic morbidity a CT-scan-based dosimetric study was undertaken in which treatment was confined mainly or exclusively to supratentorial sites considered at high risk for disease recurrence. METHODS AND MATERIALS: A comparative dosimetric study is presented in which a three field (two laterals and one posterior) proton plan (spot scanning method) is compared with a two-field conventional WBI 6 MV x-ray plan, to a 6-field "hand-made" 6 MV x-ray plan, and to a computer-optimized 9-field "inverse" 15 MV x-ray plan. For favorable patients, 30 Gy were delivered to the ventricles and main cisterns, the subfrontal and subtemporal regions, and the posterior fossa. For the unfavorable patients, 10 Gy WBI preceeded a boost to 30 Gy to the same treatment volume chosen for favorable patients. The dose distribution was evaluated with dose-volume histograms to examine the coverage of the targets as well as the dose to the nontarget brain and optical structures. In addition, the risks of radiation-related late neuropsychologic effects after WBI were collected from the literature and used to predict normal tissue complication probabilities (NTCPs) for an intelligence quotient deficit after treatment with photon or proton beams. RESULTS: Proton beams succeeded better in reducing the dose to the brain hemispheres and eye than any of the photon plans. A 25.1% risk of an IQ score <90 was predicted after 30 Gy WBI. Almost a 10% drop in the predicted risk was observed when using proton beams in both favorable and unfavorable patients. However, predicted NTCPs for both optimized photon plans ("hand made" and "inverse") were only slightly higher (0.3-2.5%) than those of proton beams. An age-modifying factor was introduced in the predictive NTCP model to assess for IQ differences in relation with age at irradiation. Children with ages between age 4 to 8 benefitted most from the dose reduction in this exercise (similar NTCP predictions for both proton and "inverse" plans). CONCLUSION: Modulated proton beams may help to significantly reduce the irradiation of normal brain while optimally treating the supratentorial subsites at higher risk for relapse. A decrease in morbidity can be expected from protons and both optimized proton plans compared to WBI.

Brain Neoplasms↗
PubMed1998

Comparative treatment planning between proton and x-ray therapy in esophageal cancer.

PURPOSE: Conformal treatment planning with megavoltage x-rays and protons for five patients with esophageal cancer has been studied in an attempt to determine if there are advantages of using protons instead of x-rays. METHODS AND MATERIALS: For each of the five patients, two different proton plans, one x-ray plan, and one mixed plan with x-rays and protons were made. A three-dimensional treatment planning system, TMS, was used. The evaluation of the different plans was made by applying the tumor control probability (TCP) model proposed by Nahum and Webb and the normal tissue complication (NTCP) model proposed by Lyman on the dose distributions in terms of dose-volume histograms (DVHs). RESULTS: The comparison shows advantages of using protons instead of x-rays for all five patients. The dose-limiting organs at risk are the spinal cord, the lungs, and the heart, but the proton plans also spare the kidneys better than the x-ray plan does. At 5% NTCP in any risk organ, the calculated mean TCP value for the five patients is increased by an average of 20%-units (from 2 to 23%-units) with the best proton plan compared with x-rays only. However, if we assume maximally a 1% risk in the spinal cord and a total NTCP for the two lungs of 100%, the mean TCP value for the five patients is increased from 6 to 49% with the best proton plan compared with x-rays only. The corresponding figure for the mixed plan is 27%. These gains are relatively insensitive to variations within reasonable limits in the biological parameters. CONCLUSIONS: Protons appear to have clear therapeutic advantages over conventional external radiotherapy when treating esophageal carcinoma.

Aged↗
PubMed1995

ATP synthase of chloroplasts: selective action of agents binding to F1 on partial reactions of proton transfer in F0.

We studied the basic steps of proton transfer through ATP synthase of chloroplasts, CF0CF1, under conditions of proton slip, a conducting stage in the absence of added nucleotides. On the background of a steady transmembrane pH difference, voltage steps were induced by flashing light. Proton intake, transfer, and release by CF0CF1 were kinetically resolved by spectrophotometric probes. Kinetic disparities between these three steps were observed. Rapid but limited proton intake from the thylakoid lumen and proton release into the medium (tau 1/2 approximately 5 ms) preceded charge transfer across the dielectric barrier in the enzyme (tau 1/2 approximately 30 ms). The saturation behavior under multiple flashes suggested a sequential reaction mechanism. ADP and dequalinium, when bound to subunit beta of the catalytic portion, CF1, blocked different partial reactions involving protons. ADP in the catalytic cleft blocked the electrogenic transfer step, and dequalinium at the adjacent DELSEED sequence on the same subunit blocked the release of protons. Both effects prove a long-range conformational transmission between remote (approximately 10 nm) domains on F1 and on F0. For the normal sequence of events from protons to ATP they suggest a specific action of certain proton-transfer steps on different domains of the catalytic portion.

Binding Sites↗
PubMed2006

Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain and couples energetically the reduction of oxygen to water to proton pumping across the membrane. The results from previous studies showed that proton pumping can be uncoupled from the O2-reduction reaction by replacement of one single residue, Asn-139 by Asp (N139D), located approximately 30 A from the catalytic site, in the D-proton pathway. The uncoupling was correlated with an increase in the pK(a) of an internal proton donor, Glu-286, from approximately 9.4 to >11. Here, we show that replacement of the acidic residue, Asp-132 by Asn in the N139D CcO (D132N/N139D double-mutant CcO) results in restoration of the Glu-286 pK(a) to the original value and recoupling of the proton pump during steady-state turnover. Furthermore, a kinetic investigation of the specific reaction steps in the D132N/N139D double-mutant CcO showed that proton pumping is sustained even if proton uptake from solution, through the D-pathway, is slowed. However, during single-turnover oxidation of the fully reduced CcO the P --> F transition, which does not involve electron transfer to the catalytic site, was not coupled to proton pumping. The results provide insights into the mechanism of proton pumping by CcO and the structural elements involved in this process.

Catalysis↗
PubMed2001

Arg-735 of the 100-kDa subunit a of the yeast V-ATPase is essential for proton translocation.

The vacuolar (H(+))-ATPases (V-ATPases) are ATP-dependent proton pumps that acidify intracellular compartments and pump protons across specialized plasma membranes. Proton translocation occurs through the integral V(0) domain, which contains five different subunits (a, d, c, c', and c"). Proton transport is critically dependent on buried acidic residues present in three different proteolipid subunits (c, c', and c"). Mutations in the 100-kDa subunit a have also influenced activity, but none of these residues has proven to be required absolutely for proton transport. On the basis of previous observations on the F-ATPases, we have investigated the role of two highly conserved arginine residues present in the last two putative transmembrane segments of the yeast V-ATPase a subunit (Vph1p). Substitution of Asn, Glu, or Gln for Arg-735 in TM8 gives a V-ATPase that is fully assembled but is totally devoid of proton transport and ATPase activity. Replacement of Arg-735 by Lys gives a V-ATPase that, although completely inactive for proton transport, retains 24% of wild-type ATPase activity, suggesting a partial uncoupling of proton transport and ATP hydrolysis in this mutant. By contrast, nonconservative mutations of Arg-799 in TM9 lead to both defective assembly of the V-ATPase complex and decreases in activity of the assembled V-ATPase. These results suggest that Arg-735 is absolutely required for proton transport by the V-ATPases and is discussed in the context of a revised model of the topology of the 100-kDa subunit a.

Adenosine Triphosphate↗
PubMed1991

A potential role for guanine nucleotide-binding protein in the regulation of endosomal proton transport.

The effects of guanosine 5'-triphosphate (GTP) and GTP-gamma-S, known activators of GTP binding proteins, on proton transport were investigated in endosome-enriched vesicles (endosomes). Endosomes were prepared from rabbit renal cortex following the intravenous injection of FITC-dextran. The rate of intravesicular acidification was determined by measuring changes in fluorescence of FITC-dextran. Both GTP and GTP-gamma-S stimulated significantly the initial rate of proton transport. In contrast, GDP-beta-S, which does not activate GTP binding proteins, inhibited proton transport. The rank order of stimulation was GTP-gamma-S greater than GTP greater than control greater than GDP-beta-S. GTP-gamma-S stimulation of proton transport was also observed under conditions in which chloride entry was eliminated, i.e., 0 mM external chloride concentration in the presence of potassium/valinomycin voltage clamping. GTP-gamma-S did not affect proton leak in endosomes as determined by collapse of H+ ATPase-generated pH gradients. ADP ribosylation by treatment of endosomal membranes with pertussis toxin revealed two substrates corresponding to the 39-41 kD region and comigrating with alpha i subunits. Pretreatment of the membranes with pertussis toxin had no effect on proton transport in the absence of GTP or GTP-gamma-S. However, pretreatment with pertussis toxin blocked the stimulation of proton transport by GTP. In contrast, as reported in other membranes by others previously, pertussis toxin did not prevent the stimulation of proton transport by GTP-gamma-S. These findings, taken together, indicate that GTP binding proteins are present in endosomal membranes derived from renal cortex and that activation of G protein by GTP and GTP-gamma-S stimulates proton transport in a rank order identical to that reported for other transport pathways modulated by Gi proteins. Therefore, these studies suggest that G proteins are capable of stimulating the vacuolar H ATPase of endosomes directly.

Animals↗
PubMed2004

Ionization-induced proton transfer in model DNA base pairs: a theoretical study of the radical ions of the 7-azaindole dimer.

Proton-transfer reactions of the radical anion and cation of the 7-Azaindole (7AI) dimer were investigated by means of density functional theory (DFT). The calculated results for the dimer anion and cation were very similar. Three equilibrium structures, which correspond to the non-proton-transferred (normal), the single-proton-transferred (SPT) and the double-proton-transferred (tautomeric) forms, were found. The transition states for proton-transfer reactions were also located. The calculations showed that the first proton-transfer reaction (normal-->SPT) is exothermic and almost barrier-free; therefore, it should occur spontaneously in the period of a vibration. In contrast, the second proton-transfer reaction (SPT-->tautomer) was found to be far less-probable in terms of reaction energy and barrier. Hence, it was concluded that both (7Al)2*- and (7Al)2*+ exist in the SPT form. The conclusion was further confirmed by the calculated electron vertical detachment energy (VDE) of the SPT form of (7Al)2*-, 1.33 eV, which is very close to the experimental measurement of 1.35 eV. The calculated VDEs of the normal and tautomeric (7Al)2*- forms were too small compared to the experimental value. The proton transfer process was found to be multidimensional in nature involving not only proton motion but also intermolecular rocking motion. In addition, IR spectra were calculated and reported. The spectra of the three structures showed very different features and, therefore, can be considered as fingerprints for future experimental identifications. The implications of these results to biology and spectroscopy are also briefly discussed.

Algorithms↗
PubMed2005

Proton transfer in a Thr200His mutant of human carbonic anhydrase II.

Human carbonic anhydrase II (HCA II) has a histidine at position 64 (His64) that donates a proton to the zinc-bound hydroxide in catalysis of the dehydration of bicarbonate. To examine the effect of the histidine location on proton shuttling, His64 was replaced with Ala and Thr200 replaced with histidine (H64A-T200H HCAII), effectively relocating the proton shuttle residue 2 A closer to the zinc-bound hydroxide compared to wild type HCA II. The crystal structure of H64A-T200H HCA II at 1.8 A resolution shows the side chain of His200 directly hydrogen-bonded with the zinc-bound solvent. Different proton transfer processes were observed at pH 6 and at pH 8 during the catalytic hydration-dehydration cycle, measured by mass spectrometry as the depletion of 18O from C18O2 by H64A-T200H HCA II. The process at pH 6.0 is attributed to proton transfer between the side chain of His200 and the zinc-bound hydroxide, in analogy with proton transfer involving His64 in wild-type HCA II. At pH 8.0 it is attributed to proton transfer between bicarbonate and the zinc-bound hydroxide, as supported by the dependence of the rate of proton transfer on bicarbonate concentration and on solvent hydrogen isotope effects. This study establishes that a histidine directly hydrogen-bonded to the zinc-bound hydroxide, can adopt the correct distance geometry to support proton transfer

Amino Acid Substitution↗
PubMed1998

High-speed magic-angle spinning 13C MAS NMR spectra of adamantane: self-decoupling of the heteronuclear scalar interaction and proton spin diffusion.

We have investigated the carbon line shape of solid adamantane under high-speed magic-angle sample spinning (MAS) acquired without proton decoupling. The CH-group shows a spinning-speed-dependent line broadening while the CH2-group consists of a spinning-speed-independent sharp component and a spinning-speed-dependent broader part. These phenomena can be explained by self-decoupling of the J-interaction due to proton spin diffusion. Such a self-decoupling process can be described by a magnetization exchange process between the multiplet lines. Changing the spin-diffusion rate constant by off-resonance irradiation of the protons allows us to observe the full range from slow exchange to coalescence to fast exchange of the carbon spectra. One of the multiplet components in the CH2-group corresponds to a group spin of the protons of zero and therefore does not couple to the other protons. This gives rise to the sharp central line. The magnetization exchange rate constant between the different multiplet lines can be determined from the spectra and is a measure for the spinning-speed-dependent proton spin-diffusion rate constant. Even at an MAS speed of 30 kHz, proton spin diffusion is still observable despite the relatively weak intermolecular proton dipolar-coupling network in adamantane which results in a static proton line width of only 14 kHz (full width at half height).

Adamantane↗
PubMed1998

Protein mediated magnetic coupling between lactate and water protons.

The magnetic coupling between methyl lactate protons and water protons in samples of cross-linked bovine serum albumin (BSA) is studied. Cross-relaxation spectroscopy shows efficient magnetization transfer from immobilized BSA to both water and methyl lactate protons. Transient and steady-state NOE experiments reveal a negative intermolecular NOE between methyl lactate and water protons. Lactate is indirectly detected by selectively saturating the methyl lactate protons and measuring the decrease in water proton magnetization. Indirect detection of methyl lactate protons is an order of magnitude more sensitive than direct detection in these model systems. Lactate was indirectly imaged, via the water proton resonance, with 1.1-microliter voxels in 2 min. Immobilized BSA reduces the intermolecular correlation time between water and lactate protons into the spin-diffusion limit where the NOE is negative. Possible molecular mechanisms for this coupling and applications to in vivo spectroscopy are discussed.

Animals↗
PubMed1987

The plasma membrane ATPase of Neurospora: a proton-pumping electroenzyme.

Probably the best marker enzyme for plasma membranes of eukaryotic cells is a magnesium-dependent, vanadate-inhibited ATPase whose primary function is the transmembrane transport of cations. In animal cells, different species of the enzyme transport different cations: sodium ions released in unequal exchange for potassium ions, calcium ions extruded alone (perhaps), or protons secreted in equal exchange for potassium ions. But in plants and fungi only proton secretion has been clearly demonstrated. A useful model cell for studying the proton-secreting ATPase has been the ascomycete fungus Neurospora, in which the enzyme drives an outward current of protons that can exceed 50 microA/cm2 and can support membrane potentials greater than 300 mV. Both thermodynamic and kinetic studies have shown that the proton-pumping ATPase of Neurospora normally transports only a single proton for each ATP molecule split; and kinetic modelling studies have suggested (contrary to conventional assumptions) that the fast steps in the overall reaction are transmembrane transit of the proton and its dissociation following transport, while the slow steps are the binding of protons and/or ATP. The primary structure of the Neurospora enzyme, recently deduced by gene sequencing, is very close to that of the yeast (Saccharomyces) enzyme, and the hydropathic patterns for both closely resemble those for the animal-cell plasma-membrane ATPases. All of these enzymes appear to have 6-10 membrane-spanning alpha-helices, plus a large cytoplasmic headgroup which bears the catalytic nucleotide-binding site. Structural data, taken together with the electrical-kinetic behavior, suggest that the catalytic headgroup functions as an energized gate for protons. From a geometric point of view, action of such a gate would transfer the membrane field across the "transported" ion, rather than vice versa.

Adenosine Triphosphatases↗