Electrochemical potential of protons in vesicles reconstituted from purified,proton-translocating adenosine triphosphatase

Summary Measurements were made of the difference in the electrochemical potential of protons ( ) across the membrane of vesicles reconstituted from the ATPase complex (TF ₀ ·F ₁) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential () and pH difference across the membrane ( pH), respectively.In the presence of Tris buffer the maximal and no pH were produced, while in the presence of the permeant anion NO ₃ ^– the maximal pH and a low were produced by the addition of ATP. When the ATP concentration was 0.24mm, the was 140–150 mV (positive inside) in Tris buffer, and the pH was 2.9–3.5 units (acidic inside) in the presence of NO ₃ ^– . Addition of a saturating amount of ATP produced somewhat larger and pH values, and the attained was about 310 mV.By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4–5 during ATP hydrolysis.The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied.     

The generation of the proton electrochemical potential and its role in energy transduction

Summary The evidence that all energy transducing membranes can generate a proton electrochemical potential difference, _H, across the membrane and that this potential can be used to transfer energy among energy transducing units and to generate ATP, has increased the interest for the view that _H plays an obligatory role in energy transduction and ATP synthesis. In the present article we shall concentrate on two experimental questions related with the generation and role of _H: (a) the charge/site ratio; (b) the relation between the proton electrochemical potential on one side and the cation electrochemical potential, the phosphate potential and the redox potential on the other. We shall then discuss the view that energy transduction corresponds to a molecular energy machine rather than to a fuel cell.     

Relationship between the octanol-water partition coefficient of tertiary amines and their effect of ‘selective’ uncoupling of photophosphorylation

A series of tertiary amines was investigated for effects on the transmembrane proton potential difference ( H), on photophosphorylation and on electron-flux control related to the intrathylakoid proton potential ( H_I), using isolated chloroplasts ofSpinacia oleracea L. As indicated by 9-aminoacridine fluorescence and [14C]methylamine uptake, all amines studied inhibited a build-up of H and, in parallel, ATP synthesis. Even when H was low, strong H₁-dependent electron-flux control was observed under the influence of tertiary amines. The strength of flux control in the presence of low H and the effectiveness of inhibition of ATP synthesis linearly increased with the lipophilicity of the amines. The most effective of the amines tested caused 50% inhibition of ATP synthesis at a concentration of 6 M, which is about 1000-fold lower than the concentration required for inhibition by methylamine. The data presented indicate the existence of two proton domains in the thylakoid vesicles, one of them feeding the ATP-synthase, the other the sites of pH-dependent electron-flux control. It is concluded that tertiary amines develop their action in a lipophilic domain of the thylakoid membrane, in the vicinity of the ATP-synthase complex. A mechanism for selective uncoupling and for the maintenance of H_I-dependent electron flux control in the presence of low H is discussed.Abbreviations and symbols coefficient for pH-dependent electron flux control - 9-AA 9-aminoacridine - Chl chlorophyll - I₅₀ amine concentration producing 50% inhibition of ATP-synthesis - J_e flux of photosynthetic electron transport - k _H apparent rate constant for proton efflux - H₁ proton potential in the thylakoid lumen - H₁ transthylakoid proton potential difference - p partition coefficient - q _AA coefficient for 9-aminoacridine fluorescence quenching - PS photosystem - Q quantum flux of photosynthetically active light Dedicated to Professor Wilhelm Simonis, on the occasion of his 80th birthday     

The electrochemical H+ gradient in the yeastRhodotorula glutinis

The electrochemical gradient of protons, , was estimated in the obligatory aerobic yeastRhodotorula glutinis in the pH₀ range from 3 to 8.5. The membrane potential, , was measured by steady-state distribution of the hydrophobic ions, tetraphenylphosphonium (TPP⁺) for negative above pH₀ 4.5, and thiocyanate (SCN^–) for positive below pH₀ 4.5. The chemical gradient of H⁺ was determined by measuring the chemical shift of intracellular P_i by³¹P-NMR at given pH₀ values. The values of pH_i increased almost linearly from 7.3 at pH₀ 3 to 7.8 at pH₀ 8.5. In the physiological pH₀ range from 3.5 to 6, was fairly constant at values between 17–18 KJ mol^–1, gradually decreasing at pH₀ above 6. In deenergized cells, the intracellular pH_i decreased to values as low as 6, regardless of whether the cell suspension was buffered at pH₀ 4.5 or 7.5. There was no membrane potential detectable in deenergized cells.     

Foam behavior of biological media

Summary The influence of the alcohol concentration on the foaminess, , of-BSA-solutions is considered. This effect is calculated by means of the function (C_BSA . f), where f=1 for pure protein solutions and f>1 for alcohol solutions. f is calculated by f = 2^TT_eff. Here, where T_T is the turbidity temperature change due to solvent structure effects and T_D, the temperature correction due to alcohol-protein interaction. The constants necessary to calculate T_T and T_D are tabulated. The agreement between the calculated and measured foaminess , as a function of the n-propanol concentration is satisfactory and for methanol or ethanol excellent.     

A study on Na+-coupled oxidative phosphorylation: ATP formation supported by artificially imposed ΔpNa and ΔpK inVibrio alginolyticus cells

Addition of Na⁺ to the K⁺-loadedVibrio alginolyticus cells, creating a 250-fold Na⁺ gradient, is shown to induce a transient increase in the intracellular ATP concentration, which is abolished by the Na⁺/H⁺ antiporter, monensin. The pNa-supported ATP synthesis requires an additional driving force supplied by endogenous respiration or, alternatively, by a K⁺ gradient (high [K⁺] inside). In the former case, ATP formation is resistant to the protonophorous uncoupler. Dicyclohexylcarbodiimide and diethylstilbestrol, but not vanadate, completely inhibit Na⁺ pulse-induced ATP formation. The data agree with the assumption that Na⁺-ATP-synthase is involved in oxidative phosphorylation inV. alginolyticus. Interrelation of H⁺ and Na⁺ cycles in bacteria is discussed.Abbreviations and electrochemical gradients of H⁺ and Na⁺, respectively - transmembrane electric potential difference - pH, pNa, and pK concentration gradients of H⁺, Na⁺, and K⁺, respectively - CCCP carbonyl cyanidem-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DES diesthylstilbestrol - HQNO 2-heptyl-4-hydroxyquinolineN-oxide - Tricine N[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

On the appropriateness of use of a continuous formulation for the modelling of discrete multireactant systems following Michaëlis–Menten kinetics

The possibility of solving the mass balances to a multiplicity of substrates within a CSTR in the presence of a chemical reaction following Michaelis-Menten kinetics using the assumption that the discrete distribution of said substrates is well approximated by an equivalent continuous distribution on the molecular weight is explored. The applicability of such reasoning is tested with a convenient numerical example. In addition to providing the limiting behavior of the discrete formulation as the number of homologous substrates increases, the continuous formulation yields in general simpler functional forms for the final distribution of substrates than the discrete counterpart due to the recursive nature of the solution in the latter case.List of Symbols C{N. M} mol/m³ concentration of substrate containing N monomer residues each with molecular weight M - {N, M} normalized value of C{N. M} - C {M} mol/m³ da concentration of substrate of molecular weight M - _in normalized value of C {M} at the i-th iteration of a finite difference method - {M} normalized value of C {M} - C ₀{N.M} mol/m³ inlet concentration of substrate containing N monomer residues each with molecular weight M - {N ·M} normalized value of C₀{N. M} - _{0 i} normalized value of C ₀ {M} at the i-th iteration of a finite difference method - C ₀ {M} mol/m³ da initial concentration of substrate of molecular weight M - C _tot mol/m³ (constant) overall concentration of substrates (discrete model) - C _tot mol/m³ (constant) overall concentration of substrates (continuous model) - D deviation of the continuous approach relative to the discrete approach - i dummy integer variable - I arbitrary integration constant - j dummy integer variable - k dummy integer variable - K _m mol/m³ Michaëlis-Menten constant for the substrates - l dummy integer variable - M da molecular weight of substrate - M normalized value of M - M da maximum molecular weight of a reacting substrate - N number of monomer residues of a reacting substrate - N maximum number of monomer residues of a reacting substrate - N total number of increments for the finite difference method - Q m³/s volumetric flow rate of liquid through the reactor - S inert product molecule - S _i substrate containing i monomer residues - V m³ volume of the reactor - v _max mol/m³ s reaction rate under saturating conditions of the enzyme active site with substrate - v _max{N. M} mol/m³ s reaction rate under saturating conditions of the enzyme active site with substrate containing N monomer residues with molecular weight M - _max{N · M} dimensionless value of v_max{N. M} (discrete model) - _max{M} dimensionless value of v _max {M} (continuous model) - mol/m³ s molecular weight-averaged value of v_max (discrete model) - mol.da/m³s molecular weight-averaged value of v_max (continuous model) - v _max {M} mol.da/m³s reaction rate under saturating conditions of the enzyme active site with substrate with molecular weight M - _max {M} dimensionless value of v_max{M} - _{max, (i)} dimensionless value of v_max{M} at the i-th iteration of a finite difference method - v _max mol/m³ s reference constant value of v _max Greek Symbols dimensionless operating parameter (discrete distribution) - dimensionless operating parameter (continuous distribution) - M da (average) molecular weight of a monomeric subunit - M selected increment for the finite difference method - auxiliary corrective factor (discrete model)     

Thermodynamics of Mn2+-binding to goat α-lactalbumin

By means of reaction calorimetry we measured the apparent enthalpy change, H^app, of the binding of Mn²⁺-ions to goat -lactalbumin as a function of temperature. The observed H^app can be written as the sum of contributions resulting from a conformational and a binding process. In combination with the thermal unfolding curve of goat -lactalbumin, we succeeded in separating the complete set of thermodynamic parameters (H, G, S, C_p) into the binding and conformational contributions. By circular dichroism we showed that NH ₄ ⁺ -ions, upon binding to bovine a-lactalbumin, induce the same conformational change as do Na⁺ and K⁺: the binding constant equals 98 ± 9 M^–1.Abbreviations BLA bovine -lactalbumin - GLA goat -lactalbumin - HLA human -lactalbumin - CD circular dichroism Offprint requests to: H. Van DaelDeceased     

The electrochemical proton potential generated by the sulphur respiration of Wolinella succinogenes

Wolinella succinogenes grown on formate and elemental sulphur was found to use the polysulphide derivatives 2,2-tetrathiobispropionate (R₂S₄) or pentathionate (S₅O ₆ ⁼ ) as acceptors for formate oxidation. The specific activities of formate oxidation with these acceptors were similar to those with elemental sulphur. The main reaction products of R₂S₄ reduction were 2,2-dithiobispropionate (R₂S₂) and sulphide. Pentathionate was converted to thiosulphate and some elemental sulphur. The electrochemical proton potential across the cytoplasmic membrane of the bacterium was measured in the steady state of electron transport from formate to R₂S₄. The electrical proportion () of the determined through the distribution of labeled tetraphenylphosphonium cation was obtained as 0.17 Volt. The was zero, when a protonophore was present. The pH-difference across the membrane was negligible. Thus the generated by sulphur respiration is close to that measured earlier with fumarate as the terminal acceptor of electron transport.Abbreviations DMO 5,5-dimethyloxazolidine-2,4-dione - R₂S_n (n=2–5) 2,2-polythiobispropionate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazol - TPP tetraphenylphosphonium cation     

Bubble column bioreactors

Summary The photographic and electrical conductivity methods to measure the structure of two phase flow, especially bubble size, bubble frequency, local gas hold-up and, for the latter, the bubble velocity are described.Symbols specific interfacial area - a gas/liquid interfacial area - B constant in Eq. (4) - d diameter of the bubbles - d mean diameter of the bubbles - d_S Sauter diameter - E_G relative gas hold up - I current - k_L mass transfer coefficient across the gas/liquid interface - k_L local k_L - LT^–1 - LT^–1 - 1 longitudinal distance between the start and stop sensors - 1_B pierced length of the bubble - t time - t₁ length of the square-wave signal at the start sensor - t₂ length of the square-wave signal at the stop sensor - t₁₂ time delay between start and stop signals - V volume of the bubbling layer - V_L volume of the bubble free layer - V_B bubble volume - v_B bubble velocity     

Molecular aspects of cytochrome c oxidase: Structure and dynamics

Summary In the last few years much attention has been dedicated to the elucidation of some of the molecular aspects of cytochrome c oxidase. It has been shown conclusively that the enzyme from several sources (yeast, Neurospora, heart, liver) contains seven different subunits, which are asymmetrically inserted in the membrane. All of these are in contact with the lipid bilayer (except subunits V and VI) and to a greater or lesser extent with the water phase as well (except for subunit I). Subunit II of the enzyme appears to be involved in the formation of the binding site of cytochrome c. The location of the redox groups of the enzyme is still a matter of controversy. Their distance from the cytochrome c heme group is approximately 35 Å such that electron tunneling appears to be the only possible mechanism for transporting electrons across such a distance.A proton pump appears to be associated with electron transport and approximately one proton is extruded per electron equivalent reducing oxygen via the enzyme. N,N, dicyclohexylcarbodiimide a well-established inhibitor of H⁺-translocating ATPases inhibits the proton pump and labels specifically subunit III of the enzyme.Abbreviations CCCP carbonyl cyanide mchlorophenylhydrazone - DADH₂ diamino-durene (reduced form) - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid - NEM N-ethyl-maleimide - pH transmembrane chemical-potential gradient of H⁺ - transmembrane electricalpotential gradient - transmembrane electrochemical-potential gradient of H⁺ - Q/QH₂ oxidised/reduced form of ubiquinone - TMPD/TMPDH₂ non-protonated/protonated form of tetramethyl-p-phenylenediamine - SDS sodium dodecyl sulphate     

Sodium ions are necessary for growth and energy transduction in the marine cyanobacterium Oscillatoria brevis

The maximal growth rate of the marine cyanobacterium Oscillatoria brevis was reached at 200–400 mM NaCl and pH 9.0–9.6. NaCl was found (i) to stimulate the rate of the light-supported generation across the cytoplasmic membrane of the cells and (ii) to decrease the sensitivity of level and motility of the O. brevis trichomes to protonophorous uncouplers. The Na⁺/H⁺ antiporter, monensin, increased both and the uncoupler sensitivity of the cells. The data obtained agree with the assumption that O. brevis possesses a primary Na⁺ pump in its cytoplasmic membrane.Abbreviations ATP adenosine-5-triphosphate - TTFB tetrachlortrifluoromethylimidazol - CCCP carbonyl cyanide m-chlorophenylhydrazone - Na⁺ transmembrane electrochemical potential differences of Na⁺ - transmembrane electric potential difference - pNa transmembrane pNa difference     

Differential cardiorespiratory response to combined exercise with different combinations of forearm and calf exercise

The purpose of this study was to examine whether cardiorespiratory responses to combined rhythmic exercise (60 contractions · min^–1) was affected by different combinations of upper and lower limb exercise in seven healthy women. Six different rhythmic exercises were compared: 6-min rhythmic handgrip at 10% of isometric maximal voluntary contraction (MVC) (H10); 6-min rhythmic plantar flexion at 10% MVC (P10); exhausting rhythmic handgrip at 50% MVC (H50); exhausting rhythmic plantar flexion at 50% MVC (P50); H50 was added to P10 (P1OH50); and P50 was added to H10 (H10P50). Exercise duration, after handgrip was combined with plantar flexion (P10H50), was shorter than that of H50, although the exercise duration of HIOP50 was not significantly different from P50. No significant difference was found between the difference from rest in oxygen uptake ( O₂) during H10P50 and the sum of O₂ during H10 and P50. Also, the differences from rest in forearm blood flow ( FBF) and calf blood flow ( CBF) during H10P50 were not significantly different from FBF in H10 and from CBF in P50. In contrast, O₂ in P10H50 was lower than the sum of O₂ in P10 and H50 (P < 0.05), and J FBF in P10H50 was lower than that in H50 (P < 0.05) , while CBF was not significantly different between P1OH50 and P10. The changes in heart rate from rest (d HR) during the combined exercises were lower than the sums of HR in the corresponding single exercises (P < 0.05). These results demonstrated an inhibitory summation of several cardiorespiratory responses to combined exercise resulting in a reduction in exercise performance which would seem to occur easily when upperlimb exercise is added to lower limb exercise.     

Genetic analysis of carbon isotope discrimination and agronomic characters in a bread wheat cross

Carbon isotope discrimination () has been suggested as a selection criterion to improve transpiration efficiency (W) in bread wheat (Triticum aestivum L.). Cultivars Chinese Spring with low A (high W) and Yecora Rojo with high (low W) were crossed to develop F₁, F₂, BC₁, and BC₂ populations for genetic analysis of and other agronomic characters under well-watered (wet) and water-stressed (dry) field conditions. Significant variation was observed among the generations for only under the wet environment. Generation x irrigation interactions were not significant for . Generation means analysis indicated that additive gene action is of primary importance in the expression of under nonstress conditions. Dominance gene action was also detected for , and the direction of dominance was toward higher values of . The broad-sense and the narrow-sense heritabilities for were 61 % and 57% under the wet conditions, but were 48% and 12% under the draughted conditions, respectively. The narrow-sense heritabilities for grain yield, above-ground dry matter, and harvest index were 36%, 39%, and 60% under the wet conditions and 21%, 44%, and 20% under dry conditions, respectively. The significant additive genetic variation and moderate estimate of the narrow-sense heritability observed for indicated that selection under wet environments should be effective in changing in spring bread wheat.     

A simple method to quantitatively measure polypeptide JHNHα coupling constants from TOCSY or NOESY spectra

A simple linear relationship between the J coupling constant and the linewidth (_1/2) of in-phase NMR peaks has been identified. This relationship permits the rapid and accurate determination of polypeptide J coupling constants from a simple inspection of amide cross peaks in homonuclear ¹H TOCSY or ¹H NOESY spectra. By using the appropriate set of processing parameters we show that J = 0.5(_1/2) – MW/5000 + 1.8 for TOCSY spectra and J = 0.6(_1/2) – MW/5000 – 0.9 for NOESY spectra, where _1/2 is the half-height linewidth in Hz and MW is the molecular weight of the protein in Da. The simplicity of this relationship, combined with the ease with which _1/2 measurements can be made, means that J coupling constants can now be rapidly determined (up to 100 measurements in less than 30 min) without the need for any complex curve-fitting algorithms. Tests on 11 different polypeptides involving more than 650 separate J measurements have shown that this method yields coupling constants with an rmsd error (relative to X-ray data) of less than 0.9 Hz. Furthermore, the correlation coefficient between the predicted NMR coupling constants and those derived from high-resolution X-ray crystal structures is typically better than 0.89. These simple linear relationships have been found to be valid for peptides as small as 1 kDa to proteins as large as 20 kDa. Despite the method's simplicity, these results are comparable to the accuracy and precision of the best techniques published to date.     

Energy conservation in whole cells of the methylotrophic bacterium Methylophilus methylotrophus

Respiratory chain phosphorylation has been investigated in the methylotrophic bacterium Methylophilus methylotrophus following the addition of oxidisable substrates to aerobic, whole cell suspensions. Initial-rate experiments showed that ATP synthesis occurred at the overall expense of AMP and inorganic phosphate via the sequential action of the ATP phosphohydrolase and adenylate kinase; some of the nascent ATP was rapidly used to synthesis nonadenine nucleoside triphosphates. After being corrected for ATP turnover, Pi/O quotients of 0.46 to 0.54, 0.77 and 1.37 nmol/ng-atom O were obtained for the oxidation of methanol dehydrogenase-linked substrates (methanol, ethanol and acetaldehyde), duroquinol and formate (NAD⁺-linked) respectively. These values were proportional to the H⁺/O and/or K⁺/O quotients exhibited by these substrates, and yielded an average H⁺/ATP (H⁺/Pi) quotient of 4.2 ng-ion H⁺/nmol. Steady-state experiments showed that the extent of cellular energisation varied with the respiration rate but was always in the order methanol > duroquinol > acetaldehyde, thus indicating that under these longer-term conditions methanol was completely oxidised to yield PQQH₂ and 2NAD(P)H. These results are discussed in terms of the various reactions which lead to the generation or utilisation of the protonmotive force in this organism.Abbreviations FCCP carbonylcyanide p-trifluoromethyxyphenyl-hydrazone - bulk phase, transmembrane electrochemical potential difference of protons ( ) - pH bulk phase, transmembrane pH difference (pH_in–pH_out) - bulk phase, transmembrane electrical potential difference (_in - _out) - [P] concentration of anhydride phosphate bonds in adenine nucleotides (2[ATP]+[ADP]) - FPLC fast protein liquid chromatography - PQQ pyrroloquinoline quinone - Gp phosphorylation potential     

Stereospecificity and electrogenicity of amino acid transport in Riccia fluitans

In the aquatic liverwort Riccia fluitans, membrane depolarization (_m), change in membrane conductance (g_m), and current-voltage (I-V) characteristics in the presence of different amino acids as well as the uptake of ¹⁴C-labeled amino acids were measured. L-isomers of the tested amino acids generate larger electrical effects (_m, g_m) than D-isomers, and the I-V characteristics show that the positive electrical inward-current of 20 mA m^-2 generated by 0.5 mM D-serine is only about 50% of the current generated by adding 0.5 mM L-serine. Whereas - and -amino acids rapidly depolarize the membrane to the same extend, with -aminobutyric acid (-AB) and dipeptides no significant electrical effects have been measured. The uptake kinetics of ¹⁴C-labeled amino acids display three components: (I) A saturable high-affinity component with K_s-values of 48 M D-alanine, 12 M -aminoisobutyric acid (AIB), 9 M L-alanine, 8 M L-proline, and 6 M L-serine, respectively; (2) an apparently linear low-affinity component, and (3) an also linear but unspecific component at concentrations >20 times the given K_s-value. Uptake of ¹⁴C-labeled AIB can be inhibited competitively by all tested neutral amino acids, the L-isomers being more effective than the D-isomers, as well as by ammonium or methylamine. Vice versa, AIB competitively inhibits uptake of L-serine and L-alanine. It is concluded that an uncharged stereospecific carrier for the investigated amino acids exists in the plasmalemma of Riccia fluitans. Accumulation ratios of about 50 suggest secondary active transport driven by a transmembrane electro-chemical gradient (mainly _m) which is generated by the electrogenic proton pump. It is suggested that this carrier binds to the amino group forming either a charged binary complex with positively charged amines (Felle 1980), or an uncharged complex with -AB or dipeptides, whereas electrogenic transport of - and -amino acids is mediated by a ternary carrier complex, probably charged by a proton.Symbols and Abbreviations _m membrane potential (mV) - E_co equilibrium potential (mV) of the transport system - g_m membrane (slope) conductance (Sm^-2) - g_m change in g_m - I-V curve current-voltage curve - AIB -aminoisobutytric acid - -AB -aminobutyric acid     

Amide proton temperature coefficients as hydrogen bond indicators in proteins

Correlations between amide proton temperature coefficients (_HN/T) and hydrogen bonds were investigated for a data set of 793 amides derived from 14 proteins. For amide protons showing temperature gradients more positive than –4.6 ppb/K there is a hydrogen bond predictivity value exceeding 85%. It increases to over 93% for amides within the range between –4 and –1 ppb/K. Detailed analysis shows an inverse proportionality between amide proton temperature coefficients and hydrogen bond lengths. Furthermore, for hydrogen bonds of similar bond lengths, values of temperature gradients in -helices are on average 1 ppb/K more negative than in -sheets. In consequence, a number of amide protons in -helices involved in hydrogen bonds shorter than 2 Å show _HN/T < –4.6 ppb/K. Due to longer hydrogen bonds, 90% of amides in 3₁₀ helices and 98% in -turns have temperature coefficients more positive than –4.6ppb/K. Ring current effect also significantly influences temperature coefficients of amide protons. In seven out of eight cases non-hydrogen bonded amides strongly deshielded by neighboring aromatic rings show temperature coefficients more positive than –2 ppb/K. In general, amide proton temperature gradients do not change with pH unless they correspond to conformational changes. Three examples of pH dependent equilibrium showing hydrogen bond formation at higher pH were found. In conclusion, amide proton temperature coefficients offer an attractive and simple way to confirm existence of hydrogen bonds in NMR determined structures.     

Metabolic cost of sodium transport in toad urinary bladder

Summary The metabolic cost of active sodium transport was determined in toad bladder at different gradients of transepithelial potential, , by continuous and simultaneous measurements of CO₂ production and of transepithelial electric current. Amiloride was used to block active sodium transport in order to assess the nontransport-linked, basal, production of CO₂ and the passive permeability of the tissue. From these determinations active sodium transport,J _Na, and suprabasal CO₂ production, , were calculated. Since large transients inJ _Na and frequently accompanied any abrupt change in , steady state conditions were carefully defined.Some 20 to 40 min were required after a change in before steady state of transport activity and of CO₂ production were achieved. The metabolic cost of sodium transport proved to be the same whether the bladder expended energy moving sodium against a transepithelial electrical potential grandient of +50 mV or whether sodium was being pulled through the active transport pathway by an electrical gradient of –50 mV. In both cases the value of the ratio averaged some 20 sodium ions transported per molecule of CO₂ produced.When the Na pump was blocked by 10^–2 m ouabain, the perturbations of the transepithelial electrical potential did not elicit changes ofJ _Na nor, consequently, of .The independence of the ratio from over the range ±50 mV indicates a high degree of coupling between active sodium transport and metabolism.