A comparative study of the mechanism of Na+ and Cl? excretion by the gill ofMugil capito andFundulus heteroclitus: Effects of Stress

Summary In seawater (SW)-adaptedMugil andFundulus, gill effluxes of Na⁺ and of Cl^– and the simultaneously recorded transgill potential (P.D.) differ according to whether they are measured in stressed or rested animals.In rested animals of the two species, transfer to Ringer's solution considerably reduces the P.D. but not . InFundulus, is also decreased. Transfer of the two species from SW to fresh water (FW) reduces and by 75 to 85% and leads to a large inversion of P.D. When K⁺ is added to FW, a gill depolarization occurs, as well as a large increase of and .These results suggest that: 1) the P.D. originates primarily from the diffusion of cations, the gill permeability to Na⁺ ( ) being greater than that to Cl^– ( ), 2) a Cl^–/Cl^– exchange independent of P.D. is associated with the Cl^– pump; 3) Cl^– pump activity is linked to Na⁺/K⁺ exchange which in turn is associated to a Na⁺/Na⁺ exchange diffusion mechanism.In stressed individuals of the two species, the P.D. in SW, as well as the P.D. changes observed during transfer experiments, are considerably reduced. The decrease of and observed after transfer from SW to FW are also minimised. Changes are smaller inFundulus. The decrease of P.D. characterizing stressed animals may be at least in part due to a 3 to 4 fold increase of which becomes equal to in both species.As a result of stress, the K⁺-activated Na⁺ and Cl^– excretion mechanisms are totally inhibited inFundulus and partially so inMugil.Stress response seems more intense inFundulus and recovery from stress faster inMugil.     

Quantitative analysis of outward rectifying K+ channel currents in guard cell protoplasts fromVicia faba

Summary A quantitative analysis of the time and voltage dependence of outward-rectifying K⁺ currents ( ) in guard cells fromVicia faba is described using the whole-cell patch-clamp technique. After step depolarizations from –75 mV to potentials positive to –40 mV, time-dependent outward currents were produced, which have recently been identified as K⁺ channel currents. This K⁺ current was characterized according to its time dependence and its steady-state activation. could be described in terms of a Hodgkin-Huxley type conductance. Activation of the current in time was sigmoid and was well fitted by raising the activation variable to the second power. Deactivating tail currents were single exponentials, which suggests that only one conductance underlies this slow outward K⁺ current. Rates of channel closing were strongly dependent on the membrane potential, while rates of channel opening showed only limited voltage dependence leading to a highly asymmetric voltage dependence for channel closing and opening. The presented analysis provides a quantitative basis for the understanding of channel gating and channel functions in plant cells.     

Carbonic anhydrase activity in the blood and the gills of rainbow trout during long-term hypercapnia in hard,bicarbonate-rich freshwater

Summary Carbonic anhydrase (CA) activities in gills and venous blood, acid-base balance, and haematological variables were studied during environmental hypercapnia in rainbow trout (Salmo gairdneri). Batches of 8–10 fish were exposed to about 3 or 13 mmHg in flow-through tests of various duration from 4 h to 80 days.After initial acidosis, blood pH rose above pre-experimental values. At 3 mmHg it became normal again within 21 days, while at 13 mmHg the overshoot lasted for 80 days. In fish acclimated for 3 weeks or more to 13 mmHg , blood HCO ₃ ^– increased four to five times while plasma Cl^– levels were lower and K⁺ higher. Na⁺ levels did not show any consistent trend associated with exposure to hypercapnia. After an initial acidaemia, Hct, Hb, and RBC remained relatively constant.Patterns of change in CA activity differed between gills and erythrocytes. Initially, blood CA decreased at both levels. It then began rising after about 3 weeks and tended to reach pre-experimental values by 80 day's hypercapnia. At 13 mmHg , gill CA increased to twice the pre-experimental level. Compared with blood CA, gill CA appeared to be more specifically involved in fish acclimation to hypercapnia, which demands an increase in blood bicarbonate to provide a sufficient buffering capacity. Increased CA indicates that the gill enzyme may play a more important role than blood CA in acid-base regulation in fish during hypercapnia.Abbreviations CA carbonic anhydrase - Hb haemoglobin - Hct haematocrit value - RBC red blood cells     

The Nitric Oxide Synthase Inhibitor NG-nitro-L-Arginine Methyl Ester Potentiates Insulin Secretion Stimulated by Glucose and L-Arginine Independently of its Action on ATP-Sensitive K+ Channels

The nature of the action of the nitric oxide synthase (NOS) inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) on hormone release from isolated islets was investigated. We found that glucose-induced insulin release was potentiated by L-NAME in the absence or presence of diazoxide, a potent channel opener, as well as in the presence of diazoxide plus a depolarizing concentration of K⁺. At a low, physiological glucose concentration L-NAME did not influence insulin secretion induced by K⁺ but inhibited glucagon secretion. L-arginine-induced insulin release was potentiated by L-NAME. This potentiation was observed also in the presence of K⁺ plus diazoxide. Further, glucagon release induced by L-arginine as well as by L-arginine plus K⁺ and diazoxide was suppressed by L-NAME. The results strongly suggest that the L-NAME-induced potentiation of insulin secretion in response to glucose or L-arginine as well as the inhibitory effects on glucagon secretion are largely mediated by L-NAME directly suppressing islet NOS activity. Hence NO apparently affects insulin and glucagon secretion independently of membrane depolarization events.     

The regulation of K+ influx in excised barley roots

The electrochemical potential differences for potassium, between excised barley (Hordeum vulgare L.) roots and external media containing 0.05 mM KCl+0.5 mM CaSO₄, were determined over a 4-h period during which initially low-K⁺ roots accumulated K⁺ by pretreatment in 50 mM KCl plus 0.5 mM CaCl₂. This pretreatment resulted in increased internal [K⁺], decreased K⁺ influx (as measured from 0.05 mM KCl+0.5 mM CaSO₄) and decreased values of . These observations indicate that the decline of K⁺ influx associated with increased internal K⁺ concentration cannot be accounted for by passive adjustment to the electrochemical gradient for this ion.     

Single voltage-dependent and outward rectifying K+-channels in isolated rat heart cells

Studies on single K⁺-channel currents recorded from isolated rat heart muscle cells, in which early repolarization is known to be exceptionally fast, are reported here. A K⁺-channel which is blocked by TEA (tetraethylammonium) from the inside only has been found.The total open time of the channel, measured in steady-state after activation, indicated outward rectifying properties. The single channel conductance increases with depolarization from 25 pS at-70 mV to 75 pS at+70 mV.Selectivity of the channel has also been measured and it was found that only Rb⁺ and K⁺ can permeate the channel, whereas the permeability (P) for Li⁺, Na⁺, Cl^-, Mg²⁺, and Ca²⁺ is less than 0.05 times .Ba²⁺ and Cs⁺ block the channel activity.These results clearly demonstrate the existence of K⁺-selective outward rectifying conductance pathways in rat ventricular myocytes.     

Effects of rapid transfer from sea water to fresh water on respiratory variables,blood acid-base status and O2 affinity of haemoglobin in Atlantic salmon (Salmo salar L.)

Summary Oxygen consumption, gill ventilation, blood acid-base/ionic status and haemoglobin oxygen affinity were studied in seawater-adapted adult salmon (Salmo salar) during five weeks after transfer into fresh water. Freshwater exposure induced the following changes: Standard oxygen consumption ( ) and ventilatory flow ( ) decreased markedly during the first days after transfer, then decreased more gradually until a new steady-state was achieved at which and were about 80% and 56% of the control values, respectively. The marked increase in oxygen extraction coefficient (Ew _{O 2}) and the marked decrease in the oxygen convection requirement ( ) were associated with a reduction in the partial pressure of carbon dioxide in arterial blood (Pa _{CO 2}), in spite of a decrease of both ventilatory flow and water CO₂ capacitance. These results suggested that transfer into fresh water induced an increase in branchial diffusive conductance. A biphasic pattern was observed in the time-course of the changes in both plasma ion concentration and acid-base status. During the first 10 days, plasma Na⁺, K⁺, and Cl^– concentrations fell abruptly, then more gradually. [Cl^–] decreased more than [Na⁺] resulting in a progressive increase in the [Na⁺]/[Cl^–] ratio. During the second phase of acclimation to fresh water plasma Na⁺, K⁺, and Cl^– concentrations progressively increased. [Cl^–] increased more than [Na⁺], so that [Na⁺]/[Cl^–] ratio decreased. Transfer into fresh water did not significantly change plasma lactate concentration. Upon exposure to fresh water, blood pH increased from 7.94±0.04 to 8.43±0.06 at day 10 and then decreased to 8.08±0.03 at day 34. The increase in blood pH induced by transfer to fresh water initially represented a mixed metabolic/respiratory alkalosis. However, after 15 days Pa _{CO 2} had returned to pretransfer values and the alkalosis was purely metabolic. The metabolic component of the alkalosis was associated with appropriate changes in the plasma strong ion difference (S.I.D.). Blood alkalosis moved the oxygen dissociation curve to the left, so that P₅₀ was decreased by 30% below the value in seawater for the maximal increase in blood pH. This rise in haemoglobin affinity for O₂, associated with a marked increase in blood buffer capacity, are regarded as adaptative processes allowing the salmon to cope with the markedly increased energy expenditure required for upstream migration.     

Transport of H+, K+, Na+ and Ca++ in Streptococcus

Summary The streptococci differ from other bacteria in that cation translocations (with the possible exception of one of the K⁺ uptake systems) occur by primary transport systems, i.e., by cation pumps which use directly the free energy released during hydrolysis of chemical bonds to power transport. Transport systems in other bacteria, especially for Na⁺ and Ca⁺⁺, are often secondary, using the free energy of another ion gradient to drive cation transport. In streptococci H⁺ efflux occurs via the F₁F₀-ATPase. This enzyme is composed of eight distinct subunits. Three of the subunits are embedded in the membrane and form a H⁺ channel; this is called the F₀ portion of the enzyme. The other five subunits form the catalytic part of the enzyme, called F₁, which faces the cytoplasm and can easily be stripped from the membrane. Physiologically, this enzyme functions as a H⁺-ATPase, pumping protons out of the cell to form an electrochemical proton gradient, . The F₁F₀-ATPase, however, is fully reversible and if supplied with P_i, ADP and a + of sufficient magnitude (ca –200 mv) catalyzes the synthesis of ATP. Streptococcus faecalis can accumulate K⁺ and establish a gradient of 50 000:1 (in>out) under some conditions. Uptake occurs by two transport systems. The dominant, constitutive system requires both an electrochemical proton gradient and ATP to operate. The minor, inducible K⁺ transport system, which has many similarities to the K⁺-ATPase of the Kdp transport system found in Escherichia coli, requires only ATP to power K⁺ uptake.Sodium extrusion occurs by a Na⁺/H⁺-ATPase. Exchange is electroneutral and there is no requirement for a . The possibility that the Na⁺/H⁺-ATPase may consist of two parts, a catalytic subunit and a Na⁺/H⁺ antiport subunit, is suggested by the finding that damage to the Na⁺ transport system either through mutation or protease action leads to the appearance of -requiring Na⁺/H⁺ antiporter activity.Ca⁺⁺ like Na⁺ is extruded from metabolizing, intact cells. Transport requires no but does require ATP. Reconstitution of Ca⁺⁺ transport activity with accompanying Ca⁺⁺-stimulated ATPase activity into proteoliposomes suggests that Ca⁺⁺ is transported by a Ca⁺⁺-translocating ATPase.Where respiring organelles and bacteria use secondary transport systems the streptococci have developed cation pumps. The streptococci, which are predominantly glycolyzing bacteria, generate a much inferior to that of respiring organisms and organelles. The cation pumps may have developed simply in response to an inadequate .Abbreviations electrochemical potential of protons - membrane potential - pH pH gradient - p proton-motive force - DCCD N,Na¹-dicyclohexlcarbodiimide - TCS tetrachlorosalicylanilide - FCCP carbonylcyanide-p-trifluoromethylphenylhydrazone - CCCP carbonylcyanie-m-chlorophenylhydrazone - TPMP⁺ triphenylmethyl phosphonium ion - DDA⁺ dibenzyldimethylammonium ion - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - EGTA ethyleneglycol-bis (amino-ethyl-ether)-N,N-tetraacetic acid     

Evidence for coupling between Na+ pump activity and TEA-sensitive K+ currents in Xenopus laevis oocytes

Using the two-microelectrode voltage clamp technique in Xenopus laevis oocytes, we estimated Na⁺-K⁺-ATPase activity from the dihydroouabain-sensitive current (I _DHO) in the presence of increasing concentrations of tetraethylammonium (TEA⁺; 0, 5, 10, 20, 40 mm), a well-known blocker of K⁺ channels. The effects of TEA⁺ on the total oocyte currents could be separated into two distinct parts: generation of a nonsaturating inward current increasing with negative membrane potentials (V _M) and a saturable inhibitory component affecting an outward current easily detectable at positive V _M. The nonsaturating component appears to be a barium-sensitive electrodiffusion of TEA⁺ which can be described by the Goldman-Hodgkin-Katz equation, while the saturating component is consistent with the expected blocking effect of TEA⁺ on K⁺ channels. Interestingly, this latter component disappears when the Na⁺-K⁺-ATPase is inhibited by 10 m DHO. Conversely, TEA⁺ inhibits a component of I _DHO with a k _d of 25±4 mm at +50 mV. As the TEA⁺-sensitive current present in I _DHO reversed at –75 mV, we hypothesized that it could come from an inhibition of K⁺ channels whose activity varies in parallel with the Na⁺-K⁺-ATPase activity. Supporting this hypothesis, the inward portion of this TEA⁺-sensitive current can be completely abolished by the addition of 1 mm Ba²⁺ to the bath. This study suggests that, in X. laevis oocytes, a close link exists between the Na-K-ATPase activity and TEA⁺-sensitive K⁺ currents and indicates that, in the absence of effective K⁺ channel inhibitors, I _DHO does not exclusively represent the Na⁺-K⁺-ATPase-generated current.     

The sodium cycle: A novel type of bacterial energetics

The progress of bioenergetic studies on the role of Na⁺ in bacteria is reviewed. Experiments performed over the past decade on several bacterial species of quite different taxonomic positions show that Na⁺ can, under certain conditions, substitute for H⁺ as the coupling ion. Various primary Na⁺ pumps ( generators) are described, i.e., Na⁺-motive decarboxylases, NADH-quinone reductase, terminal oxidase, and ATPase. The formed is shown to be consumed by Na⁺ driven ATP-synthase, Na⁺ flagellar motor, numerous Na⁺, solute symporters, and the methanogenesis-linked reverse electron transfer system. InVibrio alginolyticus, it was found that , generated by NADH-quinone reductase, can be utilized to support all three types of membrane-linked work, i.e., chemical (ATP synthesis), osmotic (Na⁺, solute symports), and mechanical (rotation of the flagellum). InPropionigenum modestum, circulation of Na⁺ proved to be the only mechanism of energy coupling. In other species studied, the Na⁺ cycle seems to coexist with the H⁺ cycle. For instance, inV. alginolyticus the initial and terminal steps of the respiratory chain are Na⁺ - and H⁺-motive, respectively, whereas ATP hydrolysis is competent in the uphill transfer of Na⁺ as well as of H⁺. In the alkalo- and halotolerantBacillus FTU, there are H⁺ - and Na⁺-motive terminal oxidases. Sometimes, the Na⁺-translocating enzyme strongly differs from its H⁺-translocating homolog. So, the Na⁺-motive and H⁺-motive NADH-quinone reductases are composed of different subunits and prosthetic groups. The H⁺-motive and Na⁺-motive terminal oxidases differ in that the former is ofaa ₃-type and sensitive to micromolar cyanide whereas the latter is of another type and sensitive to millimolar cyanide. At the same time, both Na⁺ and H⁺ can be translocated by one and the sameP. modestum ATPase which is of the F₀F₁-type and sensitive to DCCD. The sodium cycle, i.e., a system composed of primary generator(s) and consumer(s), is already described in many species of marine aerobic and anaerobic eubacteria and archaebacteria belonging to the following genera:Vibrio, Bacillus, Alcaligenes, Alteromonas, Salmonella, Klebsiella, Propionigenum, Clostridium, Veilonella, Acidaminococcus, Streptococcus, Peptococcus, Exiguobacterium, Fusobacterium, Methanobacterium, Methanococcus, Methanosarcin, etc. Thus, the sodium world seems to occupy a rather extensive area in the biosphere.     

rDNA magnification in D. melanogaster: state of rDNA copies following the first step.

Summary D. melanogaster males of bb/O genetic constitution undergoing rDNA magnification were mated singly to XXbb ⁺/O females, yielding bb/O male progeny, and to X_NO-w sn bb ⁺ fameles, yielding bb/X_NO- females. The male and female offspring were scored for the bb ⁺ phenotype.Results show that there is a higher percentage of bb ⁺ flies in the bb/O male progeny than in bb/X_NO- females progeny, in single crosses as well as in the combined data. rRNA/DNA hybridization experiments agree with this observation, by showing that the rDNA content in the progeny of premagnified flies was higher in the sons than in the daughters.These data indicate that the increase of ribosomal RNA genes is not due to a stable event such as an unequal mitotic sister exchange, whereas they do not contrast with the extracopy model.     

Intestinal HCO 3 − secretion inAmphiuma: Stimulation by mucosal Cl− and serosal Na+

Summary The requirement for Na⁺ and Cl^– in the bathing media to obtain a maximal HCO ₃ ^– secretory flux ( ) across isolated short-circuitedAmphiuma duodenum was investigated using titration techniques and ion substitution. Upon substitution of media Na⁺ with choline, HCO ₃ ^– secretion was markedly reduced. Replacement of media Cl^– produced a smaller reduction of . The presence of Cl^– enhanced HCO ₃ ^– secretion only if Na⁺ was also in the media. Elevation of media Na⁺ or Cl^– in the presence of the other ion produced a saturable increase of . In the presence of Na⁺, Cl^– stimulated when added to the mucosal but not the serosal medium. In the presence of Cl^–, Na⁺ elevated when added to the serosal but not the mucosal medium. The ability of mucosal Cl^– to stimulate was not apparently dependent on mucosal Na⁺. Simultaneous addition of 10mm Cl^– to the Na⁺-free mucosal medium and 10mm Na⁺ to the Cl^–-free serosal medium stimulated above levels produced by serosal Na⁺ alone. In conclusion, intestinal HCO ₃ ^– secretion required mucosal Cl^– and serosal Na⁺ and did not involve mucosal NaCl cotransport. The results are consistent with a mucosal Cl^– absorptive mechanism in series with parallel basolateral Na⁺–H⁺ and Cl^––HCO ₃ ^– exchange mechanisms.     

Monovalent cation permeabilities of the potassium systems in the crab giant axon

Summary Permeability ratios for pairs of monovalent cations permeating the two potassium systems proposed for the giant axon of the crabCarcinus maenas (M. E. Quinta-Ferreira, E. Rojas & N. Arispe,J. Membrane Biol. 66:171–181, 1982b) were estimated from measurements of the reversal potential of the currents under voltage-clamp conditions. With K⁺ inside the axon, permeability ratios from the reversal potential of the currents through the late channel are:P _Rb/P _K=0.9, /P _K<0.2 andP _Cs/P _K=0.18. With Cs⁺ inside the ratios are:P _K/P _Cs=8.7,P _Rb/P _Cs=7.1 and /P _Cs=2.4. The analysis of the inward currents carried by Rb⁺ or NH ₄ ⁺ showed similar reversal potentials for the early transient component and the late sustained component. Whence, the sequence of permeabilities for the two types of potassium channels is:P _K>P _Rb> >P _Na=P _Cs. The time constants for the activation of the two components recorded either in K-, Rb-, or NH₄-artificial seawater are twice as large as the corresponding time constants measured in Na-artificial seawater.     

Growth yields and saturation constant of Desulfovibrio vulgaris in chemostat culture

Desulfovibrio vulgaris (strain Marburg) was grown on H₂ and sulfate as sole energy source in a chemostat limited by the sulfate supply. The biomass concentration and the sulfate concentration in the culture were determined as a function of the dilution rate. From the data a K _S (saturation constant) for sulfate of 10 M, a _max of 0.23 h^–1, and a of 13 g/mol were calculated. The organism was also grown in chemostat culture on H₂ and sulfite, H₂ and thiosulfate, and pyruvate (without sulfate). was found to be 35 g/mol, 36 g/mol, and Y _pyr ^max 10 g/mol. The growth yields are discussed with respect to ATP gains in dissimilatory sulfate reduction.     

Larmor frequency selective model free analysis of protein NMR relaxation

Summary The Lipari-Szabo dynamical formalism is extended by setting the time constants of the Lorentzian terms to and . This analysis is compared to the earlier proposed three-parameter extended model free formalism with regard to the range of equivalence and the advantages of the simplified two-parameter (S _inff ^sup2 ,S _infH ^sup2 ) and (S _inff ^sup2 ,S _infN ^sup2 ) representations. Spectral density components are calculated and compared to those obtained from the spectral density analysis formalism. Protein relaxation data, commonly analyzed in terms of the two-parameter representation, may correspond to a dynamically heterogeneous behaviour that is more appropriately represented in terms of a fast limit order parameter and a second, lower frequency order parameter.     

Prediction of individual oxygen uptake on-step transients from frequency responses

Power-oxygen uptake ( ) frequency responses can be used to predict responses to arbitrary exercise intensity patterns. It is still an open question for which range of exercise intensities such computed response patterns yield valid predictions. In the present study, we determined the power- frequency response of nine sports students by means of pseudo-randomised switching between 20 W and 80 W during upright and supine cycle exercise. Starting from a baseline of 20 W each subject also performed sustained step increases to 40 W, 80 W, 120 W, and 160 W in both positions. The individual step responses were then compared with the expected time-courses predicted on the basis of the individual frequency responses. The comparison showed a close agreement for the 20 W–40 W and 20 W–80 W steps in both positions. With larger step amplitudes the kinetics became increasingly slower than the predicted time course in both positions. During additional ramp tests (10 W · 30 s^–1) whole blood lactic acid concentration [1a^–]_b tended to be higher in the supine position at exercise intensities higher than 160 W. The mean power at 4 mmol · 1^–1 [la^–]_b amounted to 234 (SD 32) W and 253 (SD 44) W (P<5%) in the supine and the upright position, respectively. The maximal oxygen uptake relative to body mass was not found to be significantly different [upright, mean 57 (SD 10) ml · (min · kg)^–1;supine, mean 54 (SD 10) ml · (min · kg)]. These findings would suggest that for a range of mild exercise intensities kinetics are not appreciably influenced by the step amplitude or by cardiovascular changes associated with the upright and the supine position.     

A stable Na+/H+ antiporter of thermophilic bacterium PS3

As a first step in the isolation of a stable Na⁺/H⁺ antiporter, its reaction in sonicated membrane vesicles of thermophilic bacterium PS3 has been characterized. The sonicated vesicles showed quenching of quinacrine fluorescence in either NADH oxidation or ATP hydrolysis. The quenching was reversed by the addition of Na⁺, Li⁺, Mn²⁺, Cd²⁺, and Co²⁺, but not of choline⁺ or Ca²⁺, regardless of their counter anions.²²Na⁺ was taken up into the vesicles by NADH oxidation, and the²²Na⁺ uptake was inhibited by the addition of an uncoupler. H⁺ release was observed on addition of Na⁺ to sonicated vesicles. The magnitude of the pH difference across the membrane induced by NADH oxidation was constant at pH 7.0 to 9.1, but the Na⁺/H⁺ antiport was affected by the pH of the medium (optimum pH=8.5). TheK _m 's of the antiporter for Na⁺ and Li⁺ were 2.5 and 0.1 mM, respectively, but theV _max values for the two ions were the same at pH 8.0. In the presence of Li⁺, no further decrease of fluorescence quenching was observed on addition of Na⁺ andvice versa. The Na⁺/H⁺ antiporter activity in PS3 was stable at 70°C, and the optimum temperature for activity was 55–60°C. In contrast to mesophilic cation/H⁺ antiporters, this antiporter was not inhibited by a thiol reagent.Abbreviations Tricine N-tris(hydroxymethyl)methylglycine - MOPS morpholinopropane sulfonic acid - TMAHO tetramethylammonium hydroxide - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - H⁺ — ATPase proton-translocating adenosine triphosphatase - electrochemical proton gradient across membrane - electrochemical Na⁺ gradient across membrane - pH pH difference across membrane     

Kinetic Modeling of Energy Metabolism and Superoxide Generation in Hepatocyte Mitochondria

Direct nonenzymatic oxidation of semiquinone by oxygen is one of the main sources of superoxide radicals in mitochondria. Using all the known data on hepatocyte mitochondria, we have revealed the correlation between the rate of superoxide generation by the bc ₁complex and the transmembrane potential (). Assuming that the main electrogenic stage of the Qcycle is the electron transfer between the cytochrome bhemes, then the rate of superoxide generation sharply increases when grows from 150 to 180 mV. However, this interrelation is ambiguous. Indeed, the increase of the generation rate with the growth of the potential can occur faster when succinate dehydrogenase is inhibited by malonate than when external ADP is exhausted. When the potential is changed by adding phosphate or potassium (K⁺), the rate of production remains constant, although the comparison of the rates at the same reveals the effect of phosphate or potassium. It turned out that the rate of generation is a function of rather than any of its components. Phosphate and K⁺have practically no influence on , since the change in is compensated by pH. The rate of superoxide generation by the bc ₁complex is a multiple function of the electron-transfer activity of enzymes, the processes determining the membrane potential (e.g., loading), and the oxygen concentration. The kinetic model proposed in this work may serve to understand how the superoxide production is regulated.     

Determination of heteronuclear three-bond J-coupling constants in peptides by a simple heteronuclear relayed E.COSY experiment

Summary A simple heteronuclear relayed E.COSY pulse sequence with a minimum number of pulses is proposed for the quantitative determination of heteronuclear three-bond J-coupling constants in uniformly ¹³C-enriched polypeptide samples. Numerous heteronuclear three-bond coupling constants, including , , , and , can be determined for each residue from a single heteronuclear relayed E.COSY spectrum. Couplings relevant for stereospecific assignments as well as for the determination of dihedral angles in the amino acid backbone and in side chains are obtained. The method is demonstrated on the uniformly ¹³C-enriched decapeptide antamanide (-Val¹-Pro²-Pro³-Ala⁴-Phe⁵-Phe⁶-Pro⁷-Pro⁸-Phe⁹-Phe¹⁰-).     

Times to exhaustion at 100% of velocity at [(V)\dot]\textO\text2 \dot V{\text{O}}_{\text{2}} max and modelling of the time-limit / velocity relationship in elite long-distance runners

The aim of this study was to measure running times to exhaustion (Tlim) on a treadmill at 100% of the minimum velocity which elicits _{max max} in 38 elite male long - distance runners _max = 71.4 ± 5.5 ml.kg^–1.min^–1 and _max = 21.8 ± 1.2 km.h^–1). The lactate threshold (LT) was defined as a starting point of accelerated lactate accumulation around 4 mM and was expressed in _max. Tlim value was negatively correlated with _max (r = -0.362, p< 0.05) and _max (r = –0.347, p< 0.05) but positively with LT (%v _max) (r = 0.378, p < 0.05). These data demonstrate that running time to exhaustion at _max in a homogeneous group of elite male long-distance runners was inversely related to _max and experimentally illustrates the model of Monod and Scherrer regarding the time limit-velocity relationship adapted from local exercise for running by Hughson et al. (1984) .