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1.
The general properties of the excitable membrane on molluscan pacemaker neurons can be described on the basis of a fair amount of experimental evidence available in the literature. The neuronal membrane exhibits under voltage clamp an initial inward current carried by both Na+ and Ca2+ ions, the time- and voltage-dependent characteristics of which are similar to that of other excitable structures. The conductance mechanism for the two ion species and the transport kinetics appear to be closely similar. The time course and amplitude of the delayed outward current carried by K+ ions shows a marked dependence on the membrane potential. Characteristic for the molluscan neurons is the existence of an additional fast transient outward current which is only activated by hyperpolarizing shifts from the membrane potential. A regular beating discharge over a wide range of frequencies can be predicted by making the assumption of a metabolically controlled driving of the Na+ conductance. Bursting pacemaker characteristics can be correctly simulated by the model if sinusoidal variations of an additional Na+ and Ca2+ conductances g Na and g Ca, and periodic variations of the K+ conductance g K, governed by the known operation of a metabolic substrate cycle are introduced. The close approximation of experimentally observed impulse bursts requires that the actual inpulse-frequency and the amplitude of the after-spike hyperpolarization are determined by the temporal pattern of g Na, while the spike amplitude is controlled by g Na which (although of similar time course) is lagging in phase behing g Na. The periodic changes in additional K+ conductance g K, are responsible for burst termination and the changes in inter-burst interval, to the effect that spike doublets, triplets and multi-spike bursts can be simulated by a suitable choice for the time characteristics of g K. The model makes use of the finding that the Ca2+ inflow associated with a spike discharge actually activates g K, so that large postburst hyperpolarizations can be obtained in high-frequency bursts.Supported by the Deutsche Forschungsgemeinschaft (Grant Ch 25/1)  相似文献   

2.
Currents generated by the Na+/K+ ATPase were measured under voltage clamp in oocytes of Xenopus laevis. The dependence of pump current on external [Na+] was investigated for the endogenous Xenopus pump as well as for wild-type and mutated pumps of electroplax of Torpedo californica expressed in the oocytes. The mutants had -subunits truncated before position Lys28 (K28) or Thr29 (T29) of the N-terminus. The currents generated by all variants of pump molecules in the presence of 5 mM K+ show voltage-dependent inhibition by external [Na+]. The apparent K1 values increase with membrane depolarisation, and the potential dependence can be described by the movement of effective charges in the electrical potential gradient across the membrane. Taking into account Na+-K+ competition for external binding to the E2P form, apparent K1 values and effective charges for the interaction of the Na+ ions with the E2P form can be estimated. For the Xenopus pump the effective charge amounts to 1.1 of an elementary charge and the K1 value at 0 mV to 44 mM. For the wild-type Torpedo pump, the analysis yields values of 0.73 of an elementary charge and 133 mM, respectively. Truncation at the N-terminus removing a lysinerich cluster of the a-subunit of the Torpedo pump leads to an increase of the effective charge and decrease of the K1 value. For K28, values of 0.83 of an elementary charge and 117 mM are obtained, respectively. If LyS28 is included in the truncation (·T29), the effective charge increases to 1.5 of an elementary charge and the apparent K1 value is reduced to 107 mM. The K, values for pump inhibition by external Na+, calculated by taking into account Na+-K+ competition, are smaller than the K/12 values determined in the presence of 5 mM [K+]. The difference is more pronounced for those pump variants that have higher Km, values. The variations of the parameters describing inhibition by external [Na+] are qualitatively similar to those described for the stimulation of the pumps by external [K+] in the absence of extracellular [Na+]. The observations may be explained by an acess channel within the membrane dielectric that has to be passed by the external Na+ and K+ ions to reach or leave their binding sites. The potential-dependent access and/or the interaction with the binding sites shows species differences and is affected by cytoplasmic lysine residues in the N-terminus.  相似文献   

3.
The initial oxidized species in the photochemical charge separation in reaction centers from Rps. viridis is the primary donor, P+, a bacteriochlorophyll dimer. Bound c-type cytochromes, two high potential (Cyt c 558) and two low potential (Cyt c 553), act as secondary electron donors to P+. Flash induced absorption changes were measured at moderate redox potential, when the high potential cytochromes were chemically reduced. A fast absorption change was due to the initial oxidation of one of the Cyt c 558 by P+ with a rate of 3.7×106s-1 (=270nsec). A slower absorption change was attributable to a transfer, or sharing, of the remaining electron from one high potential heme to the other, with a rate of 2.8×105s-1 (=3.5 sec). The slow change was measured at a number of wavelengths throughout the visible and near infrared and revealed that the two high potential cytochromes have slightly different differential absorption spectra, with -band maxima at 559 nm (Cyt c 559) and 556.5 nm (Cyt c 556), and dissimilar electrochromic effects on nearby pigments. The sequence of electron transfers, following a flash, is: Cyt c 556Cyt c 559P+. At lower redox potentials, a low midpoint potential cytochrome, Cyt c 553, is preferentially oxidized by P+ with a rate of 7×106s-1 (=140 nsec). The assignment of the low and high potential cytochromes to the four, linearly arranged hemes of the reaction center is discussed. It is concluded that the closest heme to P must be the high potential Cyt c 559, and it is suggested that a likely arrangement for the four hemes is: c 553 c 556 c 553 c 559P.Abbreviations diaminodurene 2,3,5,6-tetramethyl-p-phenylenediamine - MOPS 3-[N-morpholino]-propane-sulfonic acid - PMS methyl phenazinium methosulfate - PES ethyl phenazinium ethosulfate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - TX-100 Triton X-100  相似文献   

4.
1. Two mutants of the sodium channel II have been expressed inXenopus oocytes and have been investigated using the patch-clamp technique. In mutant E387Q the glutamic acid at position 387 has been replaced by glutamine, and in mutant D384N the aspartic acid at position 384 has been replaced by asparagine.2. Mutant E387Q, previously shown to be resistant to block by tetrodotoxin (Noda et al. 1989), has a single-channel conductance of 4 pS, that can be easily measured only using noise analysis. At variance with the wild-type, the openchannel current-voltage relationship of mutant E387Q is linear over a wide voltage range even under asymmetrical ionic conditions.3. Mutant D384N has a very low permeability for any of the following ions: Cl, Na+, K+, Li+, Rb+, Ca2+, Mg2+, NH4 + , TMA+, TEA+. However, asymmetric charge movements similar to the gating currents of the Na+-selective wild-type are still observed.4. These results suggest that residues E387 and D384 interact directly with the pathway of the ions permeating the open channel.Abbreviations TTX tetrodotoxin; Na+, sodium; K+, potassium; - NFR normal frog Ringer - HEPES N-2-hydroxylethyl piperazine-N-2-ethanesulfonic acid - EGTA ethyleneglycol-bis(-amino-ethyl ether) N,N,N',N'-tetra acetic acid - TEA tetraethylammonium - TMA tetramethylammonium;I g , gating current; , single-channel conductance  相似文献   

5.
Using a very low noise voltage clamp technique it has been possible to record from the squid giant axon a slow component of gating current (I g ) during the inactivation phase of the macroscopic sodium current (I Na ) which was hitherto buried in the baseline noise. In order to examine whether this slowI g contains gating charge that originates from transitions between the open (O) and the inactivated (I) states, which would indicate a true voltage dependence of inactivation, or whether other transitions contribute charge to slowI g , a new model independent analysis termed isochronic plot analysis has been developed. From a direct correlation ofI g and the time derivative of the sodium conductance dg Na/d the condition when only O-I transitions occur is detected. Then the ratio of the two signals is constant and a straight line appears in an isochronic plot ofI g vs. dg Na/d . Its slope does not depend on voltage or time and corresponds to the quantal gating charge of the O-I transition (q h ) divided by the single channel ionic conductance (). This condition was found at voltages above – 10 mV up to + 40 mV and a figure of 1.21e was obtained forq h at temperatures of 5 and 15°C. At lower voltages additional charge from other transitions, e.g. closed to open, is displaced during macroscopic inactivation. This means that conventional Eyring rate analysis of the inactivation time constant h is only valid above – 10 mV and here the figure forq h was confirmed also from this analysis. It is further shown that most of the present controversies surrounding the voltage dependence of inactivation can be clarified. The validity of the isochronic plot analysis has been confirmed using simulated gating and ionic currents.Abbreviations I g gating current - I Na sodium ionic current - g Na macroscopic sodium conductance - single channel conductance - C, O, I closed, open, inactivated state occupancy of channels - g h quantal charge displaced in a single O-I transition of Na channel - e equivalent electron charge - h index referring to inactivation process - S l limiting slope in isochronic plot see Eq.(3) - fractional distance, see Fig. 4 and (4, 5) - TMA tetramethylammonium - TTX tetrodotoxin - Tris tris(hydroxymethyl)aminomethane - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid  相似文献   

6.
The use-dependent phasic blockage of sodium channels by tetrodotoxin (TTX) and saxitoxin (STX) was examined in frog nodes of Ranvier using trains of depolarizing pulses. The decline of the peak Na+ current from its initial value (I 0) before the train to a stationary value (I ) after the train was more pronounced at more negative holding potentials. The relationship betweenI /I 0 and holding potential was fitted by a sigmoid function which yielded values for the steepness of the voltage dependencies of around –15 mV for TTX and – 8 mV for STX. Similar values were obtained at toxin concentrations of 4 and 8 nM. The higher voltage sensitivity of STX versus TTX is interpreted in terms of the higher charge and the faster binding kinetics of STX. These differences also explain the frequency dependence of the decline of Na+ currents with STX (between 0.5 and 2 Hz) and the frequency independence with TTX. Variation of the pulse amplitude in a train of conditioning pulses revealed that the magnitude of the use-dependent actions of STX parallels the steady-state Na+ inactivation curveh . Inhibition of inactivation, by pre-treatment with chloramine-T, did not, however, abolish the use dependence. Instead, it introduced a change in the time constants of the decline of the Na+ currents and the magnitude became independent of the holding potential.  相似文献   

7.
Summary The countertransport of Ca2+ and Na+ across the membranes of the unicellular fresh-water algaChlamydomonas reinhardtii CW-15 and twoDunaliella species differing in salt tolerance was studied. All algae used are devoid of cell walls. The calcium uptake by twoDunaliella species depended markedly on the intracellular sodium concentration. This calcium uptake was accompanied by Na+ release. For 15 and 30 s after artificial gradient formation (Naint + greater than Naext +) the ratio of released Na+ to absorbed Ca2+ was 31 and 41, respectively. For the extremely halotolerantD. salina, the apparent Michaelis constant of the Ca2+ uptake was 33 M, and for the marine halotolerant algaD. maritima, it was equal to 400 M, presuming more efficient Na+-for-Ca2+ exchange inD. salina cells. Ouabain, an inhibitor of Na+/K+-ATPase, suppressed Na+ transfer by 25%, whereas the agents blocking Ca2+-channels did not affect the transport of Ca2+ and Na+. The oppositely directed transmembrane Ca2+ and Na+ transfer was shown to depend on the external concentrations of Na+ and H+. In the fresh-water algaC. reinhardtii CW-15 (Naext + greater than Naint +), the direction of Ca2+ and Na+ fluxes across the plasma membrane was opposite to those described for Dunaliella cells. The results obtained point to the ability of the Na+-Ca2+ exchanger function in plasma membranes of algal cells.  相似文献   

8.
To gain information on extended flight energetics, quasi-natural flight conditions imitating steady horizontal flight were set by combining the tetheredflight wind-tunnel method with the exhaustion-flight method. The bees were suspended from a two-component aerodynamic balance at different, near optimum body angle of attack and were allowed to choose their own speed: their body mass and body weight was determined before and after a flight; their speed, lift, wingbeat frequency and total flight time were measured throughout a flight. These values were used to determine thrust, resultant aerodynamic force (magnitude and tilting angle), Reynolds number, total flight distance and total flight impulse. Flights in which lift was body weight were mostly obtained. Bees, flown to complete exhausion, were refed with 5, 10, 15 or 20 l of a 1.28-mol·l-1 glucose solution (energy content w=18.5, 37.0, 55.5 or 74.0 J) and again flown to complete exhaustion at an ambient temperature of 25±1.5°C by a flight of known duration such that the calculation of absolute and relative metabolic power was possible. Mean body mass after exhaustion was 76.49±3.52 mg. During long term flights of 7.47–31.30 min similar changes in flight velocity, lift, thrust, aerodynamic force, wingbeat frequency and tilting angle took place, independent of the volume of feeding solution. After increasing rapidly within 15 s a more or less steady phase of 60–80% of total flight time, showing only a slight decrease, was followed by a steeper, more irregular decrease, finally reaching 0 within 20–30 s. In steady phases lift was nearly equal to resultant aerodynamic force; tilting angle was 79.8±4.0°, thrust to lift radio did not vary, thrust was 18.0±7.4% of lift, lift was somewhat higher/equal/lower than body mass in 61.3%, 16.1%, 22.6% of all totally analysable flights (n=31). The following parameters were varied as functions of volume of feeding solution (5–20 l in steps of 5 l) and energy content. (18.5–74.0 J in steps of 18.5 J): total flight time, velocity, total flight distance, mean lift, thrust, mean resultant aerodynamic force, tilting angle, total flight impulse, wingbeat frequency, metabolic power and metabolic power related to body mass, the latter related to empty, full and mean (=100 mg) body mass. The following positive correlations were found: L=1.069·10-9 f 2.538; R=1.629·10-9 f 2.464; P m=7.079·10-8 f 2.456; P m=0.008v+0.008; P m=18.996L+0.022; P m=19.782R+0.021; P m=82.143T+0.028; P m=1.245·bm f 1.424 ; P mrel e=6.471·bm f 1.040 ; =83.248+0.385. The following negative correlations were found: V=3.939–0.032; T=1.324·10-4–0.038·10-4. Statistically significant correlations were not found in T(f), L(), R(), f(), P m(bm e), P m rel e(bm e), P m rel f(bm e), P m rel f(bm f).Abbreviations A(m2) frontal area - bl(m) body length - bm(mg) body mass - c(mol·1-1) glucose concentration of feeding solution - c D (dimensionless) drag coefficient, related to A - D(N) drag - F w(N) body weight - F wp weight of paper fragment lost at flight start - f wingbeat frequency (s-1) - g(=9.81 m·s-2) gravitational acceleration - I(Ns)=R(t) dt total impulse of a flight - L(N) lift vertical sustaining force component - P m(J·s-1=W) metabolic power - Pm ret (W·g-1) metabolic power, related to body mass - R(N) resultant aerodynamic force - Re v·bl·v -1 (dimensionless) Reynolds number, related to body length - s(m) v(t) dt virtual flight distance of a flight - s(km) total virtual flight distance - T (N) thrust horizontal force component of horizontal flight - T a (°C) ambient temperature - t(s) time - t tot (s or min) total flight time - v(m·s-1) flight velocity - v(l) volume of feeding solution - W (J) energy and energy content of V - ( °) body angle of attack between body longitudinal axis and flow direction - ( °) tilting angle ( 90°) between R and the horizont in horizontal flight v(=1.53·10-5m2·s-1 for air at 25°) kinematic viscosity - (=1.2 kg·m-3 at 25°C) air density  相似文献   

9.
The properties of cationic channels of an average unitary conductance were studied in guinea-pig ileum smooth muscle cells using a patch-clamp technique in the outside-out configuration. Cationic channels were activated by addition of 200 µM GTPS to an intrapipette solution, which resulted in stable activation of G proteins. The replacement of external cesium-containing (in the absence of Ca2+ and Mg2+ ions) solution with a more physiological sodium solution containing 2.5 mM Ca2+ and 2.0 mM Mg2+ led to smaller values of both the amplitude of currents through the unitary cationic channels under study and the probability of the stay of the channel in the open state (P o ). The drop in current amplitude was related mostly to the blocking effect of bivalent cations, while a decrease in the P o resulted from the replacement of Cs+ with Na+. Just a drop in the P o , which was responsible for approximately 85% of the inhibitory effect, played a crucial role in the suppression of the integral transmembrane current.Neirofiziologiya/Neurophysiology, Vol.36, No.4, pp.281–287, July–August, 2004.  相似文献   

10.
Whole cells of the extreme thermophile Thermus thermophilus HB8 contained a membrane-bound respiratory chain (comprised of nicotinamide nucleotide transhydrogenase, NADH dehydrogenase, menaquinone, and cytochromes b, c, aa3, o), which exhibited a maximumH+/O quotient of approximately 8 g-ion H+·g-atom O-1 for the oxidation of endogenous substrates. Whole cell respiration at 70° at the expense of endogenous substrates or ascorbate-TMPD generated a transmembrane protonmotive force (p) of up to 197 mV and an intracellular phosphorylation poteintial (Gp), measured under similar conditions, of approximately 43.9 kJ·mol-1.The measured Gp/p ratio thus indicated anH+/ATP quotient of approximately 2.3 g-ion H+·mole ATP-1. Glucose-limited continuous cultures of T. thermophilus at 60°, 70° and 78.5° exhibited extremely low moler growth yields (Y O2 max 27.6 g cells·mol O 2 -1 ; Y glucose max 64.4 g cells ·mol glucose-1) compared with mesophilic bacteria of similar respiratory chain composition and proton translocation efficiency. These low yields are probably at least partly explained by the extremely high permeability of the cytoplasmic membrane to H+, which thus causes the cells to respire rapidly in order to maintain the protonmotive force at a level commensurate with cell growth.Abbreviations TPMP+ triphenylmethylphosphonium cation - FCCP carbonylcyanide p-trifluoromethoxy phenythydrazone - TMPD N,N,N,N-tetramethyl-p-phenylene diamine  相似文献   

11.
Michael R. Blatt 《Planta》1987,170(2):272-287
The membrane electrical characteristics of stomatal guard cells in epidermal strips from Vicia faba L. and Commelina communis L. were explored using conventional electrophysiological methods, but with double-barrelled microelectrodes containing dilute electrolyte solutions. When electrodes were filled with the customary 1–3 M KCl solutions, membrane potentials and resistances were low, typically decaying over 2–5 min to near-30 mV and <0.2 k·cm2 in cells bathed in 0.1 mM KCl and 1 mM Ca2+, pH 7.4. By contrast, cells impaled with electrodes containing 50 or 200 mM K+-acetate gave values of-182±7 mV and 16±2 k·cm2 (input resistances 0.8–3.1 G, n=54). Potentials as high as (-) 282 mV (inside negative) were recorded, and impalement were held for up to 2 h without appreciable decline in either membrane parameter. Comparison of results obtained with several electrolytes indicated that Cl- leakage from the microelectrode was primarily responsible for the decline in potential and resistance recorded with the molar KCl electrolytes. Guard cells loaded with salt from the electrodes also acquired marked potential and conductance responses to external Ca2+, which are tentatively ascribed to a K+ conductance (channel) at the guard cell plasma membrane.Measurements using dilute K+-acetate-filled electrodes revealed, in the guard cells, electrical properties common to plant and fungal cell membranes. The cells showed a high selectivity for K+ over Na+ (permeability ratio PNa/PK=0.006) and a near-Nernstian potential response to external pH over the range 4.5–7.4 (apparent PH/PK=500–600). Little response to external Ca2+ was observed, and the cells were virtually insensitive to CO2. These results are discussed in the context of primary, charge-carrying transport at the guard cell plasma membrane, and with reference to possible mechanisms for K+ transport during stomatal movements. They discount previous notions of Ca2+-and CO2-mediated transport control. It is argued, also, that passive (diffusional) mechanisms are unlikely to contribute to K+ uptake during stomatal opening, despite membrane potentials which, under certain, well-defined conditions, lie negative of the potassium equilibrium potential likely prevailing.Abbreviations and symbols EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 2-(N-morpholino) propanesulfornic acid - E equilibrium potential - Gm membrane conductance - Rin input resistance - Vm membrane potential  相似文献   

12.
The ultraviolet (UV)-induced lethality in excision-proficientEscherichia coli strains WP-2 HCR+, B/r HCR+, B/r () HCR+, Blon HCR+, and WP-2 HCR is increased when chloroquine (300–500 g/ml) is added to the postirradiation medium. The degree to which chloroquine enhances the lethality of UV radiation varies for each strain, with strains B/r () and B showing a greater degree of repair inhibition than the other bacterial strains. The D10 (UV showing 10% survival) decreased in B/r () HCR+ strain grown in the presence of 500 g/ml (dose response of 2.5). InE. coli B, a dose response of 4.0 was obtained in the presence of the same concentration of chloroquine.Escherichia coli B/r, WP-2 HCR+, and WP-2 HCR strains showed less UV-induced lethality in the presence of chloroquine. The drug also inhibited liquid holding recovery (LHR) in irradiated HCR+ strains. These results suggest that chloroquine interferes with the excision repair (HCR function) of UV-induced photoproducts in irradiated bacterial populations.  相似文献   

13.
Summary Confluent monolayers of the established opossum kidney cell line were exposed to NH4Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PD m ) and membrane resistance (R m) in bicarbonate-free Ringer. The removal of extracellular NH4Cl leads to an intracellular acidification from a control value of 7.33±0.08 to 6.47±0.03 (n=7). This inhibits the absolute K conductance (g K+), reflected by a decrease of K+ transference number from 71±3% (n=28) to 26±6% (n=5), a 2.6±0.2-fold rise ofR m, and a depolarization by 24.2±1.5 mV (n=52). In contrast, intracellular acidification during a block ofg K+ by 3 mmol/liter BaCl2 enhances the total membrane conductance, being shown byR m decrease to 68±7% of control and cell membrane depolarization by 9.8±2.8 mV (n=17). Conversely, intracellular alkalinization under barium elevatesR m and hyperpolarizes PD m . The replacement of extracellular sodium by choline in the presence of BaCl2 significantly hyperpolarizes PD m and increasesR m, indicating the presence of a sodium conductance. This conductance is not inhibited by 10–4 mol/liter amiloride (n=7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na+-conductive channels with 18.8±1.4 pS (n=10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl. The low-conductive channel is 4.8 times more permeable for Na+ than for K+. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).  相似文献   

14.
Summary The activity of ALA-dehydratase from corn seedlings is affected by Mn++, Fe++, Pb++, Cu++, Zn++ and Sn+4 ions, in vivo Mn++ and Fe++ are ativators while Pb++ and Sn+4 are inhibitors; in vitro Cu++ and Zn++ are inhibitors. The kinetic parameters (Vmax and KM) support the hypothesis that Mn, Fe, Sn and Pb ions act on the biosynthesis of the enzyme and Zn and Cu ions on the enzyme-substrate affinity. Some related metal-organic compounds interrere in vivo on the ALA-dehydratase activity modifying the kinetic parameters, therefore the enzyme biogenesis and/or enzyme-sustrat affinity are affected.  相似文献   

15.
The effects of detergents on the electronic structure of the oxidized primary donor P+ and the time constant AP of the P+Q A charge recombination at ambient temperatures have been investigated in native and mutant reaction centers (RCs) from Rhodobacter sphaeroides. It is shown that N-lauryl-N,N-dimethyl-3-ammonio-1-propane sulfonate (SB12) induces a transition to a second distinct conformation of the RC. In the case of the wild type and the mutant FY(M197), in which a hydrogen bond is introduced to the 2-acetyl group of the dimer half of P that is associated with the M-subunit of the RC, the conformational change causes a more asymmetric spin density distribution between the two bacteriochlorophyll moieties of P+ in favor of the L-half. For both types of RCs the time constant AP depends on the SB12/RC ratio as does the position of the long-wavelength band of P, max. The increase of AP by 30 ms and the shift of max from 866 nm to 851 nm are indicative for the conformational change. In addition, a smaller linear increase of AP with increasing SB12/RC ratio is superimposed on the variation of AP due to the conformational change. Similar effects of SB12 on the optical spectra as well as on AP are also observed for the two heterodimer mutants HL(L173) and HL(M202), in which one of the bacteriochlorophylls of P is replaced by a bacteriopheophytin. There are no clear indications for a correlation of AP with the localization of the positive charge in P+. Furthermore, it is concluded from the dependence of AP on the SB12/RC ratio that the single-site mutations do not affect the standard free energy difference of the two conformations to a measurable extent.  相似文献   

16.
Yeast alcohol dehydrogenase (EC 1.1.1.1) catalyzes the novel reduction of p-nitro-so-N,N-dimethylaniline with NADH as a cofactor. Apparent kinetic constants for this enzymatic reaction are: V 2=2.1 s–1, K Q=456 M, K iQ=119 M, and K P=1.47 mM, at pH 8.9, 25 °C. This reaction is especially useful for the quantitative determination of NAD+ and NADH by enzymatic cycling.  相似文献   

17.
Changes in carp myosin ATPase induced by temperature acclimation   总被引:8,自引:0,他引:8  
Summary Myosins were isolated from dorsal ordinary muscles of carp acclimated to 10°C and 30°C for a minimum of 5 weeks and examined for their ATPase activities. Ca2+-ATPase activity was different between myosins from cold-and warm-acclimated carp, especially at KCl concentrations ranging from 0.1 to 0.2 M, when measured at pH 7.0. The highest activity was 0.32 mol Pi·min-1·mg-1 at 0.2 M KCl for cold-acclimated carp and 0.47 mol Pi·min-1·mg-1 at 0.1 M KCl for warm-acclimated fish. The pH-dependency of Ca2+-ATPase activity at 0.5 M KCl for both carp was, however, similar exhibiting two maxima around 0.3 mol Pi·min-1·mg-1 at pH 6 and 0.4 mol Pi·min-1·mg-1 at pH 9. K+(EDTA)-ATPase activity at pH 7.0 neither exhibited differences between both myosins. It increased with increasing KCl concentration showing the highest value of about 0.4 mol Pi·min-1·mg-1 at 0.6–0.7 M KCl. Actin-activated myosin Mg2+-ATPase activity was markedly different between cold-and warm-acclimated carp. The maximum initial velocity was 0.53 mol Pi·min-1·mg-1 myosin at pH 7.0 and 0.05 M KCl for cold-acclimated carp, which was 1.6 times as high as that for warm-acclimated carp. These differences were in good agreement with those obtained with myofibrillar Mg2+-ATPase activity between both carp. No differences were, however, observed in myosin affinity to actin. Differences in myosin properties between cold- and warm-acclimated carp were further evidenced by its thermal stability. The inactivation rate constant of myosin Ca2+-ATPase was 25·10-4·s-1 at 30°C and pH 7.0 for cold-acclimated carp, which was about 4 times as high as that for warm-acclimated carp. Light chain composition did not differ between both carp myosins. The differences in a primary structure of the heavy chain subunit was, however, clearly demonstrated between both myosins by peptide mapping.Abbreviations ATPase adenosine 5-triphosphatase - DTNB 5,5 dithio-bis-2-nitrobenzoic acid - DTT dithiothreitol - EGTA ethyleneglycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - K D inactivation rate constant - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis  相似文献   

18.
Summary Acute exposure of rainbow trout to hypoxic water ( =40 mmHg, 15 °C) caused a significant (P<0.01) increase in blood O2 affinity, from the normoxicP 50 value (at pHe 7.93) of 23.2±1.1 mmHg to about 19 mmHg, within 5 min. Specimens injected with the -antagonist propranolol showed no change in bloodP 50, despite a more pronounced reduction of arterial during the hypoxic exposure.The change in bloodP 50 coincided with an increase in plasma catecholamines, notably noradrenaline. There was no change in the molar ratios of ATPHb4 and GTPHb4. The altered bloodP 50, however, correlated with an alkalinization and an increased sodium concentration of the red cells. This red cell alkalinization can be explained by -adrenergic stimulation of a membrane bound Na+/H+ antiporter.Propranolol injection into normoxic resting trout caused a significant decrease in and increase in indicating -adrenergic control of gas exchange in the gills.  相似文献   

19.
Summary T-type calcium channels (I T channels) were studied in cell-attached patch electrode recordings from the ventricular cell membrane of 14-day embryonic chick heart. All experiments were performed in the absence of Ca2+ with Na+ (120mm) as the charge carrier.I T channels were distinguished from L-type calcium channels (I L) by their more negative activation and inactivation potential ranges; their smaller unitary slope conductance (26 pS), and their insensitivity to isoproterenol or D600. Inactivation kinetics were voltage dependent. The time constant of inactivation was 37 msec when the membrane potential was depolarized 40 mV from rest (R+40 mV), and 20 msec atR+60 mV. The frequency histogram of channel open times 0 was fit by a single-exponential curve while that of closed times c was biexponeintial. o was the same atR+40 mV andR+60 mV whereas c was shortened atR+60 mV. The open-state probability (P o) increased with depolarization: 0.35 atR+40 mV, 0.8 atR+60 mV and 0.88 atR+80 mV. This increase inP o at depolarized potentials could be accounted for by the decrease in c.  相似文献   

20.
Summary Ouabain-insensitive, furosemide-sensitive Rb+ influx (J Rb) into HeLa cells was examined as functions of the extracellular Rb+, Na+ and Cl concentrations. Rate equations and kinetic parameters, including the apparent maximumJ Rb, the apparent values ofK m for the three ions and the apparentK i for K+, were derived. Results suggested that one unit molecule of this transport system has one Na+, one K+ and two Cl sites with different affinities, one of the Cl sites related with binding of Na+, and the other with binding of K+(Rb+). A 11 stoichiometry was demonstrated between ouabain-insensitive, furosemidesensitive influxes of22Na+ and Rb+, and a 12 stoichiometry between those of Rb+ and36Cl. The influx of either one of these ions was inhibited in the absence of any one of the other two ions. Monovalent anions such as nitrate, acetate, thiocyanate and lactate as substitutes for Cl inhibited ouabain-insensitive Rb+ influx, whereas sulfamate and probably also gluconate did not inhibitJ Rb. From the present results, a general model and a specialized cotransport model were proposed: 1) In HeLa cells, one Na+ and one Cl bind concurrently to their sites and then one K+ (Rb+) and another Cl bind concurrently. 2) After completion of ion bindings Na+, K+(Rb) and Cl in a ratio of 112 show synchronous transmembrane movements.  相似文献   

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