Passive sodium fluxes across toad bladder in the presence of simultaneous transepithelial gradients of concentration and potential

Summary Bidirectional sodium fluxes were measured across toad bladder sacs after eliminating active transport with ouabain. Transepithelial potential was clamped to 100 mV or the Nernst potential, _eq, at varying sodium concentrations,C _m , in the mucosal medium. Serosal sodium concentration,C _s , was held constant. Equations were derived for permeability, partial ionic conductance, and unidirectional fluxes as functions ofC _m andC _s , based in part on the assumption that the ratio,Q, of bulk sodium permeability to tracer sodium permeability is a constant, independent of concentration and potential. The results conformed closely to these equations.     

Fluxes of 86Rb+ in “isolated” guard cells of Vicia faba L.

Steady-state ⁸⁶Rb⁺ fluxes and contents were measured in stomatal guard cells of Vicia faba L., using isolated epidermal strips in which all cells other than the guard cells had been killed by a brief ultrasound treatment. Flux experiments were carried out for a range of stomatal apertures, achieved by incubation in light or dark, or on solutions containing different concentrations of RbCl (1–30 mM). At pH 5.5 the efflux curve of ⁸⁶Rb⁺ could be fitted to the sum of two exponential terms and it was possible to calculate compartmental contents and fluxes. The data indicate that steady-state stomatal opening is achieved by regulation of both influx and efflux. Salt accumulation was not always adequate to contribute the observed osmotic requirement for opening, and this may indicate that some other solute accounts for the discrepancy.Abbreviations and Symbols A amplitude - k rate constant - Mes 2-(N-morpholino)-ethanesulphonic acid - Q_T, Q_C, Q_V total, cytoplasmic, vacuolar chemical content - Q^* tracer content - Ø_P, Ø_l plasmalemma, tonoplast fluxes This work was supported by a Research Studentship from the Science and Engineering Research Council. I thank Professor E.A.C. MacRobbie for much helpful discussion and advice.     

Sodium fluxes through the active transport pathway in toad bladder

Summary To assess the active components of sodium flux across toad bladder as a function of transepithelial potential, unidirectional sodium fluxes between identical media were measured before and after adding sufficient ouabain (1.89×10^–3 m) to eliminate active transport, while clamping transepithelial potential to 0, 100 or 150 mV. Evidence was adduced that ouabain does not alter passive fluxes, and that fluxes remain constant if ouabain is not added. Hence, the ouabain-inhibitable fluxes represent fluxes through the active path. Results were analyzed by a set of equations, previously shown to describe adequately passive fluxes under electrical gradients in this tissue, here modified by the insertion ofE, the potential at which bidirectional sodium fluxes ( ^E and ^E ) through the active pathway are equal. According to these equations, ^E and ^E are the logarithmic mean of bidirectional fluxes through the active path at any potential, and the flux ratio in this path is modified by a constant factorQ _ia, which represents the ratio of the bulk diffusion coefficient to the tracer diffusion coefficient in this pathway. The data are shown to conform closely to these equations.Q _ia averages 2.54. Hence, serosal-to-mucosal flux vanishes rapidly as potential falls belowE. MeanE in these experiments was 158±1 mV. Thus, linear dependence of net flux in both active and passive pathways on potential is present, even though the sodium fluxes in both paths fail to conform to the Ussing flux ratio equation.Q _{i p}<1 in the passive path (qualitatively similar to exchange diffusion) andQ _ia>1 in the active path (as in single file pore diffusion). Both of these features tend to reduce the change in serosal-to-mucosal sodium flux induced by depolarization from spontaneous potential to zero potential (short-circuiting).     

EVALUATION OF COMPETITIVE ABILITY OF STAURASTRUM LUETKEMUELLERII (CHLOROPHYCEAE) AND MICROCYSTIS AERUGINOSA (CYANOPHYCEAE) UNDER P LIMITATION1

The desmid Staurastrum luetkemuellerii Donat et Ruttner and the cyanobacterium Microcystis aeruginosa Kütz. showed pronounced differences in chemical composition and ability to maintain P fluxes. The cellular P:C ratio (Q_p) and the surplus P:C ratio (Q_sp) were higher in M. aeruginosa, indicating a lower yield of biomass C per unit of P. The subsistence quota (Q_p) was 1.85 μg P·mg C^?1in S. luetkemuellerii and 6.09 μg P·mg C^?1in M. aeruginosa, whereas the respective Q_p of P saturnted organisms (Q_s) were 43 and 63 μg P·mg C^?1. These stores could support four divisions in S. luetkemuellerii and three divisions in M. aeruginosa, which suggests that the former exhibited highest storage capacity (Q_s/Q₀). M. aeruginosa showed a tenfold higher activity of alkaline phosphatase than S. luetkemuellerii when P starved. The optimum N:P ratio (by weight) was 5 in S. luetkemuellerii and 7 in M. aeruginosa. The initial uptake of P_i pulses in the organisms was not inhibited by rapid (<1 h) internal feedback mechanisms and the short term uptake rote could be expressed solely as a function of ambient P_i. The maximum cellular C-based uptake rate (V_m) in P starved M. aeruginosa was up to 50 times higher than that of S. luetkemuellerii. It decreased with increasing growth rate (P status) in the former species and remained fairly constant in the latter. The corresponding cellular P-based value (U_m= V_m/Q_p) decreased with growth rate in both species and was about 10 times higher in P started M. aeruginosa than in S. luetkemuellerii. The average half saturation constant for uptake (K_m) was equal for both species (22 μg P·L^?1) and varied with the P status. S. luetkemuellerii exhibited shifts in the uptake rate of P_i that were characterized by increased affinity (U^m/K^m) at low P_i, concentrations (<4 μg P·L^?1) compared to that at higher concentrations. The species thus was well adapted to uptake at low ambient P_i, but M. aeruginosa was superior in P_i uptake under steady state and transient conditions when the growth rate was lower than 0.75 d^?1. Moreover, M. aeruginosa was favored by pulsed addition of P_i. M. aeruginosa relpased P_i at a higher rate than S. luetkemuellerii. Leakage of P_i from the cells caused C-shaped μ vs. P_i curves. Therefore, no unique K_s for growth could be estimated. The maximum growth rate (μ_m) (23° C) was 0.94 d^?1for S. luetkemuellerii and 0.81 d^?1for M. aeruginosa. The steady state concentration of P_i (P*) was lower in M. aeruginosa than in S. luetkemuellerii at medium growth rates. The concentration of P_i at which the uptake and release of P_i was equal (P_c was, however, lower in S. luetkemuellerii.     

The Permeation of Ammonium through a Voltage-independent K+ Channel in the Plasma Membrane of Rye Roots

Nitrogen is available to the plant in the form of NH⁺ ₄ in the soil solution. Here it is shown that a voltage-independent K⁺ channel in the plasma membrane of rye (Secale cereale L.) roots is permeable to NH⁺ ₄. The channel was studied following its incorporation into planar 1-palmitoyl-2-oleoyl phosphatidyl ethanolamine bilayers. The unitary conductance of the channel was greater when assayed in the presence of 100 mm NH₄Cl than 100 mm KCl. However, the probability of finding the channel open (P _o ) was lower in the presence of 100 mm NH₄Cl (P _o = 0.63) than in 100 mm KCl (P _o = 0.8), suggesting that P _o can be regulated by the (permeant) ions present in solution. When assayed in equimolar concentrations of NH₄Cl (cis) and KCl (trans), the zero-current (reversal) potential for the channel (E _rev) exhibited a complex concentration dependence. At low cation concentrations, the apparent permeability of NH⁺ ₄ relative to K⁺ (P_NH4/P_K) was greater than 1.0. However, as the cation concentration was increased, P_NH4/P_K initially decreased to a minimum of 0.95 at 3 mm before increasing again to a maximum of 1.89 at 300 mm. At cation concentrations above 300 mm, P_NH4/P_K decreased slightly. This implies that the pore of the channel can be occupied by more than one cation simultaneously. Ammonium permeation through the pore was simulated using a model which is composed of three energy barriers and two energy wells (the ion-binding sites). The model (3B2S) allowed for single-file permeation, double cation occupancy, ion-ion repulsion within the pore and surface potential effects. Results indicated that energy peaks and energy wells were situated asymmetrically within the electrical distance of the pore, that cations repel each other within the pore and that the vestibules to the pore contain negligible surface charge. The energy profile obtained for NH⁺ ₄ is compared with ones obtained for K⁺ and Na⁺. This information allows the fluxes through the K⁺ channel of the three major monovalent cations present in the soil solution to be predicted. Received: 16 October 1995/Revised 12 March 1996     

Large-conductance Calcium-activated Potassium Channels of Cultured Rat Melanotrophs

A large conductance, Ca²⁺-activated K⁺ channel of the BK type was examined in cultured pituitary melanotrophs obtained from adult male rats. In cell-attached recordings the slope conductance for the BK channel was ≈190 pS and the probability (P _o ) of finding the channel in the open state at the resting membrane potential was low (<<0.1). Channels in inside-out patches and in symmetrical 150 mm K⁺ had a conductance of ≈260 pS. The lower conductance in the cell-attached recordings is provisionally attributed to an intracellular K⁺ concentration of ≈113 mm. The permeability sequence, relative to K⁺, was K⁺ > Rb⁺ (0.87) > NH⁺ ₄ (0.17) > Cs⁺≥ Na⁺ (≤0.02). The slope conductance for Rb⁺ was much less than for K⁺. Neither Na⁺ nor Cs⁺ carried measurable currents and 150 mm internal Cs⁺ caused a flickery block of the channel. Internal tetraethylammonium ions (TEA⁺) produced a fast block for which the dissociation constant at 0 mV (K _D (0 mV)) was 50 mm. The K _D (0 mV) for external TEA⁺ was much lower, 0.25 mm, and the blocking reaction was slower as evidenced by flickery open channel currents. With both internal and external TEA⁺ the blocking reaction was bimolecular and weakly voltage dependent. External charybdotoxin (40 nm) caused a large and reversible decrease of P _o . The P _o was increased by depolarization and/or by increasing the concentration of internal Ca²⁺. In 0.1 μm Ca²⁺ the half-maximal P _o occurred at ≈100 mV; increasing Ca²⁺ to 1 μm shifted the voltage for the half-maximal P _o to −75 mV. The Ca²⁺ dependence of the gating was approximated by a fourth power relationship suggesting the presence of four Ca²⁺ binding sites on the BK channel. Received: 23 October/Revised: 15 December 1995     

Kinetic constants for intestinal transport of four monosaccharides determined under conditions of variable effective resistance of the unstirred water layer

Summary Theoretical considerations have suggested that variations in the resistance of the unstirred water layer (UWL) have a profound effect on the kinetic constants of intestinal transport. In this study, a previously validatedin vitro technique was employed to determine the unidirectional flux rate of glucose, galactose, 3-O-methyl glucose and fructose into the rabbit jejunum under carefully-defined conditions of stirring of the bulk phase known to yield different values for the effective resistance of the UWL. For each monosaccharide, uptake is much greater when the resistance of the UWL is low than when high. The maximal transport rate,J _d ^m , of glucose was half as large as theJ _d ^m of galactose and 3-O-methyl glucose (3-O-MG), and was twice as great as theJ _d ^m of fructose. The apparent affinity constant,K _m ^* ,of glucose is less than that of fructose, which was lower than theK _m ^* of galactose and 3-O-MG. The use of the Lineweaver-Burk double reciprocal plot is associated with an overestimation of bothJ _d ^m andK _m ^* .This discrepancy between the true and apparent values of the kinetic constants is much greater for lower than for higher values ofJ _d ^m andK _m ^* ;variations in the resistance of the unstirred layer influences the magnitude and direction of the discrepancy. The apparent passive permeability coefficient is similar for each sugar, but because of the different values ofJ _d ^m , passive permeation contributes relatively more to the uptake of glucose and fructose than of galactose or 3-O-MG. Under conditions of high unstirred layer resistance, differences in uptake rates of the sugars are due to differences in theirJ _d ^m rather than theirK _m ^* .Kinetic analysis is compatible with the suggestion that the glucose carriers are predominantly near the tip of the villus, whereas those for galactose and 3-O-MG are located along the entire villus and theK _m ^* of their carriers at the tip is lower than theirK _m ^* towards the base of the villus. It is proposed that there are multiple or heterogeneous intestinal carriers for glucose, galactose and 3-O-methyl glucose in the jejunum of the rabbit.Abbreviations Used in this Paper C ₁ Concentration of the probe molecule in the bulk phase - C ₂ Concentration of the probe molecule at the aqueous-membrane interface - d Effective thickness of the intestinal unstirred water layer - D Free diffusion coefficient of the probe molecule     

Aldosterone control of the density of sodium channels in the toad urinary bladder

Summary Near-instantaneous current-voltage relationships and shot-noise analysis of amiloride-induced current fluctuations were used to estimate apical membrane permeability to Na (P _Na), intraepithelial Na activity (Na _c ), single-channel Na currents (i) and the number of open (conducting) apical Na channels (N₀), in the urinary bladder of the toad (Bufo marinus). To facilitate voltageclamping of the apical membrane, the serosal plasma membranes were depolarized by substitution of a high KCl (85mm) sucrose (50mm) medium for the conventional Na-Ringer's solution on the serosal side.Aldosterone (5×10^–7 m, serosal side only) elicited proportionate increases in the Na-specific current (I _Na and inP _Na, with no significant change in the dependence ofP _Na on mucosal Na (Na _o ).P _Na and the control ofP _Na by aldosterone were substrate-dependent: In substrate-depleted bladders, pretreatment with aldosterone markedly augmented the response to pyruvate (7.5×10^–3 m) which evoked coordinate and equivalent increases inI _Na andP _Na.The aldosterone-dependent increase inP _Na was a result of an equivalent increase in the area density of conducting apical Na channels. The computed single-channel current did not change. We propose that, following aldosterone-induced protein synthesis, there is a reversible metabolically-dependent recruitment of preexisting Na channels from a reservoir of electrically undetectable channels. The results do not exclude the possibility of a complementary induction of Na-channel synthesis.     

Kinetics of K-Cl Cotransport in Frog Erythrocyte Membrane: Effect of External Sodium

In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO₃) has been shown to mediate a large fraction of the total K⁺ transport. In the present study, Cl⁻-dependent and Cl⁻-independent K⁺ fluxes via frog erythrocyte membranes were investigated as a function of external and internal K⁺ ([K⁺] _e and [K⁺] _i ) concentration. The dependence of ouabain-resistant Cl⁻-dependent K⁺ (⁸⁶Rb) influx on [K⁺] _e over the range 0–20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K _m ) of 8.2 ± 1.3 mm and maximal velocity (V _max ) of 10.4 ± 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl⁻-dependent K⁺ influx increased both K _m (12.8 ± 1.7 mm, P < 0.05) and V _max (20.2 ± 2.9 mmol/l/hr, P < 0.001). Raising [K⁺] _e above 20 mm in isotonic media significantly reduced the Cl⁻-dependent K⁺ influx due to a reciprocal decrease of the external Na⁺ ([Na⁺] _e ) concentration below 50 mm. Replacing [Na⁺] _e by NMDG⁺ markedly decreased V _max (3.2 ± 0.7 mmol/l/hr, P < 0.001) and increased K _m (15.7 ± 2.1 mm, P < 0.03) of Cl⁻-dependent K⁺ influx. Moreover, NMDG⁺ Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb⁺ uptake and K⁺ loss from red cells. Cell swelling did not affect the Na⁺-dependent changes in Rb⁺ uptake and K⁺ loss. In a nominally K⁺(Rb⁺)-free medium, net K⁺ loss was reduced after lowering [Na⁺] _e below 50 mm. These results indicate that over 50 mm [Na⁺] _e is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K⁺, Cl⁻-dependent K⁺ loss in K⁺-free media was a linear function of [K⁺] _i , with a rate constant of 0.11 ± 0.01 and 0.18 ± 0.008 hr⁻¹ (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K⁺ ions. The residual, ouabain-resistant K⁺ fluxes in NO₃ were only 5–10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K⁺ effluxes in isotonic (∼0.014 hr⁻¹) and hypotonic (∼0.022 hr⁻¹) media, but cell swelling resulted in a significant increase in the rate constants. Received: 19 November 1998/Revised: 23 August 1999     

Mode of action of amiloride in toad urinary bladder

Summary The effect of amiloride on the sensitivity to Na of the mucosal border of toad urinary bladder was investigated by recording Na concentration-dependent transepithelial potential difference (V _t ) and the intracellular potential. When mucosal Na concentration was normal, amiloride added to the mucosal solution at 10^–4 m markedly reduced the mucosal membrane potential (V _m ) and altered the potential profile from a two-step type to a well type. Similar changes were observed when Na was totally eliminated from the mucosal medium. The serosal membrane potential was insensitive to amiloride and elimination of mucosal Na. In the absence of amiloride, theV _t could be described by the Goldman-Hodgkin-Katz equation in the range of mucosal Na concentration from 0 to 16mm, and amiloride extended this concentration range. By using the Goldman-Hodgkin-Katz equation, Na permeability was calculated from the data ofV _t 's obtained in the allowed ranges of Na concentration and compared before and after the addition of amiloride. The results show that Na permeability decreases to 1/600 of control when the maximum dose of amiloride (10^–4 m) is applied. The relationship between Na permeability and amiloride concentration is well explained on the basis of assumptions that amiloride binds to the Na site of the mucosal border in one-to-one fashion and in a competitive manner with Na and that Na permeability reduces in proportion to increase in number of the sites bound with amiloride.     

Cation selectivity of the resting membrane of squid axon

Summary Permeability constant ratios among monovalent cations were studied in the resting membrane of a giant axon of a Pacific squid,Loligo opalescens, by observing the relationship between the membrane potential and the ion concentration.The average permeability ratios are: Tl, 1.8; K, 1.0; Rb, 0.72; Cs, 0.16; Na, <0.08; Li, <0.08. These permeability ratios suggest that neither valinomycin nor nonactin are adequate models for the sites producing the resting permeability in the axonal membrane.Cyclic polyetherbis(t-butyl cyclohexyl) 18-crown-6 does not increase the permeability ratioP _Cs/P _K except when applied at concentrations (5×10^–5 m) at which the surfactant properties of this molecule may become significant.     

Concentrationo-dependence of nonelectrolyte permeability of toad bladder

Summary A theoretical formulation was derived for the dependence of bulk solute permeability,P, defined as net flux :- concentration gradient, c, across any membrane in which solute concentration is controlling for net flux, . According to this formulation, is stimulated by increments in trans concentration,c ₂, in the rangec ₂/c ₁=0.0–0.1. Net flux of urea across toad bladder down concentration gradients was shown to be stimulated threefold by small increments in trans urea concentration. The theory also predicts that, in the absence of concentration gradients, tracer permeability,P ^*, defined as tracer flux :- tracer concentration, will be independent ofc provided thatP=P ^*, but will diminish with increasingc ifP/P ^*<1.P/P ^* was not significantly different from unity for urea, and bothP andP ^* were independent ofc in the absence of concentration gradients. However,P/P ^* was significantly less than unity (0.90 and 0.85) for thiourea and mannitol, respectively. In conformity with theory,P ^* (and alsoP) of these two solutes, measured asc was increased by 3–4 orders of magnitude, diminished progressively. These effects are more consistent with this formulation than with transport via a saturable carrier.     

Effects of mono- and multi-valent cations on the inward-rectifying potassium channel in isolated protoplasts from maize roots

Transport properties mediated by ionic channels were studied by the patch-clamp technique in protoplasts from cortical parenchyma cells of maize roots (CPMR). While outward currents could be seen only occasionally, macroscopic voltage- and time-dependent potassium-selective inward currents (I_K+in) were frequently observed in the whole-cell configuration. These currents increased continuously as a function of K⁺ concentration (in the range 3 – 200 mm) and the slow-saturating macroscopic chord-conductance was fitted by a Michaelis-Menten function with K_m = 195 ± 39 mm. Other ions, like sodium and lithium, did not permeate at all through the maize root inward-channel, or like ammonium (P_NH4+/ P_K+ = 0.16 0.25) and rubidium (P_Rb+/P_K+≈ 0.10) displayed a very low permeability ratio. Up to 5 mm Rb⁺ did not induce any inhibition of the K⁺ inward current, whereas submillimolar concentrations of Cs⁺ were sufficient to block, in a voltage-dependent manner, the inward currents. A decrease of the external potassium concentration favoured Cs⁺ inhibition (K_m = 89 ± 6 μm and 26 ± 2 μm in 200 and 100 mm KCl, respectively). The potassium inward-currents were reversibly and consistently inhibited by submillimolar external concentrations of the metal ions Ni²⁺, Zn²⁺ and Co²⁺, while 1 mm La³⁺ only slightly decreased (≈10%) both the single channel conductance (9.2 ± 1.2 pS in 100 mm potassium) and the macroscopic current. In contrast to the case with Cs⁺, inhibition induced by other metal ions did not show any voltage dependence. These results suggest that, as with animal potassium channels, the inward channel of maize-root cortical cells has a narrow pore of permeation and metal ions decrease the K⁺ current, possibly by acting on binding sites located outside the pore. Received: 21 February 1997 / Accepted: 27 May 1997     

Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells

Summary Usin gintracellular microelectrode technique, the response of the voltageV across the plasma membrane of cultured bovine corneal endothelial cells to changes in sodium and bicarbonate concentrations was investigated. (1) The electrical response to changes in [HCO ₃ ^– ] _o (depolarization upon lowering and hyperpolarization upon raising [HCO ₃ ^– ] _o ) was dependent on sodium. Lithium could fairly well be substituted for sodium, whereas potassium or choline were much less effective. (2) Removal of external sodium caused a depolarization, while a readdition led to a hyperpolarization, which increased with time of preincubation in the sodium-depleted medium. (3) The response to changes in [Na⁺] _o was dependent on bicarbonate. In a nominally bicarbonate-free medium, its amplitude was decreased or even reversed in sign. (4) Application of SITS or DIDS (10^–3 m) had a similar effect on the response to sodium as bicarbonate-depleted medium. (5) At [Na⁺] _o =151mm and [HCO ₃ ^– ] _o =46mm, the transients ofV depended, with 39.0±9.0 (sd) mV/decade, on bicarbonate and, with 15.3±5.8 (sd) mV/decade, on sodium. (6) After the preincubation of cells with lithium, replacement of Li by choline led to similar effects as the replacement of sodium by choline, though the response ofV was smaller with Li. This response could be reduced or reversed by the removal of bicarbonate or by the application of SITS. (7) Amiloride (10^–3 m) caused a reversible hyperpolarization of the steady-state potential by 8.5±2.6 mV (sd). It did not affect the immediate response to changes in [Na⁺] _o or [HCO ₃ ^– ] _o , but reduced the speed of regaining the steady-state potential after a change in [HCO ₃ ^– ] _o . (8) Ouabain (10^–4 m) caused a fast depolarization of –6.8±1.1 (sd) mV, which was followed by a continuing slower depolarization. The effect was almost identical at 10^–5 m. (9) It is suggested, that corneal endothelial cells possess a cotransport for sodium and bicarbonate, which transports net negative charage with these ions. It is inhibitable by stilbenes, but not directly affected by amiloride or ouabain. Lithium is a good substitute for sodium with respect to bicarbonate transport and is transported itself. In addition, the effect of amiloride provides indirect evidence for the existence of a Na⁺/H⁺-antiport. A model for the transepithelial transport of bicarbonate across the corneal endothelium is proposed.     

Cation-dependent nutrient transport in shrimp digestive tract

Purified epithelial brush border membrane vesicles (BBMV) were produced from the hepatopancreas of the Atlantic White shrimp, Litopeneaus setiferus, using standard methods originally developed for mammalian tissues and previously applied to other crustacean and echinoderm epithelia. These vesicles were used to study the cation dependency of sugar and amino acid transport across luminal membranes of hepatopancreatic epithelial cells. ³H-d-glucose uptake by BBMV against transient sugar concentration gradients occurred when either transmembrane sodium or potassium gradients were the only driving forces for sugar accumulation, suggesting the presence of a possible coupled transport system capable of using either cation. ³H-l-histidine transport was only stimulated by a transmembrane potassium gradient, while ³H-l-leucine uptake was enhanced by either a sodium or potassium gradient. These responses suggest the possible presence of a potassium-dependent transporter that accommodates either amino acid and a sodium-dependent system restricted only to l-leucine. Uptake of ³H-l-leucine was significantly stimulated (P < 0.05) by several metallic cations (e.g., Zn²⁺, Cu²⁺, Mn²⁺, Cd²⁺, or Co²⁺) at external pH values of 7.0 or 5.0 (internal pH 7.0), suggesting a potential synergistic role of the cations in the transmembrane transfer of amino acids. ³H-l-histidine influxes (15 suptakes) were hyperbolic functions of external [zinc] or [manganese], following Michaelis–Menten kinetics. The apparent affinity constant (e.g., K _m) for manganese was an order of magnitude smaller (K _m = 0.22 μM Mn) than that for zinc (K _m = 1.80 μM Zn), while no significant difference (P > 0.05) occurred between their maximal transport velocities (e.g., J _max). These results suggest that a number of cation-dependent nutrient transport systems occur on the shrimp brush border membrane and aid in the absorption of these important dietary elements.     

To what extent is a constant volume design worse than a minimum volume design for a series of CSTR's?

The balance equations for substrate in a cascade of CSTR's undergoing an enzyme-catalyzed reaction following Michaelis-Menten kinetics are developed in dimensionless form. Analytical expressions relating the intermediate concentrations are independently obtained for the cases of minimum overall volume and constant volume. The fractional deviations between the overall volumes following these two design criteria are calculated and presented for several values of the relevant parameters. For situations of practical interest, the fractional deviation is below 10%. Increasing values of the Michaelis-Menten parameter, K _m(or decreasing values of the number of reactors in the cascade, N) lead to lower values of the maximum deviation; this maximum deviation is attained at lower conversions of substrate when K _mis increased or N decreased.List of Symbols C _{S, i}mol.m^–3 concentration of substrate at the outlet of the i-th reactor - C _* ^{S, i} normalized concentration of substrate at the outlet of the i-th reactor - C _* ^{S, i, eq} normalized concentration of substrate at the outlet of the i-th reactor using the design criterion of constant volume - C _* ^{S, i, opt} normalized concentration of substrate at the outlet of the i-th reactor using the design criterion of minimum overall volume - C _{S, 0} mol.m^–3 concentration of substrate at the inlet to the first reactor - Da _i Damköhler number for the i-th reactor - Da _eq constant Damköhler number for each reactor of the cascade - Da _{tot, eq} overall Damköhler number for the cascade assuming equal-sized reactors - Da _{tot, min} minimum overall Damköhler number for the cascade - Er fractional deviation between the overall volumes using the two different design criteria - K _mmol. m^–3 Michaelis-Menten constant - K _* ^M dimensionless Michaelis-Menten constant - N number of reactors of the cascade - Q m³. s^–1 volumetric flow rate - V _im³ volume of the i-th reactor - v _max mol. m^–3. s^–1 reaction rate under saturation conditions of the enzyme with substrate - V _{tot, opt} m³ minimum overall volume of the cascade - V _{tot, eq} m³ overall volume of the cascade assuming equal-sized reactors     

Single-Channel Analysis of a Delayed Rectifier K+ Channel in Xenopus Myelinated Nerve

Single-channel properties of a delayed rectifier voltage-gated K⁺ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between −60 and −40 mV with a potential of half-maximal activation, E₅₀, at −47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal activation decreased from 80 to 1.6 msec between −60 and +40 mV. The channel inactivated monoexponentially with a time constant of 10.9 sec at −40 mV. The time constant of deactivation was 126 msec at −80 mV and 16.9 msec at −110 mV. In symmetrical 105 mm K⁺, the single-channel conductance (γ) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13–15°C. In Na⁺-rich solution with 2.5 mm extracellular K⁺γ was 7 pS and the reversal potential was negative to −80 mV, indicating a high selectivity for K⁺ over Na⁺. γ depended on extracellular K⁺ concentration (K _D = 19.6 mm) and temperature (Q ₁₀= 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel current amplitude at all potentials tested with a half-maximal inhibiting concentration (IC₅₀) of 0.6 mm. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX, IC₅₀= 6.8 nm) and mast cell degranulating peptide (MCDP, IC₅₀= 41.9 nm). In Ringer solution the membrane potential of macroscopic I-channel patches was about −65 mV and depolarized under TEA and DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane potential. Received: 2 June 1995/Revised: 13 October 1995     

Relationship between fluidity and ionic permeability of bilayers from natural mixtures of phospholipids

Summary Proton and calcium permeability coefficients of large unilamellar vesicles made from natural complex mixtures of phospholipids were measured in various conditions and related to membrane fluidity. Permeability coefficients at neutral pH and 25°C were in the range of 10⁴ cm sec¹ and 2.5×10¹¹ cm sec¹ for protons and calcium, respectively. With the exception of two cases. (H⁺)>10⁴ m and (Ca²⁺)>10³ m, fluidity increases correspond to permeability increases. Theoretical analysis shows that, for both ions, the measured values of permeability coefficients imply that the permeation process is controlled by the productD ₁ D ₂ of the diffusion coefficient from the medium into the membrane (D ₁) by the diffusion coefficient in the membrane (D ₂). Further analysis ofD ₁ values deduced from combined use of permeability and fluidity data shows that the solubilization should occur in a medium of dielectric constant of about 12, suggesting the involvement of the hydration water of membranes. High proton concentrations, although having virtually no effect on fluidity, trigger the appearance of (i) lateral heterogeneity in membranes, as seen by³¹P NMR, and (ii) large permeability increases. It is proposed that the main effect of fluidity and/or lateral heterogeneity on permeability may bevia the membrane hydration control. We conclude that the current assumption that permeability is controlled by fluidity should be regarded with caution, at least in the case of ions and natural mixtures of phospholipids.     

Kinetic behaviour of penicillin acylase stabilized by poly (ethyleneimine)

The paper presents the kinetic evaluations of poly(ethyleneimine)-penicillin acylase preparations. The comparative studies show that the investigated system is much better than the native enzyme, and slightly worse than commercially available Boenringer preparation. Additionally, the high stability of PEI-enzyme system, very easy way of its preparation, high flexibility, and possibility to set the needed enzyme concentration are particularly favourable for use of the membrane bioreactor with PEI-enzyme system immobilized in its volume. Some advantages of the use of such bioreactor are also discussed.List of Symbols C _E IU/m³ activity concentration - C _S mol/m³ substrate concentration - C _P , C_Q mol/m³ products concentration - K _A mol/m³ constant which defines the affinity of a substrate to enzyme - K _iS mol/m³ substrate inhibitory constant - K _iP mol/m³ PhAA inhibitory constant - K _iQ mol/m³ 6-APA inhibitory constant - k ₃ , mol/IU min constant rate of dissociation of the active complex - mol/m³ min rate of reaction This work was supported by Government Committee of Science: Grant KBN # 3 0321 92 01     

Experimental demonstration of the effect of the unstirred water layer on the kinetic constants of the membrane transport ofd-glucose in rabbit jejunum

Summary The rate of active transport of a probe molecule into the intestinal mucosal cells is determined by the rate of movement of the solute molecule across two barriers, the unstirred water layer and the microvillus membrane of the epithelial cell. Previously a theoretical equation has been derived which describedJ _d, the velocity of unidirectional flux, as a function of the characteristics of the transport carrier in the membrane and of the resistance of the overlying unstirred water layer (UWL). The predictions of these equations have been tested experimentally by studying the effect of the rate of stirring of the bulk phase on thein vitro uptake ofd-glucose by rabbit jejunum. These studies demonstrated that, first, alterations in the UWL have a profound effect on the magnitude of the apparent affinity constant, xK _m ^* , of the active transport process. Second, at bulk phase concentrations in excess of theK _m the passive component of the experimentally determined flux rate becomes of such magnitude as to introduce significant error into the estimate of both the maximal transport rate,J _d ^m , and the trueK _m. Third, as a result of the UWL, the use of double-reciprocal plots to determineJ _d ^m andK _m leads to the overestimation of these constants. Finally, failure to account for the UWL leads to important quantitative errors describing a number of the characteristics of the transport process: these include an underestimation of the Q₁₀ and the effect of sodium ion on the active transport of glucose in the jejunum. The results confirm that the kinetic characteristics of the uptake of an actively transported molecule are a complex function of the resistance of both the UWL and the mucosal cell membrane, and this transport process can be adequately described by a newly-derived equation. It is apparent that there are serious limitations in the interpretation of much of the previously published data dealing with active transport processes in the intestine, since these studies failed to account for the effect of the UWL.