SCN− and HSCN transport through lipid bilayer membranes: A model for SCN− inhibition of gastric acid secretion

Diffusion of thiocyanate (SCN?) and thiocyanic acid (HSCN) (pK=?1.8) through lipid bilayer membranes was studied as a function of pH. Membranes were made of egg phosphatidylcholine or phosphatidylcholine plus cholesterol (1:1 mol ratio) dissolved in decane or tetradecane. Tracer fluxes and electrical conductances were used to estimate the permeabilities to HSCN and SCN?. Over the pH range 1.0 to 3.3 only HSCN crosses the membrane at a significant rate. The relation between the total SCN flux (JA), concentrations and permeabilities is: 1/JA=1/Pul([A?]+[HA])+1/PHAm[HA], where [A?] and [HA] are the concentrations of SCN? and HSCN, Pul is permeability coefficient of the unstirred layer, and PHAm is the membrane permeability to HSCN. By fitting this equation to the data we find that PHAm = 2.6 cm · s?1 and Pul = 9.0 · 10?4 cm · s?1. Conductance measurements indicate that PA?m is 5 · 10?9 cm · s?1. Addition of cholesterol to phosphatidylcholine (1:1 mol ratio) reduces PHAm by a factor of 0.4 but has no effect on PA?m. SCN? is potent inhibitor of acid secretion in gastric mucosa, but the mechanism of SCN? action is unknown. Our results suggest that SCN? acts by combining with H+ in the mucosal unstirred layer (secretory pits) and diffusing back into the cells as HSCN, thus dissipating the proton gradient across the secretory membrane. A similar mechanism of action is proposed for some other inhibitors of gastric acid secretion, e.g. nitrite (NO2?), cyanate (CNO?) and NH4+.     

Cat Heart Muscle in Vitro : VI. Potassium exchange in papillary muscles

The exchange of cell K with K⁴², J _K, has been measured in cat right ventricular papillary muscle under conditions of a steady state with respect to intracellular K concentration. Within the limits of the measurement, all of cell K exchanged at a single rate. Cells from small cats are smaller and have larger surface/volume ratios than cells from large cats. The larger surface/volume ratio results in larger flux values. J _K increases in an approximately linear manner as the external K concentration is increased twentyfold, from 2.5 to 50 mM, at constant intracellular K concentration. The permeability for K ions, P _K, calculated from the influx and membrane potential, remains very nearly constant over this range of external K concentrations. J _K is not affected by replacement of O₂ by N₂, or by stimulated contractions at 60 per minute, but K influx decreases markedly in 10^-5 M and 10^-8 M ouabain.     

The development of osmotic flow through an unstirred layer

We investigate the errors involved in estimating the osmotic permeability of a semi-permeable membrane, from the measured osmotic flow and the difference in concentration of osmotically active solute across it, without taking account of the unstirred layer in the solution next to the membrane. In the problem solved, this layer is represented as a region of thickness δ at the far side of which a solute concentration C_b is imposed for time . The initial diffusion of solute towards the membrane causes the concentration at the membrane C_m to rise, generating an osmotic flow of water, J, whose convective effect opposes the diffusion. The problem is made non-linear by the dependence of J on C_m. Ultimately a steady state is set up, in which C_m is less than C_b. The solution is shown to depend on a single parameter β, equal to (L_pRT) δ C_b/D, where L_pRT is the osmotic permeability of the membrane and D is the diffusivity of solute. Solution of the steady state leads to a prediction of C_m/C_b as a function of β, and analysis of the decay of transient terms leads to a prediction of the decay time π, also as a function of β. Numerical data for membranes with a wide range of osmotic permeabilites, and for a reasonable range of solute, i.e. sucrose, concentrations, suggest that values of β can range from 0.001 or below to 7.5 or above. The former value implies negligible error in neglecting the unstirred layer, while the latter implies a 79%. error. For β = 0.1 and for δ = 2 × 10⁻⁴ m, π is predicted to be around 74 s. This decreases as β increases (for fixed δ); for values of β above about 27, the decay of transients is no longer monotonic but takes the form of damped oscillations.     

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.     

Bi-ionic potentials of bovine lens capsules and collodion membranes

Concentration dependencies of bi-ionic potentials of well-cleaned bovine lens capsules in vitro, of collodion and of modified collodion membranes were studied. The lens capsules have positively fixed charges, and collodion membranes have negatively fixed charges. As these membranes are partially selectively permeable, both co-ions and counter-ions exist in the membrane. However, many studies on bi-ionic potentials have been limited to systems in which the membrane has extreme ionic selectivity and co-ions are completely excluded from the membrane. Experimental results agreed with theoretical values obtained by assuming the common ion concentration to be constant throughout the membrane for systems such as KCl(C)-membrane (θ>0, or θ<0)-NaCl(C), NaNO₃(C)-membrane (θ>0)-NaCl(C) and CaCl₂(C₁)-membrane (θ>0)-NaCl(C₂) (C₂/C₁ = 2), where C is the bulk concentration. The theoretical reliability of this assumption was checked. When both electrolytes in solution were uni-univalent, the ratio of ionic mobilities of two counter-ions (or two co-ions) in all of these membranes was almost the same as the ratio obtained in bulk solution, while the ratio of ionic mobilities of the counter-ion and the co-ion was almost the same as the ratio obtained in bulk solution for the lens capsule, but different in the case of the collodion and modified collodion membranes.     

Interactions of Elevated CO2 Concentration and Drought Stress on Photosynthesis in Eucalyptus Cladocalyx F. Muell

Response of net photosynthetic rate (P _N), stomatal conductance (g _s), intercellular CO₂ concentration (c _i), and photosynthetic efficiency (F_v/F_m) of photosystem 2 (PS2) was assessed in Eucalyptus cladocalyx grown for long duration at 800 (C₈₀₀) or 380 (C₃₈₀) µmol mol^-1 CO₂ concentration under sufficient water supply or under water stress. The well-watered plants at C₈₀₀ showed a 2.2 fold enhancement of P _N without any change in g _s. Under both C₈₀₀ and C₃₈₀, water stress decreased P _N and g _s significantly without any substantial reduction of c _i, suggesting that both stomatal and non-stomatal factors regulated P _N. However, the photosynthetic efficiency of PS2 was not altered.     

Mesophyll conductance variations in response to diurnal environmental factors in Myrcia paivae and Minquartia guianensis in Central Amazonia

Mesophyll conductance (g _m) is essential to determine accurate physiological parameters used to model photosynthesis in forest ecosystems. This study aimed to determine the effects of time of day on photosynthetic parameters, and to assess the effect of using either intercellular CO₂ concentration (C _i) or chloroplast CO₂ concentration (C _c), on maximum carboxylation velocity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), V _cmax. We used Amazonian saplings of Myrcia paivae and Minquartia guianensis. Photosynthetic parameters were measured using an infrared gas analyzer (IRGA); g _m was determined using both gas exchange and chlorophyll (Chl) a fluorescence and gas-exchange data alone. Leaf thickness (L _T) and specific leaf area (SLA) were also measured. Air temperature, relative humidity or understory light did not correlate with g _m and on average daily IRGA-fluorometer-determined g _m was 0.04 mol(CO₂) m^?2 s^?1 for M. paivae and 0.05 mol(CO₂) m^?2 s^?1 for M. guianensis. Stomatal conductance (g _s), g _m, electron transport rate (J _F), and light-saturated net photosynthetic rate (P _Nmax) were lower in the afternoon than in the morning. However, no effect of time of day was observed on V _cmax. L _T and SLA did not affect any of the examined parameters. IRGA-determined g _m was almost the double of the value obtained using the IRGA-fluorescence method. V _cmax values determined using C _c were about 25% higher than those obtained using C _i, which highlighted the importance of using C _c in V _cmax calculation. Decline in P _Nmax at the end of the afternoon reflected variations in g _s and g _m rather than changes in V _cmax. Diurnal variation in g _m appeared to be associated more with endogenous than with atmospheric factors.     

Hydrochlorothiazide enhances the apical Cl− backflux in rabbit gallbladder epithelium: Radiochemical analysis

Hydrochlorothiazide (HCTZ) was shown to inhibit the transepithelial NaCl transport and the apical Na⁺-Cl^? symport and to depolarize the apical membrane potential in the rabbit gallbladder epithelium. The depolarization was likely related to the opening of a Cl^? conductance. To better understand whether an apical Cl^? leak is involved in the mechanism of action of HCTZ, the transapical Cl^? backflux was measured radiochemically by the washout technique. The gallbladder wall, pretreated with pronase on the serosal side to homogenize the subepithelium, was loaded with ³⁶Cl^? on the luminal side; mucosal and serosal ³⁶Cl^? effluxes (J _m , J _s ) were then measured every 2 min. The pretreatment with pronase did not alter the membrane potentials and the selectivity of the epithelium. Under control conditions and the tissue in steady-state, J _m and J _s time courses were each described by two exponential decays (A,B); the rate constants, k ^A and k ^B , were 0.71 ±0.03 and 0.16±0.01 min^?1, respectively, and correspondingly the half-times (t ₁ ^2A , t ₁ ^2B ) were 1.01±0.05 and 5.00±0.44 min (n=10); these parameters were not significantly different for J _m and J _s time courses. J _s was always greater than J _m (J _s /J _m =2.02±0.22 and 1.43 ±0.17 for A and B decays). Under SCN^? treatment in steady-state conditions, both J _m and J _s time courses were described by only one exponential decay, the component B being abolished. Moreover t ₁ ^2A was similar to that predictable for the subepithelium. It follows that it is the component B which exits the epithelial compartment. Based on the intracellular specific activity and ³⁶Cl^? J _m ^B at 0 min time of the washout experiment, the cell-lumen Cl^? backflux in steady-state was calculated to be equal to about 2 μmol cm^?2hr^?1, in agreement with the value indirectly computable by other techniques. The experimental model was well responsive to different external challenges (increases in media osmolalities; luminal treatment with nystatin). HCTZ (2.5 · 10^?4 m) largely increased ³⁶Cl^? J _m ^B . The increase was abolished by luminal treatment with 10^?4 m SITS, which not only brought back the efflux time courses to the ones observed under control conditions but even increased J _s /J _m of the cellular component, an indication of a reduced J _m ^B . It is concluded that HCTZ opens an apical, SITS-sensitive Cl^? leak, which contributes to dissipate the intracellular Cl^? accumulation and to inhibit the NaCl transepithelial transport. Moreover, the drug is likely to reduce the basal electroneutral Cl^? backflux supported by Na⁺-Cl^? cotransport, in agreement with the inhibition of the cotransport itself.     

Significance of mesophyll conductance for photosynthetic capacity and water-use efficiency in response to alkaline stress in Populus cathayana seedlings

Cuttings of Populus cathayana were exposed to three different alkaline regimes (0, 75, and 150 mM Na₂CO₃) in a semicontrolled environment. The net photosynthesis rate (P _N), mesophyll conductance (g _m), the relative limitations posed by stomatal conductance (L _s) and by mesophyll conductance (L _m), photosynthetic nitrogen-use efficiency (PNUE), carbon isotope composition (δ¹³C), as well as specific leaf area (SLA) were measured. P _N decreased due to alkaline stress by an average of 25% and g _m decreased by an average of 57%. Alkaline stress caused an increase of L _m but not L _s, with average L _s of 26%, and L _m average of 38% under stress conditions. Our results suggested reduced assimilation rate under alkaline stress through decreased mesophyll conductance in P. cathayana. Moreover, alkaline stress increased significantly δ¹³C and it drew down CO₂ concentration from the substomatal cavities to the sites of carboxylation (C _i-C _c), but decreased PNUE. Furthermore, a relationship was found between PNUE and C _i-C _c. Meanwhile, no correlation was found between δ¹³C and C _i/C _a, but a strong correlation was proved between δ¹³C and C _c/C _a, indicating that mesophyll conductance was also influencing the ¹³C/¹²C ratio of leaf under alkaline stress.     

An analysis of unstirred layers in series with "tight" and "porous" lipid bilayer membranes

The present experiments were designed to evaluate the effective thickness of the unstirred layers in series with native and porous (i.e., in the presence of amphotericin B) lipid bilayer membranes and, concomitantly, the respective contributions of membranes and unstirred layers to the observed resistances to the diffusion of water and nonelectrolytes between aqueous phases. The method depended on measuring the tracer permeability coefficients for the diffusion of water and nonelectrolytes (P_DDi, cm sec^-1) when the aqueous phase viscosity (η) was increased with solutes having a unity reflection coefficient, such as sucrose or dextran. The effective thickness of the unstirred layers (α^t, cm) and the true, or membrane, permeability coefficients for diffusion of water and nonelectrolytes (P_mmi, cm sec^-1) were computed from, respectively, the slope and intercept of the linear regression of 1/P_DDi on η. In both the native and porous membranes, α^t was approximately 110 x 10^-4 cm. The ratio of P_f, the osmotic water permeability coefficient (cm sec^-1) to P_mmH2O was 1.22 in the native membranes and 3.75 in the porous membranes. For the latter, the effective pore radius, computed from Poiseuille's law, was approximately 5.6 A. A comparison of P_mmi and P_DDi, indicated that the porous membranes accounted for 16, 25, and 66% of the total resistance to the diffusion of, respectively, H₂O, urea, and glycerol, while the remainder was referable to the unstirred layers.     

Electrolytes control flows of water and sucrose through collagen membranes

Summary The physical state of a collagen membrane is determined, among other factors, by the concentration of electrolytes in the bathing solutions, going from a crystalline to an amorphous phase as the concentration increases. Thus, the permeation of uncharged solutes and water is strongly dependent upon the salts in the bathing solutions, which through the induced phase transition control not only the thickness and the solvent content of the membrane but also affect the magnitudes of the frictional coefficients of transport. These changes in physical parameters are reflected in variations of several hundred per cent in the values of the phenomenological coefficients _s ,L _p and . Experiments were performed to determine the physical state and the permeability properties of the membrane as functions of the controlling electrolyte, in this instance CaCl₂, in the bathing solutions. In particular the filtration coefficientL _p , the permeability coefficient for sucrose _s , and the reflection coefficient for sucrose were determined via flow measurements at different salt concentrations. Complementary measurements of swelling and length variations were made. Data were reduced to membrane thickness, solvent volume-fraction, and the phenomenological coefficients. These in turn were reduced to the frictionsf _sm,f _sw andf _wm ; there was a direct correlation between the behavior of these frictions and the physical state of the collagen membrane as indicated by the length and volume variations.Thesis presented at the Institute of Physics of the University of Genoa, Italy as a partial requirement for the degree in physics.     

Relative Ion Permeabilities in the Crayfish Giant Axon Determined from Rapid External Ion Changes

The changes in membrane potential of isolated, single crayfish giant axons following rapid shifts in external ion concentrations have been studied. At normal resting potential the immediate change in membrane potential after a variation in external potassium concentration is quite marked compared to the effect of an equivalent chloride change. If the membrane is depolarized by a maintained potassium elevation, the immediate potential change due to a chloride variation becomes comparable to that of an equivalent potassium change. There is no appreciable effect on membrane potential when external sodium is varied, at normal or at a depolarized membrane potential. Starting from the constant field equation, expressions for the permeability ratios P _Cl/P _K, P _Na/P _K, and for intracellular potassium and chloride concentrations are derived. At normal resting membrane potential, P _Cl/P _K is 0.13 but at a membrane potential of -53 mv (external potassium level increased about five times) it is 0.85. The intracellular concentrations of potassium and chloride are estimated to be 233 and 34 mM, respectively, and it is pointed out that this is not compatible with ions distributed in a Nernst equilibrium across the membrane. It is also stressed that the information given by a plot of membrane potential vs. the logarithm of external potassium concentrations is very limited and rests upon several important assumptions.     

The Water and Nonelectrolyte Permeability Induced in Thin Lipid Membranes by the Polyene Antibiotics Nystatin and Amphotericin B

Nystatin and amphotericin B increase the permeability of thin (<100 A) lipid membranes to ions, water, and nonelectrolytes. Water and nonelectrolyte permeability increase linearly with membrane conductance (i.e., ion permeability). In the unmodified membrane, the osmotic permeability coefficient, P_f, is equal to the tagged water permeability coefficient, (P_d)_w; in the nystatin- or amphotericin B-treated membrane, P_f/(P_d)_w ≈ 3. The unmodified membrane is virtually impermeable to small hydrophilic solutes, such as urea, ethylene glycol, and glycerol; the nystatin- or amphotericin B-treated membrane displays a graded permeability to these solutes on the basis of size. This graded permeability is manifest both in the tracer permeabilities, P_d, and in the reflection coefficients, σ (Table I). The "cutoff" in permeability occurs with molecules about the size of glucose (Stokes-Einstein radius 4 A). We conclude that nystatin and amphotericin B create aqueous pores in thin lipid membranes; the effective radius of these pores is approximately 4 A. There is a marked similarity between the permeability of a nystatin- or amphotericin B-treated membrane to water and small hydrophilic solutes and the permeability of the human red cell membrane to these same molecules.     

Diurnal changes in volume and solute transport coefficients of phaseolus roots

Volume (J_v) and solute (J_s) fluxes through Phaseolus root systems were observed over a 24-hour period. The volume flux was varied in a pressure chamber by altering the hydrostatic pressure in 10 steps, from 0 to 0.41 megapascals. All root systems showed strong diurnal peaks in volume flux. The five transport coefficients (σ, ω, J_s^*, L_p, and π^*) were estimated from a nonlinear least squares algorithm. Analysis of the data revealed that all the coefficients exhibited a diurnal rhythm. When the total differential of the volume flux was considered it was possible to show that the diurnal changes in volume flux were due to a complex interaction between the diurnally shifting coefficients with the role of each highly dependent on the level of volume flux. At low volume fluxes, ω, J_s^*, and π^* accounted for nearly all the diurnal change in volume flux. At high volume fluxes, however, the major influence shifted to L_p and π^*, while ω and J_s^* became relatively unimportant. Thus, π^* was the only coefficient of interest across the entire range of J_v and appeared to be the single most important one in determining the diurnal rhythm of J_v under conditions of a constant applied pressure.     

Ionic Permeability of Thin Lipid Membranes : Effects of n-alkyl alcohols,polyvalent cations,and a secondary amine

Ultrathin (black) lipid membranes were made from sheep red cell lipids dissolved in n-decane. The presence of aliphatic alcohols in the aqueous solutions bathing these membranes produced reversible changes in the ionic permeability, but not the osomotic permeability. Heptanol (8 mM), for example, caused the membrane resistance (R_m) to decrease from >10⁸ to about 10⁵ ohm-cm² and caused a marked increase in the permeability to cations, especially potassium. In terms of ionic transference numbers, deduced from measurements of the membrane potential at zero current, T _cat/T _Cl increased from about 6 to 21 and T _K/T _Na increased from about 3 to 21. The addition of long-chain (C₈ndash;C₁₀) alcohols to the lipid solutions from which membranes were made produced similar effects on the ionic permeability. A plot of log R_m vs. log alcohol concentration was linear over the range of maximum change in R_m, and the slope was -3 to -5 for C₂ through C₇ alcohols, suggesting that a complex of several alcohol molecules is responsible for the increase in ionic permeability. Membrane permselectivity changed from cationic to anionic when thorium or ferric iron (10^-4 M) was present in the aqueous phase or when a secondary amine (Amberlite LA-2) was added to the lipid solutions from which membranes were made. When membranes containing the secondary amine were exposed to heptanol, R_m became very low (10³–10⁴ ohm-cm²) and the membranes became perfectly anion-selective, developing chloride diffusion potentials up to 150 mv.     

Photoprotective Function of Photorespiration in Several Grapevine Cultivars Under Drought Stress

Four grapevine cultivars, i.e. Cabernet Sauvignon (a member of the Western Europe cultivar group), Rizamat (a member of the East cultivar group), Red Double Taste (a hybridized cultivar from Vitis vinifera L. and V. labrusca L.), and 1103Paulsen (a hybridized rootstock), were treated by three severity orders of drought stress for 25 d. Then net photosynthetic rate (P _N), maximal photochemical efficiency (F_v/F_m), actual photochemical efficiency (_PS2) of photosystem 2, total electron transport rate (J_T), and electron transport flows used in carboxylation (J_C) and in oxygenation (J_O) reactions catalysed by ribulose-1,5-bisphosphate carboxylase/oxygenase were determined. P _N was determined again after re-watering for 2 d by gas exchange measurement. Along with the increase in severity of drought stress, P _N, F_v/F_m, _PS2, J_T, and J_C in all four cultivars decreased. The range of decrease differed among cultivars. J_O expressed various trends from cultivar to cultivar. In Rizamat that received slight and moderate drought stress, P _N evidently decreased, but J_O markedly increased, thus maintaining high values of J_T and _PS2. Prior to the moderate drought stress, the F_v/F_m was high in Rizamat, indicating that the photodamage had not happened ahead of the moderate drought stress given. Under the severe drought stress, the photorespiration rate in Rizamat decreased by 70 %, and J_T, _PS2, and F_v/F_m also dropped to very low values, i.e. the photodamage of photosynthetic apparatus has taken place. This suggested that the photorespiration has consumed the excessive assimilatory power and the photo-protective function of photorespiration is very important for Rizamat. When Cabernet Sauvignon grew under drought stress, its J_O decreased in a small range, thus maintaining higher values of J_C, J_T, _PS2, and F_v/F_m; hence no serious photodamage occurred. Despite of the fact that P _N of cv. Red Double Taste decreased markedly under the slight drought stress, J_O still increased under the severe drought stress. This suggests that photorespiration is important in photoprotection under drought stress. J_O in cv. 1103Paulsen markedly decreased under slight stress. Accordingly, P _N, F_v/F_m, _PS2, J_T, and J_C decreased to extremely low values. Thus photorespiration effectively protects the photosynthetic apparatus from photo-damage under drought, assists in maintaining a relatively high _PS2, and helps P _N to be rapidly recovered after re-watering.     

Stomatal,mesophyll conductance,and biochemical limitations to photosynthesis during induction

The dynamics of leaf photosynthesis in fluctuating light affects carbon gain by plants. Mesophyll conductance (g_m) limits CO₂ assimilation rate (A) under the steady state, but the extent of this limitation under non-steady-state conditions is unknown. In the present study, we aimed to characterize the dynamics of g_m and the limitations to A imposed by gas diffusional and biochemical processes under fluctuating light. The induction responses of A, stomatal conductance (g_s), g_m, and the maximum rate of RuBP carboxylation (V_cmax) or electron transport (J) were investigated in Arabidopsis (Arabidopsis thaliana (L.)) and tobacco (Nicotiana tabacum L.). We first characterized g_m induction after a change from darkness to light. Each limitation to A imposed by g_m, g_s and V_cmax or J was significant during induction, indicating that gas diffusional and biochemical processes limit photosynthesis. Initially, g_s imposed the greatest limitation to A, showing the slowest response under high light after long and short periods of darkness, assuming RuBP-carboxylation limitation. However, if RuBP-regeneration limitation was assumed, then J imposed the greatest limitation. g_m did not vary much following short interruptions to light. The limitation to A imposed by g_m was the smallest of all the limitations for most of the induction phase. This suggests that altering induction kinetics of mesophyll conductance would have little impact on A following a change in light. To enhance the carbon gain by plants under naturally dynamic light environments, attention should therefore be focused on faster stomatal opening or activation of electron transport.

Gas diffusional and biochemical processes impose significant limitations to CO2 assimilation during photosynthetic induction.     

Determination of the inherent kinetics of immobilized glucose oxidase using a diffusion cell

The enzyme glucose oxidase (GO) was covalently immobilized onto a poly(vinyl alcohol) hydrogel, cross-linked with glutardialdehyde and a polyazonium salt. To compare the kinetic parameters of immobilized GO with the known kinetic parameters of soluble GO, the diffusion cell method was used.Between two compartments, containing solutions with different glucose concentrations, a GO-containing hydrogel membrane was placed. Simultaneous diffusion through and enzymatic reaction in the membrane occurred. In this way diffusional effects of the membrane could be eliminated from the effective kinetic parameters to yield the inherent kinetic parameters.It appeared that the enzymatic reaction is independent of the oxygen concentration at oxygen concentrations 0.22 mol m^–3 (Michaelis constant for oxygen < 0.22 mol m^–3). Further, the Michaelis constant for glucose does not change dramatically after immobilizing the enzyme. The maximal reaction rate is depending on the enzyme concentration. As the enzyme concentration in the membrane is not exactly known (mainly due to leakage of enzyme out of the membrane during membrane preparation), only an estimation of the turnover number can be made.The diffusion cell method is easy to carry out. Still, some recommendations can be made on the performance.List of Symbols _g , _0x partition coefficient of glucose and oxygen, respectively - thickness of the wetted membrane (m) - A _m surface area of membrane (m^–2) - C constant (mol² m^–3) - c _g , c _0x concentration of glucose and oxygen, respectively (mol m^–3) - c _g,0 c _g, glucose concentration at the filter-paper/membrane interface next to compartment A and B, respectively (mol m^–3) - c _{g, A} c _{g, B} glucose concentration in compartment A and B, respectively (mol m^–3) - c _GO glucose oxidase concentration (mol m^–3) - D _eff effective diffusion coefficient (m² s^–1) - D _m , D _sl diffusion coefficient in, respectively, the membrane and the solution layer (m² s^–1) - d _dl , d _df , d _sl thickness of, respectively, the diffusion layer, the filter-paper and the solution layer (m) - h _B initial slope of concentration versus time curve of compartment B (mol m^–3 s^–1) - J flux (mol m^–2 s^–1) - J ₀ flux in the membrane at membrane/filter-paper interface next to compartment A and B, respectively (mol m^–2 s^–1) - J _A , J _B flux leaving compartment A and entering compartment B, respectively (mol m^–2 s^–1) - J _m flux through the membrane (mol m^–2 s^–1) - k total mass transfer coefficient (m s^–1) - k ₁ , k ₂ rate constant of a particular reaction step (m³ mol^–1 s^–1) - k_–1, k_–2 rate constant of a particular reaction step (s^–1) - k _cat (intrinsic) catalytic constant of turnover number (s^–1) - k _cat ^* inherent catalytic constant, determined by inserting D _m (s^–1) - k _cat ^** inherent catalytic constant, determined by inserting D _eff (s^–1) - k _m (g) (intrinsic) Michaelis constant for glucose (mol m^–3) - k _m (o) (intrinsic) Michaelis constant for oxygen (mol m^–3) - k _m ^* (g) inherent Michaelis constant for glucose (mol m^–3) - k _m ^* (o) inherent Michaelis constant for oxygen (mol m^–3) - m _GO number of moles of GO present (mol) - P _m permeability of glucose in the mebrane (m s^–1) - P _eff effective permeability (m s^–1) - V volume (m³) - v ₀ initial reaction velocity (mol m^–3 s^–1) - V _max ^** inherent maximal reaction velocity, determined by inserting D_eff (mol m^–3 s^–1) - x distance (m)     

Photosynthetic photon flux density,carbon dioxide concentration,and vapor pressure deficit effects on photosynthesis in cacao seedlings

Independent short-term effects of photosynthetic photon flux density (PPFD) of 50–400 μmol m⁻² s⁻¹, external CO₂ concentration (C _a) of 85–850 cm³ m⁻³, and vapor pressure deficit (VPD) of 0.9–2.2 kPa on net photosynthetic rate (P _N), stomatal conductance (g _s), leaf internal CO₂ concentration (C _i), and transpiration rates (E) were investigated in three cacao genotypes. In all these genotypes, increasing PPFD from 50 to 400 μmol m⁻² s⁻¹ increased P _N by about 50 %, but further increases in PPFD up to 1 500 μmol m⁻² s⁻¹ had no effect on P _N. Increasing C _a significantly increased P _N and C _i while g _s and E decreased more strongly than in most trees that have been studied. In all genotypes, increasing VPD reduced P _N, but the slight decrease in g _s and the slight increase in C _i with increasing VPD were non-significant. Increasing VPD significantly increased E and this may have caused the reduction in P _N. The unusually small response of g _s to VPD could limit the ability of cacao to grow where VPD is high. There were no significant differences in gas exchange characteristics (g _s, C _i, E) among the three cacao genotypes under any measurement conditions.