pH modulation of the kinetics of rabbit jejunal,brush-border folate transport

Summary In jejunal brush-border membrane vesicles, an outwardly directed OH^– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M. 1985.J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH^– exchange (or phenomenologically indistinguishable H⁺: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examining F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was greater at lower (pH_int/pH_ext: 5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive³H-folate uptake only at pH6.0. Since stepwise increments ininternal pH (4.57.5; pH_ext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH (4.356.5; pH_int=7.8), a 50-fold decrement in F uptake was observed (H⁺ K _m =12.8±1.2 M). Hill plots of these data suggest involvement of at least one H⁺ (OH^–) at low pH (monovalent F^– predominates) and at least 2 H⁺ (OH^–) at high pH (divalent F^–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pH_ext 4.55 or 5.8, transport of F^– and F^–2 is electroneutral. Kinetic parameters for F^– and F^–2 were calculated from uptake data at pH_ext 4.55 and 5.0. Comparison of predictedvs. experimentally determined kinetic parameters at pH_ext5.8 (K _m =1.33vs. 1.70 M;V _max=123.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV _max, but does not affect theK _m for carrier-mediated F transport. These data are consistent with similarK _i ^' s for sulfasalazine (competitive inhibitor) at pH_ext 5.35 and 5.8 (64.7 and 58.5 M, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalent F and is sensitive to external pH with a H⁺ K _m (or OH^– lC₅₀) corresponding to pH 4.89. External pH effects theV _max, but not theK _m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH^– or H⁺),rather than competitive binding that is mutually exclusive.     

pH Modulation of the kinetics of rabbit jejunal,brush-border folate transport

Summary In jejunal brush-border membrane vesicles, an out-wardly directed OH^– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M., 1985).J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH^– exchange (or phenomenologically indistiguishable H⁺: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examinin F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was geater at lower (pH_int/pH_ext:5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive³H-folate uptake only at pH6.0. Since setepwise increments ininternal pH (4.57.5; pH_ext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH(4.356.5; pH_int=7.8), a 50-fold decrement in F uptake was observed (H⁺ K _m =12.8±1.2m). Hill plots of these data suggest involvement of at least one H⁺ (OH^–) at high pH (divalent F^–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pH_ext 4.55 or 5.8, transport of F^– and F^–2 is electroneutral. Kinetic parameters for F^– and F^–2 were calculated from uptake data at pH_ext 4.55 and 5.0. Comparision of predictedvs. experimentally determined kinetic parameters at pH_ext 5.8 (K _m =1.33vs. 1.70 m;V _max=12.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV _max, but does not affect thatK _m, for carrier-mediated F transport. These data are consistent with similarK _i's for sulfasalazine (competitive inhibitor) at pH_ext 5.35 and 5.8 (64.7 and 58.5 m, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalen F and is sensitive to extermal pH with a H⁺ K _m (or OH^– IC₅₀) corresponding to pH 4.89. External pH affects theV _max, but not theK _m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH^– or H⁺) rather than competitive binding that is mutually exclusive.     

Uptake of iron byGeotrichum candidum,a non-siderophore producer

Summary Geotrichum candidum (isolate 1–9) pathogenic on citrus fruits, appears to lack siderophore production. Iron uptake byG. candidum is mediated by two distinct iron-regulated, energy-and temperature-dependent transport systems that require sulfhydryl groups. One system exhibits specificity for either ferric or ferrous iron, whereas the other exhibits specificity for ferrioxamine-B-mediated iron uptake and presumably other hydroxamate siderophores. Radioactive iron uptake from⁵⁹FeCl₃ showed an optimum at pH 6 and 35° C, and Michaelis-Menten kinetics (apparentK _m = 3 m,V _max = 0.054 nmol · mg^–1 · min^–1). The maximal rate of Fe²⁺ uptake was higher than Fe³⁺ (V _max = 0.25 nmol · mg^–1 · min^–1) but theK _m was identical. Reduction of ferric to ferrous iron prior to transport could not be detected. The ferrioxamine B system exhibits an optimum at pH 6 and 40° C and saturation kinetics (K _m = 2 M,V _max = 0.22 nmol · mg^–1 · min^–1). The two systems were distinguished as two separate entities by negative reciprocal competition, and on the basis of differential response to temperature and phenazine methosulfate. Mössbauer studies revealed that cells fed with either⁵⁷FeCl₃ or⁵⁷FeCl₂ accumulated unknown ferric and ferrous binding metabolites.     

Rat osseous plate alkaline phosphatase: mechanism of action of manganese ions

Polidocanol-solubilized osseous plate alkaline phosphatase was modulated by manganese ions in a similar way as by zinc ions. For concentrations up to 1.0 nm, the enzyme was stimulated by manganese ions, showing site-site interactions (n = 2.2). However, larger concentrations (> 0.1 m) were inhibitory. Manganese ions could play the role of zinc ions stimulating the enzyme synergistically in the presence of magnesium ions (K _d = 7.2 m; V = 1005.5 U mg^–1). Manganese ions could also play the role of magnesium ions, stimulating the enzyme synergistically in the presence of zinc ions (K _d = 2.2 m; V = 1036.7 U mg^–1). However, manganese ions could not substitute for zinc and magnesium at the same time since ion assymetry is necessary for full activity of the enzyme. A steady-state kinetic model for the modulation of enzyme activity by manganese ions is proposed.     

Supply and compartmentalization of potassium in mesophyll cells of the needles of spruce,Picea abies (L.) Karst.

Summary The water and potassium content and the relative vacuolar volume ( = V_vacuole/V_cell) of mesophyll cells of the needles of healthy 21-yearold spruce trees [Picea abies (L.) Karst.] were determined. In 5-year-old needles was 0.626 ± 0.178 (ovx ± SD). Potassium concentrations in the bulk tissue water ranged from about 65 to 105 mM. Simulations were made using this information and a simple two-compartmental model of the cell with the bulk cytoplasm and the vacuole and assuming that the minimum cytoplasmic and vacuolar K⁺ concentrations are 100–150 mM and 10–15 mM respectively. It is shown that a K⁺ content of needles below 50 mmol/1 tissue water would be precarious for maintenance of normal physiological and metabolic performance.     

Action potentials inAcetabularia: Measurement and simulation of voltage-gated fluxes

Summary Amounts and temporal changes of the release of the tracer ions K⁺ (⁸⁶Rb⁺),²²Na⁺, and³⁶Cl^– as well as of H⁺ in the course of action potentials inAcetabularia have been recorded. New results and model calculations confirm in quantitative terms the involvement of three major ion transport systemsX in the plasmalemma: Cl^– pumps, K⁺ channels, and Cl^– channels (which are marked in the following by the prefixes,P, K andC) with their equilibrium voltages _X V _e and voltage/time-dependent conductances, which can be described by the following, first approximation. Let the maximum (ohmic) conductance of each of the three populations of transporter species be about the same (_P L, _KL,_C L=1) but voltage gating be different: the pump ( _{p V e} about –200 mV) being inactivated (open,oclosed,c) at positive going transmembrane voltages,V _m; the K⁺ channels (_K V _e about –100 mV) are inactivated at negative goingV _m; and the Cl^– channels (_C V _e: around 0 mV), which are normally closed (c) at a restingV _m (near_PV_e) go through an intermediate open (o) state at more positiveV _m before they enter a third shut state (s) in series. Model calculations, in which voltage sensitivities are expressed by the factorf=exp(V _mF/(2RT)), simulate, the action potential fairly well with the following parameters (_PK_co10/f ks^–1,_PK_oc1000·f ks^–1,_KK_co200·f ks^–1,_Kk_oc2/f ks^–1,_cK_co500·f ks^–1,_CK_oc5/f ks^–1,_CK_so0.1/f ks^–1,_Ck_os20·f ks^–1). It is also shown that the charge balance for the huge transient Cl^– efflux, which frequently occurs during an action potential, can be accounted for by the observation of a corresponding release of Na⁺.     

Transport of glycine-betaine by Listeria monocytogenes

Uptake of [¹⁴C]glycine-betaine by Listeria monocytogenes was stimulated by NaCl with optimal stimulation at 0.4–0.5 M. The glycine-betaine transport system had a K _m of 22 M and a V _max of 11.7 nmol^-1 min^-1 mg^-1 protein when grown in the absence of NaCl. When grown in the presence of 0.8 M NaCl the V _max increased to 27.0 nmol^-1 min^-1 mg^-1 protein in 0.8 M NaCl. At NaCl concentrations above 0.5 M the uptake rate of glycine-betaine was reduced. Measurement of intracellular K⁺ concentrations and fluorescent dye quenching indicated that higher NaCl concentrations also led to a decrease in the electrochemical potential difference across the cytoplasmic membrane. Uptake of glycine was also observed, but this was not stimulated by NaCl.     

Electrical properties of the plasma membrane of microplasmodia ofPhysarum polycephalum

Summary Microplasmodia ofPhysarum polycephalum have been investigated by conventional electrophysiological techniques. In standard medium (30mm K⁺, 4mm Ca⁺⁺, 3mm Mg⁺⁺, 18mm citrate buffer, pH 4.7, 22°C), the transmembrane potential differenceV _m is around –100 mV and the membrane resistance about 0.25 m².V _m is insensitive to light and changes of the Na⁺/K⁺ ratio in the medium. Without bivalent cations in the medium and/or in presence of metabolic inhibitors (CCCP, CN^–, N ₃ ^– ),V _m drops to about 0 mV. Under normal conditions,V _m is very sensitive to external pH (pH _o ), displaying an almost Nernstian slope at pH _o =3. However, when measured during metabolic inhibition,V _m shows no sensitivity to pH _o over the range 3 to 6, only rising (about 50 mV/pH) at pH _o =6. Addition of glucose or sucrose (but not mannitol or sorbitol) causes rapid depolarization, which partially recovers over the next few minutes. Half-maximal peak depolarization (25 mV with glucose) was achieved with 1mm of the sugar. Sugar-induced depolarization was insensitive to pH _o . The results are discussed on the basis of Class-I models of charge transport across biomembranes (Hansen, Gradmann, Sanders and Slayman, 1981,J. Membrane Biol. 63:165–190). Three transport systems are characterized: 1) An electrogenic H⁺ extrusion pump with a stoichiometry of 2 H⁺ per metabolic energy equivalent. The deprotonated form of the pump seems to be negatively charged. 2) In addition to the passive K⁺ pathways, there is a passive H⁺ transport system; here the protonated form seems to be positively charged. 3) A tentative H⁺-sugar cotransport system operates far from thermodynamic equilibrium, carrying negative charge in its deprotonated states.     

Characteristics of ouabain binding to isolated trout hepatocytes

Summary Na⁺, K⁺ exchanges were studied in isolated hepatocytes of the rainbow trout, Salmo gairdneri. Ouabain at 10^–4 M produced maximal inhibition (95%) of K⁺ uptake and enhanced intracellular Na⁺ accumulation, showing that active fluxes account for a very large proportion of Na⁺ and K⁺ exchanges. Inhibition of the Na–K pump by ouabain was significant at low concentrations (10^–8 M). When external K⁺ concentration was reduced from 7 mM to 0.5 mM, half maximum inhibition (IC₅₀) of K⁺ uptake was obtained at a 22-fold lower concentration of ouabain confirming that ouabain and potassium compete at the same pump site. Time-course analysis of [³H]ouabain binding indicated a two-component kinetics: one component saturable and dependent on K⁺ concentration in the medium, the other linear and independent of external K⁺. The ouabain binding site number, determined by Scatchard plots, remained constant (ca. 2.5·10⁵ per cell) and independent of the external K⁺ concentration (7, 0.5 or 0 mM), while the dissociation constant (K_D) decreased from 4.2 M to 7.3 nM when K⁺ was removed from the Hank's medium. These ouabain binding sites are characterized by an exceptionally low turnover rate (400 min^–1), as estimated from ouabain-sensitive K⁺ flux, in comparison to those described in other cell types of higher vertebrates. At each external K⁺ concentration studied, the inhibition of K⁺ uptake and ouabain binding measured as a function of ouabain concentration indicated a strict correlation between the degree of K pump inhibition and the amount of bound glycoside.     

Transport of potassium inChara australis: II. Kinetics of a symport with sodium

Summary An electrogenic K⁺–Na⁺ symport with a high affinity for K⁺ has been found inChara (Smith & Walker, 1989). Under voltage-clamp conditions, the symport shows up as a change in membrane current upon adding either K⁺ or Na⁺ to the bathing medium in the presence of the other. Estimation of kinetic parameters for this transport has been difficult when using intact cells, since K⁺–Na⁺ current changes show a rapid falling off with time at K⁺ concentrations above 50 m. Cytoplasm-enriched cell fragments are used to overcome this difficulty since they do not show the rapid falling off of current change seen with intact cells. Current-voltage curves for the membrane in the absence or presence of either K⁺ or Na⁺ are obtained, yielding difference current-voltage curves which isolate the symport currents from other transport processes. The kinetic parameters describing this transport are found to be voltage dependent, withK _m for K⁺ ranging from 30 down to 2 m as membrane potential varies from –140 to –400 mV, andK _m for Na⁺ ranging between 470 and 700 m over a membrane potential range of –140 to –310 mV.Two different models for this transport system have been investigated. One of these involves the simultaneous transport of both the driver and substrate ions across the membrane, while the other allows for the possibility of the two ions being transported consecutively in two distinct reaction steps. The experimental results are shown to be consistent with either of these cotransport models, but they do suggest that binding of K⁺ occurs before that of Na⁺, and that movement of charge across the membrane (the voltage-dependent step) occurs when the transport protein has neither K⁺ nor Na⁺ bound to it.     

Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased bothK _m andV _max for GABA uptake, with an increase in theV _max/K _m ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 M GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 M ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 M veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na⁺, K⁺-ATPase acting as an ion pump.     

Kinetics of malate transport and decomposition in acid soils and isolated bacterial populations: The effect of microorganisms on root exudation of malate under Al stress

The kinetics and characteristics of malate degradation were studied in four acid soils ranging in both pH (4.30 to 5.00) and vegetation type. The breakdown of malate was rapid in all soils with a half life of approximately 1.7 h, K_m of 1.7 mM and V_max of 70 nmol g^–1 soil h^–1. No relationship was observed between malate decomposition rate and pH. Co-metabolism studies with other C and N substrates (glucose, glycine, glutamate, citrate and succinate) indicated that the microorganisms were not N limited and competitive inhibition of malate breakdown was only observed in the presence of succinate. Studies with isolated mixed bacterial cultures indicated that the bacterial malate uptake was mediated by an energy dependent, dicarboxylate transporter which can be inhibited by succinate and is independent of pH between pH 5.0 and 7.0. The K_m and V_max parameters ranged from 279–955 M and 0.1–17 mol mg^–1 protein h^–1 for the mixed bacterial cultures depending on the bacteria's previous C source. The results indicate that in acid topsoils where microbial populations are high, the microbes may provide a considerable sink for organic acids. If organic acids are being released by roots in response to an environmental stress (e.g. Al toxicity, P deficiency) it can be expected that the efficiency of these root mediated metal resistance mechanisms will be markedly reduced by rapid microbial degradation.     

Potassium-dependent,bipolar gating of K+ channels in guard cells

Summary Guard cells of higher plants control transpirational water loss and gas exchange for photosynthesis by opening and closing pores in the epidermis of the leaf. To power these turgordriven movements, guard cells accumulate (and lose) 200 to 400mm (1 to 3 pmol/cell) K⁺, fluxes thought to pass through K⁺ channels in the guard cells plasma membrane. Steady-state current-voltage (I–V) relations of intactVicia guard cells frequently show large, outward-going currents at potentials approaching 0 mV. Since this current could be carried by K⁺ channels, its pharmacology and dependence on external K⁺ (K _v ⁺ ) has been examined under voltage clamp over an extended potential range. Measurements were carried out on cells which showed little evidence of primary electrogenic transport, thus simplifying analyses. Clamping these cells away from the free-running membrane potential (V _m ) revealed an outward-rectifying current with instantaneous and time-dependent components, and sensitive to the K⁺ channel blocker tetraethylammonium chloride. The current declined also under metabolic blockade with NaCN and in the presence of diethylstilbesterol, responses which were attributed to secondary effects of these inhibitors. The putative K⁺ current rose with voltage positive toV _m but it decayed over two voltage ranges, one negative toV _m and one near +100 mV, to give steady-stateI–V relations with two regions of negative (slope) conductance. Voltage-dependent and kinetic characteristics of the current were affected by K _v ⁺ and followed the K⁺ equilibrium potential. Against a (presumably) low background of primary ion transport, the K⁺ current contributed appreciably to charge balance atV _m in 0.1mm as well as in 1 to 10mm K _v ⁺ . Thus, gating of these K⁺ channels compensates for the prevailing K⁺ conditions to ensure net K⁺ movement out of the cell.     

Modelling continuous culture of Rhodospirillum rubrum in photobioreactor under light limited conditions

Rhodospirillum rubrum was grown continuously and photoheterotrophically under light limitation using a cylindrical photobioreactor in which the steady state biomass concentration was varied between 0.4 to 4 kg m^–3 at a constant radiant incident flux of 100 W m^–2. Kinetic and stoichiometric models for the growth are proposed. The biomass productivities, acetate consumption rate and the CO₂ production rate can be quantitatively predicted to a high level of accuracy by the proposed model calculations. Nomenclature: C _X, biomass concentration (kg m^–3) D, dilution rate (h^–1) Ea, mean mass absorption coefficient (m² kg^–1) I , total available radiant light energy (W m^–2) K, half saturation constant for light (W m^–2) R _W, boundary radius defining the working illuminated volume (m) r _X, local biomass volumetric rate (kg m^–3 h^–1) <r _X>, mean volumetric growth rate (kg m^–3 h^–1) V _W, illuminated working volume in the PBR (m^–3). Greek letters: , working illuminated fraction (–) _M, maximum quantum yield (–) bar, mean energetic yield (kg J^–1).     

Properties of the phosphate and phosphite transport systems of Phytophthora palmivora

Germlings of Phytophthora palmivora possess at least two systems for the uptake of inorganic phosphate (P_i). The first is synthesized on germination in medium containing 50 M P_i and has a K_m of approx. 30 M (V_max=7–9 nmol P_i/h·10⁶ cells). The second is synthesized under conditions of P_i-deprivation and has a higher affinity for P_i (K_m=1–2 M), but a lower V_max (0.5–2 nmol P_i/h·10⁶ cells). The fungicide phosphite likewise enters the germlings via two different transport systems, the synthesis of which also depends on the concentration of P_i in the medium. The K_m of the lower affinity system is 3 mM (V_max=20 nmol phosphite/h·10⁶ cells) and that of the higher affinity system is 0.6 mM (V_max=12 nmol/h·10⁶ cells). P_i and phosphite are competitive inhibitors for each other's transport in both systems. However, whereas mM concentrations of phosphite are necessary to inhibit P_i transport, only M concentrations of P_i are required to inhibit phosphite transport. A third system of uptake for P_i also exists, since when phosphate-deprived cells are presented with mM concentrations of P_i, they transport the anion at a very high rate (around 100 nmol/h·10⁶ cells). High rates of transport of phosphite are also observed when these cells are presented with mM concentrations of this anion.     

The effect of prior exposure on sodium uptake in tadpoles exposed to low pH water

Summary Bullfrog tadpoles (Rana catesbeiana) were pre-exposed (7d) to 5 different solutions which varied in ionic composition and pH. Unidirectional sodium influx was then measured over a range of sodium concentrations (50–2000 M) and at 2 pH's (4.0 and 5.8). Tadpoles pre-treated in either distilled water or pH 4.0 soft water exhibited higher sodium influx than animals pre-treated in pH 5.8 soft water. Pre-exposure to solutions high in calcium or sodium reduced sodium influx. Tadpoles pre-treated in pH 4.0 soft water exhibited an increasedV _max for sodium transport, whileK _m was unchanged relative to tadpoles pre-treated in pH 5.8 soft water. In contrast,K _m was increased andV _max was unchanged in tadpoles pre-exposed to high concentrations of calcium or sodium. Within each pre-exposure treatment, influx was inhibited in pH 4.0 test water relative to pH 5.8 test water. However, the magnitude of inhibition was lessened with increasing external concentrations of sodium.Abbreviations ASW artificial soft water - DBM dry body mass - DW distilled water     

Determination of charge,stoichiometry and reaction constants fromI–V curve studies on a K+ transporter inNitella

Summary InNitella cells with low pump activity, the electrical characteristics of membrane transport are mainly determined by K⁺ transport. Current-voltage curves were measured at outside K⁺ concentrations ranging from 0.1 to 100 mol m^–3. Above 1 mol m^–3, current saturated at positive and at very negative potentials. It was found that theseI–V curves could be fitted by a Class 1, case 1 reaction kinetic model, which is a cyclic reaction scheme with one pair of rate constants sensitive to membrane potential (Class I) and neutral transporter (or electrically charged substrate-transporter complex, case I). The analysis revealed the relative rate constants of a 3-state model. From the linear dependence of the rate constant of substrate binding (k ₃₂) on [K⁺] _a the stoichiometry of 1 K⁺/cycle was obtained. The complex transporter substrate is very unstable (very high value ofK ₂₃) resulting in a very low density of this state and in what can be called Mitchellian behavior; namely, the driving forces resulting from the electrical and from the concentration gradient can hardly be distinguished.     

Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem

Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)^–1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52–55°C. The enzyme retained 97% activity after a 24-h incubation at 55°C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1–5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5–8 M), significantly affected phytase activity. The maximum hydrolysis rate (V_max) and apparent Michaelis-Menten constant (K_m) were 1,074 IU/mL and 606 M, respectively, with a catalytic turnover number of 3×10⁵ s^–1 and catalytic efficiency of 3.69×10⁸ M^–1 s^–1.     

Transport of malic acid in Leuconostoc oenos strains defective in malolactic fermentation: a model to evaluate the kinetic parameters

Two Leuconostoc oenos mutant strains unable to metabolize malic acid were differentiated by [U-¹⁴C]-labelled L-malate transport assays into a malolactic-enzyme-deficient mutant and a malate-transport-defective mutant. A mathematical analysis of the data from L-malic acid uptake at three pH values (5.2, 4.5, and 3.2) in the malolactic-enzyme-deficient strains suggest two simultaneous uptake mechanisms, presumably a carrier-mediated transport and a passive diffusion for the anionic and the undissociated forms of the acid, respectively. The apparent affinity constant (K _m t) and the maximal rate (V _m t) values for L-malate active transport were, 12 mM and 43 mol L-malate·mg^–1·s^–1, respectively. Active transport was constitutive and strongly inhibited by protonophores and by ATPase inhibitors. L-Lactic acid appeared to inhibit L-malic acid transport, suggesting an L-lactate/L-malate exchange. At pH values of 4.5 or above, the passive diffusion of L-malic acid was negligible. However, at pH 3.2, the mean pH of wine, the permeability of the cells to the undissociated acid by simple diffusion could represent more than 50% of total L-malic acid uptake, with a diffusion constant (K _D) of 0.1 s^–1. Correspondence to: C. Divies     

Ion permeation in a K+ channel inChara australis: Direct evidence for diffusion limitation of ion flow in a maxi-K channel

Summary We report a study of a potassium-selective channel in the membrane delineating cytoplasmic drops fromChara australis. The relatively large conductance (170 pS in 150 mol/m³ (mm) KCl), high ion selectivity (P _Cl/P _K=0.015±0.01) and voltagedependent kinetics of this channel indicate that it is a type of maxi-K channel commonly found in animal cells but not previously detected in any plant cell.The current-voltage (I/V) characteristic of these channels was examined in drop-attached and in excised outside-out patches using the patch-clamp technique, over the unusually large voltage range of –250 to 200 mV. TheI/V characteristic is nonlinear and shows saturation at extreme voltages; the current also saturates at high [K⁺]. In solutions with symmetrical KCl concentrations the saturation behavior of the current is asymmetrical. The permeability of the channel depends on whether it is observed in excised or in drop-attached membrane patches.Here we investigate the main factors affecting the permeation of K⁺ ions through this maxi-K channel. We present the first direct evidence for the importance of diffusion external to the pore in limiting ion flow through maxi-K channels. The data are consistent with an ion translocation mechanism whose current is limited (i) at high voltages by ion diffusion external to the pore and (ii) at high [K⁺] by the maximum transport rate of the channel. We fit the data to a diffusion-limited pore model in which the pore exhibits saturation described by Michaelis-Menten kinetics with aK _m=50±25 mol/m³ andG _max=300±20 pS.