Catabolite interconversion of glucose transport systems in the yeast Candida wickerhamii

Candida wickerhamii IGC 3244 growing in glucose medium transported glucose by facilitated diffusion (at 25°C and pH 5, the K_s value was 1.7 mM and the V_max value was 1.6 mmol/h per g dry wt.), while cells grown under derepressed conditions produced a glucose proton symport (at 25°C and pH 5, the K_s value was 0.18 mM and the V_max value was 1.8–1.9 mmol/h per g dry wt.). In each case, the Lineweaver-Burk plot of initial uptake rates was linear, indicating the presence of a single system. In buffer with 2% glucose, the symport suffered catabolite inactivation while the facilitated diffusion system emerged concomitantly in such a way that the combined V_max remained nearly constant. During the conversion process, the Linewaver-Burk plots were biphasic, indicating the transitory co-existence of the two systems. A model is proposed that envisions the proton symport as composed of the facilitated diffusion system in association with (an)other transport protein(s), the latter being sensitive to carbon catabolite repression and inactivation.     

Catabolite regulation in a diauxic strain and a nondiauxic strain of Streptococcus bovis

Streptococcus bovis JB1 utilized glucose preferentially to lactose and grew diauxically, but S. bovis 581AXY2 grew nondiauxically and used glucose preferentially only when the glucose concentration was very high (greater than 5 mM). As little as 0.1 mM glucose completely inhibited the lactose transport of JB1. The lactose transport system of 581AXY2 was at least tenfold less sensitive to glucose, and 1 mM glucose caused only a 50% inhibition of lactose transport. Both strains had phosphotransferase systems (PTSs) for glucose and lactose. The glucose PTSs were constitutive, but little lactose PTS activity was detected unless lactose was the energy source for growth. JB1 had approximately threefold more glucose PTS activity than 581AXY2 (1600 versus 600 nmol glucose (mg protein)⁻¹(min)⁻¹. The glucose PTS of JB1 showed normal Michaelis Menten kinetics, and the affinity constant (K _s ) was 0.12 mM. The glucose PTS of 581AXY2 was atypical, and the plot of velocity versus velocity/substrate was biphasic. The low capacity system had a K_s of 0.20 mM, but the K_s of the high capacity system was greater than 6 mM. On the basis of these results, diauxic growth is dependent on the affinity of glucose enzyme II and the velocity of glucose transport. Received: 22 January 1996 / Accepted: 18 March 1996     

Transport of hemicellulose monomers in the xylose-fermenting yeastCandida shehatae

Summary Cells ofCandida shehatae repressed by growth in glucose- or D-xylose-medium produced a facilitated diffusion system that transported glucose (K _s±2 mM,V _max±2.3 mmoles g⁻¹ h⁻¹),d-xylose (K _s±125 mM,V _max±22.5 mmoles g⁻¹ h⁻¹) and D-mannose, but neither D-galactose norl-arabinose. Cells derepressed by starvation formed several sugar-proton symports. One proton symport accumulated 3-0-methylglucose about 400-fold and transported glucose (K _s±0.12 mM,V _max ± 3.2 mmoles g⁻¹ h⁻¹) andd-mannose, a second proton symport transportedd-xylose (K _s± 1.0 mM,V _max 1.4 mmoles g⁻¹ h⁻¹) andd-galactose, whilel-arabinose apparently used a third proton symport. The stoicheiometry was one proton for each molecule of glucose or D-xylose transported. Substrates of one sugar proton symport inhibited non-competitively the transport of substrates of the other symports. Starvation, while inducing the sugar-proton symports, silenced the facilitated diffusion system with respect to glucose transport but not with respect to the transport of D-xylose, facilitated diffusion functioning simultaneously with thed-xylose-proton symport.     

High-affinity transport and phosphorylation of 2-deoxy-D-glucose in synaptosomes

2-Deoxy-d -glucose (2 DG) entered synaptosomes (from rat brain) by a high-affinity, Na⁺-independent glucose transport system with a K_m, of 0.24 mM. 3-O-methyl-glucose, D-glucose, and phloretin were competitive inhibitors of 2-DG transport with K_i's of 7 mM, 64 μM, and 0·75 μM, respectively. Insulin was without effect. 2-DG uptake was also saturable at high substrate concentrations with an apparent low affinity K_m, of 75 mM, where the K_l, for glucose was 17.5 mM. We are not certain whether the rate-limiting step for the low-affinity uptake system is attributable to transport or phosphorylation. However, the high-affinity glucose transport system probably is a special property of neuronal cell membranes and could be useful in helping to distinguish separated neurons from glial cells.     

Glucose transport and metabolism in Gymnodinium breve

Florida's red tide organism, Gymnodinium breve, utilized exogenous glucose in the light for the synthesis of cellular components. Glucose was not taken up in the dark. Kinetic parameters for glucose uptake include a K_FD of 11 μM and a V_max of 1 × 10^?10 mol of glucose taken up/mg cellular protein/hr. Glucose uptake was competitively inhibited by phloridzin (K_i = 40 μM), mannose (K_i = 12O μM), and 2-deoxy-d-glucose (K_i = 190 μM) and non-competitively inhibited by galactose (K_i = 125 μM). Kinetics and inhibition of glucose uptake are consistent with a facilitated diffusion transport system.     

β-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides

Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously over-expressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-β-d-galactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLM-family type, consisting of a small subunit of 35 kDa and a large subunit of 72 kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 °C, respectively. The enzyme is most stable in the pH range of 6.5-8.0. The K_m, k_cat and k_cat/K_m values for oNPG and lactose are 0.9 mM, 92 s⁻¹, 130 mM⁻¹ s⁻¹ and 29 mM, 98 s⁻¹, 3.3 mM⁻¹ s⁻¹, respectively. The L. plantarum β-galactosidase possesses a high transgalactosylation activity and was used for the synthesis of prebiotic galacto-oligosaccharides (GOS). The resulting GOS mixture was analyzed in detail, and major components were identified by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as capillary electrophoresis. The maximal GOS yield was 41% (w/w) of total sugars at 85% lactose conversion (600 mM initial lactose concentration). The enzyme showed a strong preference for the formation of β-(1→6) linkages in its transgalactosylation mode, while β-(1→3)-linked products were formed to a lesser extent, comprising ∼80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were β-d-Galp-(1→6)-d-Lac, accounting for 34% of total GOS, and β-d-Galp-(1→6)-d-Glc, making up 29% of total GOS.     

l-Proline transport in Saccharomyces cerevisiae

Transport of l-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a K_T of 31 μM and J_max of 40 nmol · s^?1 · (g dry wt.)^?1, the other with K_T > 2.5 mM and J_max of 150–165 nmol · s^?1 · (g dry wt.)^?1, The kinetic properties of the high-affinity system were studied in detail. It proved to be highly specific, the only potent competitive inhibitors being (i) l-proline and its analogs l-azetidine-2-carboxylic acid, sarcosine, d-proline and 3,4-dehydro-dl-proline, and (ii) l-alanine. The other amino acids tested behaved as noncompetitive inhibitors. The high-affinity system is active, has a sharp pH optimum at 5.8–5.9 and, in an Arrhenius plot, exhibits two inflection points at 15°C and 20–21°C. It is trans-inhibited by most amino acids (but probably only the natural substrates act in a trans-noncompetitive manner) and its activity depends to a considerable extent on growth conditions. In cells grown in a rich medium with yeast extract maximum activity is attained during the stationary phase, on a poor medium it is maximal during the early exponential phase. Some 50–60% of accumulated l-proline can leave cells in 90 min (and more if washing is done repeatedly), the efflux being insensitive to 0.5 mM 2,4-dinitrophenol and uranyl ions, to pH between 3 and 7.3, as well as to the presence of 10–100 mM unlabeled l-proline in the outside medium. Its rate and extent are increased by 1% d-glucose and by 10 μg nystatin per ml.     

Uncoupler-Resistant Glucose Uptake by the Thermophilic Glycolytic Anaerobe Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum)

The transport of glucose across the bacterial cell membrane of Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum) Rt8.B1 was governed by a permease which did not catalyze concomitant substrate transport and phosphorylation and thus was not a phosphoenolpyruvate-dependent phosphotransferase. Glucose uptake was carrier mediated, could not be driven by an artificial membrane potential (Δψ) in the presence or absence of sodium, and was not sensitive to inhibitors which dissipate the proton motive force (Δp; tetrachlorosalicylanilide, N,N-dicyclohexylcarboiimide, and 2,4-dinitrophenol), and no uptake of the nonmetabolizable analog 2-deoxyglucose could be demonstrated. The glucokinase apparent K_m for glucose (0.21 mM) was similar to the K_t (affinity constant) for glucose uptake (0.15 mM), suggesting that glucokinase controls the rate of glucose uptake. Inhibitors of ATP synthesis (iodoacetate and sodium fluoride) also inhibited glucose uptake, and this effect was due to a reduction in the level of ATP available to glucokinase for glucose phosphorylation. These results indicated that T. thermosulfuricus Rt8.B1 lacks a concentrative uptake system for glucose and that uptake is via facilitated diffusion, followed by ATP-dependent phosphorylation by glucokinase. In T. thermosulfuricus Rt8.B1, glucose is metabolized by the Embden-Meyerhof-Parnas pathway, which yields 2 mol of ATP (G. M. Cook, unpublished data). Since only 1 mol of ATP is used to transport 1 mol of glucose, the energetics of this system are therefore similar to those found in bacteria which possess a phosphotransferase.     

Substrate and endproduct regulation of cellobiose uptake by Candida wickerhamii

Summary Cellobiose-grown cells of Candida wickerhamii transported cellobiose as glucose by a glucose-proton symport after previous hydrolysis of the disaccharide by an exocellular -glucosidase. Both the symport and the -glucosidase were subject to glucose-induced repression and inactivation while glucose also acted as a competitive inhibitor of the enzyme (K _i 0.3 mM). Under conditions of glucose repression glucose was transported by facilitated diffusion. Cellobiose acted as a competitive inhibitor of the latter (K _i 75 mM) and is possibly a low-affinity substrate, while it inhibited non-competitively the glucoseproton symport (K _i 80 mM). The affinity of cellobiose for the cell-bound -glucosidase was much higher (K _m 4.2 mM) than for the purified enzyme as reported by others (K _m 67–225 mM). Ethanol reversibly inhibited the two glucose transport systems with exponential non-competitive kinetics. The minimum inhibitory concentrations were about 3% and 4% (w/v) for facilitated diffusion and proton symport while the respective exponential inhibition constants were 0.58 l mol^-1 and 1.65 l mol^-1. Ethanol affected the -glucosidase in a complex way, a major effect was deviation from Michaelis-Menten kinetics for ethanol concentrations higher than 4% (w/v), the Hill coefficient increasing up to 1.8 at 6% (w/v) ethanol.     

Transport of lactate and other short-chain monocarboxylates in the yeast Candida utilis

Summary Lactic acid grown cells of the yeast Candida utilis transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoicheiometry of 1:1. The accumulation ratio at pH 5.5 was about twenty. The symport accepted the following monocarboxylates (K _svalues at 25°C, pH 5.5 in brackets): d-lactate (0.06 mM), l-lactate (0.06 mM), pyruvate (0.03 mM), propionate (0.05 mM) and acetate (0.1 mM). The system was inducible and was subject to glucose repression. The affinity of the symport for lactate was not affected by pH over the range 3–6, while the maximum transport velocity was strongly pH dependent, its optimum pH being around pH 5. Undissociated lactic acid entered the cells by simple diffusion. The permeability for the undissociated acid increased exponentially with pH, the diffusion constant increasing 35-fold when the pH was increased from 3 to 5.5.     

5-thio-D-glucose is an acceptor for UDP-galactose : D-glucose 1-galactosyltransferase.

The glucose analog 5-thio-d-glucose, a potent inhibitor of glucose transport across membranes, was examined as an acceptor and/or inhibitor of lactose synthetase (UDP-galactose: D-glucose 1-galactosyltransferase, EC 2.4.1.22). Thioglucose was an effective acceptor for lactose synthetase with a K_m of 7.4 mM. Under identical conditions the K_m for D-glucose in this reaction was 5.4 mM. Thioglucose was 45 to 50% as effective an acceptor as D-glucose. Thioglucose acted as a pseudo substrate having a different K_m and V_max. Thus, thioglucose could be considered to the be a competitive substrate for lactose synthetase. thetase. The product of the lactose synthetase reaction with thioglucose as an acceptor had a thin-layer chromatographic retardation factor slightly higher than that for lactose. Upon treatment of the reaction product with β-galactosidase, galactose and thioglucose were released. These observations suggest that the product of the lactose synthetase reaction with thioglucose was thiolactose.     

Properties of glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate as an NADH-regeneration unit in enzyme reactors

Glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate (GlcDH-PEG-NAD) was prepared and its kinetic properties as an NADH-regeneration unit were investigated. The conjugate has about two molecules of active and covalently linked NAD per tetramer. The specific activity of the enzyme moiety of the conjugate in the presence of exogenous NAD is about 77% of that of the native enzyme, and this decrease is mainly due to the decrease in the V_max value. The conjugate has the same pH-stability profile as the native enzyme and an internal activity of 0.26s⁻¹ (as a monomer); its NAD moiety has similar coenzyme activity to poly(ethylene glycol)-bound NAD. These results indicate that GlcDH-PEG-NAD can be used as an NADH-regeneration unit for many dehydrogenase reactions. The coupled reaction of GlcDH-PEG-NAD and lactate dehydrogenase was then studied. The specific activity of the conjugate is 1.1 s⁻¹ (as a tetramer), the recycling rate of the active NAD moiety is 0.54 s⁻¹, and the apparent K_m value for glucose is 24 mM. Kinetically, lactate dehydrogenase behaves like a substrate with an apparent K_m value of 1.8 units·ml⁻¹ in this coupled reaction system with low coenzyme concentration. l-Lactate was continuously produced from pyruvate in a reactor with a PM10 ultrafiltration membrane, and containing GlcDH-PEG-NAD and lactate dehydrogenase. GlcDH-PEG-NAD proved to be applicable in continuous enzyme reactors as an NADH-regeneration unit with a large molecular size.     

Lactose transport in Escherichia coli cells Dependence of kinetic parameters on the transmembrane electrical potential difference

We determine the kinetic parameters $\text{V}$ and $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ of lactose transport in Escherichia coli cells as a function of the electrical potential difference (Δψ) at pH 7.3 and $\text{Δ}\text{pH}\text{= 0}$. We report that transport occurs simultaneously via two components: a component which exhibits a high $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ (larger than 10 mM) and whose contribution is independent of Δψ, a component which exhibits a low $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ independent of Δψ (0.5 mM) but whose $\text{V}$ increases drastically with increasing Δψ. We associate these components of lactose transport with facilitated diffusion and active transport, respectively. We analyze the dependence upon Δψ of $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ and $\text{V}$ of the active transport component in terms of a mathematical kinetic model developed by Geck and Heinz (Geck, P. and Heinz, E. (1976) Biochim. Biophys. Acta 443, 49–63). We show that within the framework of this model, the analysis of our data indicates that active transport of lactose takes place with a H⁺/lactose stoichiometry greater than 1, and that the lac carrier in the absence of bound solutes (lactose and proton(s)) is electrically neutral. On the other hand, our data relative to facilitated diffusion tend to indicate that lactose transport via this mechanism is accompanied by a H⁺/lactose stoichiometry smaller than that of active transport. We discuss various implications which result from the existence of H⁺/lactose stoichiometry different for active transport and facilitated diffusion.     

Characterization of a robust glucose 1-dehydrogenase,SyGDH, and its application in NADPH regeneration for the asymmetric reduction of haloketone by a carbonyl reductase in organic solvent/buffer system

To realize coenzyme regeneration in the reduction of haloketones, a codon-optimized gene Sygdh encoding glucose 1-dehydrogenase (SyGDH) was synthesized based on the putative GDH gene sequence (Ta0897) in Thermoplasma acidophilum genomic DNA, and expressed in E. coli BL21(DE3). Recombinant SyGDH was purified to homogeneity by affinity chromatography with the specific activity of 86.3 U/mg protein towards D-glucose at the optimum pH and temperature of 7.5 and 40 °C. It was highly stable in a pH range of 4.5–8.0 and at 60 °C or below, and resistant to various organic solvents. The K_m and catalytic efficiency (k_cat/K_m) of SyGDH towards NADP+ were 0.67 mM and 104.0 mM⁻¹ s⁻¹, respectively, while those towards NAD+ were 157.9 mM and 0.64 mM⁻¹ s⁻¹, suggesting that it preferred NADP+ as coenzyme to NAD+. Additionally, using whole cells of E. coli/Sygdh-Sys1, coexpressing SyGDH and carbonyl reductase (SyS1), as the biocatalyst, the asymmetric reduction of 60 mM m-chlorophenacyl chloride coupled with the regeneration of NADPH in situ was conducted in DMSO/phosphate buffer (2:8, v/v) system, producing (R)-2-chloro-1-(3-chlorophenyl)ethanol with over 99.9% ee_p and 99.2% yield. Similarly, the reduction of 40 mM α-bromoacetophenone in n-hexane/buffer (6:4, v/v) biphasic system produced (S)-2-bromo-1-phenylethanol with over 99.9% ee_p and 98.3% yield.     

Recombinant glucose oxidase from Penicillium amagasakiense for efficient bioelectrochemical applications in physiological conditions

GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (k_cat/K_M(glucose) = 93 μM⁻¹ s⁻¹) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (k_cat/K_M(glucose) = 27 μM⁻¹ s⁻¹). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FM_ox) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependant FM_ox reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(k_cat/K_M(FM_ox) = 27 μM⁻¹ s⁻¹ and 17 μM⁻¹ s⁻¹ respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions.     

Regulation of glucose transport in Candida utilis

The transport systems for glucose present in Candida utilis cells, growing in batch and continuous cultures on several carbon sources, have been studied. Two different systems were found: a proton symport and a facilitated diffusion system. The high-affinity symport (Km for glucose about 15 microM) transported one proton per mole of glucose and was partially constitutive, appearing in cells grown on gluconeogenic substrates such as lactate, ethanol and glycerol. It was also induced by glucose concentrations up to 0.7 mM and repressed by higher ones. The level of repression depended on the external glucose concentration at which cells had grown in a way similar to that shown by the maltose-uptake system, so both systems seem to be under a common glucose control. Initial uptake by facilitated diffusion, the only transport system present in cells growing at glucose concentrations higher than 10 mM, showed a complex kinetic dependence on the extracellular glucose concentration. This could be explained either by the presence of at least two different systems simultaneously active, one with a Km around 2 mM and the other with a Km of about 1 M, or by the allosteric or hysteretic behaviour of a single carrier whose apparent Km would oscillate between 2 and 70 mM.     

Effect of magnesium ions on the high-affinity binding of eosin to the (Na+ + K+)-ATPase

(1) The fluorescence of eosin Y in the presence of (Na⁺ + K⁺)-ATPase is enhanced by Mg²⁺. The enhancement by Mg²⁺ is larger than that obtained with Na⁺ (Skou, J.C. and Esmann, M. (1981) Biochim. Biophys. Acta 647, 232–240). Mg²⁺ shifts the excitation maximum from 518 to 524 nm, the emission maximum from 538 to 542 nm. Also a shoulder appears at about 490 nm on the excitation curve, as was also observed with Na⁺. (2) The Mg²⁺-dependent enhancement of fluorescence can be reversed by K⁺ as well as by ATP. In the presence of Mg²⁺ + P_i (i.e. under conditions of phosphorylation), the fluorescence enhancement can be reversed by ouabain. With Mg²⁺ and a low concentation of K⁺ (i.e. conditions for vanadate binding), the enhancement of fluorescence can be reversed by vanadate. (3) There is a low-affinity binding of eosin which increases with the Mg²⁺ concentration. This is observed as a slight increase in the fluorescence when the excitation wavelength is above 520 nm. The low-affinity binding is K⁺-, ATP-, ouabain- and vanadate-insensitive. (4) Scatchard analysis of the binding experiments suggests that there are two high-affinity eosin-binding sites per ³²P-labelling site in the presence of 5 mM Mg²⁺ both of which are ouabain-, vanadate- and ATP-sensitive. With 5 M Mg²⁺ + 0.25 P_i, the K_d values are 0.14 μM and 1.3 μM, respectively. With 5 mM Mg²⁺, 150 mM Na⁺, the K_d values are 0.45 μM and 3.2 μM, respectively. With 5 mM Mg²⁺, the addition of K⁺ gives a pronounced decrease in affinity but does not decrease the number of binding sites (which remains at two per ³²P-labelling site). With 5 mM Mg²⁺ + 150 mM K⁺, the affinities of the two binding sites become identical, at a K_d of 17 μM. (5) The rate of conformational transitions was measured using the stopped-flow method. The rate of the transition from the Mg²⁺-form to the K⁺-form is high. Oligomycin has only a small (if any) effect on the rate. Addition of Na⁺ in the presence of Mg²⁺ does not appreciably change the rate of conversion to the K⁺-form, giving a rate constant of about 110 s^?. However, the addition of oligomycin in the presence of Mg²⁺ + Na⁺ had a profound effect: the rate of conversion to the K⁺-form was decreased by a factor of 2000 to about 0.063 s^?1. This suggests that the conformation with Mg²⁺ alone is different from the conformation with Na⁺ alone. (6) The effects of K⁺, ouabain, vanadate and ATP on the high-affinity binding of eosin suggest that the two eosin molecules bound per ³²P-labelling site are bound to ATP sites.     

Characterization of Xylose Uptake in the Yeasts Pichia heedii and Pichia stipitis

The kinetics of xylose uptake were investigated in the efficient xylose fermenter Pichia stipitis and in the more readily genetically manipulated, strictly respiratory yeast Pichia heedii. Both yeasts demonstrated more than one xylose uptake system, differing in substrate affinity. The K_m of high-affinity xylose uptake in both organisms was similar to that of the efficient high-affinity glucose uptake system of Saccharomyces cerevisiae. In P. heedii, low-affinity xylose uptake was enhanced with growth on 2% but not 0.05% xylose and high-affinity uptake was reduced. In contrast to glucose uptake, xylose uptake in P. heedii was inhibited by dinitrophenol. Dinitrophenol inhibited both glucose and xylose uptake by P. stipitis. Glucose uptake was not inhibited by a 100-fold molar excess of xylose in P. heedii. It is suggested that xylose uptake in P. heedii is via a carrier system(s) distinct from those for glucose uptake.     

A study of the current-voltage relationships of electrogenetic active and passive membrane elements in riccia fluitans

Potassium- and proton-dependent membrane potential, conductance, and current-voltage characteristics (I–V curves) have been measured on rhizoid cells of the liverwort Riccia fluitans. The potential difference (E_m) measured with microelectrodes across plasmalemma and tonoplast is depolarized to the potassium-sensitive diffusion potential (E_D) in the presence of 1 mM NaCN, 1 mM NaN₃, or at temperatures below 6°C. Whereas the temperature change from 25°C to 5°C decreases the membrane conductance (g_m) from 0.71 to 0.43 S ? m^?2, 1 mM NaCN increases g_m by about 25%. The membrane displays potassium-controlled rectification which gradually disappears at temperatures below 5°C. The potassium pathway can be described by an equivalent circuit of a diode and an ohmic resistor in parallel. In the potential interval of E_D ± 100 mV the measured I-V curves roughly fit the theoretical curves obtained from a modified diode equation. ⁸⁶Rb⁺(K⁺)-influx is voltage sensitive: In the presence of 1 mM NaCN, ⁸⁶Rb⁺-influx follows a hyperbolic function corresponding to a low conductance at low [K⁺]_o and high conductance at high [K⁺]_o. On the contrary ⁸⁶Rb⁺-influx is linear with [K⁺]_o when pump activity is normal. It is believed that there are two K⁺-transport pathways in the Riccia membrane, one of which is assigned to the low conductance (0.2 S · m^?2), the other to a temperature-dependent facilitated diffusion system with a higher conductance (7.7 S · m^?2). The electrogenic pump essentially acts as a current source and consumes about 39% of the cellular ATP-turnover. In the presence of 30 μM CCCP the saturation current of 0.1 A · m^?2 is doubled to about 0.2 A · m^?2, and the electromotive force of ?360 mV switches to ?250 mV. It is suggested that this may be due to a change in stoichiometry from one to two transported charges per ATP hydrolyzed.