The influence of phosphate concentration on the kinetics of uptake by maize roots

In an experiment with native maize roots depending on different phosphorus concentration in the external solution (0.001 … 50 mM P), the multiphasic character of the kinetics of phosphate uptake has been stated. The single phases are characterized by the different values of K_m and V_max. In the wide range of concentrations the isotherm of the phosphate uptake has five evident phases. The character of kinetics for the uptake of phosphate is analogical to the kinetics of the enzymatic reactions described by the Michaelis-Menten equation. On the other hand the linear dependence for the inactivated root was determined,i.e. the uptake of phosphate versus different phosphorus concentration in the external solution. The graphic representation of the logarithmic values for the phosphorus taken up versus the different phosphorus concentration in the external solution gives the biphasic course including concentration less than 1.0 mM P and more than 1.0 mM P. Within the framework of the concentration range the following values of V_max, K_m and ϕ_in were calculated under the conditions if the concentration of phosphorus is less than 1.0mMP: V_max = 1.705 μmol P × g^-1h^-1, K_m = 0.057 mM P and ϕ_in = 0.83,i.e. if the concentration of phosphorus is more than 1.0mM P: V_max = 40 μmol P × g^-1 h^-1, K_m = 16.66 mM and ϕ_in = 20. According to these results, the phosphate concentration in the external solution influences the activity of the transport mechanisms concerning their conformative changes which discretely change their working regime of membrane transport. This is also demonstrated in the change of values V_max, K_m and ϕ_in.     

An enzyme rate equation for the overall rate of reaction of gel-immobilized glucose oxidase particles under buffered conditions. II. Two limiting substrates

The overall rate of reaction of gel-immobilized glucose oxidase particles in buffered media has been investigated theoretically under two substrate diffusion limited conditions by the numerical solution of the diffusion equations. It has been found that the Enzyme Rate Equation (Atkinson and Lester), Biotechnol. Bioeng., 16 , 1299 (1974), together with an analytical solution which describes the asymptotic conditions associated with a large particle size, provides an adequate estimation of the values resulting from the numerical solution outside the region of the parameter space defined by 0.4 < M_g′, M₀′ < 10. When the dimensionless parameter (B₀′/B_g′)(M_g′²/M₀′²) is greater than unity the overall rate of reaction is limited principally by the external concentration and when the parameter has a value less than unity, by the external oxygen concentration. The results are generally applicable to enzymes whose kinetics are similar to those of glucose oxidase or for which the equation describing glucose oxidase kinetics provides an adequate curve-fit of experimental data.     

A theory of cell hydration governed by adsorption of water on cell proteins rather than by osmotic pressure

It is postulated that cell hydration is governed by adsorption of water on cell proteins in accord with the Bradley adsorption isotherm, and that the action of a solute in the surrounding solution is to lower the vapor pressure of the solution so that cell water adsorption is decreased by moving down the Bradley isotherm. From these concepts, it is derived that cell volume (V) should be related to solute concentration (x) by the equationV=−E log₁₀ x+F whereE andF are constants which are independent of type of solute. For a non-adsorbed solute this agrees well with experimental data. For solutes which are adsorbed by cell proteins, a correction in the above equation may be necessary at higher solute concentrations, which is shown to be compatible with various experimental data. The types of experiments which are generally used to support the osmotic pressure theory of cell hydration agree equally well with the adsorption theory. The virtue of the adsorption theory is that, unlike the osmotic pressure theory of cell swelling, it is compatible with permeability of the cell membrane to solutes, which has been experimentally observed for various solutes. The opinions and conclusions contained in this report are those of the author. They are not to be construed as necessarily reflecting the views or the endorsement of the Navy Department.     

Oxygen uptake kinetics of Pseudomonas chlororaphis grown in glucose- or glutamate-limited continuous cultures

Oxygen uptake and glucose and glutamate oxidation kinetics of the heterotrophic bacterium Pseudomonas chlororaphis grown in glucose- or glutamate-limited cultures under oxygen-saturating or oxygen-limiting conditions were determined. K _m values for oxygen were 1.4– 5.6 μM. Only in the case of glucose were significantly lower K _m values and enhanced specific oxygen affinity (V _max/K _m) per cell found under oxygen-limiting conditions. Both K _m and specific affinity values for glucose and glutamate oxidation were apparently affected by oxygen concentration, although a statistically significant enhancement of the oxidation kinetics was found only for glutamate. The kinetic data found for P. chlororaphis support the conclusion that the outcome of competition for oxygen with Nitrosomonas europaea in the rhizosphere of oxygen-releasing macrophytes will primarily be determined by oxidation kinetics of the electron donor instead of the oxygen uptake kinetics of the respective organisms. Received: 20 September 1996 / Accepted: 5 February 1997     

Structural and biochemical properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense

Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety. A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE) for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not been published for glucose oxidase from P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M (NH₄)₂SO₄, which stabilised glucose oxidase 100- to 300-fold at 50 °C and pH 7–8, and 2 M KF, which stabilised the enzyme up to 36-fold at 60 °C and pH 6. In sodium acetate buffer, changes in pH (4–6) affected the affinity for glucose but had no effect on the V _max of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in V _max and a 2-fold decrease in K _m were observed. Received: 8 October 1996 / Received revision: 14 January 1997 / Accepted: 17 January 1997     

Nystatin affects zinc uptake in human fibroblasts

The mechanism(s) by which zinc is transported into cells has not been identified. Since zinc uptake is inhibited by reducing the temperature, zinc uptake may depend on the movement of plasma membrane micoenvironments, such as endocytosis or potocytosis. We investigated the potential role of potocytosis in cellular zinc uptake by incubating normal and acrodermatitis enteropathica fibroblasts with nystatin, a sterol-binding drug previously shown to inhibit potocytosis. Zinc uptake was determined during initial rates of uptake (10 min) following incubation of the fibroblasts in 50 μg nystatin/mL or 0.1% dimethyl-sulfoxide for 10 min at 37°C. The cells were then incubated with 1 to 30 μM ⁶⁵zinc. Michaelis-Menten kinetics were observed for zinc uptake. Nystatin inhibited zinc uptake in both the normal and AE fibroblasts. Reduced cellular uptake of zinc was associated with its internalization, not its external binding. In normal fibroblasts, nystatin significantly reduced theK _m 56% and theV _max 69%. In the AE fibroblasts, nystatin treatment significantly reduced theV _max 59%, but did not significantly affect theK _m. The AE mutation alone affected theV _max for cellular zinc uptake. The control AE fibroblasts exhibited a 40% reduction inV _max compared to control normal fibroblasts. We conclude that nystatin exerts its effect on zinc uptake by reducing the velocity at which zinc traverses the cell membrane, possibly through potocytosis. Furthermore, the AE mutation also effects zinc transport by reducing zinc transport.     

Biolayer interferometry of lipid nanodisc‐reconstituted yeast vacuolar H+‐ATPase

Vacuolar H⁺‐ATPase (V‐ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V‐ATPase activity is regulated by reversible disassembly, resulting in cytosolic V₁‐ATPase and membrane‐integral V₀ proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein‐protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein–protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc‐reconstituted V‐ATPase (V₁V₀ND). We show that V₁V₀ND can be immobilized on streptavidin‐coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation.     

Enhanced glucose tolerance in the Brattleboro rat

[Arg⁸]-vasopressin (AVP) plays a crucial role in regulating body fluid retention, which is mediated through the vasopressin V₂ receptor in the kidney. In addition, AVP is involved in the regulation of glucose homeostasis via vasopressin V_1A and vasopressin V_1B receptors. Our previous studies demonstrated that vasopressin V_1A receptor-deficient (V_1AR−/−) and V_1B receptor-deficient (V_1BR−/−) mice exhibited hyperglycemia and hypoglycemia with hypoinsulinemia, respectively. These findings indicate that vasopressin V_1A receptor deficiency results in decreased insulin sensitivity whereas vasopressin V_1B receptor deficiency results in increased insulin sensitivity. In addition, vasopressin V_1A and vasopressin V_1B receptor double-deficient (V_1ABR−/−) mice exhibited impaired glucose tolerance, suggesting that the effects of vasopressin V_1B receptor deficiency do not influence the development of hyperglycemia promoted by vasopressin V_1A receptor deficiency, and that the blockage of both receptors could lead to impaired glucose tolerance. However, the contributions of the entire AVP/vasopressin receptors system to the regulation of blood glucose have not yet been clarified. In this study, to further understand the role of AVP/vasopressin receptors signaling in blood glucose regulation, we assessed the glucose tolerance of AVP-deficient homozygous Brattleboro (di/di) rats using an oral glucose tolerance test (GTT). Plasma glucose and insulin levels were consistently lower in homozygous di/di rats than in heterozygous di/+ rats during the GTT, suggesting that the blockage of all AVP/vasopressin receptors resulting from the AVP deficiency could lead to enhanced glucose tolerance.     

State-Dependent Inhibition of Inactivation-Deficient CaV1.2 and CaV2.3 Channels By Mibefradil

The structural determinants of mibefradil inhibition were analyzed using wild-type and inactivation-modified Ca_V1.2 (α1C) and Ca_V2.3 (α1E) channels. Mibefradil inhibition of peak Ba²⁺ currents was dose- and voltage-dependent. An increase of holding potentials from −80 to −100 mV significantly shifted dose-response curves toward higher mibefradil concentrations, namely from a concentration of 108 ± 21 μm (n= 7) to 288 ± 17 μm (n= 3) for inhibition of half of the Ca_v1.2 currents (IC ₅₀) and from IC ₅₀= 8 ± 2 μm (n= 9) to 33 ± 7 μm (n= 4) for Ca_V2.3 currents. In the presence of mibefradil, Ca_V1.2 and Ca_V2.3 experienced significant use-dependent inhibition (0.1 to 1 Hz) and slower recovery from inactivation suggesting mibefradil could promote transition(s) to an absorbing inactivated state. In order to investigate the relationship between inactivation and drug sensitivity, mibefradil inhibition was studied in inactivation-altered Ca_V1.2 and Ca_V2.3 mutants. Mibefradil significantly delayed the onset of channel recovery from inactivation in CEEE (Repeat I + part of the I–II linker from Ca_V1.2 in the Ca_V2.3 host channel), in EC(AID)EEE (part of the I–II linker from Ca_V1.2 in the Ca_V2.3 host channel) as well as in Ca_V1.2 E462R, and Ca_V2.3 R378E (point mutation in the β-subunit binding motif) channels. Mibefradil inhibited the faster inactivating chimera EC(IS1-6)EEE with an IC ₅₀= 7 ± 1 μm (n= 3), whereas the slower inactivating chimeras EC(AID)EEE and CEEE were, respectively, inhibited with IC ₅₀= 41 ± 5 μm (n= 4) and IC ₅₀= 68 ± 9 μm (n= 5). Dose-response curves were superimposable for the faster EC(IS1-6)EEE and Ca_V2.3, whereas intermediate-inactivating channel kinetics (CEEE, Ca_V1.2 E462R, and Ca_V1.2 E462K) were inhibited by similar concentrations of mibefradil with IC ₅₀≈ 55–75 μm. The slower Ca_V1.2 wild-type and Ca_V1.2 Q473K channels responded to higher doses of mibefradil with IC ₅₀≈ 100–120 μm. Mibefradil was also found to significantly speed up the inactivation kinetics of slower channels (Ca_V1.2, CEEE) with little effect on the inactivation kinetics of faster-inactivating channels (Ca_V2.3). A open-channel block model for mibefradil interaction with high-voltage-activated Ca²⁺ channels is discussed and shown to qualitatively account for our observations. Hence, our data agree reasonably well with a ``receptor guarded mechanism' where fast inactivation kinetics efficiently trap mibefradil into the channel. Received: 14 March 2001/Revised: 25 June 2001     

Myoglobin and mitochondria: Oxymyoglobin interacts with mitochondrial membrane during deoxygenation

The rates of oxygen uptake by rat liver mitochondria (MC) (native coupled, freshly frozen, and uncoupled by FCCP) have been measured polarographically in the absence (V ₀) or presence (V ₁) of 0.11–0.25 mM sperm whale MbO₂. Under the same standard conditions, the rate of sperm whale MbO₂ deoxygenation (V ₂) has been studied spectrophotometrically in the presence of respiring MC. For freshly frozen MC, the dependence of V ₁ and V ₂ on the overall charge of MbO₂ has been investigated at pH 5.6–7.6, and the influence of other differently charged proteins (apomyoglobin, egg lysozyme, lactalbumin, and BSA) has been studied at pH 7.4. It is shown that the rate of mitochondrial respiration in the presence of MbO₂ increases by 10–30% (V ₁ > V ₀). No myoglobin effect is observed for FCCP-uncoupled MC (V _max does not change). The rate of MbO₂ deoxygenation is equal to the rate of oxygen uptake by mitochondria (V ₂/V ₁ ∼ 1 at pH 7.2–7.5). At varying pH < 7.2, the V ₂ values become markedly higher than V ₁, evidently due to the increased MbO₂ positive charge and its stronger interaction with negatively charged mitochondrial membrane. At pH 7.4, on the contrary, V ₂ is twice lower than V ₁ in the case of negatively charged CM-MbO₂ (pI 5.2), which has carboxymethylated histidines. Positively charged lysozyme (pI 11) strongly inhibits MbO₂ deoxygenation (V ₂) without affecting oxygen uptake by MC (V ₀ and V ₁). At the same time, apomyoglobin (pI 8.5), which is structurally very similar to the holoprotein, and both negatively charged lactalbumin (pI 4.4) and BSA (pI 4.7) have no substantial influence on V ₂ and V ₁. The MC membrane evidently has no specific sites for the interaction with myoglobin. Rather, the protein contacts with phospholipids of the outer membrane during MbO₂ deoxygenation, and electrostatic interactions are of great importance for this process.     

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.     

β-Fructofuranosidase and sucrose phosphorylase of rumen bacterium <Emphasis Type="Italic">Pseudobutyrivibrio ruminis</Emphasis> strain 3

The subject of this study was the fructan and sucrose degrading enzymes of bacterium Pseudobutyrivibrio ruminis strain 3. It was stated that cell extract from bacteria growing on inulin contained β-fructofuranosidase (EC 3.2.1.80 and/or EC 3.2.1.26) and sucrose phosphorylase (EC 2.4.1.7), while the bacteria maintained on sucrose showed only phosphorylase. Partially purified β-fructofuranosidase digested inulooligosaccharides and sucrose to fructose or fructose and glucose, respectively, but was unable to degrade the long chain polymers of commercial inulin and Timothy grass fructan. Digestion rate of inulooligosaccharides fit Michaelis–Menten kinetics with V_max 5.64 μM/mg/min and K_m 1.274%, respectively, while that of sucrose was linear. Partially purified sucrose phosphorylase digested only sucrose. The digestion products were fructose, glucose-1P and free glucose. The reaction was in agreement with Michaelis–Menten kinetics. The V_max were 0.599 and 0.584 μM/mg/min, while K_m were 0.190 and 0.202% for fructose release and glucose-1P formation, respectively, when bacteria grew on inulin. The V_max were, however, 1.37 and 1.023 μM/mg/min, while K_m were 0.264 and 0.156%, if bacteria were grown on sucrose. The free glucose was hardly detectable for the enzyme originated from inulin grown bacteria, but glucose levels ranged from 0.05 to 0.25 μM/mg/min, when cell extract from bacteria grown on sucrose was used. Release of free glucose was observed when no inorganic phosphate was present in reaction mixture.     

Correlation between glucose/fructose discrepancy and hexokinase kinetic properties in different <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine yeast strains

Grape juice contains about equal amounts of glucose and fructose, but wine strains of Saccharomyces cerevisiae ferment glucose slightly faster than fructose, leading to fructose concentrations that exceed glucose concentrations in the fermenting must. A high fructose/glucose ratio may contribute to sluggish and stuck fermentations, a major problem in the global wine industry. We evaluated wine yeast strains with different glucose and fructose consumption rates to show that a lower glucose preference correlates with a higher fructose/glucose phosphorylation ratio in cell extracts and a lower K _m for both sugars. Hxk1 has a threefold higher V _max with fructose than with glucose, whereas Hxk2 has only a slightly higher V _max with glucose than with fructose. Overexpression of HXK1 in a laboratory strain of S. cerevisiae (W303–1A) accelerated fructose consumption more than glucose consumption, but overexpression in a wine yeast strain (VIN13) reduced fructose consumption less than glucose consumption. Results with laboratory strains expressing a single kinase showed that total hexokinase activity is inversely correlated with the glucose/fructose (G/F) discrepancy. The latter has been defined as the difference between the rate of glucose and fructose fermentation. We conclude that the G/F discrepancy in wine yeast strains correlates with the kinetic properties of hexokinase-mediated sugar phosphorylation. A higher fructose/glucose phosphorylation ratio and a lower K _m might serve as markers in selection and breeding of wine yeast strains with a lower tendency for sluggish fructose fermentation.     

Effects of external diffusion and design geometry on the performance of immobilized glucose isomerase reactor system

Using immobilized glucose isomerase, the effects of superficial velocity of the reaction solution flowing through a packed-bed reactor on the apparent kinetic constants of reversible reaction system were studied. The results showed that the apparent kinetic constants, both V_m″ and K_m″, of the forward reaction varied significantly as the superficial velocity is changed, whereas those of the reverse reaction varied only slightly. Using the kinetic data determined experimentally, computer simulation of the enzyme reactor performance was carried out, and the importance of the external mass transfer in the proximity of immobilized-enzyme particles was recognized. The reactor performance, expressed in terms of productivity, was examined as a function of the reactor height-to-diameter ratio, H/D. The productivity of the reactor system goes through a maximum value at a H/D ratio of about 1.6. and decreases as the H/D ratio increases. Theoretical analysis of the reaction kinetics of immobilized-enzyme system that has reversible reaction kinetics is also presented. The experimental results showed good agreement with the results found from the theoretical analysis and the computer simulation studies. Based on the principles of the methods and the results presented in this paper, it is anticipated that one can predict the optimal design and operating conditions for the glucose isomerase reactor system and that application of the results could be extended to other enzyme systems with reversible reaction kinetics.     

Trehalose assimilation and turnover inTorulopsis glabrata

Torulopsis glabrata cells have a specific mechanism for endogenous trehalose mobilization that is associated with the lag phase attending new growth. The degree of internal trehalose depletion depends upon the initial concentration of glucose in the medium. Exogenous trehalose was shown to be assimilated via extra-cytoplasmic hydrolysis prior to, transport of the generated glucose into the cytoplasm. The effect on internal trehalose depletion in this case was commensurate with slower presentation of free glucose and was not indicative of the initial concentration of exterrial glucose residues as trehalose. Kinetic parameters for glucose uptake wereK _m =0.49 mM,V _max =109 μmol/min/g dry weight; those for trehalose assimilation wereK _m =2.5 mM,V _max =17.4μmol/min/g dry weight.     

Purification and characterization of an ADP-dependent phosphofructokinase from Thermococcus zilligii

The ADP-dependent phosphofructokinase (PFK) from Thermococcus zilligii has been purified 950 fold; it had a specific activity of 190 U mg⁻¹. The enzyme required Mg²⁺ ions for optimal activity and was specific for ADP. The forward reaction kinetics were hyperbolic for both cosubstrates (pH optimum of 6.4), and the apparent K _m values for ADP and fructose-6-phosphate were 0.6 mM (apparent V _max of 243 U mg⁻¹) and 1.47 mM (apparent V _max of 197 U mg⁻¹), respectively. Significantly, the enzyme is indicated to be nonallosteric but was slightly activated by some monovalent cations including Na⁺ and K⁺. The protein had a subunit size of 42.2 kDa and an estimated native molecular weight of 66 kDa (gel filtration). Maximal reaction rates for the reverse reaction were attained at pH 7.5–8.0, and the apparent K _m values for fructose-1,6-bisphosphate and AMP were 0.56 mM (apparent V _max of 2.9 U mg⁻¹) and 12.5 mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared to ATP and pyrophosphate-dependent phosphofructokinases. Received: August 14, 1998 / Accepted: December 2, 1998     

The boxing glove shape of subunit <Emphasis Type="Italic">d</Emphasis> of the yeast V-ATPase in solution and the importance of disulfide formation for folding of this protein

The low resolution structure of subunit d (Vma6p) of the Saccharomyces cerevisiae V-ATPase was determined from solution X-ray scattering data. The protein is a boxing glove-shaped molecule consisting of two distinct domains, with a width of about 6.5 nm and 3.5 nm, respectively. To understand the importance of the N- and C-termini inside the protein, four truncated forms of subunit d (d _11–345, d _38–345, d _1–328 and d _1–298) and mutant subunit d, with a substitution of Cys329 against Ser, were expressed, and only d _11–345, containing all six cysteine residues was soluble. The structural properties of d depends strongly on the presence of a disulfide bond. Changes in response to disulfide formation have been studied by fluorescence- and CD spectroscopy, and biochemical approaches. Cysteins, involved in disulfide bridges, were analyzed by MALDI-TOF mass spectrometry. Finally, the solution structure of subunit d will be discussed in terms of the topological arrangement of the V₁V_O ATPase.     

Presteady-State Currents of the Rabbit Na+/Glucose Cotransporter (SGLT1)

The rabbit Na⁺/glucose cotransporter (SGLT1) exhibits a presteady-state current after step changes in membrane voltage in the absence of sugar. These currents reflect voltage-dependent processes involved in cotransport, and provide insight on the partial reactions of the transport cycle. SGLT1 presteady-state currents were studied as a function of external Na⁺, membrane voltage V _m , phlorizin and temperature. Step changes in membrane voltage—from the holding V _h to test values, elicited transient currents that rose rapidly to a peak (at 3–4 msec), before decaying to the steady state, with time constants τ≈4–20 msec, and were blocked by phlorizin (K _i ≈30 μm). The total charge Q was equal for the application of the voltage pulse and the subsequent removal, and was a function of V _m . The Q-V curves obeyed the Boltzmann relation: the maximal charge Q _max was 4–120 nC; V _0.5, the voltage for 50% Q _max was −5 to +30 mV; and z, the apparent valence of the moveable charge, was 1. Q _max and z were independent of V _h (between 0 and −100 mV) and temperature (20–30°C), while increasing temperature shifted V _0.5 towards more negative values. Decreasing [Na⁺] _o decreased Q _max, and shifted V _0.5 to more negative voltages 9by −100 mV per 10-fold decrease in [Na⁺] _o ). The time constant τ was voltage dependent: the τ-V relations were bell-shaped, with maximal τ_max 8–20 msec. Decreasing [Na⁺] _o decreased τ_max, and shifted the τ-V curves towards more negative voltages. Increasing temperature also shifted the τ-V curves, but did not affect τ_max. The maximum temperature coefficient Q ₁₀ for τ was 3–4, and corresponds to an activation energy of 25 kcal/mole. Simulations of a 6-state ordered kinetic model for rabbit Na⁺/glucose cotransport indicate that charge-movements are due to Na⁺-binding/dissociation and a conformational change of the empty transporter. The model predicts that (i) transient currents rise to a peak before decay to steady-state; (ii) the τ-V relations are bell-shaped, and shift towards more negative voltages as [Na⁺] _o is reduced; (iii) τ_max is decreased with decreasing [Na⁺] _o ; and (iv) the Q-V relations are shifted towards negative voltages as [Na⁺] _o is reduced. In general, the kinetic properties of the presteady-state currents are qualitatively predicted by the model. Received: 12 August 1996/Revised: 30 September 1996     

Role of ionization of the phosphate cosubstrate on phosphorolysis by purine nucleoside phosphorylase (PNP) of bacterial (<Emphasis Type="Italic">E. coli</Emphasis>) and mammalian (human) origin

Kinetics of the reactions of purine nucleoside phosphorylases (PNP) from E. coli (PNP-I, the product of the deoD gene) and human erythrocytes with their natural substrates guanosine (Guo), inosine (Ino), a substrate analogue N(7)-methylguanosine (m⁷Guo), and orthophosphate (P_i, natural cosubstrate) and its thiophosphate analogue (SP_i), found to be a weak cosubstrate, have been studied in the pH range 5–8. In this pH range Guo and Ino exist predominantly in the neutral forms (pK_a 9.2 and 8.8); m⁷Guo consists of an equilibrium mixture of the cationic and zwitterionic forms (pK_a 7.0); and P_i and SP_i exhibit equilibria between monoanionic and dianionic forms (pK_a 6.7 and 5.4, respectively). The phosphorolysis of m⁷Guo (at saturated concentration) with both enzymes exhibits Michaelis kinetics with SP_i, independently of pH. With P_i, the human enzyme shows Michaelis kinetics only at pH ∼5. However, in the pH range 5–8 for the bacterial enzyme, and 6–8 for the human enzyme, enzyme kinetics with P_i are best described by a model with high- and low-affinity states of the enzymes, denoted as enzyme-substrate complexes with one or two active sites occupied by P_i, characterized by two sets of enzyme-substrate dissociation constants (apparent Michaelis constants, K _m1 and K _m2) and apparent maximal velocities (V _max1 and V _max2). Their values, obtained from non-linear least-squares fittings of the Adair equation, were typical for negative cooperativity of both substrate binding (K _m1 < K _m2) and enzyme kinetics (V _max1/K _m1 > V _max2/K _m2). Comparison of the pH-dependence of the substrate properties of P_i versus SP_i points to both monoanionic and dianionic forms of P_i as substrates, with a marked preference for the dianionic species in the pH range 5–8, where the population of the P_i dianion varies from 2 to 95%, reflected by enzyme efficiency three orders of magnitude higher at pH 8 than that at pH 5. This is accompanied by an increase in negative cooperativity, characterized by a decrease in the Hill coefficient from n _H ∼1 to n _H ∼0.7 for Guo with the human enzyme, and to n _H ∼0.7 and 0.5 for m⁷Guo with the E. coli and human enzymes, respectively. Possible mechanisms of cooperativity are proposed. Attention is drawn to the substrate properties of SP_i in relation to its structure.     

Regulation of the basolateral potassium conductance of theNecturus proximal tubule

Summary Two methods, the measurement of the response of the basolateral membrane potential (V _bl) of proximal tubule cells ofNecturus to step changes in basolateral K⁺ concentration, and cellular cable analysis, were used to assess the changes in basolateral potassium conductance (G _K) caused by a variety of maneuvers. The effects of some of these maneuvers on intracellular K⁺ activity (a _K ⁱ ) were also evaluated using double-barreled ion-selective electrodes. Perfusion with 0mm K⁺ basolateral solution for 15 min followed by 45 min of 1mm K⁺ solution resulted in a fall in basolateral potassium (apparent) transference number (t _K),V _bl anda _K ⁱ . Results of cable analysis showed that total basolateral resistance,R _b , rose. The electrophysiological effects of additional manipulations, known to inhibit net sodium reabsorption across the proximal tubular epithelium ofNecturus, were also investigated. Ouabain caused a fall int _K accompanied by large decreases ina _K ⁱ andV _bl. Lowering luminal sodium caused a fall int _K and a small reduction inV _bl. Selective reduction of peritubular sodium, a maneuver that has been shown to block sodium transport from lumen to peritubular fluid, also resulted in a significant decrease int _K. These results suggest thatG _K varies directly with rate of transport of the sodium pump, irrespective of the mechanism of change in pump turnover.Part of this material has been presented at the 10th International Conference on Biological Membranes (Cohen & Giebisch, 1984).