Temperature dependence of anion transport in the human red blood cell

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (E_a). Below 15 or 25°C (for Cl⁻ and Br⁻, respectively), E_a is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283–306). For the temperature dependence of SO₄²⁻ transport up to 37°C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO₄²⁻ transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60°C and to show that, even for the slowly permeating SO₄²⁻ in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37°C and where E_a changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using ³⁵Cl⁻-NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472–6480), we also determined the temperature dependence of Cl⁻-binding. We found no significant change over the entire range from 0 to 57°C, regardless of whether the measurements were performed in the absence or presence of competing SO₄²⁻. We conclude that the enthalpy changes associated with Cl⁻-or SO₄²⁻-binding are negligible as compared to the E_a values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl⁻, Br⁻ and SO₄²⁻ below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.     

Specific cation modulation of anion transport across the human erythrocyte membrane

The specific modulation by three cations, Ca²⁺, Mg²⁺, and tetracaine of the equilibrium exchange of SO₄²⁻ across the erythrocyte membrane was investigated. While external calcium had no effect on SO₄²⁻ exchange, internal calcium, and external calcium in the presence of 10 μM A23187 were found to be potent inhibitors of the exchange reaction. The apparent inhibition constants (K₁) for Ca²⁺ were calculated to be 6.1 μM and 5 μM for the above two conditions, respectively.Unlike Ca²⁺, Mg²⁺ was shown to be a weak activator of SO₄²⁻ exchange with an apparent dissociation constant of 3.6 μM. Competition experiments demonstrated that the Ca²⁺ and Mg²⁺ sites associated with anion transport are distinct and noninteracting.Tetracaine, a cation at neutral pH, was also found to be an inhibitor of SO₄²⁻ exchange with an apparent K₁ of 0.8 mM. Although tetracaine was observed to displace calcium from non-specific sites on the erythrocyte membrane, it showed no effect on the apparent inhibition constant of Ca²⁺ for SO₄²⁻ exchange. Thus, the Ca²⁺ and tetracaine sites also appear to be independent. The difficulty of situating three mutually independent sites on a single subunit protein, i.e., band 3, is considered.Using the experimental data obtained from five individuals, the concentration of free calcium in the red cell cytoplasm was calculated to range from 0.2 to 0.7 μM. This concentration was sufficient to reduce SO₄²⁻ exchange only 3–8%. It was concluded that calcium inhibition of anion exchange, and, hence, impairment of CO₂ transport, may be physiologically significant only in senescent cells and in certain types of anemia where calcium concentrations are significantly increased.     

Dissociation of cellular K+ accumulation from net Na+ transport by toad urinary bladder

Summary A number of published data suggest a variable stoichiometry between the rates of cellular potassium uptake and net sodium transport (J _Na) across the urinary bladder of the toad. This problem was examined by simultaneously studying the intracellular chemical activity of potassium (a _K) with open-tip K⁺-selective microelectrodes and micropipets, and monitoringJ _Na by measuring the short-circuit current (SCC). When bathed in the short-circuited state with solutions containing ana _K of 2.7mm, the mean ±sem values for intracellulara _K were 43±0.6mm.Ouabain, at a concentration of 10^–2 m, reduced intracellulara _K by 56–67% and SCC by 96–100%. At 5×10^–4 m, ouabain reversibly reduced intracellulara _K by 40–55%, and SCC by 63–68%; the inhibition of SCC was only partly reversible during the period of observation.Removal of external potassium reduced intracellulara _K by 69–80% and SCC by 51–76%. Restoration of external potassium entirely returned intracellulara _K to its control value, but only partially reversed the inhibition of SCC during the period of study. Furthermore, recovery ofa _K began 19–43 min before that of SCC; recovery ofa _K was 90–97% complete before any increase in SCC could be measured. Although other interpretations are possible, the simplest interpretation of the data is that the processes responsible for potassium accumulation and transepithelial sodium transport are not identical. We propose the existence of a separate transfer mechanism at the basolateral cell membrane, responsible for accumulating intracellular potassium, and not directly coupled to active sodium transport.     

Transepithelial Taurine Transport in Caco-2 Cell Monolayers

Here we characterized transepithelial taurine transport in monolayers of cultured human intestinal Caco-2 cells by analyzing kinetic apical and basolateral uptake and efflux parameters. Basolateral uptake was Na⁺- and Cl⁻- dependent and was inhibited by β-amino acids. Uptake by this membrane showed properties similar to those of the apical TauT system. In both membranes, taurine uptake fitted a model consisting of a non-saturable plus a saturable component, with a higher half-saturation constant and transport capacity at the apical membrane (K_m, 17.1 μmol/L; V_max, 28.4 pmol·cm⁻²·5 min⁻¹) than in the basolateral domain (K_m, 9.46 μmol/L; V_max, 5.59 pmol·cm⁻²·5 min⁻¹). The non-saturable influx component, estimated in the absence of Na⁺ and Cl⁻, showed no significant differences between apical and basolateral membranes (K_D, 89.2 and 114.7 nL·cm⁻² · 5 min⁻¹, respectively). Taurine efflux from the cells is a diffusive process, as shown in experiments using preloaded cells and in trans-stimulation studies (apical K_D,72.7 and basolateral K_D, 50.1 nL·cm⁻²·5 min⁻¹). Basolateral efflux rates were significantly lower than passive influx rates. We conclude that basolateral taurine uptake in Caco-2 cells is mediated by a transport mechanism that shares some properties with the apical system TauT. Moreover, calculation of unidirectional and transepithelial taurine fluxes reveals that apical influx of this amino acid is higher than basolateral efflux rates, thereby enabling epithelial cells to accumulate taurine against a concentration gradient.     

Transport of neutral, cationic and anionic amino acids by systems B, b, XAG, and ASC in swine small intestine

Amino acid influx across the brush border membrane of the intact pig ileal epithelium was studied. It was examine whether in addition to system B, systems ASC and b^o,+ were involved in transport of bipolar amino acids. The kinetics of interactions between lysine and leucine demonstrates that system b^o,+ is present and accessible also to -glutamine. -aspartate (K_1/2 0.3 mM) and -glutamate (K_i 0.5 mM) share a high affinity transporter with a maximum rate of 1.3 μmol cm⁻² h⁻¹, while only -glutamate with a K_1/2 of 14.4 mM uses a low affinity transporter with a maximum rate of 2.7 μmol cm⁻² h⁻¹, system ASC, against which serine has a K_i of 1.6 mM. In the presence of 100 mM lysine, -glutamine (A), leucine (B), and methionine (C) fulfilled the criteria of the ABC test for transport by one and the same transporter. However, serine inhibits not only transport of -glutamate but also of glutamine (K_i 0.5 mM), and -glutamate inhibits part of the transport of glutamine. The test does, therefore, only indicate that the three bipolar amino acids have similar affinities for transport by systems B and ASC. Further study of the function of system B must be carried out under full inhibition by lysine and glutamate.     

Studies on molybdenum absorption and transport in bean and rice

The patterns of molybdenum (MoO₄²⁻) absorption and transport were investigated in intact bean (Phaseolus vulgaris L.) and rice (Oryza sativa L. cv. I.R.8) plants. The mobility of MoO₄²⁻ absorbed by roots and by leaves was compared with that of a freely mobile element, Rb⁺. Although MoO₄²⁻ absorption by bean roots was nearly as high as that of Rb⁺, its transport to the shoot was considerably less. When MoO₄²⁻ was fed to one of the primary leaves, most of it was transported to the stem and root. Evidence obtained here showed that MoO₄²⁻ was mobile. Experiments with intact rice seedlings revealed large differences in the absorption and transport of MoO₄²⁻ between the plants grown in CaSO₄ and those in Hoagland solution. Molybdate uptake by excised rice roots was suggested to be an active process since it was greatly inhibited by a metabolic inhibitor. The presence of Mn²⁺, Zn²⁺, Cu²⁺, CI⁻, or SO₄²⁻ in the absorption medium reduced MoO₄²⁻ uptake which was markedly enhanced by the presence of Fe²⁺.     

Chloride conductance and mitochondria-rich cell density in isolated skin of Rana catesbeiana acclimated to various environments

The Cl⁻ conductance in isolated skin of frogs (Rana catesbeiana) acclimated to 30 mM solutions of NaCl, Na₂SO₄, MgCl₂ and distilled water (DW) was studied. Transepithelial potential difference (PD_trans), short-circuit current (I_SC) and total conductance (G_t) were measured under conditions such that there was Cl⁻ flux in the presence and absence of Na⁺ transport. The Cl⁻ content of the mucosal solution was acutely replaced with SO₄²⁻ or gluconate to evaluate the effect of removal of Cl⁻ conductance on electrophysiological parameters. Mitochondria-rich cell density (D_MRC) was also measured. Skins from frogs acclimated to NaCl and Na₂SO₄ showed the lowest and the highest D_MRC, respectively, but no difference could be found between the skins from frogs acclimated to DW and MgCl₂ indicating that D_MRC is not unconditionally dependent on environmental Cl⁻ in this species. Frogs acclimated to NaCl showed marked differences when compared to the other groups: the highest G_t, probably represented by a higher paracellular conductance; the lowest transepithelial electrical potential difference which remained invariant after replacement of mucosal Cl⁻ with SO₄²⁻ or replacement of mucosal Cl⁻ with gluconate and an inwardly oriented positive current in the absence of bilateral Na⁺.     

Evidence of multiple operational affinities for d-glucose inside the human erythrocyte membrane

1. 1. The Michaelis-Menten parameters of labelled d-glucose exit from human erythrocytes at 2°C into external solution containing 50 mM d-galactose were obtained. The K_m is 3.4 ± 0.4 mM, V 17.3 ± 1.4 mmol · 1⁻¹ cell water · min⁻¹ for this infinite-trans exit procedure.

2. 2. The kinetic parameters of equilibrium exchange of d-glucose at 2°C are K_m = 25 ± 3.4 mM, V 30 ± 4.1 mmol · 1⁻¹ cell water · min⁻¹.

3. 3. The K_m for net exit of d-glucose into solutions containing zero sugar is 15.8 ± 1.7 mM, V 9.3 ± 3.3 mol 9.3 ± 3.3 mol · 1⁻¹ cell water · min⁻¹.

4. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126–132] that there are sites with both high and low operational affinities for d-glucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport.

Facilitated water transport in cyanobacterium Synechococcus sp. PCC 7942 studied by phycobilisome-sensitized chlorophyll a fluorescence

Water transport across plant cell membranes is difficult to measure. We present here a model assay, based on chlorophyll (Chl) a fluorometry, with which net water transport across the cell membrane of freshwater cyanobacterium Synechococcus sp. PCC7942 (S7942) can be followed kinetically with millisecond-time resolution. In cyanobacteria, the phycobilisome (PBS)-sensitized Chl a fluorescence increases when cells expand (e.g., in hypo-osmotic suspension) and decreases when cells contract (e.g., in hyper-osmotic suspension). The osmotically-induced Chl a fluorescence changes are proportional to the reciprocal of the suspension osmolality (ΔF ∝ Osm⁻¹; Papageorgiou GC and Alygizaki-Zorba A (1997) Biochim Biophys Acta 1335: 1–4). In our model assay, S7942 cells were loaded with NaCl (passively penetrating solute) and shrunk in hyper-osmotic glycine betaine (nonpenetrating solute). Upon injecting these cells into hypo-osmotic medium, the PBS-sensitized Chl a fluorescence rose to a maximum due to the osmotically-driven water uptake. The rise of Chl a fluorescence (water uptake) was partially inhibited by HgCl₂, at micromolar concentrations. Arrhenius plots of the water uptake rates gave activation energies of E_A=4.9 kcal mol⁻¹, in the absence of HgCl₂, and E_A=11.9 kcal mol⁻¹ in its presence. These results satisfy the usual criteria for facilitated water transport through protein water pores of plasma membranes (aquaporins), namely sensitivity to Hg²⁺ ions and low activation energy.     

Partitioning of small organic molecules in aqueous biphasic systems

Aqueous biphasic systems (ABS) are suitable for the separation of small organic molecules in industrial and environmental applications and thus, it is important to correlate partitioning behavior of model organic solutes with their structure in order to develop predictive models. The partitioning behavior of five, uncharged, substituted benzenes (benzene, toluene, chlorobenzene, 1,4-dichlorobenzene and 1,2,4-trichlorobenzene) were studied in ABS prepared from stock solutions of 40% (w/w) PEG-2000 and increasing concentrations of four water-structuring salts (K₃PO₄, K₂CO₃, (NH₄)₂SO₄ and NaOH). For a given solute and a defined concentration of salt, the partition coefficients increase as the ΔG_hyd value of the salt anion becomes more negative (e.g., D_benzene increases in the order OH⁻<SO₄²⁻<CO₃²⁻<PO₄³⁻). In a given salt, the distribution ratios increase in the order benzene<toluene<chlorobenzene<1,4-dichlorobenzene<1,2,4-trichlorobenzene. The partitioning behavior of the solutes in PEG–salt ABS was found to be strongly correlated with their partitioning coefficients in 1-octanol–water biphasic systems.     

Long-Term Temperature Acclimation of Photosynthesis in Steady-State Cultures of the Polar Diatom <Emphasis Type="Italic">Fragilariopsis cylindrus</Emphasis>

Cultures of the obligate psychrophilic diatom Fragilariopsis cylindrus (Grunow) were grown for 4 months under steady-state conditions at −1 °C and +7 °C (50 μmol photons m⁻² s⁻¹) prior to measurements in order to investigate long-term acclimation of photosynthesis to both temperatures. No differences in maximum intrinsic quantum yield of PS II (F_V/F_M) and relative electron transport rates could be detected at either temperature after 4 months of acclimation. Measurements of photosynthesis (relative electron transport rates) vs. irradiance (P vs. E curves) revealed similar values for relative light utilization efficiency (α = 0.57 at −1 °C, α = 0.60 at +7 °C) but higher values for irradiance levels at which photosynthesis saturates (E_K) at −1 °C and, therefore, higher maximum photosynthesis (P_MAX = 54 (relative units) at −1 °C, P_MAX = 49 at +7 °C). Nonphotochemical quenching (NPQ) measurements at 385 μmol photons m⁻² s⁻¹ indicated higher (37%) NPQ for diatoms grown at −1 °C compared to +7 °C, which was possibly related to a 2-fold increase in the concentration of the pigment diatoxanthin and a 9-fold up-regulation of a gene encoding a fucoxanthin chlorophyll a,c-binding protein. Expression of the D1 protein encoding gene psbA was ca. 1.5-fold up-regulated at −1 °C, whereas expression levels of other genes from Photosystem II (psbC, psbU, psbO), as well as rbcL, the gene encoding the Rubisco large subunit were similar at both temperatures. However, a 2-fold up-regulation of a plastid glyceraldehyde-P dehydrogenase at −1 °C indicated enhanced Calvin cycle activity. This study revealed for the first time that a polar diatom could efficiently acclimate photosynthesis over a wide range of polar temperatures given enough time. Acclimation of photosynthesis at −1 °C was probably regulated similarly to high light acclimation.     

Effect of ions on phospholipid layer structure as indicated by Raman Spectroscopy

Various anions and cations are found to induce changes in the layered structure of phosphatidylcholine-water systems as indicated by Raman Spectroscopy. From the ratio of Raman intensities, , it is inferred that dispositive ions decrease the proportion of gauche character in the hydrocarbon chains, with the relative influence being: Ba²⁺ < Mg²⁺ < Ca²⁺ ≈ Cd²⁺. Unipositive ions (Li⁺, K⁺ and Na⁺) produce no observed changes in the Raman spectrum of the lecithin dispersion. The proportion of gauche character of the hydrocarbon chains is found to be nearly independent of the anion for: Br⁻, Cl⁻, acetate⁻, I⁻, ClO₄⁻, CNS⁻ and SO₄²⁻. Dispersions prepared with a solution of KI + I₂ produced Raman spectra in which the 1089 cm⁻¹ peak, which is characteristic of random lipid chains, was greatly intensified, presumably because of the presence of I₃⁻ which is known to penetrate the lipid lamellae. The observed trends are discussed.     

Sulphate transport by rat ileum. Effect of molybdate and other anions

Kinetic constants for SO₄^2? transport by upper and lower rat ileum in vitro have been determined by computer fitting of rate vs concentration data obtained using the everted sac technique. MoO₄^2? inhibition of this transport is competitive, and kinetic constants for the inhibition were similarly determined. Transport is also inhibited by the anions WO₄^2?, S₂O₃^2? and SeO₄^2?, in the order $\text{S}{}_2\text{O}{}_3{}^{\mathrm{2?}}\text{> SeO}{}_4{}^{\mathrm{2?}}\text{≧ MoO}{}_4{}^{\mathrm{2?}}\text{> WO}{}_4{}^{\mathrm{2?}}$. These anions have no effect on the transport of l-valine. Low SO₄^2? transport rates were observed in sacs from animals fed a high-molybdenum diet. The significance of the results with respect to the problem of molybdate toxicity in animals is discussed, and related to the known protective effect of SO₄^2?.     

Remediation of depleted soils by addition of ion exchange resins

Parallel investigations with fertility were carried out in standard garden soil with ion exchange substrate BIONA 111 as well with mixtures in different proportions. The ion exchange substrate was a mixture of ion exchange resins saturated in certain proportions with a complete set of biogenic ions. Plant productivity in the ion exchange substrate in a 6-week vegetation period was 950 g/kg of the green biomass compared with 29 g/kg in soil. Productivity linearly depended on the mass fraction of the ion exchange substrate in the mixtures with the garden soil. Addition of 1% of the ion exchange substrate is sufficient for starting vegetation in completely depleted soil and barren sand. Addition of different ionic forms of ion exchange resins (K⁺, Ca²⁺+Mg²⁺+K⁺, NO₃⁻, NO₃⁻+H₂PO₄²⁻+SO₄²⁻) caused pronounced positive effects on soil productivity though these effects were less significant than those of ion exchange substrate. Addition of ion exchange substrates can be an efficient means for remediation of destroyed soils and fruitless rocks.     

Laser raman studies of molecular interactions with phosphatidylcholine multilayers. II. Effects of mono- and divalent ions on bilayer structure

Laser Raman spectroscopy was applied to characterize structural behavior of dipalmitoyl phosphatidylcholine multibilayer systems in the presence of several cations (K⁺, Na⁺, Cs⁺, Rb⁺, Ca²⁺, Mg²⁺, Cd²⁺, Ba²⁺) and anions (Cl⁻, Br⁻, I⁻, NO₃⁻, SO₃²⁻, SO₄²⁻, S₂O₃²⁻, S₂O₈²⁻). To evaluate the Raman-spectroscopical data quantitatively, characteristic intensity ratios, lateral and trans order parameters were used and compared. It was shown that the different trans order parameters are rather sensitive to ion-polar head group interactions and thus, they cannot give unequivocal information on the trans-gauche isomerization of hydrocarbon chains of phospholipids. The observed effects of ions on Raman spectra of phospholipid multilayers could not be explained simply on the basis of electrostatic interactions. The possible involvement of other factors (changes in polarizability, hydrogen bonds, etc.) is also discussed. It was demonstrated that the order parameters defined in different ways may result in different effectiveness sequences of ions. Of monopositive ions Na⁺ was found to be the most effective to influence the bilayer structure. For dipositive ions, of which Ca²⁺ proved to be the most effective, concentration-dependent effectiveness sequences were obtained. A plausible interpretation and some consequences of the concentration-dependent two-step binding of divalent cations were also outlined. Bilayered phospholipid structures turned out to be more responsive to anions than to most cations investigated. Interdependent actions of cations and anions, as well as the possible relevance of the charge distribution on anions are postulated.     

Kinetic analyses for bindings of concanavalin A to dispersed and condensed mannose surfaces on a quartz crystal microbalance

A biotinylated mannotriose (Man3-bio) was dispersively immobilized in the matrix of biotinylated lactose (Gal-Glc-bio) on a streptavidin-covered, 27-MHz quartz crystal microbalance (QCM), and binding kinetics of concanavalin A (Con A) to Man3-bio in the Gal-Glc-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. Association constants (K_a) and binding and dissociation rate constants (k_on and k_off) could be determined separately as the 1:1 and 1:2 bindings of Con A to Man3-bio on the surface. When Man3-bio was immobilized with content of 1 to 5 mol% in the matrix, the 1:1 binding of Con A to Man3-bio was obtained as K_a = (4 ± 1) × 10⁶ M⁻¹, k_on = (4 ± 1) × 10⁴ M⁻¹ s⁻¹, and k_off = (12 ± 2) × 10^–3 s⁻¹. On the contrary, when Man3-bio was immobilized with content of 20 to 100 mol% in the matrix, the 1:2 binding of Con A to Man3-bio was obtained as K_a = (14 ± 2) × 10⁶ M⁻¹, k_on = (14 ± 2) × 10⁴ M⁻¹ s⁻¹, and k_off = (7 ± 2) × 10^–3 s⁻¹. Thus, K_a for the 1:2 binding was 10 times larger than that for the 1:1 binding, with a three times larger binding rate constant (k_on) and a three times smaller dissociation rate constant (k_off). This is the first example to obtain separate kinetic parameters for the 1:1 and 1:2 bindings of lectins to carbohydrates on the surface.     

Membrane Permeability Characteristics of Metaphase II Mouse Oocytes at Various Temperatures in the Presence of Me2SO

In this study, the hydraulic conductivity (L_p), Me₂SO permeability ( _Me2SO), and the reflection coefficients (ς) and their activation energies were determined for Metaphase II (MII) mouse oocytes by exposing them to 1.5 M Me₂SO at temperatures of 30, 20, 10, 3, 0, and −3°C. These data were then used to calculate the intracellular concentration of Me₂SO at given temperatures. Individual oocytes were immobilized using a holding pipette in 5 μl of an isosmotic PBS solution and perfused with precooled or prewarmed 1.5 M Me₂SO solutions. Oocyte images were video recorded. The cell volume changes were calculated from the measurement of the diameter of the oocytes, assuming a spherical shape. The initial volume of the oocytes in the isoosmotic solution was considered 100%, and relative changes in the volume of the oocytes after exposure to the Me₂SO were plotted against time. Mean (means ± SEM) L_pvalues in the presence of Me₂SO ( ^Me₂SO_p) at 30, 20, 10, 3, 0, and −3°C were determined to be 1.07 ± 0.03, 0.40 ± 0.02, 0.18 ± 0.01, 7.60 × 10⁻²± 0.60 × 10⁻², 5.29 × 10⁻²± 0.40 × 10⁻², and 3.69 × 10⁻²± 0.30 × 10⁻²μm/min/atm, respectively. The _Me2SOvalues were 3.69 × 10⁻³± 0.3 × 10⁻³, 1.07 × 10⁻³± 0.1 × 10⁻³, 2.75 × 10⁻⁴± 0.15 × 10⁻⁴, 7.83 × 10⁻⁵± 0.50 × 10⁻⁵, 5.24 × 10⁻⁵± 0.50 × 10⁻⁵, and 3.69 × 10⁻⁵± 0.40 × 10⁻⁵cm/min, respectively. The ς values were 0.70 ± 0.03, 0.77 ± 0.04, 0.81 ± 0.06, 0.91 ± 0.05, 0.97 ± 0.03, and 1 ± 0.04, respectively. The estimated activation energies (E_a) for ^Me₂SO_p, _Me2SO, and ς were 16.39, 23.24, and −1.75 Kcal/mol, respectively. These data may provide the fundamental basis for the development of more optimal cryopreservation protocols for MII mouse oocytes.     

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na⁺,K⁺)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg⁻¹ with K_0.5=1.2±0.1 mmol l⁻¹; stimulation by potassium (V=121.0±6.1 U mg⁻¹; K_0.5=2.1±0.1 mmol l⁻¹) and magnesium ions (V=125.3±6.3 U mg⁻¹; K_0.5=1.0±0.1 mmol l⁻¹) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (n_H=2.7) to a rate of V=126.1±4.8 U mg⁻¹ with K_0.5=13.7±0.5 mmol l⁻¹. However, K⁺-phosphatase activity was not stimulated further by K⁺ plus NH₄⁺ ions. Sodium ions (K_I=36.7±1.7 mmol l⁻¹), ouabain (K_I=830.3±42.5 μmol l⁻¹) and orthovanadate (K_I=34.0±1.4 nmol l⁻¹) completely inhibited K⁺-phosphatase activity. The competitive inhibition by ATP (K_I=57.2±2.6 μmol l⁻¹) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K⁺-phosphatase activity corresponds strictly to a (Na⁺,K⁺)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K⁺-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.     

The Effect of Different Salts of Sodium and Potassium on the Accumulation of Glycinebetaine in Atriplex Prostrata

Atriplex prostrata was grown for one month in nutrient solutions with NaCl, KCl, Na₂SO₄, and K₂SO₄ (at osmotic potentials of 0, –0.75, –1.00, and –1.50 MPa). Plants treated with K₂SO₄ had less glycinebetaine at –1.0 and –1.50 MPa than those treated with Na⁺ salts, probably due to the inhibitory effects of K⁺ on glycinebetaine accumulation.     

A spectroscopic model for the high pH form of sulfite oxidase

The syntheses and spectroscopic properties of two monomeric oxo-Mo(V) complexes [NMe₄][MoO(SC₆H₄---CH=N---C₆H₄O)(SAr)₂] (Ar=Ph (2a), PhCH₃ (2b)) exhibiting a novel S₃NO coordination site are described. The EPR parameters of the Mo(V) complexes are almost identical with the parameters for the high pH form of sulfite oxidase, allowing further predictions for the unknown coordination site of the molybdenum cofactor in such molybdenum-containing enzymes. The Mo(V) compounds react with water to form a μ-oxo-bridged Mo(V) dimer, which is readily oxidized by oxygen to give the monomeric dioxo-Mo(VI) complex MoO₂(SC₆H₄---CH=N---C₆H₄O)(sol) (4) (sol=acetonitrile; dimethyl sulfoxide (DMSO)). The structure of 4 has been determined by X-ray crystallography (space group P (No. 2), a=7.855(4), b=9.530(3), c=11.676(6) Å, α=103.96(3), β=99.03(3), γ=100.73(3)°, V=814.5 ų, ρ_calc=1.79 g cm⁻³, Z=2, R(F)=0.026, R(wF)=0.026). The geometry about the molybdenum is distorted octahedral, with two terminal oxo groups cis to each other.