Cell and Solution Velocity Constants for the Reaction CO + Hb → COHb at Different Temperatures in Mammals with Different Red Cell Sizes

Using a double beam stopped-flow apparatus, measurements were made of the velocity constant of the reaction CO + Hb → COHb in solution and in the red cells of human beings, rabbits, horses, and goats. The solution constant (l'') at 37°C for human beings was 362 mM ^-1 sec.^-1; in other species l'' was somewhat lower. Two rabbits, despite having apparently identical hemoglobins had significantly different values for l''. The energy of activation (E) of l'' was between 8 and 11 kcal/mole in all cases. The cell reaction constant (l''_c) at 37° was between 61 and 73 mM ^-1 sec.^-1 in all cases; at 37° the trend was for the smaller cells to have the higher l''_c. This cell size effect was much less than previously found for the faster oxygen reaction. This showed that by merely increasing the rate of chemical reaction, it was not possible to increase cell uptake rate beyond a certain level, this level being dependent on the size and membrane properties of the cell. At lower temperatures l'' was a more important factor in determining l''_c than was cell size. The cell membrane was a barrier to gas diffusion in all species. The effect of temperature on l''_c was also measured and was less than its effect on l'' at most temperatures. Temperature effect increased in small cells at low temperatures. Both these findings are in accordance with predictions based on differentiation of Roughton''s equations.     

Water-relation Parameters of Individual Mesophyll Cells of the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana

Water-relation parameters of leaf mesophyll cells of the CAM plant Kalanchoë daigremontiana have been determined directly in cells of tissue slices using the pressure-probe technique. Turgor pressures measured in cells of the second to fourth layer from the cut surface showed an average of 1.82 ± 0.62 bar (mean ± sd; n = 157 cells). This was lower than expected from measurements of the osmotic pressure of the cell sap. The half-time (T_1/2) for water-flux equilibration of individual cells was 2.5 to 8.8 seconds. This is the fastest T_1/2 found so far for higher-plant cells. The calculated values of the hydraulic conductivity were in the range of 0.20 to 1.6 × 10⁻⁵ centimeters second⁻¹ bar⁻¹, with an average of (0.69 ± 0.46) × 10⁻⁵ centimeters second⁻¹ bar⁻¹ (mean ± sd; n = 8 cells). The T_1/2 values of water exchange of individual cells are consistent with the overall rates of water-flux equilibration measured for tissue slices.The volumetric elastic moduli (∈) of individual cells were in the range 13 to 128 bar for turgor pressures between 0.0 and 3.4 bar; the average ∈ value was 42.4 ± 27.7 bar (mean ± sd; n = 21 cells). This ∈ value is similar to that observed for other higher-plant cells.The water-storage capacity of individual cells, calculated as C_c = V/(∈ + πⁱ) (where V = cell volume and πⁱ = internal osmotic pressure) was 9.1 × 10⁻⁹ cubic centimeters bar⁻¹ per cell, and the capacity for the tissue was 2.2 × 10⁻² cubic centimeters bar⁻¹ gram⁻¹ fresh weight. The significance of the water-relation parameters determined at the cellular level is discussed in terms of the water relations of whole leaves and the high water-use efficiency characteristic of CAM plants.     

Kinetic analyses of calcium movements in cell cultures. 3. Effects of calcium and parathyroid hormone in kidney cells

Calcium compartments and fluxes were measured by kinetic analyses in kidney cell suspensions in a three-compartment closed system. The fast phase influx and compartment size increase linearly with the medium calcium and the half-time of exchange is only 1.3 min which suggests that the fast component is extracellular. The slow phase compartment rises linearly from 0.1 to 0.5 mmole calcium/kg cell water when the medium calcium is raised from 0.02 to 2.5 mM. The slow phase calcium influx exhibits the pattern of saturation kinetics with a V _max of 0.065 µµmole cm^-2 sec^-1 and a K_m of 0.3 mM indicating that it is a carrier-mediated transport process. PTH has no effect on the fast phase of calcium influx, but increases both calcium influx and the calcium pool size of the slow component. The maximum effect is obtained at medium calcium concentration of 1.3 mM. Below 0.3 mM extracellular calcium, the effects of the hormone cannot be demonstrated. PTH increases the V _max of calcium influx from 0.065 to 0.128 µµmole cm^-2 sec^-1 while the K_m rises from 0.3 to 1.15 mM. These findings suggest that PTH increases the translocation of the calcium-carrier complex across the membrane and not the carrier concentration or its binding affinity for calcium.     

Thymidine transport by cultured Novikoff hepatoma cells and uptake by simple diffusion and relationship to incorporation into deoxyribonucleic acid

The initial rate of thymidine-³H incorporation into the acid-soluble pool by cultured Novikoff rat hepatoma cells was investigated as a function of the thymidine concentration in the medium. Below, but not above 2 µM, thymidine incorporation followed normal Michaelis-Menten kinetics at 22°, 27°, 32°, and 37°C with an apparent K_m of 0.5 µM, and the V_max values increased with an average Q₁₀ of 1.8 with an increase in temperature. The intracellular acid-soluble ³H was associated solely with thymine nucleotides (mainly deoxythymidine triphosphate [dTTP]). Between 2 and 200 µM, on the other hand, the initial rate of thymidine incorporation increased linearly with an increase in thymidine concentration in the medium and was about the same at all four temperatures. Pretreatment of the cells with 40 or 100 µM p-chloromercuribenzoate for 15 min or heat-shock (49.5°C, 5 min) markedly reduced the saturable component of uptake without affecting the unsaturable component or the phosphorylation of thymidine. The effect of p-chloromercuribenzoate was readily reversed by incubating the cells in the presence of dithiothreitol. Persantin and uridine competitively inhibited thymidine incorporation into the acid-soluble pool without inhibiting thymidine phosphorylation. At concentrations below 2 µM, thymidine incorporation into DNA also followed normal Michaelis-Menten kinetics and was inhibited in an apparently competitive manner by Persantin and uridine. The apparent K_m and K_i values were about the same as those for thymidine incorporation into the nucleotide pool. The over-all results indicate that uptake is the rate-limiting step in the incorporation of thymidine into the nucleotide pool as well as into DNA. The cells possess an excess of thymidine kinase, and thymidine is phosphorylated as rapidly as it enters the cells and is thereby trapped. At low concentrations, thymidine is taken up mainly by a transport reaction, whereas at concentrations above 2 µM simple diffusion becomes the principal mode of uptake. Evidence is presented that indicates that uridine and thymidine are transported by different systems. Upon inhibition of DNA synthesis, net thymidine incorporation into the acid-soluble pool ceased rapidly. Results from pulse-chase experiments indicate that a rapid turnover of dTTP to thymidine may be involved in limiting the level of thymine nucleotides in the cell.     

Effects of acriflavine on the mitochondria and kinetoplast of Crithidia fasciculata. Correlation of fine structure changes with decreased mitochondrial enzyme activity

The effects of acriflavine on the fine structure and function of the mitochondria and the kinetoplast in Crithidia fasciculata have been investigated. A mitochondrial fraction was prepared by differential centrifugation of cells broken by grinding with neutral alumina. Isolated mitochondria or intact cells revealed by spectrophotometric measurements the presence of cytochromes a + a ₃, b, c ₅₅₅ and o. After cells were grown in acriflavine for 3–4 days, the fine structure of the mitochondria and their cytochrome content were affected. Cells grown in 5.0 µM acriflavine had a threefold decrease in cytochrome a + a ₃ and decreased respiratory activity. The mitochondrial preparation from these cells had a fivefold decrease in cytochrome a + a ₃ and a less but significant decrease of other cytochromes present. There was also a decrease in the mitochondrial enzyme activities of NADH, succinic and L-α-glycerophosphate oxidases, and succinic and L-α-glycerophosphate dehydrogenases. Dyskinetoplastic cells could be demonstrated after growth in 1.0 µM acriflavine. At 5 µM, 80–90% of the cells were dyskinetoplastic. The kinetoplastic DNA was condensed, nonfibrillar, and did not incorporate thymidine-³H. The mitochondria in these cells had few cristae and were shorter and more swollen than the controls. Acriflavine may induce the fine structure effects we have observed and may affect the formation of the mitochondria in C. fasciculata.     

Hydroxyl ion movements across the human erythrocyte membrane. Measurement of rapid pH changes in red cell suspensions

A stopped flow rapid reaction apparatus capable of following changes of ±0.02 pH unit in 0.1 ml of solution in less than 0.005 sec has been developed, utilizing a commercially available pH-sensitive glass electrode. Using this instrument, extracellular pH at 37°C was followed from less than 0.025 sec to 300 sec after mixing equal volumes of the following CO₂-free solutions: (A) normal human red cells, washed three times and resuspended in 150 mM NaCl at pH 7.2 with a hematocrit of 18%; and, (B) 150 mM NaCl adjusted with HCl or NaOH to pH 2.1 to pH 10.3. A minimum of 2 ml of mixture had to flow through the electrode chamber to ensure complete washout. The mixing process produced a step change in the pH of the extracellular fluid, after which exchanges across the red cell membrane and buffering by intracellular hemoglobin caused it to return toward pH 7.2 with an approximately exponential time course. Under the assumption that pH changes after mixing represent exchanges of hydroxyl for chloride ions across the cell membrane, hydroxyl ion permeabilities (P _OH ^- in cm/sec) were calculated and found to vary from 2 x 10^-4 at pH 9 to 4 x 10^-1 at pH 4 according to the empirical relationship P _OH ^- = 170 exp (-1.51 pH). The form of the dependence of P _OH ^- on extracellular pH does not appear compatible with a simple fixed charge theory of membrane permselectivity.     

Decreased K+ conductance produced by Ba++ in frog sartorius fibers

The action of Ba⁺⁺ on membrane potential (E_m) and resistance (R_m) of frog (R. pipiens) sartorius fibers was studied. In normal Cl^- Ringer''s, Ba⁺⁺ (<9 mM) did not depolarize or induce contractions, but increased R_m slightly above the control value of 3.8 ± 0.6 KΩ-cm². In Cl^--free Ringer''s (methane sulfonate) R_m was 28.8 ± 2.8 KΩ-cm², and low concentrations of Ba⁺⁺ (0.05–5.0 mM) depolarized and induced spontaneous contractions (fibrillation), even in tetrodotoxin. To stop disturbance of the microelectrodes, contractions were prevented by using two Cl^--free solutions: (a) twice hypertonic with sucrose (230 mM), or (b) high K⁺ (83 mM) partially replacing Na⁺. In the hypertonic solution, the fiber diameters decreased, E_m increased slightly, and R_m decreased to 9.0 ± 0.6 KΩ-cm² (perhaps due to swelling of sarcotubules). Ba⁺⁺ (0.5 mM) rapidly increased R_m to 31.3 ± 3.8, decreased E_m (e.g., to -30 mv), and induced spontaneous "action potentials;" Sr⁺⁺ had no effect. In the high K⁺ solution, the fibers were nearly completely depolarized, and R_m was decreased markedly to 1.5 ± 0.2 KΩ-cm²; Ba⁺⁺ increased R_m to 6.7 ± 0.5 KΩ-cm². The Ba⁺⁺ actions usually began within 0.5 min and reached a maximum within 5 min. Addition of SO₄ ⁼, to precipitate the Ba⁺⁺, rapidly reversed the increase in R_m. Ba⁺⁺ must act by decreasing K⁺ conductance (g_K). In Cl^- Ringer''s, the high g_Cl/g_K ratio masked the effect of Ba⁺⁺ on g_K. Thus, small concentrations of Ba⁺⁺ specifically and rapidly decrease g_K.     

Evidence that the uptake of tri-iodo-l-thyronine by human erythrocytes is carrier-mediated but not energy-dependent

We investigated 3,3′,5-tri-iodo-l-thyronine transport by human erythrocytes and by `ghosts'' prepared from these cells. Uptake of tri-iodothyronine by erythrocytes at 37°C was time-dependent with a maximum reached after 60min. Tracer analysis after incubation for 1min revealed only one saturable binding site, with K<sub>m</sub> 128±19nm (mean±s.e.m.; n=7) and V<sub>max.</sub> 4.6±0.7pmol of tri-iodothyronine/min per 6×10<sup>7</sup> cells. After 10min incubation K<sub>m</sub> 100±16nm (n=10) was found with V<sub>max.</sub> 7.7±1.2pmol of tri-iodothyronine/10min per 6×10<sup>7</sup> cells. At 0°C the uptake system is still active, with K<sub>m</sub> 132±26nm and V<sub>max.</sub> 1.8±0.3pmol of tri-iodothyronine/10min per 6×10<sup>7</sup> cells. The V<sub>max.</sub> with intact cells is 5-fold greater than the V<sub>max.</sub> with membranes derived from the same amount of cells when uptake studies are performed in media with similar free tri-iodothyronine concentrations. This indicates that at least 80% of tri-iodothyronine taken up by the intact erythrocytes enters the cell. This saturable uptake system can be inhibited by X-ray-contrast agents in a dose-dependent fashion. (±)-Propranolol, but not atenolol, has the same effect, indicating that the membrane-stabilizing properties of (±)-propranolol are involved. Furthermore, there is no inhibition by ouabain or vanadate, which indicates that tri-iodothyronine uptake is not dependent on the activity of Na<sup>+</sup>+K<sup>+</sup>-dependent adenosine triphosphatase. We have prepared erythrocyte `ghosts'', resealed after 2.5min with 0mm-, 2mm- or 4mm-ATP inside. Inclusion of ATP and integrity of the membrane of the erythrocyte `ghosts'' were verified on the basis of an ATP-concentration-dependent functioning of the Ca<sup>2+</sup> pump. No difference was found in the uptake of tri-iodothyronine by erythrocyte `ghosts'' with or without ATP included, indicating that uptake of tri-iodothyronine is not ATP-dependent. The following conclusions are drawn. (1) Tri-iodothyronine enters human erythrocytes. (2) There is only one saturable uptake system present for tri-iodothyronine, which is neither energy (i.e. ATP)-dependent nor influenced by the absence of an Na⁺ gradient across the plasma membrane. This mode of uptake of tri-iodothyronine by human erythrocytes is in sharp contrast with that of rat hepatocytes, which uptake system is energy-dependent and ouabain-sensitive [Krenning, Docter, Bernard, Visser & Hennemann (1978) FEBS Lett. 91, 113–116; Krenning, Docter, Bernard, Visser & Hennemann (1980) FEBS Lett. 119, 279–282]. (3) X-ray-contrast agents inhibit tri-iodothyronine uptake by erythrocytes in a similar fashion to that by which they inhibit the uptake of tri-iodothyronine by rat hepatocytes [Krenning, Docter, Bernard, Visser & Hennemann (1982) FEBS Lett. 140, 229–233].     

Uptake of aluminium into Arabidopsis root cells measured by fluorescent lifetime imaging

Background and Aims

Measuring the Al³⁺ uptake rate across the plasma membrane of intact root cells is crucial for understanding the mechanisms and time-course of Al toxicity in plants. However, a reliable method with the sufficient spatial and temporal resolution to estimate Al³⁺ uptake in intact root cells does not exist.

Methods

In the current study, fluorescent lifetime imaging (FLIM) analysis was used to quantify Al³⁺ uptake in the root-cell cytoplasm in vivo. This was performed via the estimation of the fluorescence lifetime of Al–lumogallion {5-chloro-3[(2,4-dihydroxyphenyl)azo]-2-hydroxybenzenesulfonic acid} complexes and measurements of intracellular pH while exposing arabidopsis seedlings to acidic and Al³⁺ stresses.

Key Results

The lifetime of Al–lumogallion complexes fluorescence is pH-dependent. The primary sites for Al³⁺ entry are the meristem and distal elongation zones, while Al³⁺ uptake via the cortex and epidermis of the mature root zone is limited. The maximum rates of Al uptake into the cytoplasm (2–3 µmol m⁻³ min⁻¹ for the meristematic root zone and 3–7 µmol m⁻³ min⁻¹ for the mature zone) were observed after a 30-min exposure to 100 µm AlCl₃ (pH 4·2). Intracellular Al concentration increased to 0·4 µm Al within the first 3 h of exposure to 100 µm AlCl₃.

Conclusions

FLIM analysis of the fluorescence of Al–lumogallion complexes can be used to reliably quantify Al uptake in the cytoplasm of intact root cells at the initial stages of Al³⁺ stress.Key words: Acid stress, Al³⁺, aluminium toxicity, Arabidopsis thaliana, low pH, fluorescent lifetime imaging (FLIM), lumogallion     

Sodium flux in the smooth muscle of frog stomach

Sodium efflux from rings of frog stomach muscle was measured at 5° and 15°C in three different steady states. After incubation in normal, K-free, or ouabain (10^-4 M) solutions, intracellular cations stabilized at markedly differing levels. At 5°C, inhibition of Na extrusion was shown in the rate coefficients for ²²Na efflux, which were slightly smaller in K-free than in normal solutions, and much smaller in ouabain. Due to the intracellular Na concentration differences, total Na efflux was similar in K-free and ouabain solutions, and only ⅕ as large in normal solution. At 15°C, normal total Na flux was only 1/7;–1/10 inhibitors, and may be underestimated. The total flux differences may involve dependence of the Na pump and Na permeation on internal Na concentration. The Q ₁₀ of the steady-state fluxes was 3.7 in ouabain, 2.8 in K-free solution, and 1.9 in normal solution. The high temperature dependence of influx as well as efflux suggests transport mechanisms other than simple diffusion. Sodium turnover in the cell water was 46–66 mM/hr in inhibitors at 15°C, and a high rate of Na extrusion in normal muscle is suggested. However, cell volume:surface ratio is only 1.6 µ and all estimates of Na flux were under 3 pmoles/cm² per sec, indicating low Na permeability.     

Barley beta‐glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment

Manganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley-derived (1.3) β-d-glucan (β-d-glucan) is a natural water-soluble polysaccharide with antioxidant properties. To explore the effects of β-d-glucan on MnSOD-related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo. Long-term treatment of HUVECs with 3% w/v β-d-glucan significantly increased the level of MnSOD by 200% ± 2% compared to control and by 50% ± 4% compared to untreated H₂O₂-stressed cells. β-d-glucan-treated HUVECs displayed greater angiogenic ability. In vivo, 24 hrs-treatment with 3% w/v β-d-glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O₂⁻) and an increased survival under oxidative stress. In addition, β-d-glucan prevented the rise of hypoxia inducible factor (HIF)1-α under oxidative stress. The level of histone H4 acetylation was significantly increased by β-d-glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD-related angiogenesis and did not impair β-d-glucan effects. In conclusion, 3% w/v β-d-glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O₂⁻, cell survival and angiogenic response to oxidative stress. The identification of dietary β-d-glucan as activator of MnSOD-related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodelling and heart failure.     

Potassium fluxes in dialyzed squid axons

Measurements have been made of K influx in squid giant axons under internal solute control by dialysis. With [ATP]_i = 1 µM, [Na]_i = 0, K influx was 6 ± 0.6 pmole/cm² sec; an increase to [ATP]_i = 4 mM gave an influx of 8 ± 0.5 pmole/cm² sec, while [ATP]_i 4, [Na]_i 80 gave a K influx of 19 ± 0.7 pmole/cm² sec (all measurements at ∼16°C). Strophanthidin (10 µM) in seawater quantitatively abolished the ATP-dependent increase in K influx. The concentration dependence of ATP-dependent K influx on [ATP]_i, [Na]_i, and [K]_o was measured; an [ATP]_i of 30 µM gave a K influx about half that at physiological concentrations (2–3 mM). About 7 mM [Na]_i yielded half the K influx found at 80 mM [Na]_i. The ATP-dependent K influx responded linearly to [K]_o from 1–20 mM and was independent of whether Na, Li, or choline was the principal cation of seawater. Substances tested as possible energy sources for the K pump were acetyl phosphate, phosphoarginine, PEP, and d-ATP. None was effective except d-ATP and this substance gave 70% of the maximal flux only when phosphoarginine or PEP was also present.     

Transport of glucose and galactose in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25° was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S]_i) and extracellular ([S]_o) glucose concentrations was increased by 0·4mm-phlorrhizin and 0·3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S]_i/[S]_o fell below 1·0 only at [S]_o higher than 0·5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K_m 1·16mm; V_max. 4·5μmoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0·1mm-2,4-dinitrophenol, 0·4mm-phlorrhizin and by the absence of external Na⁺. 6. The kinetic parameters of galactose entry into the cells were: K_m 1·5mm; V_max 10μmoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0°, was inhibited by 0·4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na⁺-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na⁺-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

The effect of amphotericin B on the water and nonelectrolyte permeability of thin lipid membranes

This paper reports the effects of amphotericin B, a polyene antibiotic, on the water and nonelectrolyte permeability of optically black, thin lipid membranes formed from sheep red blood cell lipids dissolved in decane. The permeability coefficients for the diffusion of water and nonelectrolytes (P_DDi) were estimated from unidirectional tracer fluxes when net water flow (J_w) was zero. Alternatively, an osmotic water permeability coefficient (P_f) was computed from J_w when the two aqueous phases contained unequal solute concentrations. In the absence of amphotericin B, when the membrane solutions contained equimolar amounts of cholesterol and phospholipid, P_f was 22.9 ± 4.6 µsec^-1 and P _DDHDH2O was 10.8 ± 2.4 µsec^-1. Furthermore, P_DDi was < 0.05 µsec^-1 for urea, glycerol, ribose, arabinose, glucose, and sucrose, and σ_i, the reflection coefficient of each of these solutes was one. When amphotericin B (10^-6 M) was present in the aqueous phases and the membrane solutions contained equimolar amounts of cholesterol and phospholipid, P _DDHDH2O was 18.1 ± 2.4 µsec^-1; P_f was 549 ± 143 µsec^-1 when glucose, sucrose, and raffinose were the aqueous solutes. Concomitantly, P_DDi varied inversely, and σ_i directly, with the effective hydrodynamic radii of the solutes tested. These polyene-dependent phenomena required the presence of cholesterol in the membrane solutions. These data were analyzed in terms of restricted diffusion and filtration through uniform right circular cylinders, and were compatible with the hypothesis that the interactions of amphotericin B with membrane-bound cholesterol result in the formation of pores whose equivalent radii are in the range 7 to 10.5 A.     

Active transport of chloride by the giant neuron of the Aplysia abdominal ganglion

Internal chloride activity, aⁱ _Cl, and membrane potential, E_m, were measured simultaneously in 120 R2 giant neurons of Aplysia californica. aⁱ _Cl was 37.0 ± 0.8 mM, E_m was -49.3 ± 0.4 mv, and E _Cl calculated using the Nernst equation was -56.2 ± 0.5 mv. Such values were maintained for as long as 6 hr of continuous recording in untreated neurons. Cooling to 1°–4°C caused aⁱ _Cl to increase at such a rate that 30–80 min after cooling began, E _Cl equalled E_m. The two then remained equal for as long as 6 hr. Rewarming to 20°C caused aⁱ _Cl to decline, and E _Cl became more negative than E_m once again. Exposure to 100 mM K⁺-artificial seawater caused a rapid increase of aⁱ _Cl. Upon return to control seawater, aⁱ _Cl declined despite an unfavorable electrochemical gradient and returned to its control values. Therefore, we conclude that chloride is actively transported out of this neuron. The effects of ouabain and 2,4-dinitrophenol were consistent with a partial inhibitory effect. Chloride permeability calculated from net chloride flux using the constant field equation ranged from 4.0 to 36 x 10^-8 cm/sec.     

Substrate specificity of the Bacillus licheniformis lyxose isomerase YdaE and its application in in vitro catalysis for bioproduction of lyxose and glucose by two-step isomerization

Enzymatic processes are useful for industrially important sugar production, and in vitro two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by ydaE of Bacillus licheniformis was found to have broad substrate specificities and has shown high catalytic efficiency on d-lyxose, suggesting that the enzyme is d-lyxose isomerase. Escherichia coli BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn²⁺. The apparent K_m values for d-lyxose and d-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (k_cat/K_m) for lyxose (3.2 ± 0.1 mM⁻¹ s⁻¹) was higher than that for d-mannose (1.6 mM⁻¹ s⁻¹). The purified protein was applied to the bioproduction of d-lyxose and d-glucose from d-xylose and d-mannose, respectively, along with the thermostable xylose isomerase of Thermus thermophilus HB08. From an initial concentration of 10 mM d-lyxose and d-mannose, 3.7 mM and 3.8 mM d-lyxose and d-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for in vitro catalysis and can be applied to industrial production.     

Oxidative Half-reaction of Arabidopsis thaliana Sulfite Oxidase: GENERATION OF SUPEROXIDE BY A PEROXISOMAL ENZYME*

Vertebrate forms of the molybdenum-containing enzyme sulfite oxidase possess a b-type cytochrome prosthetic group that accepts reducing equivalents from the molybdenum center and passes them on to cytochrome c. The plant form of the enzyme, on the other hand, lacks a prosthetic group other than its molybdenum center and utilizes molecular oxygen as the physiological oxidant. Hydrogen peroxide is the ultimate product of the reaction. Here, we present data demonstrating that superoxide is produced essentially quantitatively both in the course of the reaction of reduced enzyme with O₂ and during steady-state turnover and only subsequently decays (presumably noncatalytically) to form hydrogen peroxide. Rapid-reaction kinetic studies directly following the reoxidation of reduced enzyme demonstrate a linear dependence of the rate constant for the reaction on [O₂] with a second-order rate constant of k_ox = 8.7 × 10⁴ ± 0.5 × 10⁴ m⁻¹s⁻¹. When the reaction is carried out in the presence of cytochrome c to follow superoxide generation, biphasic time courses are observed, indicating that a first equivalent of superoxide is generated in the oxidation of the fully reduced Mo(IV) state of the enzyme to Mo(V), followed by a slower oxidation of the Mo(V) state to Mo(VI). The physiological implications of plant sulfite oxidase as a copious generator of superoxide are discussed.     

Cholinesterase activity per unit surface area of conducting membranes

According to theory, the action of acetylcholine (ACh) and ACh-esterase is essential for the permeability changes of excitable membranes during activity. It is, therefore, pertinent to know the activity of ACh-esterase per unit axonal surface area instead of per gram nerve, as it has been measured in the past. Such information has now been obtained with the newly developed microgasometric technique using a magnetic diver. (1) The cholinesterase (Ch-esterase) activity per mm² surface of sensory axons of the walking leg of lobster is 1.2 x 10^-3 µM/hr. (σ = ± 0.3 x 10^-3; SE = 0.17 x 10^-3); the corresponding value for the motor axons isslightly higher: 1.93 x 10^-3 µM/hr. (σ = ± 0.41 x 10^-3; SE = ± 0.14 x 10^-3). Referred to gram nerve, the Ch-esterase activity of the sensory axons is much higher than that of the motor axons: 741 µM/hr. (σ = ± 73.5; SE = ± 32.6) versus 111.6 µM/hr. (σ = ± 28.3; SE = ± 10). (2) The enzyme activity in the small fibers of the stellar nerve of squid is 3.2 x 10^-4 µM/mm²/hr. (σ = ± 0.96 x 10^-4; SE = ± 0.4 x 10^-4). (3) The Ch-esterase activity per mm² surface of squid giant axon is 9.5 x 10^-5 µM/hr. (σ = ± 1.55 x 10^-5; SE = ± 0.38 x 10^-5). The value was obtained with small pieces of carefully cleaned axons after removal of the axoplasm and exposure to sonic disintegration. Without the latter treatment the figurewas 3.85 x 10^-5 µM/mm²/hr. (σ = ± 3.24 x 10^-5; SE = ± 0.93 x 10^-5). The experiments indicate the existence of permeability barriers in the cell wall surrounding part of the enzyme, since the substrate cannot reach all the enzyme even when small fragments of the cell wall are used without disintegration. (4) On the basis of the data obtained, some tentative approximations are made of the ratio of ACh released to Na ions entering the squid giant axon per cm² per impulse.     

Sucrose Octasulfate Selectively Accelerates Thrombin Inactivation by Heparin Cofactor II

Inactivation of thrombin (T) by the serpins heparin cofactor II (HCII) and antithrombin (AT) is accelerated by a heparin template between the serpin and thrombin exosite II. Unlike AT, HCII also uses an allosteric interaction of its NH₂-terminal segment with exosite I. Sucrose octasulfate (SOS) accelerated thrombin inactivation by HCII but not AT by 2000-fold. SOS bound to two sites on thrombin, with dissociation constants (K_D) of 10 ± 4 μm and 400 ± 300 μm that were not kinetically resolvable, as evidenced by single hyperbolic SOS concentration dependences of the inactivation rate (k_obs). SOS bound HCII with K_D 1.45 ± 0.30 mm, and this binding was tightened in the T·SOS·HCII complex, characterized by K_complex of ∼0.20 μm. Inactivation data were incompatible with a model solely depending on HCII·SOS but fit an equilibrium linkage model employing T·SOS binding in the pathway to higher order complex formation. Hirudin-(54–65)(SO₃⁻) caused a hyperbolic decrease of the inactivation rates, suggesting partial competitive binding of hirudin-(54–65)(SO₃⁻) and HCII to exosite I. Meizothrombin(des-fragment 1), binding SOS with K_D = 1600 ± 300 μm, and thrombin were inactivated at comparable rates, and an exosite II aptamer had no effect on the inactivation, suggesting limited exosite II involvement. SOS accelerated inactivation of meizothrombin 1000-fold, reflecting the contribution of direct exosite I interaction with HCII. Thrombin generation in plasma was suppressed by SOS, both in HCII-dependent and -independent processes. The ex vivo HCII-dependent process may utilize the proposed model and suggests a potential for oversulfated disaccharides in controlling HCII-regulated thrombin generation.