Jeffrey D. Owen

The determination of the stability constant for calcium EGTA

Abstract The stability constant for calcium-EGTA, KCaEGTA, was determined in imidazole, Tris-maleate and phosphate buffered-solutions. The value for KCaEGTA was found to be independent of the buffer solution, in contrast with earlier reports (Ogawa, Y. (1968) J. Biochem. 64, 225; Godt, R.E. (1974) J. Gen. Physiol. 63, 722). Using a newly developed Ca2+-selective electrode (Brown, H.M., Pemberton, J.P. and Owen, J.D. (1976) Anal. Chim. Acta, in the press) the Ca2+ activities in solutions of different [CaEGTA]/[EGTA] ratio were found to fit acalibration plot of mV vs. log calcium activity using the KCaEGTA value of Schwarzenbach, G., Senn, H. and Anderegg, G. ((1957) Helv. Chim. Acta 40, 1886).

The effect of phloretin on red cell nonelectrolyte permeability.

SummaryThe effect of phloretin on permeability of small nonelectrolytes into human red cells was shown to be bimodal for hydrophilic molecules and nonbimodal for lipophilic molecules. At low phloretin concentrations (<0.1mm) hydrophilic as well as lipophilic nonelectrolyte permeation was increased. At high phloretin concentrations (>0.1mm) the permeability of hydrophilic molecules was decreased, whereas lipophilic molecule permeability continued to be increased. These results suggest that the mechanism for phloretin acting on red cell nonelectrolyte pathways is different for hydrophilic molecules, as compared to lipophilic permeant molecules. The pH of the red cell buffer suspension also influenced the effect of phloretin on nonelectrolyte permeability and the keto or un-ionized form of phloretin (present at low pH), which is known to have a greater affinity for the membrane, had a larger effect on hydrophilic nonelectrolyte permeability than the ionized form of phloretin.

The Effect of phloretin on the potassium conductance inAplysia giant neurons

SummaryPhloretin perfusedAplysia giant neurons exhibited a rapid and reversible increased potassium conductance. When the cell was voltage-clamped at the resting potential of about −40 mV, the phloretin-induced conductance increase was accompanied by an outward current, which reversed to an inward current below the potassium equilibrium potential of approximately −77 mV. The phloretin effect was not affected by variations in the sodium, chloride, calcium, or magnesium concentrations, but it was sensitive to varied potassium concentrations. The increased potassium conductance persisted at pH=5, when pH≪pK, where according to a dose-response curve, only the un-ionized from of the phloretin molecule could be responsible for the effect. Phloretin increased the membrane potassium conductance as if the neuron were depolarized. The mechanism for this phloretin-induced depolarization may involve an increased surface potential due to an induction of a dipole moment or a selective increase in the number of membrane potassium sites.

Light scattering temperature jump relaxations in mixed solvent suspensions of phosphatidylcholine vesicles

Abstract Lipid vesicles were prepared from hen egg yolk phosphatidylcholine according to Huangs method. A relaxation of approx. 0.4 msec was observed in the intensity of white light scattered from an aqueous suspension of these vesicles following a rapid Joule heating temperature jump. This relaxation was observed to lengthen to about 1.5 msec as non-electrolyte (glycol, glycerol, 1,3-propanediol, or 1,4-butanediol) was added to the medium. The relaxation was attributed to passage of small molecules through the lipid membrane. The solvent viscosity was found to have a more pronounced effect upon the measured relaxation than did the size of the added non-electrolyte molecules.

Temperature jump relaxations in aqueous saline suspensions of human erythrocytes

The Joule heating temperature jump relaxation method utilizing scattered white light intensity detection has been found suitable for measuring kinetic properties of whole cells suspended in aqueous saline solutions. Human erythrocytes suspended in aqueous 0.9% NaCl and subjected to a rapid 8° temperature jump to 25° yield two relaxation; τ1 of approx. 0.9 msec and τ2 of approx. 120 msec. The latter appears to be characteristic of intact cells only. On the other hand, τ1 is obtained with ghosts as well as with whole erythrocytes. Similar experiments were carried out at several cell dilutions, at different scattering angles, at several NaCl concentrations, with other salts, in 2H2O, and with added sodium lauryl sulfate or 1,5-difluoro-2,4-dinitrobenzene. The 0.9-nisec relaxation appears to be the time constant for passage of water through the erythrocyte membrane. The principal obstacle to a firmer attribution of τ1 is the unknown time constant for temperature equilibrium of the erythrocyte interior with the heated saline solution.

Intracellular changes of h+ and ca2+ activities in Aplysia giant neurons as measured with ion selective microelectrodes

Abstract 1. 1. Intracellular pH (pH i ) and Ca 2+ activity (a i Ca ) were measured in giant cells (R 2 ) of Aplysia californica with recessed tip pH electrodes and diaryl-phosphate-PVC Ca electrodes. 2. 2. The intracellular pH with the cells bathed in Tris buffered saline (pH 0 7.65) was 7.25 ± 0.05 (SD) in four different cells. 3. 3. An acid load produced by 5% CO 2 13.25 mM HCO − 3 , reduced pH i to 6.95 ± 0.05 (SD) in approx 15 min. 4. 4. The buffer power of the cell (β) was approx 33 mM HCO − 3 /l.pH unit. 5. 5. Bright white illumination which produces a membrane hyperpolarization was without measureable effect on pH i or(a i Ca ). CO 2 -HCO 3 saline (5% CO 2 , 13.25 mM HCO 3 ) was also without effect on (a i Ca ). 6. 6. Sustained activation of the cell by outward current pulses from another microelectrode produced no measureable change in (a i Ca ). 7. 7. Pressure injection of Ca 2+ from a CaCl 2 -filled microelectrode into the cell cytoplasm raised (a i Ca ) to 1.4 × 10 −3 M. The time course of (a i Ca ) recovery was considerably longer than the membrane hyperpolarization.

Dimerization kinetics of aqueous gallium(III) perchlorate

Temperature jump relaxation times between 5 and 10 msec observed in pH 3·6 aqueous ∼10−2M gallium(III) perchlorate solutions are ascribed to the equilibrium 2 GAOH2+⇌kaGa2(OH)24+. The specific rate of dimerization at 25° and 0·5 M ionic strength is found to be k3 = 3·4 ± 0·6 × 103 M−1 sec−1. The first order rate constant k0 = 2·7 × 105 sec−1 for the rate determining step in the dimerization of GaOH2+ (deduced from the k3 assuming an ion contact distance of 5 A) is considerably larger than k0∗ = 1·2 × 103 sec−1 reported for inner coordination sphere water loss from GaOH2+ in the course of complexation by SO42−. Thus unlike the cases of FeOH2+ and VOOH+ dimerzation persuasive evidence is lacking for rate determining inner coordination sphere water loss in the dimerization of aqueous GaOH2+.

Computer-simulated urea reflection coefficients in human red cells.

Relative volume curves for human red cells were generated with a computer program to simulate the experimental results obtained by rapidly mixing red cells and urea. Known values for the permeability variables were used in the Kedem and Katchalsky (Biochim. Biophys. Acta (1958) 27, 229-246) solute and solvent flux equations. The different values used for the reflection coefficient, o, were 0.62, 0.75 and 1.0. The theoretical computer curves with o = 0.75 compared closely with the experimental stopped-flow curves.

Effects of potassium-free solutions on membrane current—voltage relations of Aplysia giant neurons

Abstract 1. Cooling, ouabain and K + -free solutions are thought to produce inhibition of an electrogenic Na + -pump in Aplysia giant neurons. 2. Current-voltage relations of the membrane in ouabain and K + -free solution became increasingly dissimilar at membrane potentials more negative than the resting potential. 3. Ouabain produced a constant depolarization over the physiological range of membrane potentials. 4. K + -free solutions elicited a potential change that reversed sign at the normal potassium equilibrium potential, E k ⋍ −80 mV. 5. Measurements of external K + -activity (a o k ) next to the membrane indicate that a o k remains relatively constant even in K + -free solutions. 6. K + -free solutions produce membrane depolarization by K + -conductance decrease and ouabain inhibits the pump.

Reflection coefficients in red cells.

Milgram and Solomon (1977) reported Owen and Eyrings (1975) value for reflection coefficients, o, in human red cells were in error by 33 ~. Their concern is similar to Levitts (1974) and Owen and Eyrings when they re-examined Goldstein and Solomons (1960) o data, i.e., a will be incorrect unless determined at time zero (Kedem & Katchalsky, 1958). Determining cr at times greater than zero should tend to underestimate cr, and Solomon, Milgram and Kirkwood (1975) reported that Goldstein and Solomons cr value was underestimated by 10 20 ~. This corresponds to Goldstein and Solomons a for urea of 0.62 being corrected to 0.68 0.74. This revised a is very close to Owen and Eyrings o of 0.79, so it is difficult to see where there is any controversy. Also, computer-simulated red cell shrinking/swelling data (Owen, 1976) for a a other than 0.79 does not agree with the experimental results, and similar theoretical red cell data (Owen & Galey, 1977) does not rule out the possibility that o is 0.79.