Effects of temperature on water diffusion in human erythrocytes and ghosts--nuclear magnetic resonance studies

The temperature-dependence of water diffusion across human erythrocyte membrane was studied on isolated erythrocytes and resealed ghosts by a doping nuclear magnetic resonance technique. The conclusions are the following: (1) The storage of suspended erythrocytes at 2 degrees C up to 24 h or at 37 degrees C for 30 min did not change the water exchange time significantly, even if Mn2+ was present in the medium. This indicates that no significant penetration of Mn2+ is taking place under such conditions. (2) In case of cells previously incubated at 37 degrees C for longer than 30 min with concentrations of p-chloromercuribenzene sulfonate (PCMBS) greater than 0.5 mM, the water-exchange time gradually decreased if the cells were stored in the presence of Mn2+ for more than 10 min at 37 degrees C. (3) When the Arrhenius plot of the water-exchange time was calculated on the basis of measurements performed in such a way as to avoid a prolonged exposure of erythrocytes to Mn2+ no discontinuity occurred, regardless of the treatment with PCMBS. (4) No significant differences between erythrocytes and resealed ghosts regarding their permeability and the activation energy of water diffusion (Ea,d) were noticed. The mean value of Ea,d obtained on erythrocytes from 35 donors was 24.5 kJ/mol. (5) The value of Ea,d increased after treatment with PCMBS, in parallel with the percentage inhibition of water diffusion. A mean value of 41.3 kJ/mol was obtained for Ea,d of erythrocytes incubated with 1 mM PCMBS for 60 min at 37 degrees C and 28.3 kJ/mol for ghosts incubated with 0.1 mM PCMBS for 15 min, the values of inhibition being 46% and 21% respectively.     

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XIV. Little Penguin, Eudyptula minor

As part of a programme of comparative measurements of diffusional water permeability (Pd) the red blood cells (RBC) from Little Penguin (Eudyptula minor) were studied. The cell dimensions were measured with light and electron microscopy, and by a newly described non-invasive technique, NMR q-space analysis. In view of its relative novelty for cell biologists, an overview of this technique is presented. The RBC revealed an ellipsoidal shape that is characteristic of avian RBC, with axis lengths ("diameters") estimated to be: a=16.0 microm; b=9.6 microm; c=5.0 microm. The values of P(d)were: 2.0 x 10(-3)cm s(-1)at 5 degrees C, 3.3 x 10(-3)cm s(-1)at 10 degrees C, 4.6 x 10(-3)cm s(-1)at 15 degrees C and approximately 5.4 x 10(-3)cm s(-1)at 20, 25, 30, 37 and 42 degrees C.There was a lack of inhibition of water permeability by p-chloromercuribenzensulfonate (PCMBS), the well-known inhibitor of RBC aquaporin. It was notable that in the temperature range 5-20 degrees C the NMR parameters, and hence the permeability, varied linearly as is found for other species, but at temperatures higher than 20 degrees C there was no temperature-dependence of Pd. Consequently, there was an obvious break at approximately 20 degrees C in the Arrhenius plot, of the mean residence life time of water inside the cells, 1/Te, versus temperature. For temperatures less than 20 degrees C the activation energy E(a,d) was 45.6 +/- 6.6 kJ/mol. For temperatures higher than 25 degrees C E(a,d) was zero. The lack of inhibition of water permeability by PCMBS and the very high value of E(a,d) for diffusive water exchange suggests that the water permeation occurs primarily via the membrane bilayer per se, i.e., there is no aquaporin in Little Penguin RBC. The discontinuity at approximately 20 degrees C in the Arrhenius plot is an interesting finding, not seen before in other species, and we suggest that it reflects a phase transition in the membrane lipids.     

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XVI Dingo (Canis familiaris dingo) and dog (Canis familiaris)

As part of a programme of comparative measurements of P _d (diffusional water permeability) the RBCs (red blood cells) from dingo (Canis familiaris dingo) and greyhound dog (Canis familiaris) were studied. The morphologies of the dingo and greyhound RBCs [examined by light and SEM (scanning electron microscopy)] were found to be very similar, with regard to aspect ratio and size; the mean diameters were estimated to be the same (～7.2 μm) for both dingo and greyhound RBCs. The water diffusional permeability was monitored by using an Mn²⁺‐doping ¹H NMR technique at 400 MHz. The P _d (cm/s) values of dingo and greyhound RBCs were similar: 6.5×10^?3 at 25°C, 7.5×10^?3 at 30°C, 10×10^?3 at 37°C and 11.5×10^?3 at 42°C. The inhibitory effect of a mercury‐containing SH (sulfhydryl)‐modifying reagent PCMBS (p‐chloromercuribenzene sulfonate) was investigated. The maximal inhibition of dingo and greyhound RBCs was reached in 15–30 min at 37°C with 2 mmol/l PCMBS. The values of maximal inhibition were in the range 72–74% when measured at 25°C and 30°C, and ～66% at 37°C. The lowest value of P _d (corresponding to the basal permeability to water) was ～2–3×10^?3 cm/s in the temperature range 25–37°C. The E _a,d (activation energy of water diffusion) was 25 kJ/mol for dingo RBC and 23 kJ/mol for greyhound RBCs. After incubation with PCMBS, the values of E _a,d increased, reaching 46–48 kJ/mol in the condition of maximal inhibition of water exchange. The electrophoretograms of membrane polypeptides of the dingo and greyhound RBCs were compared and seen to be very similar. We postulate that the RBC parameters reported in the present study are characteristic of all canine species and, in particular in the two cases presented here, these parameters have not been changed by the peculiar Australian habitat over the millennia (as in the case of the dingo) or over shorter time periods, decades or centuries (as in the case of the domestic greyhound).     

Water exchange through erythrocyte membranes: Biochemical and nuclear magnetic resonance studies re-evaluating the effects of sulfhydryl reagents and of proteolytic enzymes on human membranes

Summary The water permeability of human red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on intact cells and resealed ghosts following exposure to various sulfydryl-reacting (SH) reagents and proteolytic enzymes. The main conclusions are the following: (i) When appropriate conditions for exposure of erythrocytes or ghosts to mercury-containing SH reagents (concentration, temperature and duration of incubation) were found, the maximal inhibition of water diffusion could be obtained with all mercurials (including HgCl₂ and mersalyl that failed to show their inhibitory action on RBC water permeability in some investigations). While previous studies claimed that long incubation times are required for the development of maximal inhibition of water diffusion by mercurials, the present results show that it can be induced in a much shorter time (5–15 min at 37°C) if relatively high concentrations of PCMBS (2–4mm) are used and no washings of the inhibitor are performed after incubation. Higher than optimal concentrations of mercurials and/or longer incubation times result in lower values of inhibition, sometimes a loss of inhibition, or can even lead to higher values of permeability compared to control RBCs. (ii) The conditions for inhibition by mercurials are drastically changed by preincubation of erythrocytes with noninhibitory SH reagents (such as NEM or IAM) or by exposure to proteolytic enzymes. If the cells are digested with papain, the duration of incubation with PCMBS should be decreased in order for inhibition to occur. This explains the lack of inhibition reported previously, when a relatively long duration of incubation with PCMBS was used subsequent to papain digestion. (iii) The degree of inhibition of water diffusion induced by mercurials appeared to be dependent upon the temperature of which the water permeability was measured. The values of maximal inhibition ranged from 45–50% at 37°C, increased 10–15% at 20°C and further increased at lower temperatures, reaching values above 75% below 10°C; these results clarify the conflicting reports of various authors. (iv) The inhibition of water diffusion, either reversible, or irreversible, was not accompanied by significant changes in the pattern of RBC membrane polypeptides fractionated by polyacrylamide gel electrophoresis. (v) The mean value of the activation energy of water diffusion (E _a,d) obtained on 42 donors was 25.6 kJ/mol. The values ofE _a,d increased in parallel with the values of the inhibition of water diffusion induced by PCMBS until the maximal inhibition was reached (whenE _a,d=41 kJ/mol) and then both sets of values decreased in parallel.     

Diffusional water permeability of human erythrocytes and their ghosts

The diffusional water permeability of human red cells and ghosts was determined by measuring the rate of tracer efflux by means of an improved version of the continuous flow tube method, having a time resolution of 2-3 ms. At 25 degrees C, the permeability was 2.4 x 10(3) and 2.9 x 10(3) cm s-1 for red cells and ghosts, respectively. Permeability was affected by neither a change in pH from 5.5 to 9.5, nor by osmolality up to 3.3 osmol. Manganous ions at an extracellular concentration of 19 mM did not change diffusional water permeability, as recently suggested by NMR measurements. A "ground" permeability of 1 x 10(3) cm s-1 was obtained by inhibition with 1 mM of either p- chloromercuribenzoate (PCMB) or p-chloromercuribenzene sulfonate (PCMBS). Inhibition increased temperature dependence of water permeability for red cells and ghosts from 21 to 30 kJ mol-1 to 60 kJ mol-1. Although diffusional water permeability is about one order of magnitude lower than osmotic permeability, inhibition with PCMB and PCMBS, temperature dependence both before and after inhibition, and independence of osmolality showed that diffusional water permeability has qualitative features similar to those reported for osmotic permeability, which indicates that the same properties of the membrane determine both types of transport. It is suggested that the PCMB(S)- sensitive permeability above the ground permeability takes place through the intermediate phase between integral membrane proteins and their surrounding lipids.     

Effects on water diffusion of inhibitors affecting various transport processes in human red blood cells.

The water permeability of human red blood cells has been monitored by nuclear magnetic resonance (NMR) following exposure to inhibitors of various transport processes across their membranes. No significant inhibition of water diffusion could be detected after the treatment of red blood cells with the anion exchange transport inhibitor dihydro-4,4'-diisothiocyano-stilbene-2,2'-disulfonate (H2DIDS) or the glucose transport inhibitors diallyl-diethyl-stilbestrol (DADES), cytochalasin B, or 30 mM iodoacetamide. It is for the first time that the effects of glucose transport inhibitors has been studied in detail by the NMR approach. A special case proved to be phloretin, an inhibitor of anion, nonelectrolyte and glucose permeability. A small but statistically significant inhibition of water permeability (around 12% at 20 degrees C) was induced by exposure to 2 mM phloretin (for 60 min at 37 degrees C); after a pretreatment of cells with 12 mM N-ethylmaleimide (NEM), for 60 min at 37 degrees C, the degree of inhibition induced by phloretin increased (becoming 17% at 20 degrees C). None of the inhibitors prevented or potentiated the strong inhibitory effect on water diffusion of a mercurial, p-chloromercuribenzene sulfonate (PCMBS). No increase in the activation energy of water diffusion occurred by treatment with the reagents used (exception the effect of PCMBS). The present results clarify some conflicting reports concerning the effects on water permeability of inhibitors of various transport processes in red blood cells and indicate that in addition to the drastic inhibition induced by mercurials other reagents may also have inhibitory effects.     

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. V—Rabbit (Oryctolagus Cuniculus)

• 1.1. The diffusional water permeability (P_d) of rabbit red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulfonate (PCMBS).
• 2.2. The values of P_d were around 6.3 × 10⁻³ cm/sec at 15°C, 7.0 × 10⁻³cm/sec at 20°C, 8.0 × 10⁻³ cm/sec at 25°C, 9.1 × 10⁻³ cm/sec at 30°C and10.7 × 10⁻³ cm/sec at 37°C.
• 3.3. Systematic studies on the effects of PCMBS on water diffusion indicated that the maximal inhibition was reached in 15 min at 37°C with 0.5 mM PCMBS.
• 4.4. The values of maximal inhibition were around 71–74% at all temperatures.
• 5.5. The basal permeability to water was estimated as 1.6 × 10⁻³cm/sec at 15°C, 2.0 × 10⁻³cm/sec at 20°C, 2.4 × 10⁻³cm/sec at 25°C, 2.6 × 10⁻³cm/sec at 30°C, and 3.1× 10^{−3 cm/secat 37°C}.
• 6.6. The activation energy of water diffusion was around 18 kJ/mol and increased to 27 kcal/mol after incubation with PCMBS in conditions of maximal inhibition of water diffusion.
• 7.7. The membrane polypeptide electrophoretic pattern of rabbit RBCs has been compared with its human counterpart.
• 8.8. The rabbit membrane contained a higher amount of spectrin (bands 1 and 2), while the band 6 (glyceraldehyde-3-phosphate dehydrogenase) was markedly less intense.
• 9.9. Considerable differences in the electrophoretic patterns of the two sources of RBC membranes appeared in the bands migrating in the band 4.5 region and in front of band 7, where some polypeptides were apparent in higher amounts in the rabbit RBC membrane.

     

On measuring the diffusional water permeability of human red blood cells and ghosts by nuclear magnetic resonance

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes and ghosts by a doping nuclear magnetic resonance technique. In contrast to all previous investigations, systematic measurements were performed on blood samples obtained from a large group of donors. The mean values of P ranged from 2.2 X 10(-3) cm.s-1 at 5 degrees C to 8.1 X 10(-3) cm.s-1 at 42 degrees C. The reasons for some of the discrepancies in the permeability coefficients reported by various authors were found. In order to estimate the basal permeability, the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzenesulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 1.3 X 10(-3) cm.s-1 at 20 degrees C, 1.6 X 10(-3) cm.s-1 at 25 degrees C, 1.9 X 10(-3) cm.s-1 at 30 degrees C and 3.2 X 10(-3) cm.s-1 at 37 degrees C. The results reported here represent the largest series of determinations of water diffusional permeability of human red blood cells (without or with exposure to mercurials) available in the literature, and consequently the best estimates of the characteristics of this transport process. The values of P can be taken as references for the studies of water permeability in various cells or in pathological conditions.     

The basal permeability to water of human red blood cells evaluated by a nuclear magnetic resonance technique

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes by a doping nuclear magnetic resonance technique. In order to estimate the basal permeability the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzene sulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 0.7×10^–3 cm s^–1 at 10°C, 1.2×10^–3 cm s^–1 at 15°C, 1.4×10^–3 cm s^–1 at 20°C, 1.8×10^–3 cm s^–1 at 25°C, 2.1×10^–3 cm s^–1 at 30°C and 3.5×10^–3 cm s^–1 at 37°C. The mean value of the activation energy of water diffusion (E_a,d) was 25 kJ/mol for control and 43.7 kJ/mol for PCMBS-inhibited erythrocytes. The values of P and E_a,d obtained after induction of maximal inhibition of water diffusion by PCMBS can be taken as references for the basal permeability to water of the human red blood cell membrane.     

Urea permeability of human red cells

The rate of unidirectional [14C]urea efflux from human red cells was determined in the self-exchange and net efflux modes with the continuous flow tube method. Self-exchange flux was saturable and followed simple Michaelis-Menten kinetics. At 38 degrees C the maximal self-exchange flux was 1.3 X 10(-7) mol cm-2 s-1, and the urea concentration for half-maximal flux, K1/2, was 396 mM. At 25 degrees C the maximal self-exchange flux decreased to 8.2 X 10(-8) mol cm-2 s-1, and K1/2 to 334 mM. The concentration-dependent urea permeability coefficient was 3 X 10(-4) cm s-1 at 1 mM and 8 X 10(-5) cm s-1 at 800 mM (25 degrees C). The latter value is consonant with previous volumetric determinations of urea permeability. Urea transport was inhibited competitively by thiourea; the half-inhibition constant, Ki, was 17 mM at 38 degrees C and 13 mM at 25 degrees C. Treatment with 1 mM p-chloromercuribenzosulfonate inhibited urea permeability by 92%. Phloretin reduced urea permeability further (greater than 97%) to a "ground" permeability of approximately 10(-6) cm s-1 (25 degrees C). This residual permeability is probably due to urea permeating the hydrophobic core of the membrane by simple diffusion. The apparent activation energy, EA, of urea transport after maximal inhibition was 59 kJ mol-1, whereas in control cells EA was 34 kJ mol-1 at 1 M and 12 kJ mol-1 at 1 mM urea. In net efflux experiments with no extracellular urea, the permeability coefficient remained constantly high, independent of a variation of intracellular urea between 1 and 500 mM, which indicates that the urea transport system is asymmetric. It is concluded that urea permeability above the ground permeability is due to facilitate diffusion and not to diffusion through nonspecific leak pathways as suggested previously.     

On the water and proton permeabilities across membranes from erythrocyte ghosts

The diffusional permeability of water across membranes from bovine and human erythrocyte ghosts was measured by a recently developed method which is based on the different indices of refraction of H2O and 2H2O. Resealed erythrocyte ghosts were prepared by a gel-filtration technique. Pd (2H2O/H2O) values of 1.2 X 10(-3) cm/s (human) and 1.7 X 10(-3) cm/s (bovine) were calculated at 20 degrees C. The activation energies of the water exchange were 23.5 kJ/mol (human) and 25.4 kJ/mol (bovine). Treatment of the ghosts with p-chloromercuribenzenesulfonic acid (PCMBS) led to a 60-70% inhibition of the diffusional water exchange. The pH equilibration across membranes of erythrocyte ghosts was measured by intracellular carboxyfluorescein. The rates of proton flux after pH-jumps (pH 7.3 to pH 6.1) were about 100-fold lower than those of the water exchange and dependent on the kind of anions present (Cl-, NO-3, SO2-4). The activation energies of proton flux were 60-70 kJ/mol. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibited the exchange by 97-98% and lowered the activation energy. The inhibitor of water exchange, PCMBS, increased the proton-permeation rate by a factor of 4-5. It is assumed that the rate-limiting step for the proton permeation is determined by the anion exchange. Under this condition our results are not in accord with one channel as a common pathway for both the passive water and anion transport.     

Water channels in membranes

A systematic programme of comparative nuclear magnetic resonance measurements of the membrane permeability for water (Pd) and of activation energy (E_a,d) of this process in red blood cells of various wild, laboratory and domestic animals was carried out here. The RBC from humans, cow, sheep and kangaroos had P_d values around 5·10^?3 cm/s at 25 °C, 7 · 10^?3 cm/s at 37 °C with E_a,d values around 25 kJ/mol. For RBC from other ten marsupial species and from mouse, rat and rabbit, the P_d values were more than twice as high as for human RBC. For mosr RBC a high value of Pd was associated with a low value of E_a,d (range from 15 to 21 kJ/mol), pointing to specialized channels for water diffusion incorporated in membrane proteins. Recently a channel-forming integral protein of 28 kDa (CHIP 28) was identified as a major water channel protein in the RBC membrane. A procedure for quantitating the purified CHIP 28 by densitometry of silver-stained polyacrylmide gel electrophoreograms was developed. The analysis of a purified fraction of CHIP 28 showed that the 28 kDa component represents approximately two-thirds of the sample with the remainder comprising the glycosylated high-molecular-weight component. A correlation between the content in CHIP 28 and the relative water permeability among RBC from different vertebrate species was attempted.     

The influence of temperature upon the hydrolysis of cellobiose by beta-1,4-glucosidases from Aspergillus niger

We have made experimental studies into the enzymatic hydrolysis of cellobiose within the temperature range of 40 degrees C to 70 degrees C at pH 4.9, by using beta-1,4-glucosidase from Aspergillus niger. At 70 degrees C there was significant enzyme deactivation, which could be fitted to a potential deactivation model with values of n equal to 1.09 and k(d) to 0.1564 (g/l)(-0.09) min(-1), whereas the rate of hydrolysis could be fitted to the Michaelis-Menten equation. Between 40 degrees C and 60 degrees C we noted a substrate inhibition and that the CEC compound formed contributed to glucose production. The apparent activation energies had values of 4.66, 8.45, 4.82, and 3.99 kJ/mol for the kinetic constants k(a) and k(a2) the Michaelis constant and the substrate inhibition constant, respectively.     

Loss of surface galactosyl receptor activity on isolated rat hepatocytes induced by monensin or chloroquine requires receptor internalization via a clathrin coated pit pathway

We studied the effect of hyperosmotic inhibition of the clathrin coated pit cycle on the monensin- and chloroquine-dependent loss of surface galactosyl (Gal) receptor activity on isolated rat hepatocytes. Cells treated for 60 min without ligand at 37 degrees C with 25 microM monensin or 300 microM chloroquine in normal medium (osmolality congruent to 275 mmol/kg) bound 40-60% less 125I-asialo-orosomucoid (ASOR) at 4 degrees C than untreated cells. Cells exposed to monensin or chloroquine retained progressively more surface Gal receptor activity, however, when the osmolality of the medium was increased above 400 mmol/kg (using sucrose as osmolite) 10 min prior to and during drug treatment. Cells pretreated for 10 min with hyperosmolal media (600 mmol/kg) alone internalized less than or equal to 10% of surface-bound 125I-ASOR. Thus, the ligand-independent loss of surface Gal receptor activity on monensin- and chloroquine-treated hepatocytes requires internalization of constitutively recycling receptors via a coated pit pathway.     

Store-operated calcium channels in HL-60 cells effects of temperature,differentiation and loperamide

The effect of temperature on calcium release and influx has been compared in differentiated and undifferentiated HL-60 cells. Receptor-mediated release of intracellular calcium by ATP was little affected by temperature in HL-60 cells. In differentiated HL-60 cells the store-operated calcium (SOC) channel-dependent sustained elevation of calcium levels after ATP was maximal at 25-29 degrees C; at higher temperatures calcium levels returned relatively rapidly towards basal levels. In undifferentiated cells, a SOC channel-dependent sustained elevation of calcium levels was not observed with levels returning to basal levels much more rapidly than in differentiated cells. The initial thapsigargin-initiated elevation of calcium did not become maximal until about 25 degrees C in both differentiated and undifferentiated HL-60 cells. In differentiated cells, the SOC channel-dependent sustained elevation of calcium after thapsigargin was maximal at 30-37 degrees C, while in undifferentiated cells, the sustained elevation was maximal at 25-30 degrees C. Loperamide, which augments the SOC channel-dependent sustained elevation of calcium, showed a temperature-dependent response that was maximal at about 22 degrees C after either ATP or thapsigargin and was minimal at 37 degrees C. In contrast, inhibition of SOC channel-dependent elevation of calcium by miconazole or trifluoperazine was not greatly affected by temperature.     

Temperature dependence of ADP/ATP translocation in mitochondria

The temperature dependence of the adenine nucleotide exchange in mitochondria has been determined by employing a rapid mixing, quenching and sampling apparatus and the inhibitor quench-back exchange method. Thus the exchange is resolved down to 0.1 s. Rates are evaluated from accumulating the time-dependent progress at about 10 points. The exchange rate in liver mitochondria was determined from -10 degrees C to + 10 degrees C in the presence of 20% glycol, from 0 degrees C to 25 degrees C, and from 20 degrees C to 40 degrees C under partial inhibition by carboxyatractylate. The total range between -10 degrees C to + 40 degrees C has only one temperature break at 13 degrees C. From the Arrhenius plot between -10 degrees C to + 13 degrees C, EA approximately equal to 140 kJ and above 13 degrees C, EA approximately equal to 56 kJ is evaluated, corresponding to a Q10 of 8 and 2 respectively. In beef heart mitochondria the exchange rate was measured between 0 degrees C and 20 degrees C, and between 15 degrees C and 30 degrees C under partial inhibition with carboxyatractylate. There is a temperature break around 14 degrees C with EA approximately equal to 143 kJ between 0 degrees C and 14 degrees C and EA approximately equal to 60 kJ from 15 degrees C to 30 degrees C. The extrapolated translocation rates at 37 degrees C are 500 and 1800 mumol min-1 (g protein)-1 for rat liver and for beef heart mitochondria respectively. The temperature break is suggested to reflect a conformation change since there is no reversed break at low temperature, the temperature break changes in sonic particles and no lipid phase transition at 14 degrees C in mitochondria has been reported.     

Cell-to-cell adhesion of dissociated embryonic brain cells; the effects of metabolic inhibitors and temperature

Brain cells from 16 to 18-day-old mice embryos were dissociated by mild trypsinization and rotated for 120 min. The area and density of of the adhesive complexes formed were registered using the method described previously. The adhesiveness of dissociated embryonic brain cells (measured during the 120 min of rotation) was diminished in the presence of inhibitors of protein synthesis (puromycin, cycloheximide and inhibition of mRNA synthesis actinomycin D). The inhibition was, however, not distinct, because 1 microgram/ml of cycloheximide and actinomycin was without any significant effect, and the degree of inhibition evoked by 10 micrograms/ml and 25 micrograms/ml of puromycin bordered on significance. However, protein synthesis inhibitors in long-term aggregation experiments had a pronounced inhibitory effect and/or induced destruction of the aggregates. Metabolic inhibitors (KCN and NaN3) caused an inhibition at the lowest level of significance (p less than 0.05) 10(-3) mol/l KCN reduced the final adhesive product significantly. Cells rotated at room temperature and at +5 degrees C adhere to the same extent as in control experiments (37 degrees C). The adhesion was significantly inhibited at +60 degrees C and also after freezing at -80 degrees C with subsequent thawing. The adhesion of cells exposed for 30 min to between +80 degrees C and 100 degrees C was completely abolished. The process of embryonic brain cell adhesion requires a low energy supply, and is relatively independent of biosynthetic processes and of temperature changes between +5 degrees C and +50 degrees C.     

Cytochalasin B inhibition and temperature dependence of 3-O-methylglucose transport in fat cells

The transport of 3-O-methylglucose in white fat cells was measured under equilibrium exchange conditions at 3-O-methylglucose concentrations up to 50 mM with a previously described method (Vinten, J., Gliemann, J. and Osterlind, K. (1976) J. Biol. Chem. 251, 794--800). Under these conditions the main part of the transport was inhibitable by cytochalasin B. The inhibition was found to be of competitive type with an inhibition constant of about 2.5 . 10(-7) M, both in the absence and in the presence of insulin (1 micrometer). The cytochalasin B-insensitive part of the 3-O-methylglucose permeability was about 2 . 10(-9) cm . s-1, and was not affected by insulin. As calculated from the maximum transport capacity, the half saturation constant and the volume/surface ratio, the maximum permeability of the fat cell membrane to 3-O-methylglucose at 37 degrees C and in the presence of insulin was 4.3 . 10(-6) cm . s-1. From the temperature dependence of the maximum transport capacity in the interval 18--37 degrees C and in the presence of insulin, an Arrhenius activation energy of 14.8 +/- 0.44 kcal/mol was found. The corresponding value was 13.9 +/- 0.89 in the absence of insulin. The half saturating concentration of 3-O-methylglucose was about 6 mM in the temperature interval used, and it was not affected by insulin, although this hormone increased the maximum transport capacity about ten-fold to 1.7 mmol . s-1 per 1 intracellular water at 37 degrees C.     

Thermodynamic dependence of DNA/DNA and DNA/RNA hybridization reactions on temperature and ionic strength

The thermodynamics of 5'-ATGCTGATGC-3' binding to its complementary DNA and RNA strands was determined in sodium phosphate buffer under varying conditions of temperature and salt concentration from isothermal titration calorimetry (ITC). The Gibbs free energy change, DeltaG degrees of the DNA hybridization reactions increased by about 6 kJ mol(-1) from 20 degrees C to 37 degrees C and exhibited heat capacity changes of -1.42 +/- 0.09 kJ mol(-1) K(-1) for DNA/DNA and -0.87 +/- 0.05 kJ mol(-1) K(-1) for DNA/RNA. Values of DeltaG degrees decreased non-linearly by 3.5 kJ mol(-1) at 25 degrees C and 6.0 kJ mol(-1) at 37 degrees C with increase in the log of the sodium chloride concentration from 0.10 M to 1.0 M. A near-linear relationship was observed, however, between DeltaG degrees and the activity coefficient of the water component of the salt solutions. The thermodynamic parameters of the hybridization reaction along with the heat capacity changes were combined with thermodynamic contributions from the stacking to unstacking transitions of the single-stranded oligonucleotides from differential scanning calorimetry (DSC) measurements, resulting in good agreement with extrapolation of the free energy changes to 37 degrees C from the melting transition at 56 degrees C.     

Permeability of human blood platelets to glycerol

The permeability of human platelets to glycerol was determined at 37 degrees C, 25 degrees C, and 0 degrees C from the rate of change of cell volume after abrupt addition of 0.5 mol/liter glycerol in phosphate-buffered saline. Intracellular water volume was measured employing both tritiated water and a photometric method. Intracellular glycerol was measured employing tritiated glycerol. The glycerol permeability coefficient derived from the tracer cell volume data was 4.0 +/- 0.7 X 10(-7) cm/s at 37 degrees C, and 1.1 +/- 0.4 X 10(-7) cm/s at 25 degrees C, and the photometric data gave a permeability coefficient of 5.4 +/- 0.4 X 10(-7) cm/s at 37 degrees C. The activation energy between 23 degrees C and 37 degrees C for glycerol permeation was 19.8 kcal/mol. The cells were virtually impermeable to glycerol at 0 degrees C. The minimum intracellular water volume attained after the addition of 0.5 mol/liter glycerol at 37 degrees C determined by the photometric method was 47.8% of normal water volume, whereas the minimum water volume calculated assuming that glycerol exerted its full osmotic effect (i.e., sigma = 1) was 45.6%. The reflexion coefficient was therefore assumed to be unity. Neither method of cell volume determination could be used with 1 or 2 mol/liter glycerol: adequate separation of the cells from the labeled medium could not be achieved in the tracer method; in the photometric method, it was apparent that transmittance (660 nm) was influenced by one or more variables in addition to cell volume.