Keitaro Kiyosawa

Volumetric properties of polyols (ethylene glycol, glycerol, meso-erythritol, xylitol and mannitol) in relation to their membrane permeability: Group additivity and estimation of the maximum radius of their molecules

The relationship of molecular volume and maximum molecular radii to the ability of some polyols, ethylene glycol, glycerol and meso-erythritol, to permeate the Chara cell membrane and to the inability of one of the polyols, mannitol, to permeate the Chara cell membrane, was examined by measuring the partial molar volumes of the polyols, Vm(2). Analysis of Vm(2) at infinite dilution showed that group additivity is maintained for all the groups, i.e., CH2OH and CHOH, of the polyols tested. However, as the permeability and impermeability could not be related to the geometrical properties of the polyol molecules based only on the thermodynamic quantities, molecular models of the polyol molecules were constructed using the CPK(Corey-Pauling Koltum) molecular model, which is designed to have the van der Waals radius of 1 A equivalent to 1.25 cm. The results showed that the maximum radius of the water-filled pore (hydrophilic channel) should be 3.2-3.3 A, and the longer the axial length and maximum radius of the polyol molecule, the lesser was its permeative ability. All the experimental and analytical results and inferences support the idea that water molecules pass across the cell membrane through a narrow pore in a single-file fashion.

Hydraulic conductivity of tonoplast-freeChara cells

SummaryThis study is the first trial to measure the osmotic water permeability or the hydraulic conductivity of the plasmalemma alone of a plant cell. For this purpose tonoplast-free cells were prepared from intenodal cells ofChara australis and their hydraulic conductivities were measured by the transcellular osmosis method.The transcellular hydraulic conductivity did not change after removing the tonoplast. The transcellular hydraulic conductivity of the tonoplast-free cells was dependent on the internal osmotic pressure as is the case in the tonoplast-containing normal cells. The hydraulic conductivities for both endosmosis and exosmosis of the tonoplast-free cells were equal to respective values of the normal cells. Consequently the ratio between the inward and outward hydraulic conductivities did not change due to the loss of the tonoplast. The results indicate that the resistance of the tonoplast to water flow is negligibly small as compared with that of the plasmalemma and further that the tonoplast is not a factor responsible for the direction-dependency of hydraulic conductivity. The hydraulic conductivity of the plasmalemma is invariable for wide variations of K+ and Ca2+ in the cytoplasm.

Theoretical and experimental studies on freezing point depression and vapor pressure deficit as methods to measure osmotic pressure of aqueous polyethylene glycol and bovine serum albumin solutions

For survival in adverse environments where there is drought, high salt concentration or low temperature, some plants seem to be able to synthesize biochemical compounds, including proteins, in response to changes in water activity or osmotic pressure. Measurement of the water activity or osmotic pressure of simple aqueous solutions has been based on freezing point depression or vapor pressure deficit. Measurement of the osmotic pressure of plants under water stress has been mainly based on vapor pressure deficit. However, differences have been noted for osmotic pressure values of aqueous polyethylene glycol (PEG) solutions measured by freezing point depression and vapor pressure deficit. For this paper, the physicochemical basis of freezing point depression and vapor pressure deficit were first examined theoretically and then, the osmotic pressure of aqueous ethylene glycol and of PEG solutions were measured by both freezing point depression and vapor pressure deficit in comparison with other aqueous solutions such as NaCl, KCl, CaCl(2), glucose, sucrose, raffinose, and bovine serum albumin (BSA) solutions. The results showed that: (1) freezing point depression and vapor pressure deficit share theoretically the same physicochemical basis; (2) theoretically, they are proportional to the molal concentration of the aqueous solutions to be measured; (3) in practice, the osmotic pressure levels of aqueous NaCl, KCl, CaCl(2), glucose, sucrose, and raffinose solutions increase in proportion to their molal concentrations and there is little inconsistency between those measured by freezing point depression and vapor pressure deficit; (4) the osmotic pressure levels of aqueous ethylene glycol and PEG solutions measured by freezing point depression differed from the values measured by vapor pressure deficit; (5) the osmotic pressure of aqueous BSA solution measured by freezing point depression differed slightly from that measured by vapor pressure deficit.

Studies on the effects of alcohols on membrane water permeability ofNitella

SummaryWater permeability of theNitella internode was measured by means of transcellular osmosis. The internode was partitioned into two chambers, one filled with water or alcohol solution and the other with mannitol solution or mannitol-alcohol solution. In this experimental system, the osmotic driving force is equal to the osmotic pressure due to mannitol.Water permeability of the internode was decreased by aliphatic alcohols. The reciprocal of the permeability coefficient,i.e., the resistance of the membranes to water flow, increased linearly with increasing alcohol concentration; the slope of the linear relationship became steeper with increasing carbon chain length.As causes for the decrease in water permeability, the following two possibilities were checked: whether or not driving force for transcellular osmosis is reduced by the alcohols; whether or not viscosity of the liquid in the pores increases by coexistence of the alcohols. The former was completely excluded since osmotic pressure due to mannitol did not change by coexistence of the alcohols. As for the latter possibility, it was found that, although viscosity of the alcohol solution increased with increasing alcohol concentration, the magnitude was not large enough to explain the decrease in water permeability.Apparent activation energy for water permeability was nearly equal to that for the fluidity of water and they were scarcely affected by the alcohols. Thus, we concluded that alcohol molecules interact with the membranes to make the equivalent pore radius of the membranes narrower without changing the nature of the water flow.

Influence of intracellular and extracellular tonicities on water permeability in Characean cells

SummaryThe effect of the concentration of the central vacuolar sap on water permeability previously demonstrated onNitella internode (Tazawa and Kamiya 1966), has been further studied. By using a technique of vacuole perfusion the ionic concentration of the cell sap has been modified independently of its tonicity. Transcellular water permeability has been measured by means of a double-chamber osmometer.When the tonicities of artificial saps were adjusted to that of the natural cell sap, wide variations in the concentration of K+, Na+, or Ca++ in the vacuole did not bring about any change in the magnitude of water permeability. On the other hand, water permeability was strongly influenced by varying the tonicity of the vacuolar medium by addition of mannitol. It increased when the tonicity was lowered from the normal level, while it decreased when tonicity was heightened. Water permeability was also decreased by increase in the tonicity of the external medium.Analysis of the results showed that the specific resistance to water flow across the plasmalemma and the tonoplast in series (the reciprocal of the water permeability k′p) was related to the osmotic pressures of the intracellular (πi) and the extracellular (π0) medium by the empirical formula, l/k′p=0.088 + 0.015 π. + 0.0074π0. Thus, intra- and extracellular tonicities influence the water permeability of theNitella internode independently of each other. The decrease in water permeability by increase in tonicity of the intra- or extracellular medium may be explained in terms of the effect of these tonicities on hydration of the cell membranes.The water permeability ofLamprothamnium, a brackish water Characeae was only one fourth that ofNitella, a fresh water Characeae. The lower permeability inLamprothamnium may be accounted for in terms of the high tonicities of its cell sap and external medium.

Rectification characteristics ofNitella membranes in respect to water permeability

SummaryOne of the membrane characteristics of plant cells, rectification, or the direction dependence of water permeability, was investigated inCharaceae internodes using the procedures we developed (Tazawa andKiyosawa 1973) for determining the endosmotic (kpen) and exosmotic (kpex) water permeabilities of the membranes (plasmalemma and tonoplast) in the transcellular osmosis system. Bothkpen andkpex were dependent on the osmotic pressure (πo) of the mannitol solution, which is the driving force for the transcellular osmosis. Thus, kpen increased andkpex decreased with πo. The rectification parameter, or the polarity (ρp), defined askpen/kpex tended to unity when πo approached zero.InNitella flexilis the specific resistances of the membranes to endosmosis and exosmosis,kpen−1 andkpex−1, were linearly dependent on π0. When the cell was partitioned into two equal halves,kpen−1=4.2×104−1.1×103π0,kpex−1=4.2×104+2.9×103π0, where the specific resistances are represented in cm−1 sec atm. When πo is 0.1, 0.2, 0.3, 0.4, and 0.5 M mannitol eq., the rectification parameter is calculated as 1.3, 1.6, 1.9, 2.4, and 2.9, respectively. Essentially the same results were also obtained withChara australis.Results were discussed on the basis of changes in the hydration of the cytoplasm. Assuming that the driving force across the protoplasmic layer can be divided into two forces; one driving water across the plasmalemma and the other driving water across the tonoplast, we deduced that the cytoplasm on the endosmosis side is hydrated, while the cytoplasm on the exosmosis side is dehydrated. Analysis showed that changes in hydration depend on the rate of flow.

Analysis of transcellular water movement inNitella

SummaryThe mechanism of transcellular osmosis was analyzed on the assumption that the driving force, which is equal to the osmotic pressure of the mannitol solution given to the exosmosis side, is divided into two parts; one causing the inward water flow on the water side, the other causing the outward water flow on the solution side, when each force drives an equal amount of water. Based on this analysis a new procedure was developed to measure the endosmotic and exosmotic water permeabilities of the membranes independently. It involved measurement of volume of water transported transcellularly, change in turgor pressure, and water permeability of the cell wall alone.Experiments following the new procedure revealed that in aNitella internode positioned across a partition wall with equal length both the endosmotic and exosmotic water permeabilities remained constant during transcellular osmosis induced with 0.4M mannitol, at least for the first minute. It was found that the permeability coefficient for endosmosis (3.9 × 10−5 cm sec−1 atm−1) was very much higher than that for exosmosis (1.4× 10−5 cm sec−1 atm−1). Treatment of the endosmotic cell part with 5% ethanol conspicuously decreased the water permeability of the cell on this side down to 1/2.4 the value obtained without ethanol but never affected the permeability on the other side (exosmosis side).

Freezing-point of mixtures of H 2 16 O and H 2 18 O

Freezing-point depression of mixtures of H216O and H218O were measured. The results showed that the freezing point of the mixture rose linearly with an increase in the molal concentration of H218O. The results suggested the formation of a solid solution of H216O and H218O by freezing, similar to that formed by H2O−D2O, and that H218O behaves as a different molecule than H216O.

Barrier components acting against osmotic water flow inNitella

In order to check whether or not the layer of chloroplasts densely arranged in the cortical gel of aNitella internode offers substantial resistance to osmotic water flow, a material was prepared which had the cortical gel layer freed from chloroplasts by centrifugation either longitudinal or lateral to the cell axis. The water permeability of the cell remained the same as normal even though the chloroplasts were exfoliated from the cell cortex to the extent of 50% of the total area, showing that the chloroplast layer plays hardly any significant part as a barrier to osmotic flow. Since it is known that the layer of the streaming endoplasm is also negligible as resistance against osmotic water flow (Tasawa and Kamiya, 1965), it is concluded that the major barrier components against osmotic flow in theNitella internode are the cell wall, plasmalemma and/or tonoplast.

Permeabilities of theChara cell wall to saccharides, albumin and ficoll

A method was developed to enable the determination of the permeability coefficient of theChara cell wall to various solutes from a measurement of the water flow occurring in the solution-cell wall-water system. For this method, the cell wall tube, closed at one end with the natural septum, was connected to a pipette, which serves as a volumeter, by using a glass capillary and a needle.Permeability coefficientsks of the cell wall to glucose (M.W.=180.2), mannitol (M.W.=182.2), sucrose (M.W.=342.3), lactose (M.W.=342.3), raffinose (M.W.=504.5) and melezitose (M.W.=504.4) were 2.27, 2.36, 1.43, 1.38, 1.11 and 1.09×10−4 cm sec−1, respectively. The reciprocal ofks is expressed as a linear function of molecular weight,M, by the equation 1/ks=16M+1.5×103 (cm−1 sec)Albumin (M.W.=68,000) passed through the cell wall fairly well. Ficoll (M.W.=400,000±100,000) for practical purposes could not permeate the cell wall.