Preferential Resistance of Phosphodiester Bonds between Deoxycytidine and 5'-Adjacent Bases to Chlamydomonas Nuclease C

Chlamydomonas Ca²⁺-dependent nuclease (nuclease C) has beenshown to be polymorphic (Ogawa and Kuroiwa 1985a). Aged preparationsof crude extract obtained in the absence of protease inhibitorcontained little nuclease C1&2. Instead, most of the activitiesappeared in association with smaller molecules similar in sizeto nuclease C3 and C4 that are members of nuclease C isozymes,suggesting that the polymorphism may be caused by the actionof an endogenous protease. Both the original form and the active fragments showed base-specificendo-exonucleolytic activity, and liberated an extremely lowlevel of 3'-dCMP as compared with the other three 3'-dNMPs.We compared the extent of hydrolysis of phosphodiester bondsbetween two bases of all possible combinations. The result showedpreferential resistance of phosphodiester bonds between dC and5' -adjacent bases to nuclease C. This explains the paucityof 3'-dCMP in the digests of DNA. The base-specific action ofnuclease C1&2 was preserved during the modification by endogenousprotease. (Received September 6, 1986; Accepted December 23, 1986)     

Nuclease C Polymorphism of Calcium-Dependent Nucleases in Chlamydomonas reinhardtii

DNases were extracted from cells of Chlamydomonas reinhardtii.Their polymorphism and metal ion requirements were investigated.Most of the nucleases from this organism required Ca²⁺ for fullactivation; therefore, the name nuclease C has been given tothem. Zn²⁺ and Mn²⁺ restored activation, but their respectivepotencies were 1/8 and 1/32 that of Ca²⁺. An in situ nucleaseassay revealed that there are at least 6 species of nucleaseC. The molecular weights of the components were 26,000 (C1),25,000 (C2), 22,000 (C5), 21,000 (C6), 18,000 (C3) and 17,000(C4) by SDS PAGE. Ca²⁺-dependent nuclease activity was slightlyhigher in the female gamete than in the male gamete. There wasno marked change in the apparent nuclease activity during thefirst 3 h after mating. An in situ assay, however, showed thatnuclease C6 and C4 are present in smaller amounts in the malegamete in comparison to the female gamete, and that the contentsor activities of nuclease C5 and C6 diminish during the first30–60 min after mating. This evidence is discussed interms of the possible participation of nuclease C in the maternalinheritance of chloroplast genes in this organism. (Received October 8, 1984; Accepted January 21, 1985)     

Glucose 6-phosphate dehydrogenase from sweet potato: Effect of various ions and their ionic strength on enzyme activity

Activity of glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate:NADP oxidoreductase, EC 1.1.1.49 [EC] ) preparation from sweet potatoroot tissue was markedly altered in the presence of variousions. Cations or anions were effective in the following order:Na^$, K^$>Tris^$>NH₄^$>Mg^2$>Ca^2$, or Cl^–>NO₃^–,HPO₄^2–>SO₄^2–>HCO₃^–. Activity was inhibitedat high concentrations of Ca^2$, and HCO₃^–,. In an investigationon the dependence of the activity on pH, two activity peakswere clearly observed at low ionic strength. Ionic strength altered both the Km and Vmax for glucose 6-phosphate(G6P). A Lineweaver-Burk plot for the enzyme, with respect toG6P, showed a bimodal nature at low ionic strength; suggestingnegative cooperativity. Deviation from linearity of the plotwas less with an increase in the ionic strength. ¹ Present address: Institute of Applied Microbiology, Universityof Tokyo, Bunkyo-ku, Tokyo 113. (Received September 18, 1971; )     

SPHAERIUM CORNEUM (L.) AND PISIDIUM SPP. PFEIFFER -- THE ECOLOGY OF FRESHWATER BIVALVE MOLLUSCS IN RELATION TO WATER CHEMISTRY

Populations of Sphaerium corneum (L.) and Pisidium spp. weresampled monthly for 13 months, at 11 sites with a wide rangeof water chemistry in N.W. England. Both genera showed almostyear-round reproduction, but the periods of maximum productionshowed between-population variation. S. corneum adults containedspat at different stages of development and spat size was relatedto number in the brood. Multiple regression analysis with 16water physicochemical variables showed the following to be highlysignificant factors (P<0.01) in the distribution of the molluses(factors in order of importance):- S. corneum: HCO₃, K^$, Cl^–, Mg^2$, temperature, Ca^2$, month; Pisidium spp: PO₄^3–, Mg^2$, mud, oxygen, temperature, month. (Received 25 January 1978;     

Reactivation of Cytoplasmic Streaming in a Tonoplast-Permeabilized Cell Model of Acetabularia

To find whether cytoplasmic streaming in Acetabularia is controlledby Ca²⁺, a tonoplast-permeabilized cell model was prepared usinga vacuolar perfusion technique. The cytoplasmic streaming remainedalmost normal after perfusion with EGTA medium (10 mM EGTA,40 mM PIPES, 5mM MgCl₂ and 800 mM sorbitol, pH 6.9), but stoppedwithin 10 min when saponin medium (EGTA medium plus 50 µg/mlsaponin, 50 µg/ml hexokinase and 5 mM glucose) was perfused.This model system was reactivated with a solution containing0.5 mM ATP and different concentrations of Ca²⁺ (reactivationmedium). With the reactivation medium at pCa 6–5, theresumed streaming lasted for about 10 min before the cytoplasmaggregated. At pCa 4–3, the streaming was observed onlyfor a few minutes because the cytoplasm aggregated quickly.At pCa 7, no reactivated movement was observed. Reactivationwas not induced in an ATP- or Mg²⁺-deficient medium even inthe presence of an adequate concentration of Ca²⁺, and was inhibitedby 50 µg/ml cytochalasin B or 1 mM N-ethylmaleimide. We concluded from these observations that the cytoplasmic streamingin Acetabularia is very likely to be driven by the actomyosinsystem in the presence of Mg-ATP and Ca²⁺ at pCa 6–5. (Received October 31, 1984; Accepted April 1, 1985)     

The Ca2+-Induced Phase Transformation in Soybean Root Microsomal Membranes is a Consequence of Phospholipase Activation

Microsomes from soybean (Glycine max L.) roots contain a Ca²⁺-activatedphospholipase D which hydrolyzes phosphatidylethanolamine andphosphatidylcholine to phosphatidic acid. The phosphatidic acidbinds Ca²⁺ in the medium and undergoes a liquid crystal to gelphase transformation (measured by wide-angle x-ray diffraction).The gel phase formation in the microsomes half-saturates at1 to 10 mM Ca²⁺. The upper limit for the transition temperaturefor the microsomes is –10 to 10°C in the native state.After treatment with Ca²⁺, the transition temperature increasesto 35 to 50°C. Under the same experimental conditions, incubationwith 10 mM Ca²⁺ at 4°C causes an increase in phosphatidicacid content from 8 mole % to 49% with a concomitant decreasein phosphatidylethanolamine and phosphatidylcholine from about22% and 41%, respectively, to 14% each. These results suggest that Ca²⁺ effects in systems where Ca²⁺plays a multifunctional role be interpreted with caution. ³Present address: Department of Physiology, Yale UniversitySchool of Medicine, New Haven, CT 06510, U.S.A. (Received June 15, 1985; Accepted June 13, 1986)     

Distinct roles of PMCA isoforms in Ca2+ homeostasis of bladder smooth muscle: evidence from PMCA gene-ablated mice

We previously showed that plasma membrane Ca²⁺-ATPase (PMCA) activity accounted for 25–30% of relaxation in bladder smooth muscle (8). Among the four PMCA isoforms only PMCA1 and PMCA4 are expressed in smooth muscle. To address the role of these isoforms, we measured cytosolic Ca²⁺ ([Ca²⁺]_i) using fura-PE3 and simultaneously measured contractility in bladder smooth muscle from wild-type (WT), Pmca1^+/–, Pmca4^+/–, Pmca4^–/–, and Pmca1^+/–Pmca4^–/– mice. There were no differences in basal [Ca²⁺]_i values between bladder preparations. KCl (80 mM) elicited both larger forces (150–190%) and increases in [Ca²⁺]_i (130–180%) in smooth muscle from Pmca1^+/– and Pmca1^+/–Pmca4^–/– bladders than those in WT or Pmca4^–/–. The responses to carbachol (CCh: 10 µM) were also greater in Pmca1^+/– (120–150%) than in WT bladders. In contrast, the responses in Pmca4^–/– and Pmca1^+/–Pmca4^–/– bladders to CCh were significantly smaller (40–50%) than WT. The rise in half-times of force and [Ca²⁺]_i increases in response to KCl and CCh, and the concomitant half-times of their decrease upon washout of agonist were prolonged in Pmca4^–/– (130–190%) and Pmca1^+/–Pmca4^–/– (120–250%) bladders, but not in Pmca1^+/– bladders with respect to WT. Our evidence indicates distinct isoform functions with the PMCA1 isoform involved in overall Ca²⁺ clearance, while PMCA4 is essential for the [Ca²⁺]_i increase and contractile response to the CCh receptor-mediated signal transduction pathway. PMCA; bladder smooth muscle; gene-altered mice     

Cytoplasmic Ca2+ has No Effect on the Excitability of Chara Plasmalemma

Internodal cells of Chara australis were subjected to two consecutiveintracellular perfusions with a Ca²⁺-free EGTA medium whichdisintegrated the tonoplast within about 10 minutes and thenwith a Ca²⁺-buffered medium. All perfusion media usually contained1 mM ATP. To stop the electrogenic pump, the internode was depletedof intracellular ATP. The excitability of the plasmalemma wasnot significantly influenced by intracellular free Ca²⁺ concentrationsup to 10^–4 M. To trigger action potentials, minimum currentdensities of 1 to 2 µA cm^–2 had to be applied atall tested Ca²⁺ concentrations. In the absence of cytoplasmicATP, excitability was completely lost at all Ca²⁺ concentrations. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, D-5300 Bonn, FRG. (Received September 22, 1984; Accepted March 6, 1985)     

Effect of Ca2+ on Tonoplast Potential of Permeabilized Characeae Cells

Using permeabilized characean cells in which the ionic conditionsat the cytoplasmic side of the tonoplast are easily controlled,effects of Ca²⁺ ion on tonoplast potential were examined. Whenthe cell was treated with 1 µM Ca²⁺, the tonoplast potential(E_M became positive in a complicated manner in Chara corallinawhile it simply became negative in Nitella axilliformis. Whenthe cell was treated with 9-antracenecarboxylic acid, a Cl^–-channelinhibitor, E_m became more negative and the response of E_m toCa²⁺ was significantly suppressed. It is suggested that Ca²⁺activates Cl^–-channel at a low concentration and inactivatesat a higher one in C. corallina while it simply inactivate Cl^–-channelin N. axilliformis. ¹Present address: Biological Laboratory, The University of theAir, Wakaba 2-11, Wakaba, 260 Japan. (Received August 22, 1988; Accepted December 26, 1988)     

The NADP$-specific isocitrate dehydrogenase of Rhodospirillum rubrum

The NADP^$-specific isocitrate dehydrogenase was partially purifiedfrom photosynthetically-grown Rhodospirillum rubrum. The pHoptimum is between 7.5 and 9.0 in phosphate buffer. The apparentKm is 3.1x10^–5 M for isocitrate, 5.1x10^–5 M forNADP^$, 1.7x10^–5 M for manganese, 1.5x10^–4 M formagnesium, and 3.5x10^–3 M for inorganic orthophosphate.Arsenate exerts a slight inhibition. The Q₁₀ between 17.5°Cand 40°C is 1.62, and the energy of activation at 25°Cis 9.74 Kcal/mole. Glyoxylate and oxalacetate cause concertedinhibition of the enzyme activity. Various nucleotides inhibitthe activity. The kinetics of inhibition by ATP was found tobe mixed type with respect to NADP^$ and isocitrate, the K_i valuesbeing 1.17x10^–3 M and 1.10x10^–3 M respectively.The inhibition between ATP and orthophosphate is competitivewith a K_i of 10^–4M. Thiol binding reagents are inhibitory;this inhibition is reversed by cysteine or reduced glutathione. (Received October 1, 1971; )     

Effect of Cytoplasmic Ca2+ on the Membrane Potential and Membrane Resistance of Chara Plasmalemma

Effects of cytoplasmic Ca²⁺ on the electrical properties ofthe plasma membrane were investigated in tonoplast-free cellsof Chara australis that had been internally perfused with media,containing either 1 mM ATP to fuel the electrogenic pump orhexokinase and glucose to deplete the ATP and stop the pump. In the presence of ATP, cytoplasmic Ca²⁺ up to 2.5?10^–5M did not affect the membrane potential (about -190 mV), butmembrane resistance decreased uniformly with increasing [Ca²⁺]_i.In the absence of ATP, the membrane potential, which was onlyabout -110 mV, was depolarized further by raising [Ca²⁺]_i from1.4?10^–6 to 2.5?10^–5 M. Membrane resistance, whichwas nearly the twofold that of ATP-provided cells, decreasedmarkedly with an increase in [Ca²⁺]_i from zero to 1.38?10^–6M, but showed no change for further increases. Internodal cellsof Nitellopsis obtusa were more sensitive to intracellular Ca²⁺with respect to membrane potential than were those of Charaaustralis, reconfirming the results obtained by Mimura and Tazawa(1983). The effect of cytoplasmic Ca²⁺ on the ATP-dependent H⁺ effluxwas measured. No marked difference in H⁺ effluxes was detectedbetween zero and 2.5?10^–5 M [Ca²⁺]_i; but, at 10^–4M the ATP-dependent H⁺ efflux was almost zero. Ca²⁺ efflux experimentswere done to investigate dependencies on [Ca²⁺]_i and [ATP]_i.The efflux was about 1 pmol cm^–2 s^–1 at all [Ca²⁺]_iconcentrations tested (1.38?10^–6, 2.5?10^–5, 10^–4M).This value is much higher than the influx reported by Hayamaet al. (1979), and this efflux was independent of [ATP]_i. Thepossibility of a Ca²⁺-extruding pump is discussed. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, 5300 Bonn, F.R.G. (Received September 22, 1984; Accepted February 19, 1985)     

Direct Effects of Ca2+-Channel Blockers on Plasma Membrane Cation Channels of Amaranthus tricolor Protoplasts

Ca²⁺-channel blockers at concentrations greater than 1 mmolm^–3, directly affect the activity of K ⁺selective channelsin the plasma membrane of Amaranthus tricolor protoplasts. Theseeffects are not mediated by the blockade of Ca²⁺ channels. Blockers tested included 1, 4-dihydropyridines (nifedipine,nicardipine), verapamil, bepridil, Gd³⁺ and La³⁺, applied towhole-cell and detached outside-out patches of plasma membraneat concentrations from 50µmol m^–3 to 100 mmol m^–3.For certain experiments the concentration of Ca²⁺ on the cytoplasmicside of the plasma membrane ([Ca²⁺]cyt) was buffered at either50ftmol m^–3 or 500 µmol m^–3. The principal currents observed in whole-cells flowed throughcation outward rectifier (OR) channels. Each blocker causedan immediate reduction of time-dependent outward currents atdoses down to 1 mmol m^–3 and produced a different, reversible,kinetic block of the outward current, independent of the levelof [Ca²⁺]cyt. Verapamil also activated a sustained inward cationcurrent at negative p.d. The same effects were found with individualchannels in detached outside-out patches. Conductance and selectivityof the cation OR channels were unchanged by the drugs. [Ca²⁺]ex, was varied over a range from 0 to 10 mol m^–3.Progressively lower [Ca²⁺]eI, increasingly enhanced the maximumamplitude of the time-dependent currents. Time-constants fordecay of inward tail currents were increased at low [Ca²⁺]eit.These effects were rapidly reversible. Although there was noevidence that the cation ORs in plasma membrane of Amaranthustricolor were dependent on [Ca²⁺]cyl for their activation, theywere sensitive to the concentration of free Ca²⁺ in the extracellularmedium. Key words: Verapamil, blocker, cation channels, Amaranthus, protoplasts     

Calcium Inhibits Ion-Stimulated Stomatal Opening in Epidermal Strips of Commelina communis L.

Inoue, H. and Katoh, Y. 1987. Calcium inhibitsion-stimulatedstomatal opening in epidermal strips of Commelina communis L.—J.exp. Bot. 38: 142–149. Ca²⁺ suppressed both the ion-stimulated stomatal opening andH⁺ extrusion of pre-illuminated epidermal strips isolated fromCommelina communis L. In the absence of Ca²⁺, the rate of H⁺release was 18 nmol H⁺ cm^–2 h^–1 per epidermal stripunit area in 150 mol m^–3 KCL at pH 7?4. Half-maximum inhibitionof stomatal opening was observed with 220 mmol m^–3 ofCa²⁺. The hexavalent dye, ruthenium red, showed concentration-dependentprevention of the inhibition by Ca²⁺ of the ion-stimulated stomatalopening. The effect of ruthenium red was non-competitive, andthe K₁ for the calcium inhibition was found to be 3?6 mmol m^–3.The calcium inhibition of H⁺ extrusion was also prevented byruthenium red. These results suggest that Ca²⁺ inhibits theactivity of electrogenic H⁺ translocating ATPase of the guardcell plasma membrane and leads to the suppression of stomatalopening. Key words: Calcium, Commelina communis, ruthenium red, stomata     

Calcium Activation of Potassium Channels in the Plasmalemma of Eremosphaera viridis

The role of cytoplasmic calcium activity in activation of K⁺-channelsin the unicellular green alga Eremosphaera viridis has beenstudied. As reported previously, after a ‘light off’signal a voltage independent opening of K⁺-channels in the plasmalemmais observed. This effect is indicated by a transient polarization(TP) with a simultaneous increase of the membrane conductance.TPs can also be triggered by different treatments, which allowinvestigations within a ‘short-circuited’ signalchain. (i) After incubation with EGTA a single extended TP canbe released by a sudden increase of the external calcium concentration.The Ca²⁺-channel inhibitors nifedipine (10 ^–2 mol m^–3)and verapamil (5 ? 10^–2 mol m^–3) suppress the releaseof this TP. (ii) In the presence of external calcium the additionof the ionophore A23187 [GenBank] (10–3 mol m^–3) causes anextremely prolonged TP. (iii) Low external concentrations ofbarium (10^–2 mol m^–3) induce repetitive TPs in thepresence of external calcium. In this case the Ca²⁺-channelinhibitors are less effective. (iv) Strontium (0.1–1.0mol m^–3) is able to trigger repetitive TPs even withoutexternal calcium. Whereas barium may stimulate a calcium influx,strontium can serve as a substitute for calcium to induce anopening of K⁺-channels. These results indicate strongly a Ca²⁺-dependentand voltage-independent activation of K⁺-channels in the plasmalemmaof Eremosphaera. The participation of cytoplasmic calcium inthe signal transduction chain after a ‘light off’signal is discussed. Key words: Ca²⁺-dependent K⁺-channels, Ca²⁺-channel effectors, A23187, transient membrane potential, Eremosphaera     

Base-Specific Endo-Exonucleolytic Activity of Chlamydomonas Nuclease C1&2

The reaction kinetics of nuclease C1&2 from Chlamydomonasreinhardtii were studied. It showed endo-exonucleolytic activitywith sugar non-specificity. The relative rates of RNA breakdownwere in order of poly(U) > poly(A) > yeast sRNA. In contrast,poly(G) and poly(C) released almost no acid-soluble materialsafter reacting with nuclease C1&2. The major products ofa 100% limit digest of synthetic RNA homopolymers were mononucleotideswith 3'-phosphate termini. Large oligonucleotides produced duringendo-exonucleolytic degradation also appeared carrying 3'-phosphatetermini. Nuclease C1&2 hydrolyzed single stranded DNA 20times faster than double stranded DNA by endo-exonucleolyticaction, releasing acid-soluble materials. High performance liquidchromatography of a 100% limit digest of salmon testes DNA demonstratedthat the major products were deoxymononucleotides with phosphateat 3'-position. Furthermore, the level of 3'-dCMP among themwas found to be extremely low. Poly(dC) and poly(me⁵dC) werehydrolyzed much more slowly than single stranded (or denatured)DNA, releasing acid-soluble materials. The present results suggestthat nuclease C1&2 is a base-specific nucleate 3'-oligonucleotidohydrolasedifferent from the restriction enzymes. (Received January 13, 1986; Accepted March 25, 1986)     

A Ca2+- and Voltage-Dependent Cl- -Sensitive Anion Channel in the Chara Plasmalemma: A Patch-Clamp Study

The inside-out patch-clamp technique was applied to the plasmolyzedplasmalemma of inter-nodes of Chara corallina without enzymatictreatment. We found two different types of channel activitythat were CP^–-sensitive. Both types of channel were Ca²⁺-dependent.However, the one that exhibited greater dependence on Ca²⁺ ionswas the focus of our studies, and we named it the Ca²⁺-dependentCP^–-sensitive anion channel. When the concentration ofCa²⁺ ions on the cyto-plasmic side was 1.0 µM, the Ca²⁺-dependentCP^–-sensitive channel opened most frequently between approximately–80 and –100 mV. At 10 µM Ca²⁺, it openedless frequently, and at 0.1 µM Ca²⁺ it scarcely openedat all. These observations indicate that the anion channel ofinterest is voltage-dependent over a restricted range of concentrationsof Ca²⁺ ions. The dependence on Ca²⁺ and voltage of the channelcan explain the behavior of the excitable Ca²⁺-activated Cl^–channel in the Chara plasmalemma. The channel activity was blockedby several antagonists of calmodulin. ⁴ Present Address: Department of Biology, College of GeneralEducation, Osaka University, Toyonaka, 560 Osaka, Japan (Received October 8, 1990; Accepted April 4, 1991)     

Studies on the Hill reaction of membrane fragments of blue-green algae III. Fluorescence characteristics of membrane fragments of Anabaena variabilis and Anabaena cylindrica

Fluorescence spectra of the pigment system at –196°Cin membrane fragments of Anabaena variabilis and A. cylindricawere investigated. The fluorescence spectra of membrane fragments having four emissionbands at 645–655, 685, 695 and 725 nm were basically similarto those reported for intact cells of blue-green algae, thoughthe emission from phycocyanin (645–655 nm) was far strongerwith membrane fragments than with intact algal cells. Incubation of membrane fragments of A. variabilis in a dilutebuffer (10^–2M, pH 7.5) caused an increase in the 645 nmfluorescence and slight decreases in the 685 and 695 nm fluorescences,but had no influence on the 725 nm fluorescence. The decreasein the 685 and 695 nm fluorescences of A. cylindrica was moremarked and had the same kinetics as the inactivation of photosystemII reaction measured by DPIP-photoreduction. When membrane fragments of A. cylindrica were incubated in thebuffer solution at room temperature or in the presence of MgCl₂(10^–3M) at 0°C; phycobilin aggregates, which emittedthe 655 and 685 nm fluorescence, were solubilized. This solubilizationwas not observed with membrane fragments of A. variabilis. (Received August 31, 1972; )     

Ca2+ Increases the Cl- Efflux of the Permeabilized Chara

An internode of Chara was permeabilized as described by Shimmenand Tazawa [(1983) Protoplasma 117:93]. The Cl^– effluxof the permeabilized cell increased when the extracellular Ca²⁺concentration was increased, and the degree of the increasewas dependent on the Ca²⁺ concentration. This suggests thatthe Cl^– channel in the tonoplast was activated by Ca²⁺. (Received May 22, 1987; Accepted October 21, 1987)     

The Effects of Cytokinins and ABA on Stomatal Behaviour of Maize and Commelina

Epidermal strips and leaf fragments of Commelina and leaf fragmentsof maize were incubated on solutions containing naturally-occurringor synthetic cytokinins and/or ABA. The effects of these treatmentson stomatal behaviour were assessed. Cytokinins alone did notpromote stomatal opening in either species but concentrationsof both zeatin and kinetin from 10^–3 to 10^–1 molm^–3 caused some reversal of ABA-stimulated closure ofmaize stomata. The reversal of the ABA effect increased withincreasing cytokinin concentration. Cytokinins had no effecton ABA-stimulated closure of Commelina stomata. When appliedalone, at high concentration (10^–1 mol m^–3), toCommelina epidermis or leaf pieces both zeatin and kinetin restrictedstomatal opening. Key words: ABA, Cytokinins, Stomata, Maize, Commelina     

The Osmotic Pressure of Concentrated Solutions of Polyethylene Glycol 6000, and its Variation with Temperature

The vapour pressures of aqueous solutions of polyethylene glycol6000 have been measured (by equilibration with sucrose solutions)up to the saturation point at 25 °C (1.45 g g^–1 water).The reduced-osmotic-pressure (/c), when plotted versus concentration(c), rapidly and linearly increased up to a concentration ofabout 0.8 g g^–1 (crossing the similar plot for sucrose).Above this concentration, the reduced-osmotic-pressure rosemore slowly, but still more rapidly than sucrose. The maximumosmotic pressure achieved at saturation was nearly 18 MPa. Usingthe virial equation: /c= RT/M + RTA₂c, the calculated secondvirial coefficient (A₂) for the linear part is 4.5 x 10^–3mol g^–1, a value slightly greater than most literaturevalues at 25 °C. Data are cited showing that A₂ varies linearlyfrom 5–6 x 10^x3 at 0 °C, to zero at 80–90 °C