Estimation of the Anion-Exchange Capacity of the Cell Wall of Nitella flexilis

Diffusion potentials in KCI and triphenyl phosphonium chloride(TPP) solutions have been measured across an isolated cell wallof Nitella flexilis either after pretreatment by difluoronitrobenzene(DFNB), a compound which reacts with the protein amino groups,or after alkalization of the external solutions. In both cases,it appears that the cationic transport numbers are enhancedwith respect to the controls. These data suggest that the maineffect of the two treatments was to deprive the wall of positivecharges carried by protonized amino groups. The changes in t_Kwere used to roughly estimate the anion-exchange capacity ofthe wall.     

Conformational Transitions and Modifications in Ionic Exchange Properties Induced by Alkaline Ions in the Nitella flexilis Cell Wall

The decrease in the cationic exchange capacity (CEC) concomitantto the replacement in the Nitella wall of adsorbed Mn²⁺ ionswas measured in different mixtures of alkaline ions. At lowexternal concentrations, the loss of CEC is enhanced in presenceof Li⁺ ions but is weaker when Na⁺ ions are present in the exchangemixtures. The relative affinity of the wall exchange sites foralkaline ions was Na⁺>K⁺>Rb⁺Cs⁺>Li⁺. As the CEC isprogressively reduced, the wall discrimination between the differentalkaline ions tends to cancel out except for the Na⁺-K⁺ pair.The wall preference for K⁺ is then increased. A diminution ofthe effective pK of the polygalacturonic acids constitutiveof the wall is also observed, while increasing the CEC loss.The simple disruption of divalent cation crosslinks cannot fullyexplain the CEC leakage at low monovalent concentrations. Itis suggested that the alkaline ions also cleave H bonds or solvatation-likebonds between the cell wall polyuronides and then cause a concomitantunfolding of the short pectic chains which involves their solubilization. (Received May 6, 1993; Accepted November 8, 1993)     

Identification of myosin in Nitella flexilis.

A myosin B-like protein was extracted from the alga Nitella flexilis. SDS-polyacrylamide gel electrophoresis revealed the presence of myosin heavy chain and actin as the main components. At high ionic strength, its ATPase [EC 3.6.1.3] reaction was activated by EDTA or Ca2+ and inhibited by Mg2+. At low ionic strength, superprecipitation was induced by the addition of ATP. Myosin was purified from Nitella myosin B. The molecular weight of the heavy chain of Nitella myosin, estimated by SDS-gel electrophoresis, was slightly higher than that of skeletal muscle myosin. At low ionic strength, Nitella myosin aggregated to form bipolar filaments about 0.2 micron long. At high ionic strength, its ATPase reaction was activated by EDTA or Ca2+, and inhibited by Mg2+. The Mg2+-ATPase reaction of Nitella myosin was activated by skeletal muscle F-actin.     

Vacuolation in Nitella flexilis (Characeae)

In Nitella flexilis, a species belonging to the section Anarthrodactylaeof the genus Nitella (Characeae), some mature cells, with theexception of those of the nodal complex and the rhizoid, shownumerous spherical vacuoles occupying the entire central regionof the cell. These vacuoles are fairly uniform in size measuring30–40 µm in diameter. Evidence is presented frominvestigations with the light and electron microscopes suggestingthat some mature cells of a certain group of Nitella speciesmay not have a huge central vacuole. Vacuolation in young cellsof the apex apparently begins by progressive dilation of theendoplasmic reticulum. The Golgi complex may also be involvedin vacuolation. The necessity for re-examination of certainbiological activities in the giant internode cell is explored.     

Selectivity characteristics of cation channels in Nitella flexilis plasmalemma

Ionic selectivity of Nitella flexilis plasmalemma cation channels is studied by voltage-clamp method with consecutive replacing of cations in the bathing medium. The selectivity sequence received by measuring the ionic current reversal potentials, psi alpha is: Ba++ approximately equal to Sr++ approximately equal to Ca++ greater than Mg++ greater than Cs+ approximately equal to K+ greater than Na+ greater than Li+. An analysis of results based on the three-barrier channel model suggests that when ions of the same valency are compared, the channel selectivity is determined by specific interactions between the ion and the nearest water molecules, which is possible both in a narrow and wide pore. On the other hand, when monovalent and divalent ions are compared the effects of ions binding in the channel or near the membrane surface prevail, thus causing the channel preference for divalent cations.     

Correlation Between the Weight Loss Induced by Alkaline Ions and the Cationic Exchange Capacity of the Nitella Cell Wall

There is a linear relationship between loss of weight and lossof cationic exchange capacity of Nitella cell walls, inducedby treatment with alkaline ions. As the rate of CEC loss dependsto a much greater extent on the ionic species than does themaximal loss quantity, it is suggested that the alkaline ionscompete with the bivalent ions initially adsorbed for a particulartype of site. These are different from the carboxylate exchangesites since the equivalent fractions of the alkaline ions adsorbedon to these sites are about the same for external concentrationswhere the effect of the different ions on weight and CEC lossis, nevertheless, quite different. Such sites give to the matrixpectin its structural coherence by preventing the action ofelectrostatic repulsive forces. In fact, it also appears thatCEC loss is a linear function of the inverse of the square rootof the internal ionic strength, a parameter proportional tothe Debye length. Key words: Cationic exchange capacity, cell wall, Nitella flexilis     

Ionic Diffusion Through the Nitella Cell Wall in the Presence of Calcium

Diffusion potentials (concentration and bi- or multi-ionic potentials)in KC1, NaCl, or LiCl solutions have been measured across anisolated cell wall of Nitella, with or without the same concentrationof CaCl² on either side of the cell wall. The absolute valueof the potentials decreases as the external Ca²⁺concentrationincreases and it may happen that an inversion of the sign ofthe concentration potentials results when the external Ca²⁺solution reaches 1 mM. Dosages of K⁺ and Ca²⁺ in the cell wallhave shown that Ca²⁺ easily displaces the monovalent ion fromthe exchange sites and tends to neutralize the cationic exchanger.However, in most cases, the measured potentials are still morenegative than the theoretical potentials which would be setup by a neutral-site membrane in the same conditions. Theseresults suggest that Ca²⁺ largely reduces the discriminationproperties of the cell wall between cations and anions.     

Light-induced hyperpolarization of membrane potential in Nitella flexilis call plasmalemma

Light-induced hyperpolarization of the membrane potential in Nitella flexilis cell plasmalemma was investigated by the clamping method. It is shown that this response is of an excitation character. The equivalent electric diagram used in the work and the electromotive force included on its basis allowed consideration of two possible (from the common point of view) mechanisms of the response: the action of the electrogenic ion pump and membrane specific permeability to a definite ion. The membrane being in the hyperpolarization state is not capable of acting as a bicarbonate electrode. It is suggested that the response under study is defined by the action of the electrogenic pump, the role of which is performed by HCO2(3)-dependent H+-ATPase with the ion channel as an exciting one.     

Deterination of Effective Partition Coefficients of Alkali Chlorides by Measurements of Concentration Potential across the Nitella flexilis (L.) Ag. Isolated Cell Wall

We report a study in which we estimate by means of non-equilibriumprocesses the value of an effective partition coefficient, x,a parameter that we use to take into account the non-ideal behaviourof the electrolyte of the external medium in the cell wall space.This parameter is defined by the ratio of the salt partitioncoefficient to the activity of the non-diffusing charges. Tothat purpose, concentration potential in NaCl, LiCI and CuCl₂solutions was measured across an isolated cell wall of Nitellaflexilis at different pH. No significant difference appearedbetween the potential values measured in the presence of NaClor LiCl (pH 5.5, 4.0 and 3.0). At pH 3.0, in CuCl₂ solutions,the Nitella wall behaved as an uncharged membrane. Two methods—high concentrations and least-squares approximation-wereused to calculate from these results the value of the effectivepartition coefficient. Both gave similar values of x At a givenpH, the values of x are independent of the concentration ofthe external medium over a large range of concentration ratios.The activity of the exchange sites was estimated by the experimentallydetermined fraction of absorbed monovalent cations. The ionicconcentrations in the wall space were calculated at pH 5.5 and3.0 by introducing the values of x in the relations which correlatethem to the ionic concentrations in the wall for that givenvalue of the activity of the carboxylate sites. From their values,it appears that the negative fixed charges of the wall werefunctionally shaded by the condensation of a part of the adsorbedmonovalent cations. The activity of the former was, therefore,reduced to a value considerably lower than that estimated bythe cationic exchange capacity during equilibrium processes. Key words: Nitella flexilis (L.) Ag., cell wall, concentration potential, partition coefficient, ion condensation     

Involvement of Water Channels in the Hydration-Induced Calcium Release in Nitella flexilis

An internodal cell of Nitella flexilis treated with 10 mM KC1was vacuole-perfused with an isotonic solution containing ethyleneglycol-bis-(rß-aminoethylether)N,N,N,N-tetraaceticacid (EGTA) and its content including cytoplasm was squeezedout into a vessel and covered with silicone oil. When the hypotonicsolution was added into the cytoplasmic drop which had beenmixed with aequorin, a significant increase in the light emissionfrom aequorin was detected with the photomultiplier, indicatinga release of Ca²⁺ from some cell organdies storing Ca²⁺. Thisincrease in the light emission was strongly inhibited by treatingcells with 0.1 mM HgCl₂ which is known to inhibit water channelsin the plasma membrane. The inhibition was completely recoveredby washing HgCl₂ with 2-mer-captoethanol. This suggests thatwater channels may exist in the membrane of Ca²⁺ stores andplay an essential role in the hydration-induced Ca²⁺ release. (Received February 12, 1998; Accepted May 21, 1998)     

Release of Small Polyuronides from Nitella Cell Walls during Ionic Exchange

Mono-divalent ion exchange in isolated cell walls of Nitella flexilis (L.) Ag. induces a marked loss of wall polymers and a decrease in the wall cationic exchange capacity. These data correlate with the replacement in the walls of adsorbed Mn²⁺ by Na⁺ ions. Boiling wall samples in methanol for 1 h or keeping the ionic solutions chilled to 4°C does not inhibit the cell wall polymer leakage but modifies the kinetics both of the ionic exchange and of the released polymers. These data are more compatible with physical rather than enzymic induced processes. The extracted polymers in the successively renewed NaCl solutions initially belong to the wall protein and pectin fractions and mainly to pectic fractions subsequently. Determination of the average degree of polymerization shows that the average molecular size of the lost acidic polysaccharides increases with extraction time up an average polymerization degree of 25. Enzyme-linked immunosorbent assay inhibition tests show the presence of homopolymer blocks equal to or higher than 10 in the released polymer fragments. Compositional analysis of released polysaccharides suggests that the pectin lost by action of monovalent ions was largely composed of rhamnogalacturonans whose acidic residue fraction is approximately 60% in association with galactose chains. Small quantities of glucuronylated xylans are also found.     

Mechanosensitive Ca2+ release from intracellular stores in Nitella flexilis

We found previously that the cytoplasmic drop isolated from internodal cells of Nitella flexilis releases Ca2+ in response to hypotonic treatment and named the phenomenon hydration-induced Ca2+ release (HICR). The HICR is assumed to be a result of activation of Ca2+ permeable channels in the membrane of Ca2+ stores in a stretch-activated manner. To prove this idea, mechanical stimulus was applied to the drop by means of shooting isotonic/hypnotic medium or silicon oil into the drop, or compressing the drop. All these mechanical stimuli induced a rapid increase in the Ca2+ concentration of the drop. The chloroplast fraction isolated from the cytoplasmic drop released Ca2+ on compression, while the chloroplast-free cytoplasm did not. In Chara corallina, the cytoplasmic drop, which shows a very weak HICR, also responded weakly to the mechanical stimulus, but the chloroplast fraction was inert. When chloroplasts from Chara were added to the chloroplast-free cytoplasm of N. flexilis, the cytoplasm recovered the mechanoresponse. Starch grains were as effective as chloroplasts. The data indicate that Ca2+ permeable channels in the membrane of Ca2+ stores in N. flexilis are really mechano-sensitive.     

Growth and the Current Pattern Around Internodal Cells of Nitella flexilis L.

The ionic currents that traverse the internodal cells of thegreen alga Nitella flexilis L. have been measured with an extracellularvibrating probe. In adult interriodes illuminated with whitelight a pattern of self-generated currents exists along thecell, featuring alternating zones of inward and outward current.At inward current zones current densities of up to 25 µAcm^–2 were measured, at outward current zones the maximaldensity recorded was 7.5 µA cm^–2. The zones withinward current had an average length of 1.0 mm, and those withoutward current averaged 1.5 mm. When the light was turned offthe overall current density decreased drastically and the patternchanged. Currents first appear in growing internodal cells thatare about 1.0 mm long. With increasing length a current patterndevelops, with the zones of inward and outward current at firstshifting laterally along the surface. Only in adult cells dothe zones become stationary and form a typical current pattern.In addition to the current pattern different chloroplast volumescan be observed along the internode. In outward current zonesthe volumes of chloroplasts are 3.2 times those of inward currentzones. The natural current pattern observed in Nitella internodescauses loops of electric current that extend outward from thecell surface into the bathing medium. We speculate that thesecurrent loops might provide a mechanism of electrophoretic iontransport through the medium. Such a mechanism could increasethe supply of for the internodal cells in their natural stagnant water habitat. Key words: Nitella flexilis, Internode growth, Current pattern, Vibrating probe     

The effects of calcium deficiency on the electrical activity of Nitella flexilis.

Calcium chelators such as ethylenediaminetetraacetic acid and sodium citrate produce repetitive activity and prolong the spike of internodal cells of Nitella flexilis. Removal of Ca2+, Mg2+, Na+, and K+ from the outside of the cell by washing the preparation with Tris propionate or Tris chloride hyperpolarizes the cells but does not initiate repetitive activity or increase the duration of the spike appreciably. It was concluded that cell-bound Ca2+ controls the threshold for stimulation and the duration of the spike, and that the removal of Ca2+ from the cell membrane, either by chelation or displacement, changes the normal behaviour of the cell by altering its permeability to some other ion or ions.     

45Ca displacement related to pharmacologically induced prolonged action potentials in Nitella flexilis

Nitella cells were loaded with 45Ca2+ to an activity of 2 X 10(5) cpm. Insertion of two glass-capillary electrodes into each of six cells released varying amounts of Ca2+ in the order of 1 mumol per cell, but hyperpolarizing and depolarizing pulses up to 500 ms in duration caused no measurable loss (less than 57 pmol) of Ca2+ even when the latter elicited action potentials. Addition of 10 mumol of Ba2+ or tetraethylammonium (TEA) caused losses up to 1200 pmol of Ca2+ from the cells and prolonged the action potentials by a factor of three or more. Subsequent addition of Ba2+ or TEA to treated cells caused no further losses of Ca. Because prolonged action potentials can apparently only be elicited after the chelation or displacement of Ca2+, we propose that, as in many animal cells, the K+ channels responsible for the normal brief repolarizing phase of the action potential are controlled by Ca2+ in these electrically excitable plant cells.