Plasmalemma Chloride Transport in Chara corallina

Plasma membrane was isolated in a uniform population and with a high purity from chilling-sensitive etiolated young seedlings of Vigna radiata (mung bean) utilizing an aqueous two polymer phase separation system and subsequent sucrose density gradient. The isolated plasma membrane was associated with vanadate-sensitive and KNOrinsensitive ATPase. The ATPase has high specificities both for substrate and Mg2 ion with optimum pH at 6.5. It was slightly stimulated by monovalent anions, especially Cl-. Proton ionophores such as gramicidin D and carbonyl cyanide j-trifluoromethoxyphenylhydrazone did not stimulate the enzyme activity. The ATPase is apparently latent and highly stimulated by the addition of detergents such as Triton X-100. A maximum stimulation was achieved by the addition of 0.02% Triton X-100. After treatment with proteinase K in an isotonic buffer solution, the enzyme activity was less affected, whereas the peptides were specifically digested. Based on these facts, the isolated plasma membrane vesicles appear to be tightly sealed and in a right-side-out orientation. The plasma membrane ATPase had two inflection points at higher (18.9C) and lower (6.7°C) temperatures on the Arrhenius plots of the activity. The lower inflection temperature apparently coincided with that of the anisotropy parameter of embedded 1,6-diphenyl-1,3,5-hexatriene, indicating that the membrane bound ATPase activity was affected by a phase transition of membrane lipids and/or temperature-dependent conformational changes in the enzyme molecules per se. Considering the fact that the plant material used here is highly sensitive to chilling temperatures and injured severely by exposure to temperatures below 5°C for a relatively short period, the thermotropic properties of membrane molecules are considered to be involved in the mechanism of chilling injury.Despite numerous studies on chilling injury of plants in the past decade (6,14,15), the mechanism is still obscure. In our previous studies using highly chilling-sensitive, cultured cells derived from Cornus stolonifera L. (18,30) logical symptoms which might be considered as the cause or the result of chilling injury.Based on these results, our attention should be focused more or less on the direct or indirect effects of chilling temperatures upon whole structures of cells, including plastids, mitochondria, nuclei, ER, vacuoles, Golgi apparatus, plasma membrane, and cytoskeletons, and on the metabolic processes of membrane biogenesis as influenced by the chill temperatures.For this purpose, it would be necessary to prepare cell organelles in a high purity and in a large quantity. A series of experiments to establish the isolation techniques are going on in our laboratory using chilling-sensitive etiolated young seedlings of Vigna radiata.In the present study, we will be dealing mainly with the isolation of plasma membrane utilizing the published method of an aqueous two-polymer phase partition system and subsequent sucrose density gradient centrifugation. The isolated plas...

“…It was also slightly inhibited by DIDS (30 gM) which is an inhibitor for anion transport channels in plasma membranes (4,9). Azide and KNO3 exhibited no inhibitory effect.…”

Section: Resultsmentioning

confidence: 90%

Properties of Plasma Membrane Isolated from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L.

Yoshida¹,

Kawata²,

Uemura³

et al. 1986

“…DIDS is an inhibitor of Cl-transport into red cells (3). corn protoplasts (13), and Chara (12). We have shown that DIDS calculated from the data presented in Figure 7 by subtracting the activity with K2S04 from that with KCI.…”

Section: )mentioning

confidence: 99%

Separation of Two Types of Electrogenic H⁺-Pumping ATPases from Oat Roots

Churchill¹,

Holaway²,

Sze³

1983

ABSTRACrMicrosomal vesicles of oat roots (Avena sativa var Lang) were separated with a linear dextran (0.5-10%, w/w) or sucrose (2545%, w/w) gradient to determine the types and membrne identity of proton-pumping ATPases associated with plant membranes. ATPase activity stimulated by the H'/K' exchange ionophore nigericin exhibited two peaks of activity on a linear dextran gradient. ATPase activities or ATP-generated membrane potential ( of proton transport an exciting research topic. Until recently, there was no direct evidence for a membrane constituent that pumped H+. Within the last few years, evidence for electrogenic, H+-pumping ATPases has appeared from our laboratory (4-6, 27-31) and several other laboratories (1,7,8,10,17,18,23,24,26,32).H+-pumping ATPases have been identified in nonmitochondrial membranes of several plant tissues. These transport ATPases exhibit three types ofactivities in sealed microsomal vesicles: (a) ionophore-stimulated ATPase activity (24, 27, 28), (b) ATPdependent generation of a membrane potential (positive inside the vesicle) (1,23,26,30), and (c) ATP-dependent pH gradient formation (acid inside) (1,6,8,10,18,26,29,31,32).Although several laboratories have demonstrated a vanadateresistant, H+-pumping ATPase (8,10,18,32), one report using oat roots (26) and our studies with sealed microsomal vesicles from tobacco callus or oat roots have suggested the presence of at least two types of H+-pumping ATPases, one sensitive and one insensitive to vanadate (5,6,28,31). This paper demonstrates partial separation of two types of electrogenic, H+-pumping ATPases using a linear dextran or sucrose gradient. The two types of ATPases can be distinguished by their relative densities, K+ or Cl-sensitivities and sensitivity to inhibitors. The Cl--stimulated, proton pump appears to be enriched in vacuolar membranes and the K+-sensitive, proton pump is enriched in plasma membrane-type vesicles. Preliminary reports of these results have been presented (5, 31).MATERIALS AND METHODS Plant Material. Oat (Avena sativa L. var Lang) seedlings were germinated in the dark over an aerated solution of 0.5 mm CaSO4. After 5 to 6 days of growth, the apical tips (3-4 cm) of the roots were harvested. Lang oats were generously provided by the Agronomy Department of Kansas State University.Isolation and Separation of Sealed Microsomal Vesicles. Sealed microsomal vesicles were prepared as described by Churchill and Sze (6) using a 6% (w/w), 10%, or 12% dextran cushion. In one case, a two-step dextran gradient of 6% and 15% was used to separate vesicles at the 0/6% and 6/15% dextran interfaces.Microsomal vesicles (60,000g pellet) were sometimes separated with a linear dextran gradient (0.5-10%) as described before (6).

“…More recently, DIDS has been used to inhibit Cl-and SOt-uptake in corn root protoplasts (16) and was shown to be a reversible inhibitor of Cl-influx in Chara corallina (12). To investigate the effect of DIDS on the kinetics of both Cl-and K+ influx in low-salt grown corn roots, the following experiments Points which lack error bars do so because standard errors were smaller than symbols used.…”

Section: References Therein)mentioning

confidence: 99%

Potassium Transport in Corn Roots

Kochian¹,

Xin-Zhi²,

Lucas³

1985

Self Cite

103

A detailed examination was conducted on the linear, or first-order kinetic component for K'("Rb) influx into root segments of both lowand high-salt grown corn seedlings (Zea mays 1A632 x Oh 431). In tissue from both low-and high-salt grown. roots, replacement of Cl-in the uptake solution by either SO3-, H2PO0, or NO! caused a significant (50-60%) and specific inhibition of the linear component of K' influx. The anion transport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid, was found to abolish saturable Cl-influx in corn roots while causing a significant (50-60%) and specific inhibition of the linear K' uptake system; this inhibition was identical to that observed when Cl was replaced by other anions in the K' uptake solution. Additionally, the quaternary ammonium cation, tetraethylammonium, which has been shown to block K' channels in nerve axons, also caused a dramatic (70%) and specific inhibition of the linear component of K influx, but this was obtained only in high-salt roots. The reasons for this difference are discussed with respect to the differing abilities of low-and high-salt roots to absorb tetraethylammonium.Our present results indicate that the linear component of K' influx may occur by a passive process involving transmembrane K' channels. Fluxes through these K' channels may be partly coupled to a saturating C1-influx mechanism.In studies involving the kinetics of uptake for various plant tissues, the majority of the work has focused on the generation of discontinuous isotherms, via either the operation of multiple Michaelis-Menten transport systems, or by complex, multisite carriers. In many of these studies, the presence of a linear component was either ignored or considered to reflect diffusion across the plasma membrane (13 and references therein).Recently there has been a growing interest in nonsaturating transport kinetics for organic solutes and ions. In contrast to earlier investigations, the more recent literature suggests that the nonsaturating or linear transport component may be a relatively complex process (2, 6, 7, 13-15, 20, 21, 31 KI and ClO Influx Experiments. Short-term (10 min) 86Rb+ and 3Cl-influx experiments were performed using2-cm root segments as previously described (13). Briefly, experiments were performed with 2-cm root segments cut from the 1st through 8th cm of the primary root. Root segments were washed (4 h) in solutions of identical composition to their growth solutions to allow for recovery from excision. Uptake was initiated by the addition of 86Rb+ (as RbCl, New England Nuclear, Boston) or 3Cl-(as NaCl, ICN Biochemicals, Irvine, CA) and terminated by the vacuum withdrawal of radioisotope solution. Free space radiolabel was removed by either two 8-min washes for 'Rb+ or two 5-min washes for 'Cl-in ice-cold 0.5 mM CaSO4 + 1 mM KCI. We have found that these desorption regimes removed approximately 95% ofthe free space radiolabel while exchanging only about 5% of the intracellular label. Following desorption, root segments were centrifuged (300g) for...