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1.
Protoplasts were enzymically isolated from suspension cultured cells of Nicotiana glutinosa L. and aspects of transport selectivity and kinetics were studied. In the presence of Ca2+, transport was selective for K+ (86Rb) over Na+. 36Cl transport was inhibited by Br or I but not by H2PO4. The kinetic data for short term (30 minutes) K+ influx over the range of 0.05 to 100 millimolar KCl were complex but similar to those observed in other plant tissues. In contrast, the kinetic data for Cl and H232PO4 over the same concentration range were different from those observed for K+, and could be accounted for by a single isotherm in the range of 0.05 to 4 millimolar and by an almost linear increase in influx rate above 4 millimolar. The kinetic data for Cl transport into intact cultured cells were identical in character to those observed for isolated protoplasts. The results support the view that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast.  相似文献   

2.
With the enzymatically isolated Nitella protoplasts, sufficientinsertions of micro-electrodes to make a stable measurementof the membrane potential by the conventional method could notbe made because of an ‘elasticity’ of the outermembrane. We developed an effective method in which a micro-electrodecould be inserted after the outer membrane was punctured bypassing an electrical impulse through the micro-electrode. Inthis method, Ca ions play a crucial role in the ‘punching’and ‘healing’ processes of the protoplast membrane. The effects of the cations K+, Na+, Ca2+ and the anions Cl,, , on the membrane potentials of Nitella expansa protoplasts were compared with those of intactcells. The membrane potential of protoplasts was less negativethan that of intact cells when concentrations of Na or K, inthe presence of Ca, were below certain levels which increasedwith increasing Ca concentration; and it tended to become identicalto that of intact cells when Na or K concentrations were beyondthose levels. Beyond those levels for K the membrane potentialsof both protoplasts and intact cells typically seemed to bethe Nernst potentials in the presence of 0•1 to 30 molm–3 Ca2+. However, for Na, the difference in potentialsbetween intact cells and protoplasts decreased at much higherconcentrations than for K. Increase of Ca always gave less negativeprotoplast potentials than those in intact cells. Replacementof Ca by Mg did not change the membrane potential of intactcells, although it was deleterious to protoplasts. The cellwall potential of intact cells was also measured by the micro-electrodetechnique and was revealed as a simple Donnan potential, assumingthe fixed negative charge density of 0•8 equivalent perdm3. The membrane potential of intact cells seems to be a significantreflection of the plasmalemma potential which is thought tobe measured directly in their protoplasts in terms of ionicselectivity and concentration dependency of the ion speciesexamined. In addition, increased sensitivity to calcium in protoplastpotentials compared to intact cells is suggested, though themembrane potential of intact cells seems to be largely preservedin their enzymatically isolated protoplasts. Key words: Membrane potential, protoplasts, Nitella expansa, cell wall potential  相似文献   

3.
4.
Protoplasts isolated from maize (Zea mays L.) suspension cells were used to study transport of paraquat. [14C]Paraquat uptake was measured in 400-[mu]L centrifuge tubes using silicon oil centrifugation techniques. Approximately 50% of accumulation from a 100 [mu]M paraquat solution occurred in the first 10 s, and net accumulation reached a maximum after about 10 min. Membrane binding accounted for about 30% of apparent accumulation. Concentration-dependent uptake kinetics were characterized by a non-saturating curve, which was resolved into a linear and a saturable component. The Km of the saturable component was 132 [mu]M, and the Vmax was 0.512 nmol [mu]L of protoplasts-1 min-1. In the absence of sucrose, the Vmax of the saturable component was reduced by 52%, suggesting that paraquat uptake across the plasmalemma is energy dependent. Measurement of concentration-dependent binding of paraquat to burst protoplasts showed a linear response. This suggests that the linear component from intact protoplast concentration kinetics represented paraquat binding to the plasmalemma surface. Calcium inhibited the saturable component, and this inhibition was shown by Lineweaver-Burk analysis to be noncompetitive. Putrescine, a divalent cationic polyamine with a charge distribution similar to that of paraquat, competitively inhibited paraquat uptake. These results show that paraquat transport characteristics at the plasmalemma of maize protoplasts are similar to those reported earlier for paraquat transport in roots of intact maize seedlings.  相似文献   

5.
The effects of fluoride on the tonoplast type ATPase and transport activities associated with sealed membrane vesicles isolated from sugarbeet (Beta vulgaris L.) storage tissue were examined. This anion had two distinct effects upon the proton-pumping vesicles. When ATP hydrolysis was measured in the presence of gramicidin D, significant inhibition (approximately 50%) only occurred when the fluoride concentration approached 50 millimolar. In contrast, the same degree of inhibition of proton transport occurred when the fluoride concentration was about 24 millimolar. Effects on proton pumping at this concentration of fluoride could be attributed to an inhibition of chloride movement which serves to dissipate the vesicle membrane potential. Valinomycin could partially restore ATPase activity in sealed vesicles which were inhibited by fluoride and this restoration occurred with a reduction in the membrane potential. Fluoride demonstrated a competitive interaction with chloride-stimulation of proton transport and inhibited the uptake of radioactive chloride into sealed vesicles. When the vesicles were allowed to develop a pH gradient in the absence of KCl, and KCl was subsequently added, fluoride reduced enhancement of the existing pH gradient by KCl. The results are consistent with a chloride carrier that is inhibited by fluoride.  相似文献   

6.
火炬松(PinustaedaL.)是我国亚热带和部分热带地区最重要的绿化和造林树种。它的生长周期长,杂合程度高,难以用常规的杂交方法进行品种改良。建立火炬松的原生质体胚胎发生体系,有可能、进而以遗传转化为基础进行火炬松等针叶树的品种改良。本研究以湖南省绍阳市的火炬松成熟种子为材料,建立胚性细胞悬浮系。将其培养至对数生长期,用1%RS、2.5%R10和0.2%Y23的酶混和液分离原生质体,活力达90%以上(Fig.1)。纯化原生质体,在再生培养基上培养2天后,细胞壁再生(Fig.2);;6天后,65%的原生质体第1次分裂(Fig.3);培养3周后,形成小细胞团(Fig.4);6周后,形成大细胞团(Fig.5);8周后,形成胚性胚柄细胞团(ESM)(Fig.6);10周后,形成早期体细胞胚(ESE)(Fig.7);12周后,形成后期体细胞胚(LSE)(Fig.8)火炬松原生质体胚形成过程中的关键结构是ESM,ESE和LSE。它们形成的最佳基本培养基是1.25LP培养基。再生培养基中高浓度的BA和低浓度的肌醇有利于ESM的形成,但不利于ESE和LSE的形成。反之,低浓度的BA和高浓度的肌醇不利于ESM的形成,  相似文献   

7.
The mechanisms involved in the transport of malate into isolated vacuoles of Catharanthus roseus (L.) cells were investigated with special reference to the effects of induced changes in membrane potential and surface charges of the tonoplast. For this purpose, thiocyanate (SCN?), a highly permeant anion often used as a membrane potential probe, was extensively exploited. In the absence of Mg-ATP, the low accumulation ratio of 14C SCN? could be related to the presence of negative charges at the outer surface of the tonoplast exerting a screening effect on the displacement of lipophilic anionic species. Nevertheless, malate was taken up continuously by vacuoles supporting the concept of a transport component which facilitates its transfer through the tonoplast. From experiments showing the pH dependence of malata uptake, it is suggested that the protonated form of the transporter is implicated in this process. Moreover, when the vacuoles are energized by Mg-ATP, the study of the equilibrium distribution of 14C SCN? indicated an inside positive membrane potential difference. Advantage was taken of these results to modulate the membrane potential with high levels of thiocyanate. The data obtained demonstrate that malate uptake results from electrophoretic movement in response to the positive potential difference.  相似文献   

8.
Apoptosis is characterized by the programmed activation of specific biochemical pathways leading to the organized demise of cells. To date, aspects of the intracellular signaling machinery involved in this phenomenon have been extensively dissected and characterized. However, recent studies have elucidated a novel role for changes in the intracellular milieu of the cells as important modulators of the cell death program. Specially, intracellular ionic homeostasis has been reported to be a determinant in both the activation and progression of the apoptotic cascade. Several apoptotic insults trigger specific changes in ionic gradients across the plasma membrane leading to depolarization of the plasma membrane potential (PMP). These changes lead to ionic imbalance early during apoptosis. Several studies have also suggested the activation and/or modulation of specific ionic transport mechanisms including ion channels, transporters and ATPases, as mediators of altered intracellular ionic homeostasis leading to PMP depolarization during apoptosis. However, the role of PMP depolarization and of the changes in ionic homeostasis during the progression of apoptosis are still unclear. This review summarizes the current knowledge regarding the causes and consequences of PMP depolarization during apoptosis. We also review the potential electrogenic ion transport mechanisms associated with this event, including the net influx/efflux of cations and anions. An understanding of these mechamisms could lead to the generation of new therapeutic approaches for a variety of diseases involving apoptosis.  相似文献   

9.
10.
Hormonal Control of Elongation of Tobacco Cells Derived from Protoplasts   总被引:4,自引:0,他引:4  
Spherical protoplasts (30–40 µm in diameter), isolatedfrom cultured tobacco cells, elongated rapidly during a cultureperiod of 3 to 8 days and finally assumed a long cylindricalform (300–400 µm in length). The optimum concentrationof a-naphthaleneacetic acid and benzyladenine for the elongationwas 0.1 mg/liter and 1 mg/liter respectively. (Received August 10, 1982; Accepted November 12, 1982)  相似文献   

11.
Abstract: The role of Na+ channels and membrane potential in stimulus secretion coupling in adrenal medulla cell cultures was investigated. Veratridine, aconitine, batrachotoxin (BTX), and scorpion venom, which increase the flux of ions through tetrodotoxin(TTX)-sensitive Na+ channels, all evoke secretion of catecholamines that is blocked by TTX. TTX partially inhibits secretion induced by low concentrations of nicotine in Locke's solution but has no effect on high concentrations of nicotine (20 μM). In Ca2+-sucrose media TTX has no effect on secretion at either high or low concentrations of nicotine. Replacement of Na+ with Li+ in Locke's solution reduces the response to nicotine and to veratridine. Complete replacement of Na+ with hydrazine, diethanolamine, TRIS, and choline completely inhibits the response to nicotine and almost completely inhibits the response to veratridine. Following exposure of cells to 50 mM-100 mM-K+, nicotine does not stimulate catecholamine secretion unless the cells are resuspended in media containing less than 50 mM-K+. Neither dibutyryl-cyclic AMP nor dibutyryl-cyclic GMP evokes secretion. α-Bungarotoxin (1 μM) did not inhibit nicotine-induced secretion. These studies indicate that Na+ channels and acetylcholine (ACh) receptor ion channels are independently coupled to the influx of Ca2+. The membrane potential appears to affect nicotine- and veratridine-evoked secretion.  相似文献   

12.
13.
苹果胚性细胞原生质体培养获得再生新梢   总被引:4,自引:0,他引:4  
苹果原生质体再生愈伤组织已有报道,但再生植株的报道仅有一例。以往的工作,多采用茎、叶、叶愈伤组织或悬浮培养物分高原生质体,培养成愈伤组织后不易发生器官分化。本文首次报道从新红星苹果胚珠愈伤组织建立的胚性悬浮细胞系分离原生质体,经培养获得无根绿苗的结果。 1.材料及悬浮细胞系的建立试材为中国农科院果树所果园内14年生新红星苹  相似文献   

14.
The stability and respiratory and permeability properties ofprotoplasts isolated from maize and tobacco tissues have beeninvestigated. Oxygen uptake rates of maize leaf protoplastsshowed the expected response to a variety of metabolic inhibitors.Studies of leakage from preloaded tissues during protoplastisolation and of phosphate and potassium uptake byprotoplastsindicated that isolation produced a marked increase in membranepermeability. Ageing of protoplasts for 24 h produced a markedchange in the ability of maize leaf protoplasts to absorb potassium.  相似文献   

15.
The effects of metabolic inhibitors, pH, and temperature on the kinetics of sucrose uptake protoplasts isolated from developing soybean Glycine max L. cv Wye cotyledons were studied. Structural requirements for substrate recognition by the sucrose carrier were examined by observing the effects of potential alternate substrates for the saturable component on sucrose uptake.  相似文献   

16.
Although model protocellular membranes consisting of monoacyl lipids are similar to membranes composed of contemporary diacyl lipids, they differ in at least one important aspect. Model protocellular membranes allow for the passage of polar solutes and thus can potentially support cell-to functions without the aid of transport machinery. The ability to transport polar molecules likely stems from increased lipid dynamics. Selectively permeable vesicle membranes composed of monoacyl lipids allow for many lifelike processes to emerge from a remarkably small set of molecules.Lipid bilayer membranes are an integral component of living cells, providing a permeability barrier that is essential for nutrient transport and energy production. It is reasonable to assume that a similar boundary structure would be required for the origin of cellular life (Szostak et al. 2001). Even though bilayer membranes are a cellular necessity, they also pose a significant obstacle to early cellular functions, the most obvious being that the permeability barrier would inhibit chemical exchange with the environment. Such an exchange is important not only for acquiring nutrient substrates for primitive metabolic processes, but also for the release of inhibitory side-products.Contemporary cells circumvent the permeability problem by incorporating complex transmembrane protein machinery that provides specific transport capabilities. It is unlikely that Earth’s first cells assembled bilayer membranes together with specific membrane protein transporters. Rather, intermediate evolutionary steps must have existed in which simple lipid molecules provided many of the characteristics of contemporary membranes without relying on advanced protein machinery. What seems to have been necessary was the appearance of a simple membrane system capable of retaining and releasing specific molecules. In short, a protocell needed to be selectively permeable.  相似文献   

17.
Binding of tobacco mosaic virus (TMV) to disrupted tobacco leaf membrane was studied. Membrane isolated from tobacco leaves was treated successively with (NH4)2SO4, Li-diiodosalicylate and then pronase. TMV-binding substance was thus isolated in a soluble form. From enzymatic digestion experiments, it was suggested that the binding substance was composed of lipid and carbohydrate.  相似文献   

18.
Sterile shoot cultures of Nicotiana tabacum L. cv. Sansum proved to be a highly appropriate source for the isolation of viable protoplasts. High yields of isolated protoplasts and high plating efficiencies are guaranteed by the absence of contaminaction, by controlled culture conditions and by the rhythmic rejuvenation of the shoots at each transfer to fresh agar medium.  相似文献   

19.
Ion channel activity in cell-attached patch recordings shows channel behavior under more physiological conditions than whole-cell and excised patch measurements. Yet the analysis of cell-attached patch measurements is complicated by the fact that the system is ill defined with respect to the intracellular ion activities and the electrical potential actually experienced by the membrane patch. Therefore, of the several patch-clamp configurations, the information that is obtained from cell-attached patch measurements is the most ambiguous. The present study aims to achieve a better understanding of cell-attached patch measurements. Here we describe a method to calculate the intracellular ion concentration and membrane potential prevailing during cell-attached patch recording. The first step is an analysis of the importance of the input resistance of the intact cell on the cell-attached patch measurement. The second step, and actual calculation, is based on comparison of the single channel conductance and reversal potential in the cell-attached patch and excised patch configurations. The method is demonstrated with measurements of membrane potential and cytosolic K+ concentrations in Vicia faba guard cells. The approach described here provides an attractive alternative to the measurement of cytosolic ion concentrations with fluorescent probes or microelectrodes. Received: 3 April 1998/Revised: 6 August 1998  相似文献   

20.
Membrane potentials of protoplasts isolated from Vigna mungohypocotyl segments were measured using the fluorescent probediS-C3-(5). The fluorescence intensity changed in response tothe external K+ concentration. Membrane potential was estimatedto be inside negative (–85?8 mV at 0.1 mM KCl) from theNernst equation for K+. The membrane potential was not affectedby DCCD (50 µM) or low temperature (5?C). Addition of0.5 mM Ca2+ to the protoplast suspension markedly depolarizedthe membrane potential, and subsequent EDTA treatment repolarizedit to the initial level. The Ca2+ effect on the membrane potentialmay be due to change in the permeability ratio of Clto K+. (Received December 16, 1986; Accepted April 22, 1987)  相似文献   

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