The K+ channel in the plasma membrane of rye roots has a multiple ion residency pore

The permeation of K⁺ and Na⁺ through the pore of a K⁺ channel from the plasma membrane of rye roots was studied in planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers. The pore contains at least two ion-binding sites which can be occupied simultaneously. This was indicated by: (i) biphasic relationships with increasing cation concentration of both channel conductance at the zero-current (reversal) potential of the channel (E _rev) and unitary-current at a specified voltage and (ii) a decline in E _rev in the presence of equimolar Na⁺ (cis):K⁺ (trans) as the cation concentration was increased. To determine the spatial characteristics and energy profiles for K⁺ and Na⁺ permeation, unitary-current/ voltage data for the channel were fitted to a three energy-barrier, two ion-binding site (3B2S) model. The model allowed for simultaneous occupancy of binding sites and ionic repulsion within the pore, as well as surface potential effects. Results suggested that energy peaks and energy wells (ion binding sites) were situated asymmetrically within the electrical distance of the pore, the trans energy-well being closer to the center of the pore than its cis counterpart; that the energy profile for K⁺ permeation differed significantly from that of Na⁺ in having a higher cis energy peak and a deeper cis energy well; that cations repelled each other within the pore and that vestibule surface charge was negligible. The model successfully simulated various aspects of K⁺ and Na⁺ permeation including: (i) the complexities in current rectification over a wide range of contrasting ionic conditions; (ii) the biphasic relationships with increasing cation concentration of both channel conductance at E _rev and unitary-current at a specified voltage; (iii) the decline in E _rev in equimolar Na⁺ (cis):K⁺ (trans) as cation concentrations were increased and (iv) the complex relationships between mole fraction and E _rev at total cation concentrations of 100 and 300 mm.We thank Prof. O. Alvarez (Universidad de Chile, Santiago, Chile) for supplying the computer program AJUSTE and Prof. D. Sanders (University of York, UK), Prof. D. Gradmann and Dr. G. Thiel (University of Göttingen, Germany) for stimulating ideas. This work was supported by the Agricultural and Food Research Council.     

Lysophosphatidic acid induces inositol phosphate and calcium signals in exocrine cells from the avian nasal salt gland

We tested lysophosphatidic acid (LPA), known to induce inositol phosphate generation and calcium signals as well as rearrangements of the cytoskeleton and mitogenic responses in fibroblasts, for its ability to activate phospholipase C in an exocrine cell system, the salt-secreting cells from the avian nasal salt gland. LPA (>10 nmol/l) caused the generation of inositol phosphates from membrane-bound phosphatidylinositides. The resulting calcium signals resembled those generated upon activation of muscarinic receptors, the physiological stimulus triggering salt secretion in these cells. However, close examination of the LPA-mediated calcium signals revealed that the initial calcium spike induced by high concentrations of LPA (>10 μmol/l) may contain a component that is not dependent upon generation of inositol (1,4,5)-trisphosphate (Ins(1,4,5)P₃) and may result from calcium influx from the extracellular medium induced by LPA in a direct manner. Low concentrations of LPA (<10 μmol/l), however, induce inositol phosphate generation, Ins(1,4,5)P₃-mediated release of calcium from intracellular pools and calcium entry. These effects seem to be mediated by a specific plasma membrane receptor and a G protein transducing the signal to phospholipase C in a pertussis-toxin-insensitive manner. Signaling pathways of the muscarinic receptor and the putative LPA-receptor seem to merge at the G-protein level as indicated by the fact that carbachol and LPA trigger hydrolysis of the same pool of phosphatidylinositol (4,5)-bisphosphate (PIP₂) and mobilize calcium from the same intracellular stores.     

菜豆热激蛋白在生物膜上的定位

选用菜豆 Phaseolus vulgris L. 下胚轴 ,运用35S- Met标记放射自显影和二维电泳技术 ,研究热激蛋白 HSPs 的表达和在生物膜组分中的定位 .实验结果表明 ,盐溶蛋白中主要HSPs为 70 k D HSPs和小分子量 HSPs,而小分子量组 HSPs大量富集在质膜和液泡膜组分中 .     

Hydrogen fluoride effects on plasma membrane composition,ATPase activity and cell structure in needles of eastern white pine (Pinus strobus) seedlings

Eastern white pine (Pinus strobus L.) seedlings were grown in controlled environment growth cabinets and fumigated with 0.4 and 1.6 g m^–3 hydrogen fluoride for 2–28 days. Plasma membranes were isolated from needles of treated and control seedlings and their chemical composition and ATPase activity examined to determine early effects of hydrogen fluoride action. In plants treated for 2 days with both fluoride levels, ratios of plasma membrane free sterols:phospholipids and sterols:proteins were drastically higher than ratios in control plants. Seedlings treated with hydrogen fluoride for 8 days contained plasma membranes with elevated phospholipid:protein and sterol:protein ratios and their plasma membrane ATPase activity was higher than that of control plants. Prolonged, 28-day hydrogen fluoride treatment with 1.6 g m^–3 level was the only treatment which produced a drastic inhibition of plasma membrane ATPase activity. During the initial stages of hydrogen fluoride treatment, treated cells did not show alterations of ultrastructure which were previously shown in cells of plants treated with soil applied sodium fluoride. The results of the present study indicate that the plasma membranes may be among the initial sites of hydrogen fluoride injury to plants as well as initial sites of defense reaction.     

A link between transport and plasma membrane redox system(s) in carrot cells

Carrot (Daucus carota L.) cells grown in suspension culture oxidized exogeneous NADH. The NADH oxidation was able to stimulate K⁺ (⁸⁶Rb⁺) transport into cells, but it did not affect sucrose transport.N,N'-Dicyclohexyl-carbodiimide, diethylstilbestrol, and oligomycin, which only partially inhibited NADH oxidation, almost completely collapsed the K⁺ (⁸⁶Rb⁺) transport. Vanadate, which is less effective as an ion transport inhibitor, was less effective in inhibiting the NADH-driven transport of K⁺ (⁸⁶Rb⁺).p-Fluormethoxycarbonylcyanide phenylhydrazone inhibits the K⁺ transport over 90% including that induced by NADH. The results are interpreted as evidence that a plasma membrane redox system in root cells is closely associated with the ATPase which can drive K⁺ transport. Because of the inhibitor effects, it appears that membrane components common to the redox system and ATPase function in the transport of K⁺.     

Partial characterization of the glucose transport activity in the golgi-rich fraction of fat cells

The glucose transport activity solubilized from the basal and plus insulin forms of the Golgi-rich fraction of adipocytes was partially characterized, and the results were compared with those of the activity obtained from the plus insulin form of the plasma membrane-rich fraction. The transport activity was determined in a cell-free, reconstituted, system. Prior to reconstitution, the activities in the three preparations were all (a) stable at 0°C for at least 4 h, but not at 37°C or above; (b) most stable at pH 7–9, and (c) less stable in Tes than in Tris buffer. After reconstitution, the three activities were all (d) stable at 0°C, (e) most active at pH 5.5, (f) mildly stimulated by divalent cations, (g) unaffected by insulin or 1 mM of several SH-blocking agents, (h) inhibited by heavy metal ions, 10–100 mM of monovalent salts, organic solvents, several sugar isomers, and specific sugar-transport inhibitors. The rates of d-glucose uptake by the three liposome preparations were all inhibited more strongly by 2-deoxy-d-glucose or $\text{3}\text{-O-}\text{methyl-}\text{d}\text{-glucose}$ than by d-glucose. These data indicate that the general properties of the glucose transport activity in the Golgi-rich fraction are similar to those of the activity in the plasma membrane-rich fraction.     

Evidence for a highly asymmetric arrangement of ether- and diacyl-phospholipid subclasses in the plasma membrane of Krebs II ascites cells

(1) Krebs II ascites cells were taken as a model of the neoplastic cells to investigate the transverse distribution of phospholipids in the plasma membrane. The experimental procedure was based on non-lytic degradation of phospholipids in the intact cell by Naja naja phospholipase A₂ and Staphylococcus aureus sphingomyelinase C and on phopholipid analysis of purified plasma membranes. It was shown that the three major phospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, are randomly distributed between the two halves of the membranes, whereas phosphatidylserine remains located in the inner leaflet. (2) The membrane localization of phosphatidylcholine and phosphatidylethanolamine subclasses (diacyl, alkylacyl and alkenylacyl) was also examined, using a new procedure of ether-phospholipid determination. The method involves a selective removal of diacyl species by guinea pig pancreas phospholipase A₁ and of alkenylacyl species by acidolysis. This analysis revealed a 50% increase of ether phospholipids in the plasma membrane as compared to the whole cell (36.5 and 23.1% of total phospholipid, respectively). Furthermore, a strong membrane asymmetry was demonstrated for the three phosphatidylcholine subclasses, since 1-alkyl-2-acyl-sn-glycerol-3-phosphocholine (alkylacyl-GPC) was entirely found in the inner leaflet, whereas both diacyl- and alkenylacyl-GPC displayed an external localization. The same pattern was observed for phosphatidylethanolamine subclasses, except for 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine, which was found randomly distributed. These results are discussed in relation to the process of cell malignant transformation and to the biosynthesis of platelet-activating factor (PAF-acether or 1-alkyl-2-acetyl-GPC).     

Evidence that neutral spingomyelinase of cultured murine neuroblastoma cells is oriented externally on the plasma membrane

The activity of the neutral, Mg²⁺-stimulated sphingomyelinase of cultured neuroblastoma cells (N1E-115) is enriched in the plasma membrane fraction and is reduced following treatment of intact or broken cells with trypsin, α-chymotrypsin, papain, and protease. Two protease-sensitive enzymes of the cell interior (lactate dehydrogenase and NADPH-cytochrome c reductase) are not affected by protease treatment of intact cells. These results indicate that the neutral, Mg²⁺-stimulated sphingomyelinase is oriented externally on the plasma membrane of the cultured neuroblastoma cell.     

β-1-4-and β-1-3-glucan synthases are associated with the plasma membrane of the fungus Saprolegnia

Cytoplasmic membranes from mycelium or protoplasts of Saprolegnia monoica (a cellulosic cell-wall fungus) were separated by continuous sucrose-density-gradient centrifugation. Glucan synthases assayed at low (micromolar uridine 5-diphosphate (UDP) glucose for -1-4-glucan synthase) and high (millimolar UDP glucose for -1-3-glucan synthase) substrate concentrations were associated with membranes exhibiting vanadate-sensitive, oligomycin-insensitive ATPase and equilibrating at density 1.16 g cm^-3. Synthase activities were also bound to membranes of lower density (1.10 and 1.145 g cm^-3). Plasma membranes were stabilized by coating protoplasts with concanavalin A. After lysis of the protoplasts, plasma membranes recovered by low centrifugal forces were isolated in continuous isopycinic gradients. Both synthase activities peaked with [³H]concanavalin A and Na-vanadate ATPase indicating that the synthetases are located at the plasma membrane. Treatments of intact protoplasts with cold glutaraldehyde or proteases before disruption lead to a diminution of glucan-synthase activities indicating that at least part of the enzymes of plasma membrane face the outside of the cell.Abbreviations ConA concanavalin A - ER endoplasmic reticulum - GSI -1,4-glucan synthase - GSH -1,3-glucan synthase - UDP uridine 5-diphosphate     

Interactions of galactose-binding lectins with plant protoplasts

Summary Protoplasts isolated from cell suspension cultures of carrot (Daucus carota L.) and leaves of tobacco (Nicotiana tabacum L.) were treated with three lectins specific for galactosyl residues. After incubation with RCA I (Ricinus communis agglutinin, molecular weight 120,000) conjugated to ferritin or fluorescein, freshly isolated protoplasts displayed heavy labeling of their surfaces. Moreover, they agglutinated rapidly when exposed to low concentrations of RCA I. In parallel studies, PNA (peanut agglutinin) also bound extensively to the protoplast plasma membranes whileBandeiraea simplicifolia lectin I attached relatively weakly. When protoplasts were cultured for two days and then incubated with conjugates of RCA I and PNA, additional binding sites were revealed on the regenerating walls.The results indicate that galactosyl residues are distributed densely over the surface of plant protoplasts. They also allow inferences to be made regarding the positions and linkages of the galactose groups being recognized by the lectins. Moreover, they open up the question whether the galactosyl moieties detected in the wall derive from those labeled on the plasma membrane. To conclude, we make comparisons with binding by concanavalin A, and predict that galactose-recognizing lectins will join and in certain respects prove superior to concanavalin A as probes of the plant cell surface.