Basic Protein Dissociating from Myelin Membranes at Physiological Ionic Strength and pH Is Cleaved into Three Major Fragments

Experiments were performed with isolated human myelin membrane preparations to analyse factors that may modulate association of myelin basic protein (MBP) with the membranes and could contribute to demyelinating processes. Transfer of membranes (5 mg protein ml-1) at 37 degrees C and pH 7.4 from a hypotonic medium, in which they were relatively stable, to one of physiological ionic strength produced three major effects: (1) initial dissociation of MBP from the membranes by a nonenzymatic process that was doubled in the presence of millimolar Ca2+/Mg2+; (2) within 10 min, the appearance in the medium of three major MBP fragments (14.4, 10.3, and 8.4 kilodaltons); and (3) progressive acidification of dissociated MBP and its fragments, probably due to deamidation. Between 1 and 6 h a steady state was apparent in which protein equivalent to 15% of the MBP originally bound to the membranes was found in the medium. The three major MBP fragments formed two-thirds of this solubilised material and appeared metabolically stable for 24 h. The kinetics of peptide formation suggested that dissociated, rather than membrane-bound, MBP was cleaved by myelin-associated neutral proteases. Two-dimensional electrophoresis and immunoblotting using two monoclonal antibodies indicated that proteolysis occurred in the vicinity of residues 35 and 75. Evidence was also obtained for removal of C-terminal arginines and relatively rapid deamidation in the C-terminal half of MBP. These modifications of MBP might also occur if extracellular fluid gained access to the compacted cytoplasmic space of the myelin sheath.     

Ionic channels formed byStaphylococcus aureus alpha-toxin: Voltage-dependent inhibition by divalent and trivalent cations

Summary The interaction ofStaphylococcus aureus -toxin with planar lipid membranes results in the formation of ionic channels whose conductance can be directly measured in voltage-clamp experiments. Single-channel conductance depends linearly on the solution conductivity suggesting that the pores are filled with aqueous solution; a rough diameter of 11.4±0.4 Å can be estimated for the pore. The conductance depends asymmetrically on voltage and it is slightly anion selective at pH 7.0, which implies that the channels are asymmetrically oriented into the bilayer and that ion motion is restricted at least in a region of the pore. The pores are usually open in a KCl solution but undergo a dose- and voltage-dependent inactivation in the presence of diand trivalent cations, which is mediated by open-closed fluctuations at the single-channel level. Hill plots indicate that each channel can bind two to three inactivating cations. The inhibiting efficiency follows the sequence Zn²⁺>Tb³⁺>Ca²⁺>Mg²⁺>Ba²⁺. suggesting that carboxyl groups of the protein may be involved in the binding step. A voltage-gated inactivation mechanism is proposed which involves the binding of two polyvalent cations to the channel, one in the open and one in the closed configuration, and which can explain voltage, dose and time dependence of the inactivation.     

Elevation of ionic conductance between insect epidermal cells by β-ecdysone in vitro

Electrophysiological methods reveal that the cell-to-cell movement of inorganic ions in the epidermis of the beetle larva is facilitated by exposing the tissue to β-ecdysone in vitro. After exposure to 2 × 10^?6 M β-ecdysone for 24 hr, the resistivity of the intercellular pathway drops by 30%, from 389 Ωcm to 264 Ωcm. This response does not occur when α-ecdysone is used for extended incubation periods. As the resistivity of the epidermal cytoplasm in the absence (64 Ωcm) and presence of β-ecdysone (65 Ωcm) is constant, the hormone must exert its effect at the cell junctions. A simple geometrical model for the epidermal monolayer allows one to calculate that the ionic permeability of the junctional membrane increases by 66% in cells exposed to β-ecdysone for 24 hr in vitro.     

Lipid vesicle-mediated alterations of membrane cholesterol levels: Effects on Na+ and K+ currents in squid axon

Summary We demonstrate that cholesterol can exchange from sonicated lipid vesicles to a perfused squid giant axon membrane and that vesicles with varying cholesterol/phospholipid (C/P) mole ratios can be used to achieve either net loading or net depletion of axon membrane cholesterol. Two types of evidence were obtained which show that net loading or depletion of cholesterol was achieved: (i) changes in the cholesterol/phospholipid (C/P) mole ratios of axons, and (ii) visualization of cholesterol depleted from the preparation by cholesterol-free vesicles by thin-layer chromatography. The C/P mole ratios indicate that cholesterol levels in the preparation were increased or decreased by 30–40%. Increasing or decreasing membrane cholesterol levels were ineffective in altering the Na⁺ or K⁺ occurrents in voltage-clamped axons. In addition, we determined that cholesterol flip-flop across the axonal membrane occurred with at ^1/2 of 7.3 to 15.3 min.     

Ion fluxes inAcetabularia: Vesicular shuttle

Summary Ion flux relations in the unicellular marine algaAcetabularia have been investigated by uptake and washout kinetics of radioactive tracers (²²Na⁺,⁴²K⁺,³⁶Cl^– and⁸⁶Rb⁺) in normal cells and in cell segments with altered compartmentation (depleted of vacuole or of cytoplasm). Some flux experiments were supplemented by simultaneous electrophysiological recordings. The main results and conclusions about the steady-state relations are: the plasmalemma is the dominating barrier for translocation of K⁺ with influx and efflux of about 100 nmol·m^–2·sec^–1×K⁺ passes three- to sevenfold more easily than Rb⁺ does. Under normal conditions, Cl^– (the substrate of the electrogenic pump, which dominates the electrical properties of the plasmalemma in the resting state) shows two efflux components of about 17 and 2 mol·m^–2·sec^–1, and a cytoplasmic as well as vacuolar [Cl^–] of about 420mm ([Cl^–] _o =529mm). At 4°C, when the pump is inhibited, both influx and efflux, as well as the cellular [Cl^–], are significantly reduced. Na⁺ ([Na⁺] _i : about 70mm, [Na⁺] _o : 461mm), which is of minor electrophysiological relevance compared to K⁺, exhibits rapid and virtually temperature-insensitive (electroneutral) exchange (two components with about 2 and 0.2 mol·m^–2·sec^–1 for influx and efflux). Some results with Na⁺ and Cl^– are inconsistent with conventional (noncyclic) compartmentation models: (i) equilibration of the vacuole (with the external medium) can be faster than equilibration of the cytoplasm, (ii) absurd concentration values result when calculated by conventional compartmental analysis, and (iii) large amounts of ions can be released from the cell without changes in the electrical potential of the cytoplasm. These observations can be explained by the particular compartmentation of normalAcetabularia cells (as known by electron micrographs) with about 1 part cytoplasm, 5 parts central vacuole, and 5 parts vacuolar vesicles. These vesicles communicate directly with the central vacuole, with the cytoplasm and with the external medium.     

Distribution of single-channel conductances in cultured rat hippocampal neurons

1. The nonhomogeneous spatial distribution of ionic channels in neurons has been implied from intracellular recordings at somatic and dendritic locations. These reports indicate that Na- and Ca-dependent regenerative currents are distributed differently throughout the neuron. Although a variety of K conductances and a noninactivating Na conductance have been described in intracellular studies, little is known about the spatial distribution of inward and outward currents throughout different regions of the neuron. 2. We recorded from cell-attached patches from cultured hippocampal cells from 1-day-old rats. The cells were cultured for 3-21 days. The spatial distribution of a variety of ionic channels was determined by comparing the conductances from somatic and dendritic membranes. Single-channel currents obtained from cell-attached patches were identified by the time course of ensemble (averaged) responses, voltage dependence, and the effect of channel blocking agents. 3. We consistently observed that only the rapidly inactivating inward current was localized to the soma. The other channel types that we studied, including an inward noninactivating, delayed rectifier and transient A-type currents, were observed in both the somatic and dendritic regions. 4. We suggest that the distribution of ionic conductances that we have observed may be functional in limiting excitability during development of neurons.     

Ecophysiological investigations of Chara vulgaris L. grown in a brackish water lake: ionic changes and accumulation of sucrose in the vacuolar sap during sexual reproduction

Abstract Chara vulgaris L. growing in a brackish water lake was investigated in a field study during the main growth season (May to October 1985). Sucrose content and the ionic concentrations of the cations Na⁺, K⁺, Mg²⁺ and Ca²⁺ and the anions Cl^? and SO₄^2- of the vacuolar sap were estimated. Sucrose concentration in the vacuolar sap of vegetative growing plants was negligible, but with the beginning of the sexual reproduction period (fructification) the sucrose content increased from about 2 mol m^?3 to 110 mol m^?3. This level remained constant until the end of the fructification period. In spite of the increase of the sucrose concentration the osmotic potential of the vacuolar sap was constant. This was achieved by changing the ionic concentrations accordingly; in old or vegetative growing plants the ionic content accounted for about 80% of the vacuolar osmotic potential, but was about 63% during fructification. Sucrose is considered as a major photosynthate to supply the developing antheridia and oogonia and to serve as a precursor for the starch stored in the eggs.     

绵羊心浦肯野纤维d-受体和β-受体激动时离子流Isi和Ix的变化

当绵羊心浦肯野纤维α-和β-受体分别激动时,用双微电极法电压箝制术研究慢内向离子流Isi和延迟整流外向离子流Ix的变化。心得安阻断β-受体时,苯肾上腺素5.0×10~(-6)mol/L使Isi的峰值由17.5增加到26nA(n=6,P<0.05).并使Isi由失活到完全恢复的时间由293±51ms延长到441±109ms(n=4,P<0.05),但对Ix无明显影响。酚妥拉明阻断α-受体时,异丙肾上腺素4×10~(-7)mol/L使Isi峰值由27.7增加到40.8nA(n=7,P<0.05),对Isi的动力过程无明显影响,却使Ix尾电流的幅值由6.7增加到14,4nA(n=6,P<0.05)。表明α-和β-受体激动剂作用的差异在于对延迟整流离子流的影响不同。     

The bioreduction of a β-tetralone to its corresponding alcohol by the yeast Trichosporon capitatum MY1890 and bacterium Rhodococcus erythropolis MA7213 in a range of ionic liquids

The stereospecific reduction of 6-Br-β-tetralone to its corresponding alcohol (S)-6-Br-β-tetralol was carried out by the yeast Trichosporon capitatum MY1890 and by the bacterium Rhodococcus erythropolis MA7213, using a range of ionic liquids chosen for the diversity of their composition. The decrease in cell viability of both types of cell upon exposure to ionic liquids was found to be between that determined for cells residing purely in fermentation media, and cells residing in a two-phase mixture of media and organic solvent (toluene). For T. capitatum MY1890 bioconversions, the water miscible hydrophilic ionic liquid [Emim][TOS] gave a reaction profile comparable to that observed in the previously studied water-ethanol (10% v/v) system, in terms of overall rate of reaction (0.2 g (prod) L^-1 h^-1) and conversion (100%). Of the hydrophobic ionic liquids evaluated, [Oc₃MeN][BTA] gave the best conversion of 60%, but at a much reduced rate, suggesting solute mass transfer from the ionic liquid phase was rate limiting. For bioconversions carried out with R. erythropolis MA7213 employing 20% v/v [Emim][TOS] as a co-solvent, the conversion yield doubled, and a four-fold increase in initial rate was found compared to the standard ethanol co-solvent. This was attributed to improved cell viability and reduced aggregation of the R. erythropolis MA7213 compared to T. capitatum MY1890. Overall, this study demonstrates the feasibility of using ionic liquids for whole cell biocatalysis, however, no obvious link is apparent between the physico-chemical properties of ionic liquids, their influence on cell viability, and their efficacy as media for bioconversions.