Effects of tricyclohexylhydroxytin on the kinetics of adenosine triphosphatase system and protection by thiol reagents

Tricyclohexylhydroxytin, commonly known as Plictran® inhibited Na⁺, K⁺ -ATPase activity of rat brain synaptosomes in a concentration-dependent manner with median inhibitory concentration (IC-50) of 2 μM. Both K⁺ -stimulated para-nitrophenylphosphatase and [3-H]-ouabain binding to synaptosomes were also inhibited by Plictran with IC-50 values of 11 and 30 μM, respectively. Altered pH and Na⁺, K⁺ -ATPase activity curves demonstrated comparable inhibition in buffered neutral and alkaline pH ranges, and no inhibition was observed in acidic pH. The inhibition of Na⁺, K⁺ -ATPase was independent of temperature. Kinetic studies of substrate (ATP) activation of Na⁺, K⁺ -ATPase indicated uncompetitive inhibition. Results also showed noncompetitive inhibition for p-nitrophenylphosphate and uncompetitive inhibition for K⁺ activations of p-nitrophenylphosphatase. Preincubation of synaptosomes with dithiothreitol, a sulfhydryl (SH) agent, resulted in the complete protection of Plictran inhibition of Na⁺, K⁺ -ATPase, K⁺ -para-nitrophenylphosphatase, and [3-H]-ouabain binding. The protection was specific and concentration dependent since cysteine and glutathione did not afford protection. These results indicate that Plictran inhibited Na⁺, K⁺ -ATPase by interacting with dephosphorylation of the enzyme-phosphoryl complex and exerted a similar effect to that of SH-blocking agents.     

Tetracyclines,verapamil and nifedipine induce callose deposition at specific cell sites in Riella helicophylla

The Ca²⁺ indicator 7-chlorotetracycline has been shown to bind to a pore complex on both outer surfaces of all non-meristematic cells in the unistratose thallus of Riella (chlorotetracycline-binding surface region=CSR; Grotha, 1983, Planta 158, 473–481). Prolonged treatment of the thallus with 7-chlorotetracycline, 5-hydroxytetracycline, verapamil and desmethoxyverapamil induces the deposition of callose at the same region. The influence of various treatments on verapamil-induced CSR-callose was measured in situ by microfluorometry of aniline-blue-stained material. Callose deposition is maximal at 10^-4M verapamil or 5·10^-5M desmethoxyverapamil with 2·10^-4M Ca²⁺ or Mg²⁺ in the medium. The reaction is completely inhibited at pH 5.5 and is optimal between pH 6.5 and 7.5. The production of CSR-callose is absolutely light-dependent with callose being first visible after 30 min of light. La³⁺, ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid and amiprophosmethyl, antagonists of Ca²⁺ functions, and 2-deoxy-D-glucose suppress the verapamil induction of CSR-callose. Furthermore the ionophores A 23187, valinomycin and monensin effectively block the reaction. The deposition of CSR-callose is diminished at increasing external osmolarity and is abolished at osmotic values that stimulate plasmolysis-callose. Wounding causes the formation of wound-callose but inhibits the induction of CSR-callose in cells of the wound edge. Nifedipine increases or prolongs callose synthesis in cell plates. The Ca²⁺-channel blocker diltiazem is completely ineffective. It is suggested as a working hypothesis that verapamil-induced CSR-callose synthesis is caused by a local change in membrane permeability, possibly as a consequence of the opening of Ca²⁺ channels being involved in Golgi-vesicle mediated exocytosis (A. Kramer and H. Lehmann, 1986, Ber. Dtsch. Bot. Ges. 99, 111–121).Abbreviations APM amiprophosmethyl - APW artificial pond water - CSR chlorotetracycline-binding surface region - CTC 7-chlorotetracycline - DDG 2-deoxy-D-glucose - EGTA ethylone glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid - OTC 5-hydroxytetracycline - Pipes 1,4-piperazinediethane sulfonic acid Dedicated to Professor Luise Stange on the occasion of her 60^th birthday     

Salt requirements for membrane transport and solute retention in some moderate halophiles

Abstract Many species of bacteria isolated from saline environments require Na⁺ specifically for membrane transport. Transport occurs by a Na⁺ symport process energized by an electrochemical gradient of Na⁺ ions. The gradient at neutral pH appears to be produced by a primary electrogenic extrusion of protons coupled to a secondary, outwardly directed Na⁺ pump, a Na⁺/proton antiporter. At alkaline pH Vibrio alginolyticus may also produce the gradient by an energy-dependent primary extrusion of Na⁺ ions. Alteromonas haloplanktis and Vibrio costicola require salts in the medium to retain intracellular solutes. For A. haloplanktis the effects of the salts are primarily osmotic. For V. costicola , only NaCl is effective in retaining solutes and Na⁺ is required by this organism to maintain the membrane potential. In Escherichia coli a single substitution in the nucleotide sequence of the gene coding for the melibiose transport protein changed the cation specificity of the transport system. The possible ecological significance of this finding has been considered.     

Effects of terbium on the hemocyanin pore formation rate in phosphatidylcholine planar bilayers

Megathura crenulata hemocyanin forms ionic channels in planar lipid bilayer membranes. It was found that hemocyanin is more potent as a channel former if TbCl₃ is added to the bathing solution. Furthermore membranes separating symmetrical TbCl₃ solutions show a pore formation rate which depends exponentially on the applied voltage, positive potentials favouring the insertion of new channels. The slope of this voltage dependence, which gives a measure of the effective charge displaced during the incorporation of one channel, increases and saturates with TbCl₃ concentration. The dose response curve indicates that binding of Tb³⁺ to the phosphatidylcholine bilayer is involved in creating the effective charge.     

Monoclonal antibodies modify acetylcholine-induced ionic channel properties in cultured chick myoballs

Summary Monoclonal antibodies directed against the cholinergic binding site of the acetylcholine receptor were found to alter the ion channel properties in cultured chick myoballs. Time and dose dependent reduction in acetylcholine sensitivity was observed. Noise analysis experiments indicated a decrease in the mean single channel conductance and an increase in the mean single channel open time.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila,Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90°C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70°C) and Escherichia coli (max. growth temp., 47°C) with respect to (a) bihelical content of rRNA; (b) G·C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. 1. Subunits of C. acidophila ribosomes (T_m = 90–93°C) exhibit considerable thermal tolerance over their B. acidocaldarius (T_m = 77°C) and E. coli counterparts (T_m = 72°C). 2. Based on the ‘melting’ hyperchromicities of the intact ribosomal subunits a 51–55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67–70%, appears to be involved in hydrogen bonding in the naked rRNA species. 3. The G·C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63–64% G·C, compared to 58.5% G·C for B. acidocaldarius and 55% G·C for E. coli. 4. The increment in ribosome T_m values with increasing thermophily is greater than the increase in T_m for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). 5. The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. 6. Compared to E. coli, the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.     

Entropy effects on the ion-diffusion rate in transmembrane protein channels

We treat the transport of univalent cations through pore-like protein channels in biological membranes analytically, using two models (A + B) for the channel and the ion-channel interaction. A Lennard-Jones-type repulsion between the ions and the pore wall is introduced. We also include Van der Waals- and coulomb-type interactions between polar ligands of the pore-forming protein (e.g., carbonyl groups directed towards the axis of the channel) and the migrating particles. In model A, the polar groups are assumed to occur in pairs of dipoles pointing in opposite directions (as in the gramicidin A channel), while in model B the channel is treated as a pore with a radially isotropic charge distribution. In both models the ion-channel interaction leads to the occurrence of periodic potentials, corresponding to quasi-equilibrium and transition state sites of the ion in the pore. The diffusion rate can be calculated employing rate-theoretical concepts on the basis of microscopic parameters. It is demonstrated that the anomaly (inversion of the normal mass effect) for the transport rates of different ions can be related to differences in the activation entropy. The latter quantity is estimated analytically for both models. As a test, we performed numerical calculations with parameters based on the gramicidin A model. The results are in good agreement with experimental data and data from computer simulations. This shows that simple analytic expressions are well suited for predicting trends in the ionic conductivity of protein channels on the basis of microscopic interactions.     

Ion-specific diffusion rates through transmembrane protein channels. A molecular dynamics study

The migration of different alkali metal cations through a transmembrane model channel is simulated by means of the molecular dynamics technique. The parameters of the model are chosen in close relation to the gramicidin A channel. Coulomb- and van der Waals-type potentials between the ions and flexible carbonyl groups of the pore-forming molecule are used to describe the ion channel interaction. The diffusion properties of the ions are obtained from three-dimensional trajectory calculations. The diffusion rates for the different ions Li+, Na+, K+ and Rb+ are affected not only by the mass of the particles but also very strongly by their size. The latter effect is more pronounced for rigid channels, i.e., for binding vibrational frequencies of the CO groups with v greater than 400 cm-1. In this range the selectivity sequence for the diffusion rates is the inverse of that expected from normal rate theory but agrees with that found in experiments for gramicidin A.     

Knockdown resistance to dichlorodiphenyl-trichloroethane and pyrethroid insecticides in the napts mutant of Drosophila melanogaster is correlated with reduced neuronal sensitivity

Resistance to pyrethroid insecticides and dichlorodiphenyltrichloroethane (DDT) was investigated in the nap^ts (no action potential, temperature sensitive) mutant of Drosophila melanogaster. In surface contact bioassays, the nap^ts strain showed threefold resistance to deltamethrin at the LC₅₀ level when compared to susceptible Canton-S flies. Cross-resistance was also observed to DDT and the pyrethroids NRDC 157 [3-phenoxybenzyl [1R,cis]-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate], fenfluthrin, and MTI-800 [1-(3-phenoxy-4-fluorophenyl)-4-(4-ethoxyphenyl)-4-methylpentane]. The onset of intoxication by pyrethroids in nap^ts flies was markedly delayed, a finding that is consistent with the existence of a resistance mechanism involving reduced neuronal sensitivity. Resistance at the level of the nerve was confirmed by electrophysiological recordings of spontaneous and evoked activity in the dorsolongitudinal flight muscles of poisoned flies. Preparations from nap^ts insects treated with fenfluthrin displayed longer latencies to the appearance of spontaneous activity and also an absence or reduction in burst discharges compared to equivalent preparations from susceptible individuals. These results are discussed in light of competing hypotheses concerning the mechanism underlying knockdown resistance and reduced nerve sensitivity in insects.