Alkali ion transport through lipid bilayer membranes mediated by enniatin A and B and beauvericin

Summary Stationary conductance measurements with lipid bilayer membranes in the presence of enniatin A and B and beauvericin were performed. For comparison, some valinomycin systems were investigated. It was found that the conductance in the case of enniatin A and B is caused by a carrier ion complex with a 11 stoichiometry, whereas for beauvericin, a 31 carrier ion complex has to be assumed to explain the dependence of the conductance on carrier and ion concentration in the aqueous phase. The current-voltage curves measured with dioleoyl phosphatidylcholine membranes show a superlinear behavior for the three carriers in the presence of potassium. On the other hand, supralinear current-voltage curves were observed with membranes from different monoglycerides, except for beauvericin. The results obtained with enniatin A and B are in a satisfactory agreement with an earlier proposed carrier model assuming a complexation between carrier and ion at the membrane water interface.The discrimination between potassium and sodium ions is much smaller for the enniatins than for valinomycin. This smaller selectivity as well as the fact that potassium ions cause the highest conductance with lipid bilayer membranes may be due to the smaller size of the cyclic enniatin molecules, which contain 6 residues in the ringvs. 12 in the case of valinomycin. Charge-pulse relaxation studies were performed with enniatin A and B, beauvericin, and valinomycin. For monoolein membranes only in the case of valinomycin, all three relaxations predicted by the model could be resolved. In the case of the probably more fluid membranes from monolinolein (^{9, 12}-C_{18: 2}) and monolinolenin (^{9, 12, 15}-C_{18: 3}) for all carrier systems except for beauvericin, three relaxations were observed.The association rate constantk _R , the dissociation rate constantk _D , and the two translocation rate constantsk _MS andk _s for complexed and free carrier, respectively, could be calculated from the relaxation data. The carrier concentration in the aqueous phase had no influence on the rate constants in all cases, whereas a strong saturation of the association rate constantk _R with increasing ion concentration was found for the enniatins. Because of the saturation,k _R did not exceed a value of 4×10⁵ m ^–1 sec^–1 with 1m salt irrespective of carrier, ion, or membrane-forming lipid.A similar but less pronounced saturation behavior was also observed for the translocation rate constantk _S of the free carrier. The other two rate constants were independent of the ion concentration in the aqueous phase. In the case of the enniatins, the translocation rate constantk _MS was not independent from the kind of the transported ion. In the series K⁺, Rb⁺ and Cs⁺,k _MS increases about threefold. The turnover numbers for the carriers as calculated from the rate constants range between 10⁴ sec^–1 and 10⁵ sec^–1 and do not show a strong difference between the individual carriers. The conductance difference in the systems investigated here is therefore mainly caused by the partition coefficients, which are smaller for the enniatins than for valinomycin.     

Das unterschiedliche Verhalten der Exopeptidasen des Hepatopankreas und Magensaftes vom Flußkrebs Astacus astacus (L.) und Cambarus affinis (Say) bei der Gelfiltration auf Sephadex sowie gegenüber Effektoren

Zusammenfassung Das Hepatopankreas (HP) der Flußkrebse Astacus astacus (L.) und Cambarus affinis (Say) enthält eine hochmolekulare Carboxypeptidase A-Wirkung (Substrat: Carbobenzoxyglycyl-l-phenylalanin) (K _d-Wert auf Sephadex G-200=0,04) und eine Arylamidase-Wirkung [Substrat: l-Leucin--naphthyl-amid·HCl (LNA)], sowie Dipeptidase-Wirkung (Substrat: Glycyl-l-leucin) (K _d-Werte auf Sephadex G-200=0,46 bzw. 0,39). Carboxypeptidase B (Substrat: Hippuryl-l-arginin)-Aktivität wurde im HP nicht gefunden. Im Gegensatz zum HP ist die Carboxypeptidase A des Magensaftes (MS) niedriger molekular (K _d-Wert auf Sephadex G-150=0,62; Molekulargewicht: ca. 30000), die LNA-ase des MS höher molekular (K _d-Wert auf Sephadex G-150=0,26). Außerdem enthält der MS eine hochaktive Carboxypeptidase B-ähnliche Wirkung, die sich auf Sephadexgel wie die Carboxypeptidase A verhält. Chelatbildner (,-Dipyridyl, o-Phenanthrolin) hemmen die Hippurylarginin-Wirkung nicht. Die Carboxypeptidase A des HP wird durch EDTA und Hydrozimtsäure deutlich, durch p-Chlormercuribenzoat gering aktiviert und durch 2-Mercaptoäthanol (10^–3 m und höhere Konzentrationen) stark gehemmt.

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The differential behaviour of the exopeptidases from hepatopancreas and gastric juice of the crayfish Astacus astacus (L.) and Cambarus affinis (Say) during gelfiltration on sephadex and towards effectors

Summary Hepatopancreas (HP) of the crayfishes Astacus astacus (L.) and Cambarus affinis (Say) contains a high molecular carboxypeptidase A like action (substrate: carbobenzoxyglycyl-l-phenylalanine) (K _d-value on Sephadex G-200 =0,04), an arylamidase like action (substrate: l-leucine- -naphthylamide·HCl; LNA), and a dipeptidase (substrate: glycyl-l-leucine) (K _d-values on Sephadex G-200 0,46 and 0,39 respectively). Carboxypeptidase B (substrate: hippuryl-l-arginine) activity was absent in HP. Contrary to the exopeptidases of HP the carboxypeptidase A of the gastric juice is of lower molecular weight (K _d-value on Sephadex G-150=0,62; molecular weight approx. 30.000), and the arylamidase of the gastric juice is of higher molecular weight (K _d-value on Sephadex G-150=0,26). Moreover gastric juice contains a highly active carboxypeptidase like activity, with identical behaviour on Sephadexgel as carboxypeptidase A. ,-dipyridyl and o-phenanthroline are without effect on the hippurylarginine splitting activity. Carboxypeptidase A of HP is significantly activated by EDTA and hydrocinnamic acid, and slightly activated by p-chloromercuribenzoate. 2-mercaptoethanol (10^–3 molar and higher concentrations) inhibits strongly the carboxypeptidase A of HP.

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Herrn Prof. Dr. Hanson möchte ich für sein Interesse an dieser Arbeit sowie für die kritische Durchsicht des Manuskriptes vielmals danken. — Der medizinisch-technischen Assistentin Frau Johanna Scheel danke ich für ihre wertvolle Mit-arbeit bei den Versuchen.     

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models

Summary In this paper, the results of the preceding electrophysiological study of sodium-alanine cotransport in pancreatic acinar cells are compared with kinetic models. Two different types of transport mechanisms are considered. In the simultaneous mechanism the cotransporterC forms a ternary complexNCS with Na⁺ and the substrateS; coupled transport of Na⁺ andS involves a conformational transition between statesNCS andNCS with inward- and outward-facing binding sites. In the consecutive (or ping-pong) mechanism, formation of a ternary complex is not required; coupled transport occurs by an alternating sequence of association-dissociation steps and conformational transitions. It is shown that the experimentally observed alanine- and sodium-concentration dependence of transport rates is consistent with the predictions of the simultaneous model, but incompatible with the consecutive mechanism. Assuming that the association-dissociation reactions are not rate-limiting, a number of kinetic parameters of the simultaneous model can be estimated from the experimental results. The equilibrium dissociation constants of Na⁺ and alanine at the extracellular side are determined to beK _N <-64mm andK _S <-18mm. Furthermore, the ratioK _N /K _N ^S of the dissociation constants of Na⁺ from the binary (NC) and the ternary complex (NCS) at the extracellular side is estimated to be <-6. This indicates that the binding sequence of Na⁺ andS to the transporter is not ordered. The current-voltage behavior of the transporter is analyzed in terms of charge translocations associated with the single-reaction steps. The observed voltage-dependence of the half-saturation concentration of sodium is consistent with the assumption that a Na⁺ ion that migrates from the extracellular medium to the binding site has to traverse part of the transmembrane voltage.     

Influence of membrane structure on ion transport through lipid bilayer membranes

Summary Charge-pulse relaxation studies with the positively charged PV-K⁺ complex (cyclo-(d-Val-l-Pro-l-Val-d-Pro)₃) and the negatively charged lipophilic ion dipicrylamine (DPA^–) have been performed in order to study the influence of structural properties on ion transport through lipid bilayer membranes. First, the thickness of monoolein membranes was varied over a wide range using differentn-alkanes and slovent-free membranes. The thickness (d) of the hydrocarbon core of these membranes varied between 4.9 and 2.5 nm. For both transport systems the partition coefficient was found to be rather insensitive to variations ind. The same was valid for the translocation rate constantk _MS of PV-K⁺, whereas a strong increase of the translocation rate constantk _i of DPA-with decreasingd was observed. In a further set of experimental conditions the structure of the lipids, such as number and position of the double bonds in the hydrocarbon chain and its chain length as well as the nature of the polar head group, was varied. The translocation constantk _MS of PV-K⁺ transport was found to be much more sensitive to these variations thank _i of DPA-.Much larger variations ink _i andk _MS were observed in membranes made from lipids with ether instead of ester linkages between glycerol backbone and hydrocarbon chain. The results are in qualitative agreement with the surface potentials of monolayers made from corresponding lipids. Increasing amounts of cholesterol in membranes of dioleoylphosphatidylcholine caused a strong decrease ofk _MS (PV-K⁺), whereask _i was found to be rather insensitive to this variation.In monoolein membranes cholesterol causes a decrease ofk _MS up to sixfold and a increase ofk _i up to eightfold. The partition coefficient of DPA^– was insensitive to cholesterol, whereas of PV-K⁺ was found to decrease about eightfold in these membranes. The influence of cholesterol onk _MS is discussed on the basis of viscosity changes in the membrane and the change ink _i of DPA^– and of PV-K⁺ on the basis of a possible change of the dipole potential of the membranes. The other sterols, epicholesterol and ergosterol cause no change in the kinetics of the two probes.The different influence of membrane properties like thickness, viscosity, and dipole potential on the two transport systems is discussed under the assumption that the adsorption planes of the two probes have different positions in a membrane. Possibly because of a larger hydrophobic interaction, the adsorption plane of PV-K⁺ is located more towards the hydrocarbon side and that of DPA^– more towards the aqueous side of the dipole layer.     

CCCP activation of the reconstituted NaK-pump

Summary In the NaK-ATPase proteoliposomes (PLs), the NaK-pump activity, Na⁺ uptake, and ATP hydrolysis were apparently enhanced by carbonyl cyanidem-chlorophenylhydrazone (CCCP) and other ionophores without ion gradients. These ionophore effects were not cation specific. Without ionophores, the PL's ATPase activity fell to its steady-state value within 3 sec at 15°C. This decrease in activity disappeared in the presence of CCCP. Since CCCP is believed to enhance proton mobility across the lipid bilayer and dissipate membrane potential (V _m ), we postulated that aV _m build-up partially inhibits the PLs by changing the conformation of the NaK-pump, and that CCCP eliminated this partial inhibition. Since this activation required extracellular K⁺ and high ATP concentration in the PLs, CCCP must affect the conversion between the phosphorylated forms of NaK-ATPase (EP); this step has been suggested by Goldschlegger et al. (1987) to be the voltage-sensitive step (J. Physiol. (London) 387:331–355). Although cytoplasmic K⁺ accelerated the change of ADP-and K⁺-sensitive EP (E^*P) to K⁺-sensitive ADP-insensitive EP (E₂P), CCCP did not compete with cytoplasmic K⁺ when cytoplasmic Na⁺ was saturated. When the PLs were phosphorylated with 20 m ATP and 20 m palmitoyl CoA instead of with high concentration of ATP, CCCP increased the E^*P content and decreased the ADP-sensitive K⁺-insensitive EP (E₁P). The results described above suggest that CCCP affects the E₁P to E^*P change in the E₁PE^*PE₂P conversion and that this reaction step is inhibited byV _m .     

Regeneration of resistance and ion transport in rabbit corneal epithelium after induced surface cell exfoliation

Summary Exposure of the rabbit corneal surface to a 20-m digitonin-0.9% NaCl solution leads to permeabilization of the most superficial cells of the stratified epithelium. The devitalized cells exfoliate spontaneously from the corneal surface. Detergent exposure limited to 4–8 min leads to permeabilization and rapid exfoliation of a monolayer of surface cells. Consistent with the presence of the epithelial paracellular permeability barrier in this cell layer, their permeabilization results in complete loss of transepithelial resistance (R _t ). Within minutes after detergent removal an initial recovery ofR _t can be noticed indicating generation of a new paracellular permeability barrier by the viable subsurface cells. This recovery proceeds rapidly andR _t reaches within 70 min a maximum equal to > 90% of the preexfoliation values (=2.43 k·cm²,n=22). TheR _t recovery is fully blocked in a reversible manner by 10 m dihydrocytochalasin B. The recovery is not affected by inhibition of protein synthesis with 5 m cycloheximide. When the ocular surface is treated again with digitonin the permeabilization and exfoliation of a monolayer of cells and loss ofR _t are repeated. After the second detergent exposure an initial recovery ofR _t occurs as before within minutes. However, the pace ofR _t recovery is much slower: 4–5 hr are required to reach a stable maximalR _t values amounting to about 73% of initial control. This recovery can be fully blocked by 5 m cycloheximide indicating that protein synthesis is required for generation of tight junctions by the second subcellular layer. With only a fraction ofR _t recovered, short-circuit currents amounting to, at least, 50% of control values and attributable in part to cell-to-tear movement of Cl^– through the apical surface can be measured. This suggests that apical-type Cl^– channels are either present in the apically facing membrane of subsurface cells or that they are rapidly inserted in it from preexisting intracellular pools immediately following the devitalization of the surface cells by digitonin.     

Prerequisite for His4 in deltorphin A for highδ opioid receptor selectivity

Summary Analysis of deltorphin A position 4 analogues included: backbone constrained N MeHis, spinacine (Spi), N MePhe and the tetrahydroisoquinoline-3-carboxylic acid (Tic); spatially confined side-chain (Phg); and imidazole alkylation ofl- andd-His⁴ enantiomers. High selectivity was lost with the following replacements: N MeHis⁴, N MePhe⁴ and Phg⁴ reduced binding and the constrained residues also increasedµ binding; ring closure between the side-chain and amino group to yield Spi⁴ or Tic⁴ increasedµ affinity. Imidazole methylation of His⁴ marginally affected opioid binding and doubled selectivity; alkylatedd-His⁴-derivatives generally maintained selectivity in spite of decreased affinities. Thus, His⁴ imidazole preserves selectivity by facilitating high binding and by repulsion at theµ receptor. Several low energy conformers of deltorphin A indicated that the His⁴ imidazole preferred a spatial orientation parallel to the phenolic side-chain of Tyr¹ suggestive that this conformation might contribute to high affinity and selectivity.     

Folding versatility of the C-terminal tetrapeptide amide sequence of the lipopeptaibol antibiotics trichodecenin and trichogin

Summary An X-ray diffraction analysis ofZ-l-Leu-Aib-Gly-l-Ile-l-Leu-OMe, containing the N-acylated tetrapeptide amide sequence-l-Leu-Aib-Gly-l-Ile-, showed that in the crystal state the carbonyl group preceding thel-Leu¹ residue acts as the acceptor of two C=OH–N intramolecular H-bonds, which give rise to an-l-Leu¹-Aib²-type-III' -turn and an-l-Leu¹-Aib²-Gly³-l-Ile⁴--turn, respectively. A second (type-I') -turn encompasses the-Aib²-Gly³-sequence. This is the third type of folding motif known for that tetrapeptide sequence, considering also those already published for the C-terminal segment of the lipopeptaibol antibiotics trichodecenin I and trichogin A IV.     

Characterization of sodium and pyruvate interactions of the two carrier systems specific of mono- and di- or tricarboxylic acids by renal brush-border membrane vesicles

Summary The experiments reported in this paper aim at characterizing the carboxylic acid transport, the interactions of pyruvate and citrate with their transport sites and specificity. The study of these carriers was performed using isotopic solutes for the influx measurements in brush-border membrane vesicles under zerotrans conditions where the membrane potential was abolished with KCl preloading with valinomycin or equilibrium exchange conditions and =0.Under zerotrans condition and =0, the influence of pyruvate concentrations on its initial rates of transport revealed the existence of two families of pyruvate transport sites, one with a high affinity for pyruvate (K _t =88 m) and a low affinity for sodium (K _t =57.7mm) (site I), the second one with a low affinity for pyruvate (K _t =6.1mm) and a high affinity for sodium (K _t =23.9mm) (site II). The coupling factor [Na]/[pyruvate] stoichiometry were determined at 0.25mm and 8mm pyruvate and estimated at 1.8 for site I, and 3 when the first and the second sites transport simultaneously.Under chemical equilibrium (0) single isotopic labeling, transport kinetics of pyruvate carrier systems have shown a double interaction of pyruvate with the transporter; the sodium/pyruvate stoichiometry also expressed according to a Hill plot representation wasn=1.7. The direct method of measuring Na⁺/pyruvate stoichiometry from double labeling kinetics and isotopic exchange, for a time course, gives an=1.67.Studies of transport specificity, indicate that the absence of inhibition of lactate transport by citrate and the existence of competitive inhibition of lactate and citrate transports by pyruvate leads to the conclusion that the low pyruvate affinity site can be attributed to the citrate carrier (tricarboxylate) and the high pyruvate affinity site to the lactate carrier (monocarboxylate).     

Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula

We purified an extracellular thermostable -galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°_20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted -d-galactopyranosides such as lactose [a Michaelis constant K _m=0.75 mm and molecular activity (k _cat)= 63.1 s^–1 at pH 7.2 and 55° C] and p-nitrophenyl -d-galactopyranoside (K _m=0.04 mm k _cat= 55.8 s^–1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)_c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula -galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 m MnCl₂) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 m MnCl₂). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C <) for efficient production of the oligosaccharides from lactose. Correspondence to: T. Nakayama     

Kinetic analysis of the inhibition of sulfate transport in human red blood cells by isothiocyanates

Summary A kinetic analysis of anion self-exchange in human red blood cells, in the presence of an irreversible inhibitor, is presented and applied to the study of the inactivation of sulfate transport by three isothiocyanates: 3-isothiocyano-1,5-naphthalenedisulfonic acid, disodium salt (INDS), 1-isothiocyano-4-naphthalene sulfonic acid, sodium salt, monohydrate (INS), and 1-isothiocyano-4-benzenesulfonic acid, sodium salt, monohydrate (IBS). The time dependence of the inhibition of sulfate transport by the isothiocyanates used could be described by a single exponential and could be shown to contain a reversible and an irreversible component. In each case a portion of sulfate efflux was found to be resistant to inactivation. The residual portion of the sulfate efflux varied with inhibition: 4% for INS, 16% for INDS, and 34% for IBS. INS showed the largest reversible inhibitory effect (12% of the flux remaining at 0.2mm inhibitor concentration), while INDS showed the weakest effect (92% of the flux remaining at 0.3mm inhibitor concentration). IBS had the highest rate of inactivation while INDS had the lowest. The kinetic analysis further suggests that all three isothiocyanates bind reversibly to an inhibitory site on the membrane before they bind covalently, and therefore irreversibly, to the same site on the membrane. The equilibrium constant for the dissociation of the reversibly-bound complex,K _i, and the rate of irreversible inactivation after all membrane sites are reversibly bound,k _max, have been computed for all three inhibitors: INDS (K _i=420m,k _max=5.04 hr^–1), INS (K _i=148 m,k _max=6.48 hr^–1), and IBS (K _i=208 m,k _max=8.11 hr^–1).     

Electrogenic 2 Na+/1 H+ exchange in crustanceans

Summary Hepatopancreatic brush border membrane vesicles of the freshwater prawn,Macrobrachium rosenbergii and the marine lobster,Homarus americanus exhibited²²Na uptake which was Cl-independent, amiloride sensitive, and stimulated by a transmembrane H gradient (H _i >H _o ). Sodium influx by vesicles of both species were sigmoidal functions of [Na] _o , yielding Hill coefficients that were not significantly different (P>0.5) than 2.0. Estimations of half-saturation constants (K _Na) were 82.2mm (prawn) and 280.1mm (lobster), suggesting a possible adaptation of this transporter to environmental salinity.Trans-stimulation andcis-inhibition experiments involving variable [H] suggested that the exchangers in both species possessed single internal cation binding sites (pK 6.5–6.7) and two external cation binding sites (prawn, pK 4.0 and 5.7; lobster pK 3.5 and 6.1). Similarcis inhibition studies using amiloride as a competitive inhibitor of Na uptake supported the occurrence of dual external sites (prawn,K _i 50 and 1520 m; lobsterK _i 9 and 340 m). Electrogenic Na/H exchange by vesicles from both crustaceans was demonstrated using equilibrium shift experiments where a transmembrane potential was used as the only driving force for the transport event. Transport stoichiometries of the antiporters were determined using Static Head analysis where driving forces for cation transfer were balanced using a 101 Na gradient, a 1001 H gradient, and a stoichiometry of 2.0. These electrogenic 2 Na/1 H exchangers appear thermodynamically capable of generating sufficient gastric acidification for organismic digestive activities.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Pleconax Rafin. — eine bis heute unbeachtete Silenoideen-Gattung (Caryophyllaceae)

Zusammenfassung Die zwischen den Arten der SektionConoimorpha Otth (UntergattungConocalyx Willk.) der GattungSilene und den übrigen Arten derselben Gattung sowie aller übrigen Gattungen der TribusLychnideae A. Br. existierenden Unterschiede berechtigen zur Abtrennung dieser Sektion (Untergattung) als selbständige GattungPleconax Rafin. Nach bisherigen Untersuchungen gehören in diese Gattung folgende Arten und Unterarten:Pleconax ammophila (Boiss.)ourková mit subsp.ammophila und subsp.carpathae (Chowdhuri)ourková,P. amphorina (Pomel)ourková,P. conica (L.)ourková mit subsp.conica und subsp.conomaritima (D.Jord. et P.Pan.)ourková,P. coniflora (Nees)ourková,P. conoidea (L.)ourková,P. lydia (Boiss.)ourková,P. macrodonta (Boiss.)ourková,P. multinervia (Wats.)ourková,P. sartorii (Boiss. etHeldr.)ourková,P. subconica (Friv. emend. D.Jord. et P.Pan.)ourková mit subsp.subconica und subsp.grisebachii (David.)ourková sowieP. tempskyana (Freyn etSint.)ourková. Die angeführten nomenklatorischen Umkombinationen werden hier zum ersten Male veröffentlicht.     

The malonyl gramicidin channel: NMR-derived rate constants and comparison of calculated and experimental single-channel currents

Summary Malonyl gramicidin is incorporated into lysolecithin micelles in a manner which satisfies a number of previously demonstrated criteria for the formation of the transmembrane channel structure. By means of sodium-23 nuclear magnetic resonance, two binding sites are observed: a tight site and a weak site with binding constants of approximately 100m ^–1 and 1m ^–1, respectively. In addition, off-rate constants from the two sites were estimated from NMR analyses to bek _off ^t 3×10⁵/sec andk _off ^w 2×10⁷/sec giving, with the binding constants, the on-rate constants,k _on ^t 3×10⁷/msec andk _on ^w 2×10⁷/m sec.Five different multiple occupancy models with NMR-restricted energy profiles were considered for the purpose of calculating single-channel currents as a function of voltage and concentration utilizing the four NMR-derived rate constants (and an NMR-limit placed on a fifth rate constant for intrachannel ion translocation) in combination with Eyring rate theory for the introduction of voltage dependence.Using the X-ray diffraction results of Koeppe et al. (1979) for limiting the positions of the tight sites, the two-site model and a three-site model in which the weak sites occur after the tight site is filled were found to satisfactorily calculate the experimental currents (also reported here) and to fit the experimental currents extraordinarily well when the experimentally derived values were allowed to vary to a least squares best fit. Surprisingly the best fit values differed by only about a factor of two from the NMR-derived values, a variation that is well within the estimated experimental error of the rate constants.These results demonstrate the utility of ion nuclear magnetic resonance to determine rate constants relevant to transport through the gramicidin channel and of the Eyring rate theory to introduce voltage dependence.     

Chloride conductive and cotransport mechanisms in cultures of canine tracheal epithelial cells measured by an entrapped fluorescent indicator

Summary To study Cl conductive and cotransport mechanisms, primary cultures of canine tracheal cells were grown to confluency on thin glass cover slips and on porous filters. Transepithelial resistance was >100 ·cm², and short circuit current (I _sc=2–20 A/cm²), representing active secretion of Cl, increased >threefold with addition of 10 m isoproterenol to the serosal solution. Cells made transiently permeable in hypotonic solution were loaded with the Cl-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (5mm, 4 min, 150 mOsm). The electrical properties of the cell monolayers were not altered by the loading procedure. Intracellular SPQ fluorescence was monitored continuously by epifluorescence microscopy (excitation 360±5 nm, emission>410 nm). SPQ leakage from the cells was <10% in 60 min at 37°C. Intracellular calibration of SPQ fluorescencevs. [Cl] (0–90mm) was carried out using high-K buffers containing the ionophores nigericin (5 m) and tributyltin (10 m); SPQ fluorescence was quenched with a Stern-Volmer constant of 13m ^–1. Intracellular Cl activity was 43±4mm. Cl flux was measured in response to addition and removal of 114mm Cl from the bathing solution. Addition of 10 m isoproterenol increased Cl efflux from 0.10 to 0.27mm/sec. The increase was inhibited by the Cl-channel blocker diphenylamine-2-carboxylic acid (1mm). In the absence of isoproterenol, removal of external Na or addition of 0.5mm furosemide, reduced Cl influx by >fourfold. In ouabain-treated monolayers, removal of external K in the presence of 5mm barium diminished Cl influx by >twofold, suggesting that Cl entry is in part K dependent. These results establish an accurate optical method for the realtime measurement of intracellular Cl activity in tracheal cells that does not require an electrically tight cell monolayer. The data demonstrate the presence of an isoproterenol-regulated Cl channel and a furosemide-sensitive cation-coupled transport mechanism.     

Identification of the anion exchange protein of ehrlich cells: A kinetic analysis of the inhibitory effects of 4,4′-diisothiocyano-2,2′-stilbene-disulfonic acid (DIDS) and labeling of membrane proteins with3H-DIDS

Summary In Ehrlich ascites tumor cells 4,4-diisothiocyano-2,2-stillbene-disulfonic acid (DIDS) inhibits the chloride exchange both reversibly and irreversibly. The reversible inhibition is practically instantaneous and of a competitive nature withK ₁ about 2 m at zero chloride concentration. This is succeeded by a slow irreversible binding of DIDS to the transporter, with a chloride dependence suggesting binding to the same site as for reversible DIDS binding/inhibition. To identify the membrane protein involved in anion exchange, cells were labeled with³H-DIDS. Incubation of cells for 10 min with 25 m DIDS at pH 8.2 leads to more than 95% inhibition of the DIDS-sensitive chloride exchange flux when the chloride concentration is low (15mm). This condition was used for the³H-DIDS-labeling experiments. After incubation the cells were disrupted, the membranes isolated and solubilized, and the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The distribution of the³H-activity in the gel showed only one major peak, which could be related to protein with a mol wt of about 30,000 Daltons. The number of transport sites was estimated at about 400,000 per cell, and from the DIDS-sensitive chloride flux under steady-state conditions we calculate a turnover number of 340 ions per sec per site.     

Determination of the electron self-exchange rates of blue copper proteins by super-WEFT NMR spectroscopy

An NMR approach for determining the electron self-exchange (ESE) rate constants in blue copper proteins is presented. The approach uses the paramagnetic relaxation enhancement of resonances in 1D ¹H super-WEFT spectra of partly oxidized (paramagnetic) proteins. These spectra allow a more precise determination of the relevant paramagnetic linebroadenings than conventional 1D ¹H spectra and, thus, permit a more detailed investigation of the applicability of the linebroadenings for determining the electron exchange rates. The approach was used to estimate the ESE rate constant of plastocyanin from Anabaena variabilis. It was found that, although the rate constant can be determined accurately from a series of resonances, precise but erroneous constants are obtained from the resonances of the copper-bound residues, unless a narrow splitting of these resonances caused by the presence of two conformations is taken into account. As demonstrated here, this complication can be overcome by a correct analysis of the paramagnetic broadening of the combined double signals. Because of the high resolution and specific sensitivity of the approach it should be generally applicable to estimate electron transfer rates, k, if the paramagnetic relaxation enhancement R _2p of the resonances can be determined, and the conditions kR _2p or _pkR _2p are fulfilled, _p being the frequency separation between corresponding diamagnetic and paramagnetic sites.     

Anion channels from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Synaptic membranes from rat brain were incorporated into planar lipid bilayers, and the characteristics of two types of anion-selective channels (type I and type II) were investigated. In asymmetric BaCl₂ buffers (cis, 100mm/trans, 25mm), single channel conductances at –40 mV were 70 pS (type I) and 120 pS (type II). Permeability ratios (P _Na:P _Ba:P _Cl) calculated from the Goldman-Hodgkin-Katz current equation for type I and type II channels were 0.230.041 and 0.050.031, respectively. Both channels exhibited characteristic voltage-dependent bursting activities. Open probability for type I channels had a maximum of 0.7 at about 0 mV and decreased to zero at greater transmembrane potentials of either polarity. Type II channels were relatively voltage independent at negative voltages and were inactivated at highly positive voltages. Type I channels showed spontaneous irreversible inactivation often preceded by sudden transition to subconducting states. DIDS blocked type I channels only from thecis side, while it blocked type II channels from either side.     

A1H NMR investigation of the hydrolysis of a synthetic substrate by KDN-sialidase fromCrassostrea virginica

The mechanism of hydrolysis of 4-methylumbelliferyl 3-deoxy-d-glycero--d-galacto-2-nonulopyranosidonic acid (KDN2MeUmb,4) by KDN-sialidase isolated from the hepatopancreas of the oysterCrassostrea virginica has been monitored by¹H NMR spectroscopy. The results of these experiments reveal that KDN-sialidase catalyses the hydrolysis of the synthetic substrate KDN2MeUmb, with initial release of -d-KDN. This is consistent with an overall mechanism for the hydrolysis which proceeds with retention of anomeric configuration. These results agree with earlier NMR studies of otherN-acetylneuraminic acid-recognising sialidases from both viral and bacterial sources.