Bending undulations and elasticity of the erythrocyte membrane: effects of cell shape and membrane organization

The undulatory excitations (flickering) of human and camel erythrocytes were evaluated by employing the previously used flicker spectroscopy and by local measurements of the autocorrelation function K (t) of the cell thickness fluctuations using a dynamic image processing technique. By fitting theoretical and experimental flicker spectra relative values of the bending elastic modulus K _c of the membrane and of the cytoplasmic viscosity were obtained. The effects of shape changes were monitored by simultaneous measurement of the average light intensity I ₀ passing the cells and by phase contrast microscopic observation of the cells. Evaluation of the cellular excitations in terms of the quasi-spherical model yielded values of K _c /R _inf0 ^sup3 and · R ₀ (R ₀=equivalent sphere radius) and allowed us to account (1) for volume changes, (2) for effects of surface tension and spontaneous curvature and (3) for the non-exponential decay of K (t). From the long time decay of K (t) we obtained an upper limit of the bending elastic modulus of normal cells of K _c = 2–3 · 10^–19 Nm which is an order of magnitude larger than the value found by reflection interference contrast microscopy (RICT, K _c , = 3.4 · 10^–20 Nm, Zilker et al. 1987) but considerably lower than expected for a bilayer containing 50% cholesterol (K _c = 5 · 10^–19 Nm, Duwe et al. 1989). The major part of the paper deals with long time measurements (order of hours) of variations of the apparent K _c and values of single cells (and their reversibility) caused (1) by osmotic volume changes, (2) by discocytestomatocyte transitions induced by albumin and triflouperazine, (3) by discocyte-echinocyte transitions induced by expansion of the lipid/protein bilayer (by incubation with lipid vesicles) and by ATP-depletion in physiological NaCI solution, (4), by coupling or decoupling of bilayer and cytoskeleton using wheat germ agglutinin or erythrocytes with elliptocytosis and (5) by cross-linking the cytoskeleton using diamide. These experiments showed: (1) K _c and are minimal at physiological osmolarity and temperature and well controlled over a large range of these parameters. (2) Echinocyte formation does not markedly alter the apparent membrane bending stiffness. (3) During swelling the cell may undergo a transient discocyte-stomatocyte transition. (4) Strong increases of the apparent K _c and after cup-formation or strong swelling and deflation are due to the effect of shear elasticity and surface tension. Our major conclusions are: (1) The erythrocyte membrane exhibits a shear free deformation regime which requires ATP for its maintenance. (2) Shape transitions may be caused by relative area changes either of the two monolayers of the lipid/protein bilayer (corresponding to the bilayer coupling hypothesis) or of the bilayer and the cytoskeleton where the latter mechanism appears to be more frequent. (3) The low bending stiffness and the shear free deformation regime are explained in terms of a slight excess area of the lipid bilayer leading to a pre-undulated surface profile. Freeze fracture electron microscopy studies provide direct evidence for a pre-undulated bilayer with an undulation wavelength of approximately 100 nm. Offprint requests to: E. Sackmann     

Ca2+-activated K+ conductance of human red cell membranes exhibits two different types of voltage dependence

Summary The voltage dependence for outward-going current of the Ca-activated K⁺ conductance (g _k (Ca)) of the human red cell membrane has been examined over a wide range of membrane potentials (V _m) at constant values of [K⁺]_ex, [K⁺]_c and pH_c, the intact cells being preloaded to different concentrations of ionized calcium. Outward-current conductances were calculated from initial net effluxes of K⁺ and the corresponding (V _m-E_k) values. The basic conductance, defined as the outward-current coductance at (V _m-E_k) 20 mV and [K⁺]_ex 3mM (B. Vestergaard-Bogind, P. Stampe and P. Christophersen,J. Membrane Biol. 95:121–130, 1987) was found to be a function of cellular ionized Ca. At all degrees of Ca activationg _K(Ca) was an apparently linear function of voltage (V _m range –40 to +70 mV), the absolute level as well as the slope decreasing with decreasing activation. In a simple two-state model the constant voltage dependence can, at the different degrees of Ca activation, be accounted for by a Boltzmann-type equilibrium function with an equivalent valence of 0.4, assuming chemical equilibrium atV _m=0 mV. Alternatively, the phenomenon might be explained by a voltage-dependent block of the outward current by an intracellular ion. Superimposed upon the basic conductance is the apparently independent inward-rectifying steep voltage function with an equivalent valence of 5 and chemical equilibrium at the givenE _K value.Abbreviations CCCP carbonyl cyanidem-chlorophenylhydrazone - DIDS 4,4-diisothiocyanostilbene-2,2-disul     

Influence of 8-azido-ATP and other anions on the activity of cytochromec oxidase

The effect of ATP and other anions on the kinetics of cytochromec oxidation by reconstituted bovine heart cytochromec oxidase was investigated. The following results were obtained: (1) ATP and other polyvalent anions increase theK _m for cytochromec and theV _max (if assayed by the photometric method). The magnitude of the effect is proportional to the charge of the anion as follows from the series of increasing effectiveness: P_iii. (2) The kinetic effects are obtained in the millimolar physiological concentration range. (3) The kinetic changes are not saturated at high concentrations. (4) A specific interaction site for ATP at the cytosolic domain of the enzyme is concluded from the increase ofK _m for cytochromec after photolabelling of proteoliposomes with 8-azido-[-³²P]-ATP, which is protected by ATP but not by ADP. (5) No specific binding site for ATP could be identified by photolabelling with 8-azido-[-³²P]-ATP. The labelling is only partly protected by ATP or ADP.Abbreviations CCP carbonylcyanide-m-chlorophenylhydrazone - TMPD N,N,N,N-tetramethyl-1,4-phenylenediamine dihydrochloride - 8-N₃-ATP 8-azido-adenosine-5-triphosphate Dedicated to Professor Dr. Friedhelm Schneider on the occasion of his 60th birthday.     

Activity coefficients of intracellular Na+ and K+ during development of frog oocytes

Summary The chemical activities, (a), of Na⁺ and K⁺ were determined in large mature and in small immature frog oocytes, using open-tipped micropipettes and ionselective microelectrodes. The average chemical concentrations,c, of Na⁺ and K⁺ were determined by spectrophotometry and by electron probe X-ray microanalysis. The apparent activity coefficient (^app) was calculated for each ion as the ratio,a/c.With development, (a _Na/a _K) decreased four to fivefold and (c _Na/c _K) increased six to sevenfold. In the large mature oocytes, _Na ^app was measured to be 0.08±0.02 and _K ^app lay within the range 1.15±0.03 to 1.29±0.04, constituting the smallest value for Na⁺ and largest value for K⁺, respectively, thus far reported. This intracellular value of _K ^app was substantially greater than the activity coefficient of K⁺ in the external medium (0.76). The data suggest that the inequality of _Na ^app and _K ^app in this and probably other cells reflects the development of subcellular compartmentalization of ions. Possible intracellular sites of ionic compartmentalization are considered.     

Recombination in the lambda repressor gene: evidence that very short patch (VSP) mismatch correction restores a specific sequence

Summary The mutation am6 in the cI gene of bacteriophage is identified as a CT transition in a 5CC _T ^A GG sequence. In four-factor crosses of am6 with nearby mutations in cI, the frequencies of cI⁺ recombinants are much higher than expected from the physical distances. A very short patch (VSP) mismatch repair system is presumed to recognize am6/am ⁺ mispairs in the heteroduplexes that accompany recombination between the outside markers. Mutation am6 is corrected to am ⁺; correction of am ⁺ to am6 was not detected. Clear-plaque mutation 1-1 in cI is a TC transition in a 5CTTGG sequence, resulting in the sequence 5CC _T ^A GG. When 1-1 was crossed with nearby mutations in gene cI, there were no excess cI⁺ recombinants, which would result from repair of CCTGG (1-1) to CTTGG (cI⁺). However, in crosses of cI⁺ phages with mutation 1-1, there was an excess of cI^- recombinants, indicating that cI⁺ was repaired to 1-1. Preferential repair does not require adenine or cytosine methylation: when repairing a mismatch, the VSP repair system apparently identifies specific mispaired bases by sequence alone.     

Carcinogenic potential and genomic instability of beryllium sulphate in BALB/c‐3T3 cells

Occupational exposure to beryllium (Be) and Be compounds occurs in a wide range of industrial processes. A large number of workers are potentially exposed to this metal during manufacturing and processing, so there is a concern regarding the potential carcinogenic hazard of Be. Studies were performed to determine the carcinogenic potential of beryllium sulfate (BeSO₄) in cultured mammalian cells. BALB/c3T3 cells were treated with varying concentrations of BeSO₄ for 72 h and the transformation frequency was determined after 4 weeks of culturing. Concentrations from 50–200 g BeSO₄/ml, caused a concentrationdependent increase (9–41 fold) in transformation frequency. Nontransformed BALB/c3T3 cells and cells from transformed foci induced by BeSO₄ were injected into both axillary regions of nude mice. All ten Beinduced transformed cell lines injected into nude mice produced fibrosarcomas within 50 days after cell injection. No tumors were found in nude mice receiving nontransformed BALB/c3T3 cells 90 days postinjection. Gene amplification was investigated in Kras, cmyc, cfos, cjun, csis, erbB2 and p53 using differential PCR while random amplified polymorphic DNA fingerprinting was employed to detect genomic instability. Gene amplification was found in Kras and cjun, however no change in gene expression or protein level was observed in any of the genes by Western blotting. Five of the 10 transformed cell lines showed genetic instability using different random primers. In conclusion, these results indicate that BeSO₄ is capable of inducing morphological cell transformation in mammalian cells and that transformed cells induced by BeSO₄ are potentially tumorigenic. Also, cell transformation induced by BeSO₄ may be attributed, in part, to the gene amplification of Kras and cjun and some BeSO₄induced transformed cells possess neoplastic potential resulting from genomic instability.     

Effect of aggregate size in cell cultures of Tagetes patula on thiophene production and cell growth

Summary The effect of the size of Tagetes patula (marigolds) cell aggregates on growth and thiophene production in MS-medium was studied. A heterogeneous aggregate suspension was aseptically divided into 7 fractions, each with a defined aggregate diameter range, with subsequent inoculation of the fractions into MS growth medium. Growth occurred in all aggregate fractions and thiophene production increased with increasing aggregate diameter starting at about 3 mm, an effect possibly due to an increasing lack of oxygen in the aggregate centre. Calculations of oxygen concentration profiles in the aggregates showed namely, that the critical aggregate diameter where the oxygen concentration in the aggregate centre becomes very low, is about 3 mm. Aggregates with a diameter exceeding 1.2 cm showed a decreased thiophene production, however, these aggregates were hollow. The thiophenes produced mainly consisted of 5-(4-hydroxy-1-butenyl)1-2,2-bithienyl, which was excreted into the medium.Nomenclature ID _e effective diffusion coefficient (m²s^-1) - c oxygen concentration (mol m^-3) - c _s substrate concentration at surface (mol m^-3) - c _s.exp experimental value of c _s (mol m^-3) - c _eq substrate concentration at equilibrium (mol m^-3) - r _s consumption rate (mol m^-3 s^-1) - d _crit critical aggregate diameter (m) - d _agg aggregate diameter (m) - L length of aggregate (m) - W width of aggregate (m) - t time (s) - r distance from aggregate centre (m) - R radius of aggregate (m) - R(c) oxygen consumption (mol m^-3 s^-1) - V _c convection velocity (m s^-1) - V _m intrinsic maximum consumption rate (mol kg^-1 s^-1) - K _m intrinsic Michaelis Menten constant (mol m^-3) - V _m apparent maximum consumption rate (mol kg^-1 s^-1) - K _m apparent Michaelis Menten constant (mol m^-3) - * multiplication sign     

Kinetics of ligand binding to a cluster of membrane-associated receptors

The process of ligand binding to a cluster of membrane-associated receptors is examined theoretically. The theoretical model proposed involves the diffusion of ligands from the solution to the disc-like cluster of receptors on the surface of the spherical cell. When the ligand hits the internal part of the disc-like cluster, it begins to move laterally until it leaves the disc through its outer surface or is bound by one of the receptors inside the disc. If the ligand leaves the cluster, it returns to the solution and hits the disc again after a certain period, etc. According to our model the transition from a diffusion-limited to a reaction-limited process of binding is determined by the dimensionless parameter Dt _c/a ², where D is the lateral diffusion coefficient,t _c is the characteristic time of reaction, anda is the radius of the disc-like cluster. The forward rate constantk _f turns out to be a function of . Comparing the results of our calculations ofk _f with some experimental data we found that agreement is achieved at high , i.e. the process of ligand binding by clustered receptors is predominantly reaction-limited.     

ATP is required for K+ active transport in the archaebacterium Haloferax volcanii

The Archaebacterium Haloferax volcanii concentrates K⁺ up to 3.6 M. This creates a very large K⁺ ion gradient of between 500- to 1,000-fold across the cell membrane. H. volcanii cells can be partially depleted of their internal K⁺ but the residual K⁺ concentration cannot be lowered below 1.5 M. In these conditions, the cells retain the ability to take up potassium from the medium and to restore a high internal K⁺ concentration (3 to 3.2 M) via an energy dependent, active transport mechanism with a K _m of between 1 to 2 mM. The driving force for K⁺ transport has been explored. Internal K⁺ concentration is not in equilibrium with m suggesting that K⁺ transport cannot be accounted for by a passive uniport process. A requirement for ATP has been found. Indeed, the depletion of the ATP pool by arsenate or the inhibition of ATP synthesis by N,N-dicyclohexylcarbodiimide inhibits by 100% K⁺ transport even though membrane potential m is maintained under these conditions. By contrast, the necessity of a m for K⁺ accumulation has not yet been clearly demonstrated. K⁺ transport in H. volcanii can be compared with K⁺ transport via the Trk system in Escherichia coli.Abbreviations CCCP Carbonylcyanide m-chlorophenyl-hydrazone - DCCD N,N-dicyclohexylcarbodiimide - MES 2-[N-morpholino] ethane sulfonic acid - MOPS 3-[N-morpholino] propane sulfonic acid - TRIS Tris (hydroxymethyl) aminomethane - TPP tetraphenyl phosphonium     

Potassium modulation of methionine uptake in astrocytes in vitro

Methionine participates in a large variety of metabolic pathways in brain, and its transport may play an important regulatory role. The properties of methionine uptake were examined in a preparation of neonatal rat brain astrocytes. Uptake is linear for 15 minutes, up to 2.5 M. At steady state conditions, methionine is concentrated 30–50-fold. Measured methionine homoexchange accounts for a significant fraction of uptake at concentrations greater than 10 M. We recently reported that methionine uptake is decreased by elevations in extracellular K⁺. Potassium induced efflux cannot account for this apparent effect; and thus for concentrations less than 2.5M, and for short times of incubation, measured rates of methionine uptake represent unidirectional flux. At extracellular concentrations of K⁺ equal to 6.9 mM, the apparentV _max of methionine transport is 182 pmol/min/mg protein, and theK _m is 1.3 M. Where K⁺ is shifted to 11.9 mM, theK _m remains unchanged, and theV _max is reduced by half.     

The effects of hydrostatic pressure on the low-K m GTPase in brain membranes from two congeneric marine fishes

To investigate, the effects of hydrostatic pressure on transmembrane signaling in cold-adapted marine fishes, we examined the high-affinity GTPase activity in two congeneric marine fishes, Sebastolobus alascanus and S. altivelis. In brain membranes there are two GTPase activities, one with a low K _m and one with a high K _m for GTP. The high-affinity GTPase activity, characteristic of the subunits of the guanine nucleotide binding protein pool, was stimulated by the A₁ adenosine receptor agonists N ⁶(R-phenylisopropyl)adenosine and N ⁶-cyclopentyladenosine, and the muscarinic cholinergic agonist carbamyl choline. Pertussis toxin-catalyzed ADP-ribosylation of the membranes for 2 h at 5°C prior to the GTPase assay decreased the basal GTPase activity 30–40% and abolished N ⁶ (R-phenylisopropyl)adenosine stimulation of GTP hydrolysis. Basal high-affinity hydrolysis of GTP, measured at 0.3 mol·1^-1GTP, was stimulated 22% in both species by 340 atm pressure. At 340 atm pressure, the apparent K _m of GTP is decreased approximately 10% in each of the species, and the V _max values are increased 11 and 15.9% in S. alascanus and S. altivelis, respectively. The apparent volume changes associated with the decreased K _m of GTP and the increased V _max ranged from-7.0 to-9.9 ml·mol^-1. Increased pressure markedly decreased the efficacy of N ⁶ (R-phenylisopropyl) adenosine, N ⁶-cylcopentyladenosine and carbamyl choline in stimulating GTPase activity. The effects of increased hydrostatic pressure on transmembrane signal transduction by the A₁ adenosine receptor-inhibitory guanine nucleotide binding protein-adenylyl cyclase system may stem, at least in part, from pressure-increased GTP hydrolysis and the concomitant termination of inhibitory signal transduction.Abbreviations [³H] DPCPX ³H cyclopentyl-1, 3-dipropylxanthine - AppNHp 5-adenylylimidodiphosphate - cpm counts per minute - CPA N ⁶-cyclopentyladenosine - EDTA ethylenediaminetetra acetic acid - EGTA ethyleneglycol-bis (-aminoethylether) N, N, N, N-totra-acctic acid - G protein guanine nucleotide binding protein - G_i inhibitory G protein - G_o other G protein, common in brain membranes - G_s stimulatory G protein - GTPase guanosine triphosphatase - K _i inhibition constant - K _m Michaelis constant - pK _a log of the dissociation constant - R-PIA N ⁶ (R-phenylisopropyl) adenosine - TRIS tris[hydroxymethyl]aminomethane - V_max maximal velocity - [-³²P]GTP [-³²P] guanosine 5-triphosphate (tetra (triethylammonium) salt)     

Characterization of a Na∶K∶2Cl cotransport system in the apical membrane of a renal epithelial cell line (LLC-PK1)

Summary ⁸⁶Rb uptake into LLC-PK₁ cells (an established renal epithelial cell line) was found to be comprised of an active ouabain-sensitive component, a loop diuretic-sensitive component which was passive and strictly dependent upon the presence of extracellular Na⁺ and Cl^– for activity, and a leak component. The diuretic-sensitive component of influx was investigated further in apical membrane vesicles derived from these cells. A large fraction of⁸⁶Rb,²²Na and³⁶Cl flux into these vesicles was sensitive to inhibition by furosemide and dependent upon the presence of the other two co-ions, in keeping with the presence of a loop diuretic-sensitive Na⁺K⁺Cl^– cotransport system. The kinetic parameters for Na⁺ and K⁺ interaction have been analyzed under initial linear zerotrans conditions. The following values were obtained:K _mNa+=0.42±0.05 mmol/liter,V _max=303±24 pmol/mg/6 sec;K _mK+=11.9±1.0 mmol/liter,V _maxK+=307±27 pmol/mg/6 sec. For Cl^– interaction evidence for two cooperative binding sites with different affinities and different specificities were obtained. Thus, a stoichiometry of 1Na⁺1K⁺2Cl^– can be calculated. It is concluded that the apical membrane of LLC-PK₁ cells contains a Na⁺K⁺2Cl^– cotransport system with properties similar to those described for the thick ascending limb of the loop of Henle.     

Blockade of cardiac outwardly rectifying K+ channels by TEA and class III antiarrhythmics — evidence against a single drug-sensitive channel site

Elementary K⁺ currents through cardiac outwardly rectifying K⁺ channels were recorded in insideout patches excised from cultured neonatal rat cardiocytes at 19 °C and at 9 °C. By studying the inhibitory effects of tetraethylammonium (TEA), quinidine and verapamil, the properties of this novel type of K⁺ channel were further characterized. Internal TEA (50 mmol/1) evoked a reversible decline of i_unit to 62.7 + 2.7% of control (at –7 mV), without significant changes of open state kinetics, indicating a blockade of the open K⁺ pore with kinetics too fast to be resolvable at 1 kHz. This TEA blockade was e-fold voltage-dependent, with a decrease of the apparent K_{D( TEA)} from 102 mmol/1 at –37 mV to 65 mmol/1 at +33 mV and, furthermore, became accentuated on lowering the internal K⁺ concentration. Thus, TEA competes with the permeant K⁺ for a site located in some distance from the cytoplasmic margin, within the K⁺ pore. Quinidine (100 mol/l), like verapamil (40 mol/1) reversibly depressed i_unit to about 80% of the control value (at –7 mV), but drug-induced fast flicker blockade proved voltage-insensitive between –27 mV and +23 mV These drugs gain access to a portion of the pore distinct from the TEA binding site whose occupancy by drugs likewise blocks K⁺ permeation. Both drugs showed a greater potency to depress P_o which, with quinidine,decreased reversibly to38.6 ± 11.1% (at –7 mV) and, with verapamil to 24.9 ± 9.1%(at –7 mV), mainly by an increase of the prolonged closed state (C,). This alteration of the gating process also includes a sometimes dramatic shortening of the open state. Most probably, cardiac K_{(outw.-rect.)} ⁺K⁺ _outw.-rect. channels possess a second drug-sensitive site whose occupancy by quinidine or verapamil may directly or allosterically stabilize their non-conducting configuration. Correspondence to: M. Kohlhardt     

Cytochromec oxidase fromParacoccus denitrificans in Triton X-100: Aggregation state and kinetics

Cytochromec oxidase fromParacoccus denitrificans was homogenously dispersed in Triton X-100. Using gel exclusion chromatography and sucrose gradient centrifugation analysis a molecular weight of the detergent-protein complex of 155,000 was determined. After subtraction of the bound detergent (111 mol/mol hemeaa ₃) a molecular weight of 85,000 resulted, which agreed well with the model of a monomer containing two subunits. This monomer showed high cytochromec oxidase activity when measured spectrophotometrically in the presence of Triton X-100 (V _max=85 s^–1). The molecular activity, plotted according to Eadie-Hofstee, was monophasic as a function of the cytochromec concentration. AK _m of 3.6×10^–6 M was evaluated, similar to theK _m observed in the presence of dodecyl maltoside [Naeczet al. (1985).Biochim. Biophys. Acta 808, 259–272].     

Characterization of inside-out oriented H+-ATPases in cholate-pretreated renal brush-border membrane vesicles

Summary Exposure of porcine renal brush-border membrane vesicles to 1.2% cholate and subsequent detergent removal by dialysis reorients almost all N-ethylmaleimide (NEM)-sensitive ATPases from the vesicle inside to the outside. ATP addition to cholate-pretreated, but not to intact, vesicles causes H⁺ uptake as visualized by the pH indicator, acridine organge. The reoriented H⁺-pump is electrogenic because permeant extravesicular anions or intravesicular K⁺ plus valinomycin enhance H⁺ transport. ATP stimulates H⁺ uptake with an apparentK _m of 93 m. Support of H⁺ uptake andP _i liberation by ATP>GTPITP> UTP indicates a preference for ATP and utilization of other nucleotides at lower efficiency. ADP is a potent, competitive inhibitor of ATP-driven H⁺ uptake,(K _i , 24 m). Mg²⁺ and Mn^2– support ATP-driven H⁺ uptake, but Ca²⁺, Ba²⁺ and Zn²⁺ do not. Imm Zn²⁺ inhibits MgATP-driven H⁺ transport completely. NEM-sensitiveP _i liberation is stimulated by Mg²⁺ and Mg^2– and, unlike H⁺ uptake, also by Ca²⁺ suggesting Ca²⁺-dependent ATP hydrolysis unrelated to H⁺ transport. The inside-out oriented H⁺-pump is relatively insensitive toward oligomycin, azide, N,N-dicyclohexylcarbodiimide (DCCD) and vanadate, but efficiently inhibited by NEM (apparentK _i , 0.77 m), and 4-chloro-7-nitro-benzoxa-1,3-diazole (NBD-Cl; apparentK _i , 0.39 m). Taken together, the H⁺-ATPase of proximal tubular brush-border membranes exhibits characteristics very similar to those of vacuolar type (V-type) H⁺-ATPases. Hence,V-type H⁺-ATPases occur not only in intracellular organelles but also in specialized plasma membrane areas.     

Ba2+-Induced conductance fluctuations of spontaneously fluctuating K+ channels in the apical membrane of frog skin (Rana temporaria)

Summary We studied the influence of mucosal Ba²⁺ ions on the recently described (Zeiske & Van Driessche, 1979a, J. Membrane Biol. 47:77) transepithelial, mucosa towards serosa directed K⁺ transport in the skin ofRana temporaria. The transport parametersG (conductance), PD (potential difference),I _sc (short-circuit current, K⁺ current), as well as the noise ofI _sc were recorded. Addition of millimolar concentrations of Ba²⁺ to the mucosal K⁺-containing solution resulted in a sudden but quickly reversible drop inI _sc.G andI _sc decreased continuously with increasing Ba²⁺ concentration, (Ba²⁺) _o . The apparent Michaelis constant of the inhibition by Ba²⁺ lies within the range 40–80 m. The apical membrane seems to remain permselective for K⁺ up to 500 m (Ba²⁺) _o . Higher (Ba²⁺) _o , however, appears to induce a shunt (PD falls,G increases). This finding made an accurate determination of the nature of the inhibition difficult but our results tend to suggest a K⁺-channel block by K⁺–Ba²⁺ competition. In the presence of Ba²⁺, the power spectrum of the K⁺ current shows a second Lorentzian component in the low-frequency range, in addition to the high-frequency Lorentzian caused by spontaneous K⁺-channel fluctuations (Van Driessche & Zeiske, 1980). Both Lorentzian components are only present with mucosal K⁺ and can be depressed by addition of Cs⁺ ions, thus indicating that Ba²⁺ ions induce K⁺-channel fluctuations. The dependence of the parameters of the induced Lorentzian on (Ba²⁺) _o , shows a rise in the plateau values to a maximum around 60 m (Ba²⁺) _o , followed by a sharp and progressive decrease to very low values. The corner frequency which reflects the rate of the Ba²⁺-induced fluctuations, however, increases quasi-linearly up to 1mm (Ba²⁺) _o with a tendency to saturate at higher (Ba²⁺) _o . Based on a three-state model for the K⁺ channel (having one open state, one closed by the spontaneous fluctuation and one blocked by Ba²⁺) computer calculations compared favorably with our results. The effect of Ba²⁺ could be explained by assuming reversible binding at the outer side of the apical K⁺ channel, thereby blocking the open channel in competition with K⁺. The association-dissociation of Ba²⁺ at its receptor site is thought to cause a chopping of the K⁺ current, resulting in modulated current fluctuations.     

The viscoelasticity of entangled actin networks: the influence of defects and modulation by talin and vinculin

Rheological measurements of the frequency-dependent complex elastic module G^*() of entangled F-actin solutions in the frequency range 10^–5 – 1 Hz were carried out in three dynamic regimes: 1.) A terminal relaxation from gel-like to liquid-like behaviour measured at frequencies < _d ^–1 2.) a rubber-type plateau and 3.) a regime determined by chain conformational transitions at frequencies > _i ^–1. A major point of interest was to clarify whether rheological, high precision measurements can yield quantitative information about the influence of talin and vinculin on the structure, chain dynamics, elasticity and viscoelasticity of actin filaments with time. We show that in the regime reflecting internal chain dynamics (10^–2 to 1 s time domain), F-actin behaves as a random coil of the Rouse type. This contrasts with dynamic light scattering and correlation spectroscopic studies of actin filament flickering, which indicate that filaments behave as semiflexible rods. The internal chain dynamics, which are determined by thermically excited bending undulations, exhibit a persistence length of 0.3–1 m Evidence is provided that this discrepancy is due to a cross-over of semiflexible rod behaviour at excitation wavelengths () below approximately 1 gm to random-coil behaviour at 1 µ (expected at a frequency 1 Hz). The random coil behaviour is largely determined by defects in actin filaments leading to sharp bends of the chain which act as semiflexible hinges. Talin produces drastic effects on the time course of viscoelasticity during actin polymerization. It promotes the rapid formation of short filament fragments ( 1 gmm, within time scales of min) which anneal slowly into long filaments (within several hours), most probably by fusion. The viscoelasticity depends on the coexistence of short and very long filaments indicated by the elongation of the rubber plateau. The most dramatic effect is a reduction of the ratio of the terminal ('Ed) to the Rouse relaxation time of _i by more than one order of magnitude (_d/_i = 100 compared to ratio _d/_i = 2000 for pure actin). From this it is concluded that talin causes a remarkable decrease in the effective segment length of the macromolecule and, thus induces an increase in chain stiffness. Vinculin on the other hand shows no such effect. Correspondence to: E. Sackmann     

Kinetic characterization of carboxypeptidase-Y-catalyzed peptide semisynthesis Prediction of yields

Summary Carboxypeptidase-Y-catalyzed peptide semisynthesis has been characterized at pH 7.5, 25°C from initial rate steady state kinetic and progress reaction studies of hydrolysis and aminolysis of-N-benzoyl-L-tyrosine 4-nitro-anilide using the natural L-amino acids and their amides as nucleophiles. The reaction mechanism previously shown to account for carboxypeptidase-Y-catalyzed aminolysis reactions (Christensen et al., 1992) was found also to account for all of the reactions studied here. It involves in addition to the classical serine proteinase mechanism: i) complex formation between the free enzyme and the nucleophile, an interaction characterized by the competitive inhibition constant,K _i, and ii) reaction of the nucleophile with the acylated enzyme forming a complex of enzyme and aminolysis product, characterized by the aminolysis kinetic parameter,K _N.A competitive inhibitory effect showing binding to the free enzyme is seen mainly with large hydrophobic amino acids and their amides i.e. the same residues as those preferred on either side of the scissile bond in carboxypeptidase-Y substrates. The stoichiometry of the inhibition is 1 : 1 and the actual binding position most likely is that of the leaving group of substrates,S ₁.Aminolysis effects are obtained with a wide range of amino acids and amino acid amides, exceptions are Pro and, probably due to their low solubility, Tyr, Trp, Asp and Glu. TheK _N-values show relatively little dependence on the chemical nature of the side groups, but a marked difference between the amino acid and its amide. The amides interact more strongly. The kinetic parameter,k _c/K_m, of the hydrolysis of the aminolysis products is another important factor in peptide semisynthesis. Thek _c/K_m-values obtained of the amidated aminolysis products are much less than those of the products formed with free amino acids. All in all this leads to rather efficient aminolysis with the L-amino acid amides and poor aminolysis with the L-amino acids.Abbreviations BzTyrNHPhNO₂ -N-benzoyl-L-tyrosinyl 4-nitro-aniline - Xaa L-amino acids - Xaaa L-amino acid amides - Z-Phe Carbobenzoxy-L-phenylalanine - Z-Met Carbobenzoxy-L-methionine - BzTyr -N-benzoyl-L-tyrosine - AlaVal L-alanyl-L-valine - ValAla L-valyl-L-alanine     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.     

Rearrangements between the operators in the bacteriophage lambda

Summary The left operator mutant v2s develops poorly during infection as a result of constitutive expression of the left operon. A revertant of v2s, designated iri, was found to contain an inversion of the cI region with the inversion endpoints to be within the lambda operators o _L and o _R. Formation of the inversion is facilitated by a translocation of right operator o _R ^c mutant sequence to the left operator in v2s. The inversion in iri positions wild-type o _R sequence at o _L returning control of the left operon to repression by the lambda cro repressor.