Kinetic Analysis of Ca2+/K+ Selectivity of an Ion Channel by Single-Binding-Site Models

Current-voltage relationships of a cation channel in the tonoplast of Beta vulgaris, as recorded in solutions with different activities of Ca²⁺ and K⁺ (from Johannes & Sanders 1995, J. Membrane Biol. 146:211–224), have been reevaluated for Ca²⁺/K⁺ selectivity. Since conversion of reversal voltages to permeability ratios by constant field equations is expected to fail because different ions do not move independently through a channel, the data have been analyzed with kinetic channel models instead. Since recent structural information on K⁺ channels show one short and predominant constriction, selectivity models with only one binding site are assumed here to reflect this region kinetically. The rigid-pore model with a main binding site between two energy barriers (nine free parameters) had intrinsic problems to describe the observed current-saturation at large (negative) voltages. The alternative, dynamic-pore model uses a selectivity filter in which the binding site alternates its orientation (empty, or occupied by either Ca²⁺ or K⁺) between the cytoplasmic side and the luminal side within a fraction of the electrical distance and in a rate-limiting fashion. Fits with this model describe the data well. The fits yield about a 10% electrical distance of the selectivity filter, located about 5% more cytoplasmic than the electrical center. For K⁺ translocation, reorientation of the unoccupied binding site (with a preference of about 6:5 to face the lumenal side) is rate limiting. For Ca²⁺, the results show high affinity to the binding site and low translocation rates (<1% of the K⁺ translocation rate). With the fitted model Ca²⁺ entry through the open channel has been calculated for physiological conditions. The model predicts a unitary open channel current of about 100 fA which is insensitive to cytoplasmic Ca²⁺ concentrations (between 0.1 and 1 μm) and which shows little sensitivity to the voltage across the tonoplast. Received: 19 February 1997/Revised: 19 May 1997     

Kinetics of high-affinity K+ uptake in plants, derived from K+-induced changes in current-voltage relationships

To investigate coupled, charge-translocating transport, it is imperative that the specific transporter current-voltage (IV ) relationship of the transporter is separated from the overall membrane IV relationship. We report here a case study in which the currents mediated by the K⁺-H⁺ symporter, responsible for high-affinity K⁺ uptake in Arabidopsis thaliana (L.) Heynh. cv. Columbia roots, are analyzed with an enzyme kinetic reaction scheme. The model explicitly incorporates changes in membrane voltage and external substrate, and enables the derivation of the underlying symport IV relationships from the experimentally obtained difference IV data. Data obtained for high-affinity K⁺ transport in A. thaliana root protoplasts were best described by a 1:1 coupled K⁺-H⁺ symport-mediated current with a parallel, outward non-linear K⁺ pathway. Furthermore, the large predictive value of the model was used to describe symport behaviour as a function of the external K⁺ concentration and the cytoplasmic K⁺ concentration. Symport activity is a complex function of the external K⁺ concentration, with first-order saturating kinetics in the micromolar range and a strong activity reduction when external K⁺ is in the millimolar range and the membrane depolarises. High cytoplasmic K⁺ levels inhibit symport activity. These responses are suggested to be part of the feedback mechanisms to maintain cellular K⁺ homeostasis. The general suitability of the model for analysis of carrier-mediated transport is discussed. Received: 23 November 1996 / Accepted: 22 April 1997     

Phosphoenolpyruvate carboxylase in developing seeds of Vicia faba L.: gene expression and metabolic regulation

To analyze the role of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) during seed development, two cDNA clones encoding two isoforms of PEPCase were isolated from a seed-specific library of Vicia faba. The two sequences (VfPEPCase1 and VfPEPCase2) have a sequence identity of 82 and 89% on the nucleotide and amino acid levels. The VfPEPCase1 mRNA was found to be predominantly expressed in roots and developing cotyledons whereas the VfPEPCase2 mRNA was more abundant in green and maternal tissues. In the cotyledons, PEPCase mRNAs accumulated from early to mid cotyledon stage and decreased thereafter. The PEPCase activity increased continuously during cotyledon development. The enzyme was strongly activated by glucose-6-phosphate, but not by glucose, fructose or sucrose. Asparagine was weakly activating whereas malate, aspartate and glutamate were inhibitory. The inhibitors became less effective with increasing pH. Aspartate was a much stronger inhibitor of cotyledonary PEPCase than glutamate at both pH 7.0 and 7.5. The sensitivity of PEPCase to malate inhibition decreased from early to mid cotyledon stage at a time when storage proteins are synthesized. This indicates activation on the protein level, possibly by protein phosphorylation. Nitrogen starvation in the presence of hexoses but not sucrose decreased mRNA levels of VfPEPCase1 and enzyme activity, indicating control on the mRNA level by both carbon and nitrogen. It is concluded that in developing cotyledons PEPCase is probably important for the synthesis of organic acids to provide carbon skeletons for amino acid synthesis. Received: 15 July 1998 / Accepted: 10 October 1998     

A study of the current-voltage relationships of electrogenetic active and passive membrane elements in riccia fluitans

Potassium- and proton-dependent membrane potential, conductance, and current-voltage characteristics (I–V curves) have been measured on rhizoid cells of the liverwort Riccia fluitans. The potential difference (E_m) measured with microelectrodes across plasmalemma and tonoplast is depolarized to the potassium-sensitive diffusion potential (E_D) in the presence of 1 mM NaCN, 1 mM NaN₃, or at temperatures below 6°C. Whereas the temperature change from 25°C to 5°C decreases the membrane conductance (g_m) from 0.71 to 0.43 S ? m^?2, 1 mM NaCN increases g_m by about 25%. The membrane displays potassium-controlled rectification which gradually disappears at temperatures below 5°C. The potassium pathway can be described by an equivalent circuit of a diode and an ohmic resistor in parallel. In the potential interval of E_D ± 100 mV the measured I-V curves roughly fit the theoretical curves obtained from a modified diode equation. ⁸⁶Rb⁺(K⁺)-influx is voltage sensitive: In the presence of 1 mM NaCN, ⁸⁶Rb⁺-influx follows a hyperbolic function corresponding to a low conductance at low [K⁺]_o and high conductance at high [K⁺]_o. On the contrary ⁸⁶Rb⁺-influx is linear with [K⁺]_o when pump activity is normal. It is believed that there are two K⁺-transport pathways in the Riccia membrane, one of which is assigned to the low conductance (0.2 S · m^?2), the other to a temperature-dependent facilitated diffusion system with a higher conductance (7.7 S · m^?2). The electrogenic pump essentially acts as a current source and consumes about 39% of the cellular ATP-turnover. In the presence of 30 μM CCCP the saturation current of 0.1 A · m^?2 is doubled to about 0.2 A · m^?2, and the electromotive force of ?360 mV switches to ?250 mV. It is suggested that this may be due to a change in stoichiometry from one to two transported charges per ATP hydrolyzed.     

Pharmacology of the SV channel in the vacuolar membrane of chenopodium rubrum suspension cells

Single channel performance and deactivation currents have been analyzed in the presence of cation channel blockers to reveal pharmacological properties of the slow-activating (SV) cation-selective ion channel in the vacuolar membrane (tonoplast) isolated from suspension cells of Chenopodium rubrum L. At a holding potential of –100 mV, the SV channel showed half-maximal inhibition with 20mm tetraethylammonium (TEA), 7 m 9amino-acridine, 6 m (+)-tubocurarine, 300nm quinacrine, and 35 m quinine, respectively. The SV channel is also blocked by charybdotoxin (20nm at –80 mV) but not by apamine. 9-Amino-acridine, (+)-tubocurarine and quinacrine act in a voltage-dependent fashion, binding to the open channel and to different sites along the transmembrane voltage profile according to Woodhull (J. Gen. Physiol. 61:687–708, 1973). No binding site could be specified for charybdotoxin, which binds to the closed channel, and for quinine. Except for quinine, all tested blockers were effective only if added to the cytoplasmic side of the tonoplast. A structural relationship between the SV channel and Maxi-K channels in animal systems is inferred.We are grateful to Prof. F. Dreyer and Dr. J. Beise from the Pharmacology Department of the Justus-Liebig-Universität Giessen for continuous interest and helpful suggestions. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Be 466/21-5) and the Bundesminister für Forschung und Technologie, Bonn.     

Characterization of a high-conductance,voltage-dependent cation channel from the plasma membrane of rye roots in planar lipid bilayers

Plasma-membrane vesicles were purified by aqueous-polymer two-phase partitioning of a microsomal membrane fraction from rye (Secale cereale L.) roots and incorporated into planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers. A high-conductance cation channel (a maxi cation channel) was characterized from single-channel electrical recordings. The channel was incorporated into the bilayer with its cytoplasmic surface facing the trans compartment and voltages were referenced cis with respect to trans. The channel was permeable to both monovalent and divalent cations. The unitary conductance was 451 pS in symmetrical 100 mM KCl and 213 pS in symmetrical 100 mM BaCl₂. The permeability ratio P_KP_Ba was 1.002.56. Unitary conductances declined in the order K⁺Rb⁺>Cs⁺>Na⁺> Li⁺ (monovalent cations) and Ba²⁺>Sr²⁺>Ca²⁺> Mg²⁺>Co²⁺>Mn²⁺ (divalent cations). The relative permeabilities of monovalent cations mirrored their conductivity sequence, whereas the permeabilities of all divalent cations were similar. The maxi cation channel showed complex kinetics, exhibiting both voltage- and time-dependent inactivation and voltage-dependent gating. The voltage dependence of the kinetics shifted in parallel with changes in the reversal potential of the channel. In symmetrical 100 mM KCl, following a voltage step from zero to the test voltage, the channel inactivated and the active-channel lifetime ( _i) shortened exponentially as the test voltage was increased. The channel always opened immediately upon depolarization to zero volts, indicating that inactivation of the channel did not result from the loss of any intrinsic factor. The probability of finding an active channel in the open state (P₀) exhibited a bell-shaped relationship with membrane potential. At voltages between -40 and 80 mV, P₀ exceeded 0.99, but p₀ declined abruptly at more extreme voltages. Under ionic conditions which approximated physiological conditions, in the presence of 100 mM KCl on the trans (cytoplasmic) side and 1 mM KCl plus 2 mM CaCl₂ on the cis (extracellular) side, the reversal potential was 15.6 mV and the kinetics approximated those observed in symmetrical 100 mM KCl. Thus, the channel would open upon depolarization of the plasma membrane in vivo. If the channel functioned physiologically as a Ca²⁺ channel it might be involved in intracellular signalling: the channel could open in response to a variety of environmental, developmental and pathological stimuli which depolarize the plasma membrane, allowing Ca²⁺ into the cytoplasm and thereby initiating a physiological response.Abbreviations E_K Nernst (equilibrium) potential for potassium - E_rev zero-current (reversal) potential - I/V current/voltage - _c apparent mean lifetime of the activated-channel closed state - _i apparent mean lifetime of the activated channel following a voltage step from zero volts - ₀ apparent mean lifetime of the activated-channel open state - PE 1-palmitoyl-2-oleoyl phosphatidylethonlamine - P₀ probability of finding the activated channel in an open state - TEA⁺ tetraethylammonium This work was supported by the Agriculture and Food Research Council and by a grant from the Science and Engineering Research Council Membrane Initiative (GR/F 33971) to Prof. E.A.C. MacRobbie (University of Cambridge, UK).     

Increases in cytosolic Ca2+ are not required for abscisic acid-inhibition of inward K+ currents in guard cells of Vicia faba L.

 The inward K⁺ channels (I_Kin) of guard cells are inhibited upon application of abscisic acid (ABA). It has been postulated that I_Kin inhibition requires an elevation in cytosolic free Ca²⁺ levels ([Ca²⁺]_c) because: (i) experimental increases in [Ca²⁺] _c can mimic the ABA effect, and; (ii) ABA can trigger an elevation of [Ca²⁺]_c in guard cells. However, not all guard cells respond to ABA with a [Ca²⁺]_c increase, and the magnitude of the increases that do occur is variable. Therefore, an obligate role for Ca²⁺ in the regulation of downstream effectors of ABA response, such as the I_Kin channels, remains in question. In this study, we developed a methodology for simultaneous patch clamping and confocal ratiometric Ca²⁺ imaging of Vicia faba L. guard-cell protoplasts. This allowed us to directly assess the relationship between ABA-induced changes in [Ca²⁺]_c and I_Kin inhibition. In the presence of extracellular Ca²⁺, the extent of [Ca²⁺]_c elevation correlated with the extent of I_Kin inhibition. However, upon chelation of either extracellular Ca²⁺, [Ca²⁺]_c, or both, extracellular Ca²⁺ and [Ca²⁺]_c, [Ca²⁺]_c elevation did not occur in response to ABA yet I_Kin currents were still strongly inhibited. These data illustrate that Ca²⁺-independent regulation is involved in ABA-inhibition of stomatal opening processes. Received: 17 September 1999 / Accepted: 26 October 1999     

A kinetic analysis of cyanine selectivity: CCyan2 and Cyan40 intercalation into poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC)

A T-jump investigation of the binding of Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC) is performed at I = 0.1M (NaCl), 25 degrees C and pH 7. Two kinetic effects are observed for both systems. The binding process is discussed in terms of the sequence D + P <==> P,D <==> PD(I) <==> PD(II), which leads first to fast formation of a precursor complex P,D and then to a partially intercalated complex PD(I) which converts to the fully intercalate complex PD(II). Concerning CCyan2 the rate parameters depend on the polymer nature and their analysis shows that in the case of poly(dG-dC) x poly(dG-dC) the most stable bound form is the fully intercalated complex PD(II), whereas in the case of poly(dA-dT) x poly(dA-dT) the partially intercalated complex PD(I) is the most stable species. Concerning Cyan40, the rate parameters remain unchanged on going from A-T to G-C indicating that this dye is unselective.     

Kinetics of Ca2+- and ATP-dependent, voltage-controlled anion conductance in the plasma membrane of mesophyll cells of Pisum sativum

Whole-cell patch-clamp techniques were used to measure anion currents through the plasma membrane of protoplasts of mesophyll cells of expanding pea (Pisum sativum L.) leaves. Voltage-induced changes of the currents could be modelled with single exponential activation and deactivation kinetics. The anion currents were activated at negative membrane potentials. The time constant of activation, τ_act, increased from 145 ms at −140 mV to 380 ms at −20 mV. A Boltzmann fit to the activation curve, n_∞ (ΔG_Vm/ΔG_max), yielded a half-activation voltage of +27 mV. Opening and closing rate constants, α and β respectively, were calculated from the values of τ and n_∞. The currents depended on the presence of cytoplasmic Ca²⁺ concentrations higher than 10⁻⁶ M. Including 3 mM MgATP in the intracellular solution resulted in a voltage-dependent inactivation of the anion current. The conductance-voltage relation resulting from the voltage-dependent activation and inactivation had a maximum at about −25 mV. The relations of the current in pea are discussed with respect to the anion currents in guard cells and suspension-cultured tobacco cells, and its possible role in growing leaf cells. Received: 1 March 1996 / Accepted: 16 September 1996     

Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.)

The temporal and spatial patterns of storage-globulin mobilization were immunohistochemically pursued in the embryonic axis and cotyledons of vetch seed (Vicia sativa L.) during germination and early seedling growth. Embryonic axes as well as cotyledons of mature seeds contain protein bodies with stored globulins. Prevascular strands of axes and cotyledons, the radicle and epidermal layers of axis organs were nearly exclusively stained by vicilin antibodies whereas the cotyledonous storage mesophyll gave similar staining for vicilin and legumin. Globulin breakdown started locally where growth and differentiation commenced in the axis. There, vicilin mobilization preceded legumin mobilization. Thus vicilin represents the initial source of amino acids for early growth and differentiation processes in vetch. Legumin presumably only serves as a bulk amino acid source for subsequent seedling growth during postgerminative globulin degradation. During the first 2–3 d after the start of imbibition the axis was depleted of globulins whereas no decrease in immunostainability was detected in the cotyledons except in their vascular strands where immunostainability was almost completely lost at this time. Continuous vascular strands were established at the third day when globulin breakdown was finished in the axis but had just started in the cotyledon mesophyll. Protein mobilization proceeded in a small zone from the epidermis towards the vascular strands in the center of the cotyledons. In this zone the storage cells, which initially appeared densely packed with starch grains and protein bodies, concomitantly transformed into cells with a large central vacuole and only a thin cytoplasmic layer attached to the cell wall. These results agree well with the hypothesis that during the first 2 d after imbibition the axis is autonomous in amino acid provision. After the endogenous reserves of the axis are depleted and the conductive tissue has differentiated, globulins are mobilized in the cotyledons, suggesting that then the amino acid supply is taken over by the cotyledons. For comparison with other degradation patterns we used garden bean (Phaseolus vulgaris L) and rape (Brassica napus L.) as reference plants. Received: 3 August 1999 / Accepted: 11 December 1999     

Influence of Ca on the plasma membrane potential and electrogenic uptake of glycine by myeloma cells. Involvement of a Ca-activated K channel

The involvement of Ca²⁺-activated K⁺ channels in the regulation of the plasma membrane potential and electrogenic uptake of glycine in SP 2/0-AG14 lymphocytes was investigated using the potentiometric indicator 3,3′-diethylthiodicarbocyanine iodide. The resting membrane potential was estimated to be −57 ± 6 mV (n = 4), a value similar to that of normal lymphocytes. The magnitude of the membrane potential and the electrogenic uptake of glycine were dependent on the extracellular K⁺ concentration, [K⁺]_o, and were significantly enhanced by exogenous calcium. The apparent V_max of Na⁺-dependent glycine uptake was doubled in the presence of calcium, whereas the K_0.5 was not affected. Ouabain had no influence on the membrane potential under the conditions employed. Additional criteria used to demonstrate the presence of Ca²⁺-activated K⁺ channels included the following: (1) addition of EGTA to calcium supplemented cells elicited a rapid depolarization of the membrane potential that was dependent on [K⁺]_o; (2) the calmodulin antagonist, trifluoperazine, depolarized the membrane potential in a dose-dependent and saturable manner with an IC₅₀ of 9.4 μM; and (3) cells treated with the Ca²⁺-activated K⁺ channel antagonist, quinine, demonstrated an elevated membrane potential and depressed electrogenic glycine uptake. Results from the present study provide evidence for Ca²⁺-activated K⁺ channels in SP 2/0-AG14 lymphocytes, and that their involvement regulates the plasma membrane potential and thereby the electrogenic uptake of Na⁺-dependent amino acids.     

Cytoplasmic calcium affects the gating of potassium channels in the plasma membrane ofChara corallina: a whole-cell study using calcium-channel effectors

The action of a wide range of drugs effective on Ca²⁺ channels in animal tissues has been measured on Ca²⁺ channels open during the action potential of the giant-celled green alga,Chara corallina. Of the organic effectors used, only the 1,4-dihydropyridines were found to inhibit reversibly Ca²⁺ influx, including, unexpectedly, Bay K 8644 and both isomers of 202–791. Methoxyverapamil (D-600), diltiazem, and the diphenylbutylpiperidines, fluspirilene and pimozide were found not to affect the Ca²⁺ influx. Conversely, bepridil greatly and irreversibly stimulated Ca²⁺ influx, and with time, stopped cytoplasmic streaming (which is sensitive to increases in cytoplasmic Ca²⁺). By apparently altering the cytoplasmic Ca²⁺ levels with various drugs, it was found that (with the exception of the inorganic cation, La³⁺) treatments likely to lead to an increase in cytoplasmic Ca²⁺ levels caused an increase in the rate of closure of the K⁺ channels. Similarly, treatments likely to lead to a decrease in cytoplasmic Ca²⁺ decreased the rate of K⁺ channel closure. The main effect of bepridil on the K⁺ channels was to increase the rate of voltage-dependent channel closure. The same effect was obtained upon increasing the external concentration of Ca²⁺, but it is likely that this was due to effects on the external face of the K⁺ channel. Addition of any of the 1,4-dihydropyridines had the opposite effect on the K⁺ channels, slowing the rate of channel closure. They sometimes also reduced K⁺ conductance, but this could well be a direct effect on the K⁺ channel; high concentrations (50 to 100 μM) of bepridil also reduced K⁺ conductance. No effect of photon irradiance or of abscisic acid could be consistently shown on the K⁺ channels. These results indicate a control of the gating of K⁺ channels by cytoplasmic Ca²⁺, with increased free Ca²⁺ levels leading to an increased rate of K⁺-channel closure. As well as inhibiting Ca²⁺ channels, it is suggested that La³⁺ acts on a Ca²⁺-binding site of the K⁺ channel, mimicking the effect of Ca²⁺ and increasing the rate of channel closure.     

Different ratios of sucrose/raffinose-induced membrane depolarizations in the mesophyll of species with symplasmic (Catharanthus roseus, Ocimum basilicum) or apoplasmic (Impatiens walleriana, Vicia faba) minor-vein configurations

In mesophyll cells of species with a symplasmic (Ocimum basilicum, Catharanthus roseus, Magnolia denudata) or an apoplasmic (Vicia faba, Impatiens walleriana, Bellis perennis) minor-vein configuration, membrane depolarizations in response to 20 or 200 mol·m^–3 raffinose and sucrose were measured. Ageing period and resting potential marginally affected the degree of depolarization. The symplasmic species showed similar depolarization responses to 20 and 200 mol·m^–3 sucrose or raffinose. In the apoplasmic species, depolarization increased statistically significantly from 20 to 200 mol·m^–3 sucrose, whereas the depolarization response to raffinose was equal at both concentrations. In the apoplasmic species, moreover, the depolarization response to raffinose was significantly weaker than to sucrose at all concentrations. A major difference between symplasmic and apoplasmic species seems to lie in the scantiness of raffinose carriers in the mesophyll plasma membrane of species with the apoplasmic mode of phloem loading.Abbreviations 20R(200R) 20(200) mol·m^–3 raffinose - 20S(200S) 20(200) mol·m^–3 sucrose     

Impact of temperature on the relationship between respiration and nitrogen concentration in roots: an analysis of scaling relationships, Q10 values and thermal acclimation ratios

* The impact of nitrogen (N) supply on the temperature response of root respiratory O(2) uptake (R) was assessed in several herbaceous species grown in solution culture. Warm-grown (25 : 20 degrees C, day:night) plants differing in root N concentration were shifted to 13 : 8 degrees C for 7 d to cold-acclimate. * Log-log plots of root R vs root N concentration both showed that R increased with increasing tissue N concentration, irrespective of the growth temperature. Although the regression slopes of the log-log plots did not differ between the warm-grown and cold-acclimated plants, cold-acclimated plants did exhibit a higher y-axis intercept than their warm-grown counterparts. This suggests that cold acclimation of root R is not entirely dependent on cold-induced increases in tissue N concentration and that scaling relationships (i.e. regression equations fitted to the log-log plots) between root R and N concentration are not fixed. * No systematic differences were found in the short-term Q(10) (proportional change in R per 10 degrees C change in temperature), or degree of cold acclimation (as measured by the proportional difference between warm- and cold-acclimated roots) among roots differing in root N concentration. The temperature response of root R is therefore insensitive to tissue N concentration. * The insensitivity of Q(10) values and acclimation to tissue N concentration raises the possibility that root R and its temperature sensitivity can be predicted for a range of N supply scenarios.     

Steady-state fluorescence polarization in planar lipid membranes. Experimental and theoretical analysis of the fluorophores 8-anilino-1-naphthalenesulfonate, 1,6-Diphenyl-1,3,5-hexatriene, dansyllysine-valinomycin and n-(9-anthroyloxy) fatty acids

In continuation of earlier work, the steady-state fluorescence polarization in a globally oriented system of planar lipid membranes was analyzed experimentally and theoretically for the fluorophores 8-anilino-1-naphthalenesulfonate, 1,6-diphenyl-1,3, 5-hexatriene, dansyllysine-valinomycin and n-(9-anthroyloxy) fatty acids. The theoretical analyses of experiments were mainly done in terms of the mean orientation of transition moments with respect to the membrane normal, an angle describing the region of hindered rotational diffusion and the coefficients of rotational diffusion perpendicular to the membrane and around the membrane normal. The nonvanishing angle between the moments of absorption and emission was taken into account. In the case of n-(9-anthroyloxy) fatty acids it was found that the orientational disorder increases significantly with the depth of the fluorophore within the membrane. In order to compare with recent results from time-dependent fluorescent polarization in globally isotropic membrane suspensions and with 2H-NMR experiments, the second moment ('order parameter') of the steady-state orientational distribution of absorption dipoles was calculated. For all fluorophores the theoretical analysis indicates a preferred orientation of absorption moments within the membrane plane.     

The relationships between growth temperature,fatty acid composition and the physical state and fluidity of membrane lipids in Yersinia enterocolitica

The relationship between membrane lipid composition and membrane lipid phase transitions was investigated in Yersinia enterocolitica cells grown at 5, 22 and 37°C. The total phospholipid concentrations were 9.4, 7.3 and 6.3% of the cell dry weight for cells grown at 5, 22 and 37°C, respectively. The relative concentrations of the three major phospholipids, phosphatidylethanolamine (73–76%), phosphatidylglycerol (9–11%) and cardiolipin (11–13%) were essentially the same at all three growth temperatures. The ratios of unsaturated to saturated fatty acids were 2.2, 1.1 and 0.4 for cells grown at 5, 22 and 37°C, respectively. This change in the fatty acid composition in response to temperature changes is similar to the patterns reported for other organisms. Reversible thermotropic phase transitions were detected by calorimetric analysis in both pure lipid preparations and membrane preparations. The mid-points of the thermotropic phase transitions were at ?13, ?9 and 1°C for membranes from cells grown at 5, 22 and 37°C, respectively. The phase transitions of the membranes from cells grown at the three different temperatures occurred below the lowest growth temperature (5°C). The alternations in the fatty acid composition in Y. enterocolitica did not, therefore, appear to be required to adjust membrane fluidity but might rather be required for some other membrane function.     

Microcompartmentation of energy metabolism at the outer mitochondrial membrane: Role in diabetes mellitus and other diseases

Complexes made up of the kinases, hexokinase and glycerol kinase, together with the outer mitochondrial membrane voltage-dependent anion channel (VDAC) protein, porin, and the inner mitochondrial membrane protein, the adenine nucleotide translocator, are involved in tumorigenesis, diabetes mellitus, and central nervous system function. Identification of these two mitochondrial membrane proteins, along with an 18 kD protein, as components of the peripheral benzodiazepine receptor, provides independent confirmation of the interaction of porin and the adenine nucleotide translocator to form functional contact sites between the inner and outer mitochondrial membranes. We suggest that these are dynamic structures, with channel conductances altered by the presence of ATP, and that ligand-mediated conformational changes in the porin-adenine nucleotide translocator complexes may be a general mechanism in signal transduction.     

Transient expression of storage-protein genes during early embryogenesis ofVicia faba: synthesis and metabolization of vicilin and legumin in the embryo,suspensor and endosperm

Seed storage proteins are thought to be accumulated exclusively in the cell-expansion phase of embryogenesis and metabolized during germination and seedling growth. Here we show by a sensitive immunohistological technique that the two Vicia faba L. storage proteins vicilin and legumin are accumulated in substantial amounts in the suspensor and coenocytic endosperm and to a lesser extent in the mid-globular embryo. Both proteins appear and disappear at precise stages specific for each tissue. In the endosperm the accumulation starts around 12 d after pollination (DAP). After a maximum attained at 14–15 DAP, storage proteins are degraded within about 4 d. Accumulation is restricted to that part of the endosperm which covers the embryo and displays the highest levels of endoploidy (maximum 96n). In all other parts of the endosperm, storage proteins do not appear to accumulate, although storage-protein-specific mRNA synthesis takes place. In the suspensor, storage proteins are already observed at 6 DAP and disappear very quickly at approximately 10 DAP. Low amounts of legumin and vicilin are also detectable in the mid-globular embryo, but disappear completely as the embryo enters the heart stage. We conclude that storage proteins of Vicia faba accumulated transiently during early seed development are used as nutritive reserves for the growing embryo.Abbreviation DAP days after pollination Dedicated to Prof. Rigomar Rieger in the occasion of his 65th birthdayThis research was supported by the Ministry of Science and Research, Land Sachsen-Anhalt, Germany. U.W. acknowledges additional support by the Fonds der Chemischen Industrie.     

Epidermal tissue homeostasis: Apoptosis and cell emigration as mechanisms of controlled cell deletion in the epidermis,of the toad,Bufo bufo

Summary In normal, non-expanding toad epidermis more cells are produced than needed to replace cells lost by moulting. By implication, cell deletion additional to moulting must take place. This paper deals with the mechanisms by which the surplus of cells is deleted, taking advantage of the fact that the ratio between cell birth rate (K _b) and the rate of desquamation (K _d), which in normal toads is 2 to 3, can be manipulated. In toads deprived of the pars distalis of the pituitary gland it is decreased to 0.2 to 0.3, and in toads with hydrocortisone pellets implanted into the subcutaneous lymph space it is increased to 7 to 10. Thus, structures candidates for the morphological manifestation of the deletion process should occur rarely in toads in which the pars distalis has been removed and frequently in toads with hydrocortisone pellets implanted. Categorization and enumeration of such structures by light microscopy in the epidermis from operated, normal, and hormone-treated toads were performed. The incidence of structures referred to as dark cells and omega-figures were found to correlate relatively well with the K _b/K_d-ratio. A subsequent ultrastructural analysis — on a cell-by-cell basis — of dark cells showed these to reflect various stages of apoptosis. The duration of the apoptotic process was calculated to be approximately 7 h. Light- and electron microscopy of omega-figures combined with histochemical observations of PSA-lectin binding were interpreted as reflecting a release of cells from the basal epidermis and their final elimination within the dermis. It is concluded (i) that apoptosis is an important mechanism of controlled cell deletion, (ii) that emigration to, and elimination in, the dermis is a possible deletion mechanism, and (iii) that necrosis is unlikely to play a role in controlled cell deletion.Supported in part by the Danish National Science Research Council (grant no. 11-6498) (PB)Part of this work was presented at the XVth Meeting of the European Study Group for Cell Proliferation, Sundvollen, Norway, 16–20 September 1987