Cyclic AMP stimulates ouabain-insensitive ion movement in shark rectal gland

Summary When the active sodium-potassium pump (Na–K-ATPase) of shark rectal glands is blocked by ouabain, the concentration of intracellular ions changes in the direction of equilibrium with extracellular fluid. These changes were examined when isolated perfused glands were in the basal state and also when they were stimulated to secrete with cAMP and theophylline, to see whether stimulation affected the passive movement of sodium, potassium and chloride across cell membranes. In basal glands 10^–4M ouabain induced an increase of 30 meq/l in intracellular [Na⁺] and a decrease in intracellular [K⁺] of about 50 meq/l after 30 min, while intracellular [Cl^–] was unchanged. In stimulated glands, these movements were exaggerated. The increase in intracellular [Na⁺] averaged 112 meq/l, and the decrease in intracellular [K⁺], 96 meq/l (P<0.01), while mean intracellular [Cl^–] rose by 80 meq/l. Furosemide, 10^–4M, partially reversed the accelerated changes in intracellular electrolytes seen after ouabain was added to stimulated glands. These results are consistent with an action of cAMP upon a ouabaininsensitive cotransport of sodium, potassium and chloride in the rectal gland, analogous to that described in avian erythrocytes.Some of these results have been previously reported in abstract form in Bull Mt Desert Isl Biol Lab (Silva et al. 1979a).     

The amyloidogenic region of the human prion protein contains a high affinity (Met)2(His)2 Cu(I) binding site

The prion protein (PrP^c) is a cuproprotein implicated in a number of human neurodegenerative diseases. Although many physiological functions have been ascribed to PrP, its potential to act as a neuronal antioxidant, based in part on its copper binding ability, is controversial and unresolved. A number of studies have shown that copper bound to PrP^c is not redox silent, and recent data shows that the Cu(II) sites at histidines 96 and 111 display reversible electrochemistry. Reversible electrochemistry implies redox cycling whilst the metal remains bound and with the absence of permanent oxidation or reduction of the protein. Despite this indirect evidence of Cu(I) binding to PrP, the nature of the Cu(I) binding site/s is unclear, although previous extended X-ray absorption fine structure (EXAFS) data has implicated methionines in the Cu(I) binding site. Using spectroscopic techniques we find that the PrP region encompassing histidines 96 and 111 can bind a Cu(I) ion in a site comprising His 96, His 111, Met 109 and Met 112. The four-coordinate (His)₂(Met)₂ Cu(I) site has a K_d = 10⁻¹⁵–10⁻¹² M indicative of high affinity. Mutation of histidine residues reduces the Cu(I) affinity. Although alluding to the fact the PrP could act in a direct superoxide dismutase-like fashion, the Cu(I)–PrP(91–124) site and affinity is comparable to that observed for bacterial periplasmic Cu(I) transporters.     

The influence of extracellular calcium on the response of fly photoreceptors

Summary This paper presents a systematic investigation of the influence of the extracellular concentration of calcium ([Ca²⁺]₀) on the electrophysiological response of the fly's photoreceptors (R1–R6) to light. The hemisected heads of flies were perfused with a standard medium containing 10^–4 mol/1 CaCl₂ and in this medium the intracellularly recorded response of the cell was virtually identical to the normal response obtained in vivo. All the effects of changing the [Ca²⁺]₀ could be reversed within 5 min by perfusing the eye with the standard medium.Changing the [Ca²⁺]₀ did not influence the frequency with which quantum bumps occurred or the resting membrane potential, but did lead to changes in the latency and amplitude of the response and, most significantly, in the repolarization time (t _r). The plot oft _r versus the [Ca²⁺]₀ revealed that the value oft _r changes significantly in two distinct regions representing a [Ca²⁺]₀ of between 2×10^–8 and 10^–7 mol/l and 10^–4 and 10^–2 mol/l, respectively. Lowering the [Ca²⁺]₀ did not affect the amplitude of the response but did lead to a drastic increase int _r which was accompanied by an increase in latency and peak time. Raising the [Ca²⁺]₀ led to a reduction in the duration and amplitude of the response. The latter effect is evidence of reduction in the sensitivity of the photoreceptor cell which is dependent on the [Ca²⁺]₀.It is postulated that two types of binding site for calcium exist, high affinity binding sites (HABS) and low affinity binding sites (LABS), which modulate the functioning of ion channels in the cell membrane that are activated as a consequence of light absorption. The results indicate that the sensitivity of the photoreceptor cell is determined by the degree of saturation of the LABS.     

An Epitope of the Viscumin Catalytic Subunit Exposed on Its Interaction with Lipid Bilayer

It was previously shown that the catalytic subunit of the plant toxin viscumin induces aggregation of small unilamellar liposomes and this process is inhibited by the mab_TA7 monoclonal antibody produced to the denatured catalytic subunit of viscumin (Agapov, I.I. et al., FEBS Lett., 1999, vol. 464, p. 63). The interaction of the synthetic F¹⁰¹–T¹⁰⁵ and A⁹⁶–T¹⁰⁵ fragments of the viscumin catalytic subunit with the mab_TA7 monoclonal antibody was studied by ¹H NMR spectroscopy. Results of this study demonstrated that only the A⁹⁶–T¹⁰⁵ fragment is capable of binding to mab_TA7. A nuclear Overhauser effect observed in the antigen–antibody complex and registered on the resonances of the free peptide transferred from the free state to the antibody-bound state was analyzed, the mab_TA7 antigen determinant (H⁹⁹–T¹⁰⁵) was identified, and its conformation and orientation within the complex with the antibody were determined.     

Synthesis of Tc-99m labeled glucosamino-Asp-cyclic(Arg-Gly-Asp-d-Phe-Lys) as a potential angiogenesis imaging agent

Angiogenesis imaging agents for single photon emission computed tomography (SPECT) play a role in diagnosing tumor-induced angiogenesis as well as tumor metastasis. We synthesized and evaluated radiolabeled RGD glycopeptides by incorporation of the [^99mTc(CO)₃(H₂O)₃]⁺. ^99mTc labeled glucosamino-D-c(RGDfK) ([^99mTc]2) was prepared in 90–93% radiochemical yields (decay corrected). In vitro cell binding assays demonstrated selective binding [^99mTc]2 to human umbilical vein endothelial (HUVE) cells, with inhibition of binding to 37.3% of control levels by 10 μM of cold authentic compounds. In addition, [^99mTc]2 was shown to have high binding affinity to purified α_vβ₃ integrin (IC₅₀ = 1.5 nM). These results suggest that these radiolabeled RGD glycopeptides may have value for non-invasive assessment of angiogenesis.     

Allosteric effects of monoclonal antibodies on human growth hormone

We have previously shown that a monoclonal antibody (MAb) recognizing the human growth hormone (hGH) antigenic domain left exposed after binding to lactogenic receptors enhanced hGH binding probably through allosteric effects on the hormone binding site. Since receptors displaying different specificities would not recognize exactly the same hGH region, we explored whether some of our MAb could affect hGH binding to somatogenic receptors from rabbit liver and to human liver hGH-specific receptors.The effect of MAbAE5, AC8 and F11 on hGH binding was measured by determining the formation of¹²⁵I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding¹²⁵I-MAb, indicated that the hGH domain defined by epitopes AE5, AC8 and F11 is uncovered in the various hormone:receptor complexes.Procedure B was devised to reveal any alteration in the hGH molecule induced by the MAb. In this case preformed¹²⁵I-MAb:hGH complexes were added to microsomes. Data showed that¹²⁵I-MAb AE5:hGH complexes bound better to the various receptors than¹²⁵I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to¹²⁵I-MAb AC8 or¹²⁵I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding of MAb AE5 or MAb F11 to hGH 20 K (a natural hGH variant lacking residues 32–46) also enhanced its affinity to the various receptors whereas MAb AC8 did not inhibit hGH 20 K binding.Results indicated that MAb recognizing the hGH antigenic area that remains unmasked after binding to different membrane-bound receptors are able to affect hormone binding site. MAb would induce either positive or negative allosteric changes in the hormone region involved in its binding to lactogenic, somatogenic and hGH-specific receptors.     

Comparison of the binding of optically pure (−)- and (+)-[3H]nicotine to rat brain membranes

A comparison of the binding of (–)- and (+)-[³H]nicotine to rat brain membranes revealed that only the (–)-enantiomer showed high affinity binding; while the (+)-enantiomer was at least 1/10 as effective as the (–)-enantiomer when in competition with (–)-[³H]nicotine as the ligand. Positive cooperativity, which is observed with (–)-[³H]nicotine as the presence of low concentrations of (+)-nicotine, may account for the seeming paradox.     

Thermoregulatory Activity of Dermorphin Fragments

We studied the effect of C-terminal truncation of the dermorphin (DM) molecule and analogs of its N-terminal tetrapeptide, [DOrn²]-DM_1–4, [DArg²]-DM_1–4, [DAla⁴]-DM_1–4, [DArg², DAla⁴]-DM_1–4, Arg-DM_1–4, Arg-[DArg²]-DM_1–4, Arg-[DAla⁴]-DM_1–4, and Arg-[DArg², DAla⁴]-DM_1–4, on the functional status of the thermoregulation system in rats at different ambient temperatures. For the first time, we demonstrate that the N-terminal tetrapeptide is the minimal fragment with the hypothermic effect. Only the N-terminal octapeptide exerted the vasomotor effect. Amino acid substitutions in the tetrapeptide affected its hypothermic effect. [DArg²]-DM_1–4 and [DArg², DAla⁴]-DM_1–4 had the greatest effect. Addition of Arg to the N-terminus of DM_1–4 analogs changed their thermoregulatory activity. The greatest thermoregulatory effect was observed for Arg-[DArg²]-DM_1–4 and Arg-[DArg², DAla⁴]-DM_1–4.     

The production of lactic acid from whey permeate byLactobacillus helveticus

Summary The effect of pH on growth and lactic acid production ofLactobacillus helveticus was investigated in a continuous culture using supplemented whey ultrafiltrate. Maximum lactate productivity of 5 gl^–1h^–1 occurred at pH 5.5. Whey permeates concentrated up to four times were fermented using batch cultures. Maximum lactic acid concentration of 95 gl^–1 was attained, but residual sugars indicated a possible limitation in growth factors.Nomenclature D Dilution rate [h^–1] - X Biomass [gl^–1] - Glu Glucose consentration [gl^–1] - Gal Galactose consentration [gl^–1] - S Substrate, Lactose consentration [gl^–1] - P Product, Lactate consentration [gl^–1] - Y_p/s Yield, defined as P/S [gg^–1] - r_i Rate of synthesis or consumption of i [gl^–1h^–1]     

The effect of N-terminal substitutions on the biological activity of MSH fragments

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of α-melanotropin, [Glp⁵]α-MSH(5–10), [Glp⁵, -Phe⁷]α-MSH(5–10), [Sar⁵, -Phe⁷]α-MSH(5–10), [Nle⁴, -Phe⁷]α-MSH(4–10), [N-carbamoyl]α-MSH(5–10), and formyl and acetyl derivatives of α-MSH(5–10), [Gly⁵]α-MSH(5–10) and [Gly⁵, -Phe⁷]α-MSH(5–10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.     

Uptakes of iodide and chloride by primary cultures of mouse astrocytes and neurons

Primary cultures of both mouse astrocytes and neurons accumulate more¹²⁵I^– than³⁶Cl^– from the medium. The average cell/medium ratio of¹²⁵I^– of astrocytes (1.01) is greater than that of neurons (0.74), whereas the ratio of³⁶Cl^– of neurons (0.47) is greater than that of astrocytes (0.25). The equilibrium potentials of both¹²⁵I^– and³⁶Cl^– calculated from the cell/medium ratios in astrocytes and neurons are significantly lower than their corresponding resting transmembrane potentials which suggest that both iodide and chloride are actively transported into both cell types. With respect to different transport inhibitors, thiocyanate is more effective in inhibiting¹²⁵I^– uptake whereas furosemide is more effective in inhibiting³⁶Cl^– uptake. Radioiodide uptake by mouse astrocytes was directly proportional to the [Na⁺]_o but was not significantly affected by changes of [Cl^–]_o or [HCO ₃ ^– ]_o, except that it is low in bicarbonate-free medium. Radiochloride uptake by astrocytes was inversely related to [Cl^–]_o and [HCO ₃ ^– ]_o and was not affected [Na⁺]_o, except that it was low in sodium-free medium. Radioiodide uptake by neurons was directly related to [Na⁺]_o between 60 and 140 mM and inversely related to [HCO ₃ ^– ]_o between 10 and 40 mM, but it was not affected by [Cl^–]_o. Radiochloride uptake by neurons was directly related to [Cl^–]_o and to [Na⁺]_o between 60 and 140 mM and was not affected by [HCO ₃ ^– ]_o. However, in sodium-free medium both¹²⁵I^– and³⁶Cl^– uptakes into neurons were higher than those in [Na⁺]_o between 5 and 60 mM. These results indicate that uptake of¹²⁵I^– and³⁶Cl^– into astrocytes and neurons are different in their ion dependence and that they are under separate regulation.Special issue dedicated to Dr. Paola S. Timiras     

The synthesis of [26,27-C]dihydroxyvitamin D3, a tracer for positron emission tomography (PET)

1α,25-Dihydroxyvitamin D₃, an endogenous ligand with the highest affinity for the vitamin D receptor (VDR), was labeled with ¹¹C for use in biological experiments. The radionuclide was incorporated via the reaction of [¹¹C]methyllithium on a methyl ketone precursor in tetrahydrofuran at −10 °C. Deprotection of the labeled intermediate yielded 2.5–3 GBq [26,27-¹¹C]1α,25-dihydroxyvitamin D₃ [¹¹C-1,25(OH)₂ D₃] with specific radioactivity averaging 100 GBq/μmol at the end of synthesis and HPLC purification. The entire process took 48 min from the end of radionuclide production. In vitro binding experiments in rachitic chick purified VDR demonstrated the high affinity binding of this novel tracer. Thus; ¹¹C-1,25(OH)₂ D₃ is available for in vivo distribution studies and may be suitable for the positron emission tomography (PET) determination of VDR levels and occupancy in animals and humans.     

Growth,Stomatal Conductance,Photosynthetic Rate,Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Phosphoenolpyruvate Carboxylase Activities during Rooting and Acclimatisation of Rosa Hybrida Plantlets

The rooting of shoots of micropropagated Rosa hybrida cv. Madame Delbard was conducted on MS medium with 30 kg m^–3 sucrose or on hydroponic medium (containing less mineral salts), under higher photosynthetic photon flux density (PPFD) (100 in comparison with 45 µmol m^–2 s^–1) and flushed by ambient air [AC, 340 µmol(CO₂) mol^–1] or by CO₂-enriched air (EC, 2 500 µmol mol^–1) and lower relative humidity (80–90 % vs. 96–99 %). This cultivation led to plantlets with longer roots and adventitious root formation. Net photosynthetic rate and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activities, RuBPCO/phosphoenolpyruvate carboxylase activities ratio, and starch accumulation increased under these conditions. After 14 d, plantlets had functional stomata and could be acclimated on open benches without gradual decrease in relative humidity. The percentage of survival was higher when the rooting took place in EC than in AC. However, the advantage acquired during rooting phase by plantlets cultured in liquid medium was not maintained after 4 weeks of acclimatisation.     

[125I]calmodulin binding to synaptic plasma membrane from rat brain: Kinetic and arrhenius analysis

Binding of [¹²⁵I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca²⁺-dependent membrane binding of [¹²⁵I]calmodulin was found to have a B_max of 284 pmol/mg protein and an apparent affinity with a K_d of 131 nM. Kinetic analysis indicates that at 37°C, the dissociation of [¹²⁵I]calmodulinmembrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7×10^–1 min^–1 and a half-time (t_1/2) of 1.8 min for the fast component, and a k of 4.8×10^–2 min^–1 and a t_1/2 of 14.5 min for the slow component. At 0°C, substantial dissociation still occurred, with a k of 4.5×10^–2 min^–1 and a t_1/2 of 15.3 min for the fast component, and a k of 5.5×10^–3 min^–1 and a t_1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associated and dissociate calmodulin. In Arrhenius analysis of temperature effects, [¹²⁵I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (T_d) estimated to be 23.8°C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [¹²⁵I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10^–7–10^–6 M), a steroid hormone, and decreased by ethanol (50–200 mM), a centrally acting drug. Our data on the characteristics of calmodulin binding to the SPM provide groundwork for future studies on physiological and pharmacological regulation of calmodulin translocation to and from the plasma membrane in synaptic terminals.Abbreviations used CaM calmodulin - SPM synaptic plasma membrane - ATPase adenosine triphosphatase - Tris tris(hydroxymethyl)aminomethane - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - SDS sodium dodecyl sulfate - TFP trifluoperazine - K_d dissociation constant - B_max maximum binding - k first-order rate constant - t_1/2 half-time - T_d transition temperature     

Effect of [DAla4]-Dermorphin and Its Analogs on the Functional Status of the Thermoregulation System in Rats at Different Environmental Temperature

We studied the influence of [DAla⁴]-dermorphin ([DAla⁴]-DM), its fragments ([DAla⁴]-DM_2–7, [DAla⁴]-DM_3–7, and [DAla⁴]-DM_4–7) and analogs ([Trp¹, DAla⁴]-DM, [DArg², DAla⁴]-DM, and [DOrn², DAla⁴]-DM) on the functional status of the thermoregulation system in rats at different thermal environments: cold (4–6°C), thermoneutral (27–28°C) and hot (31–32°C). [DAla⁴]-DM administration proved to induce temperature- and dose-dependent hypothermia and vasomotor responses. No activity of the peptide was observed in the hot environment. N-terminal shortening of the peptide inhibits its thermoregulatory activity. Tyr¹ to Trp¹ substitution nearly completely suppressed the thermoregulatory effects. DAla² to DArg² substitution decreased the hypothermic effect and only the vasodilatory response was observed in the comfortable environment. DAla² to DOrn² substitution completely suppressed the hypothermic effect in the cold environment and considerably attenuated the vasodilatory response in the thermoneutral environment.     

Time course of metabolism of benzo[e]pyrene by hamster embryo cells and the effect of chemical modifiers

In cultures of hamster embryo cells, benzo[a]pyrene (B[a]P) is metabolized primarily in the bay region. In contrast, little or no bay region metabolism of the noncarcinogenic isomer benzo[e]pyrene (B[e]P) could be detected during 12–96-h incubations of hamster embryo cells with 4 μM [³H]B[e]P. The upper limit to 9,10-dihydro-9,10-dihydroxy-B[e]P formation is about 0.2% of the ethyl acetate-soluble metabolites ( <0.1% of the total metabolites). The major identified metabolites of B[e]P were 4,5-dihydro-4,5-dihydroxy B[e]P and the glucuronide conjugates of 3-OH-B[e]P and 4,5-dihydro-4,5-dihydroxy B[e]P. Simultaneous treatment of cells with either B[a]P or 7,8-benzoflavone (BF) did not induce bay region metabolism of [³H]B[e]P.     

Selection of a support for immobilization of a microbial lipase for the hydrolysis of triglycerides

Baterial lipase from Staphylococcus carnosus (pLipMut2) has been immobilized on various supports in order to determine a suitable immobilization technique in terms of activity and stability, when utilized for the hydrolysis of tributyrin. The hydrophobic materials PBA Eupergit and PBA Eupergit 250L prooved to be appropriate supports, when the enzyme was crosslinked with glutaraldehyde after adsorption. No desorption of the immobilized enzyme occured during operation. The pore size of the support has a strong effect on the activity but does not influence stability.The initial activity for immobilized and soluble lipase is found to follow the Arrhenius equation at low temperature, where mass transfer does not affect reaction kinetics. Activation energies for soluble and immobilized lipase were evaluated to be 21.7 kJ mol^–1 and 60.8 kJ mol^–1, respectively.Operational stability was studied in a packed bed recirculation reactor. Thermal desactivation followed first order kinetics with a half-life of 1340 h at 10°C. Model calculations for productivity showed, that optimal temperatures for high productivity are well below the temperature of maximal activity.List of Symbols E _a [kJ mol^–1] activation energy - E _d [kJ mol^–1] activation energy of desactivation - H [–] half-number - k _d [h^–1] desactivation constant - k _d, [h^–1] constant - k _N [–] desactivation constant (number) - N [–] number of runs - p [mol dm^–3] productivity - t [h] time - t _0.5 [h] half-life - T [K] absolute temperature - V [U ml^–1] activity - V(N) [Uml^–1] activity exhibited in the n-th run - V _s,O [U ml^–1] initial activity of supernatant - V _s, [U ml^–1] activity of supernatant after immobilization - V _O [U ml^–1] initial activity - V [U ml^–1] constant - _imm [–] activity yield - [ml ml^–1] ratio of volume of support to volume of supernatant Financial support of this work by the Deutsche Forschungsgemeinschaft (SFB 145, A15) is gratefully acknowledged.     

Characterisation of new symbiotic Medicago truncatula (Gaertn.) mutants, and phenotypic or genotypic complementary information on previously described mutants

From a pool of Medicago truncatula mutants—obtained by gamma-irradiation or ethyl methanesulfonate mutagenesis—impaired in symbiosis with the N-fixing bacterium Sinorhizobium meliloti, new mutants are described and genetically analysed, and for already reported mutants, complementary data are given on their phenotypic and genetic analysis. Phenotypic data relate to nodulation and mycorrhizal phenotypes. Among the five new mutants, three were classified as [Nod⁺ Fix^– Myc⁺] and the mutations were ascribed to two loci, Mtsym20 (TRV43, TRV54) and Mtsym21 (TRV49). For the two other new mutants, one was classified as [Nod^–/+ Myc⁺] with a mutation ascribed to gene Mtsym15 (TRV48), and the other as [Nod^– Myc^-/+] with a mutation ascribed to gene Mtsym16 (TRV58). Genetic analysis of three previously described mutants has shown that [Nod^–/+ Myc⁺] TR74 mutant can be ascribed to gene Mtsym14, and that [Nod^–/+ Myc^–/+] TR89 and TRV9 mutants are ascribed to gene Mtsym2 (dmi2). Using a detailed analysis of mycorrhizal phenotype, we have observed a delayed typical arbuscular mycorrhizal formation on some mutants that present thick lens-shaped appressoria. This phenotype was called [Myc^–/+] and mutants TR25, TR26, TR89, TRV9, P1 and Y6 were reclassified as [Myc^–/+]. Mutant P1 was reclassified as [Nod^–/+] because of a late nodulation observed on roots of this mutant.     

Intracellular potassium activity and the role of potassium in transepithelial salt transport in the human reabsorptive sweat duct

Summary We have measured the intracellular potassium activity, [K⁺]_i and the mechanisms of transcellular K⁺ transport in reabsorptive sweat duct (RSD) using intracellular ion-sensitive microelectrodes (ISMEs). The mean value of [K⁺]_i in RSD is 79.8±4.1mm (n=39). Under conditions of microperfusion, the [K⁺]_i is above equilibrium across both the basolateral membrane, BLM (5.5 times) and the apical membrane, APM (7.8 times). The Na⁺/K⁺ pump inhibitor ouabain reduced [K⁺]_i towards passive distribution across the BLM. However, the [K⁺]_i is insensitive to the Na⁺/K⁺/2 Cl^– cotransport inhibitor bumetanide in the bath. Cl^– substitution in the lumen had no effect on [K⁺]_i. In contrast, Cl^– substitution in the bath (basolateral side) depolarized BLM from –26.0±2.6 mV to –4.7^*±2.4 mV (n=3;^* indicates significant difference) and decreased [K⁺]_i from 76.0±15.2mm to 57.7^* ±12.7mm (n=3). Removal of K⁺ in the bath decreased [K⁺]_i from 76.3±15.0mm to 32.3^*±7.6mm (n=4) while depolarizing the BLM from –32.5±4.1 mV to –28.3^*±3.0 mV (n=4). Raising the [K⁺] in the bath by 10-fold increased [K⁺]_i from 81.7±9.0mm to 95.0^*±13.5mm and depolarized the BLM from –25.7±2.4 mV to –21.3^*±2.9 mV (n=4). The K⁺ conductance inhibitor, Ba²⁺, in the bath also increased [K⁺]_i from 85.8±6.7mm to 107.0^*±11.5mm (n=4) and depolarized BLM from –25.8±2.2 mV to –17.0^*±3.1 mV (n=4). Amiloride at 10^–6 m increased [K⁺]_i from 77.5±18.8mm to 98.8^*±21.6mm (n=4) and hyperpolarized both the BLM (from –35.5±2.6 mV to –47.8^*±4.3 mV) and the APM (from –27.5±1.4 mV to –46.0^* ±3.5 mV,n=4). However, amiloride at 10^–4 m decreased [K⁺]_i from 64.5±0.9mm to 36.0^*±9.9mm and hyperpolarized both the BLM (from –24.7±1.4 mV to –43.5^*±4.2 mV) and APM (from –18.3±0.9 mV to –43.5^*±4.2 mV,n=6). In contrast to the observations at the BLM, substitution of K⁺ or application of Ba²⁺ in the lumen had no effect on the [K⁺]_i or the electrical properties of RSD, indicating the absence of a K⁺ conductance in the APM. Our results indicate that (i) [K⁺]_i is above equilibrium due to the Na⁺/K⁺ pump; (ii) only the BLM has a K⁺ conductance; (iii) [K⁺]_i is subject to modulation by transport status; (iv) K⁺ is probably not involved in carrier-mediated ion transport across the cell membranes; and (v) the RSD does not secrete K⁺ into the lumen.     

Binding of insulin to bovine liver plasma membrane. Use of insulin analogues modified at the A1 residue

Bovine liver plasma membranes [Rösen, Ehrich, Junger, Bubenzer & Kühn (1979) Biochim. Biophys. Acta 587, 593–605] show similar insulin-binding characteristics, as evaluated by Scatchard analysis, to those of membrane systems from other species. However, the dissociation rate of bound insulin cannot be accelerated by the addition of insulin, in contrast with membranes isolated from rat liver. The dissociation rate is strongly dependent on the pH. Although dependent on temperature, the total capacity of binding sites is minimally changed, but the number of high-affinity sites is increased 2–3-fold, by lowering the incubation temperature. These data might be interpreted by assuming a single population of receptors whose distribution between different affinity states depends on temperature. In competition studies, most of the modified insulins examined show a close correlation between binding, determined in plasma membranes from bovine liver, and biological activity, measured in adipocytes. The hypothesis that a positive charge on the A1 residue may be favourable for binding is supported by experiments with an isosteric pair of insulins modified at this residue ([carbamoyl-Gly^A1]- and [amidino-Gly^A1]insulin) and with modified insulins carrying one or more positive charges on the A1 residue ([Arg-Gly^A1]-, [Arg-Arg-Gly^A1]-, [Arg-Arg-Arg-Gly^A1]- and [Lys-Arg-Gly^A1]insulin). The latter insulin derivatives show a higher binding activity for plasma membranes from bovine, porcine and rat liver than expected from their biological activities in adipocytes.