[en] This paper continues studies of physico-chemical properties of vesicle-bound flavins. Based on previous results, an advanced model system was designed in order to study the mechanisms underlying bluelight-induced redox transport across artificial membranes. The lumen of single-shelled vesicles was charged with cytochrome c, and amphiphilic flavin (AF1 3, AF1 10) was bound to the membrane. Upon bluelight irradiation redox equivalents are translocated from exogeneous 1e-(EDTA)-and 2e-(BH3CN-) donors across the membrane finally reducing the trapped cytochrome c both under aerobic and anaerobic conditions. The mechanisms involved are explored and evidence for the involvement of various redox states of oxygen, dihydroflavin and flavosemiquinone is presented

[en] The modification of the ultraviolet blocking of sodium channels and of the ultraviolet-induced potential shift of the gating parameters by means of the sulfhydryl compounds l-cysteine and 2-mercaptoethanol was investigated in the node of Ranvier of Rana esculenta under voltage-clamp conditions. The UV wavelength was 280 nm. The radiation-induced potential shift of the voltage-dependent gating parameters was prevented or even reversed by the action of the sulfhydryl compounds (internal application), while the blocking effect was not affected. It is concluded that the two radiation effects are caused by two separate photoreactions. Internally applied N-ethylmaleimide, binding specifically to protein-SH groups, exhibits an effect similar to the ultraviolet-induced potential shift, without affecting the maximum sodium permeability. Therefore, the ultraviolet-induced potential shift might be caused by a photocatalyzed oxidation of -SH groups of membrane proteins changing the surface charge density at the inner side of the nodal membrane

[en] The dependence of the rate constant of Rb⁺ efflux on extracellular cation concentration was measured. At low ionic strengths Rb⁺ efflux increased strongly. Permeability coefficients were calculated from the rate constants measured, using the Goldman flux equation, with and without making allowance for surface potentials. Only when allowance was made for surface potentials and the associated differences between ion concentrations in the bulk solutions and at the membrane surface, the permeability coefficient remained constant. Best agreement between experimental data and theoretically calculated values was obtained when an interior surface potential of -110 mV was assumed. When the surface charge of erythrocytes is reduced by neuraminidase, the rate constants for Rb⁺ efflux decreased, indicating a significant influence of surface potential

[en] Sulfate uptake by rabbit ileal brush border membrane vesicles was stimulated by a transmembrane sodium gradient, but not by a similar potassium gradient. ³⁵SO₄(⁺²) influx (JSO4 oi) from outside (o) to inside (i) these vesicles was a hyperbolic function of [SO₄⁺²]o and the affinity constant for anion transport was strongly influenced by [Na⁺]o. JSO₄ oi was a sigmoidal function of [Na⁺]o at pH 7.4 for both low (0.2 M) and high (4.0 mM) [SO₄⁺²]o. The Na⁺-dependency of JSO₄ oi was examined at pH 6.0, 7.4, and 8.0 (same pH inside and outside). When a H⁺ gradient was imposed across the vesicle wall (pHi . 8.0, pHo . 6.0), Na⁺-dependent JSO₄ oi was hyperbolic and significantly increased at each [Na⁺]o over values observed using bilateral pH 8.0. In contrast, a H⁺ gradient oriented in the opposite direction (pHi . 6.0, pHo . 8.0) led to Na⁺-dependent JSO₄ oi that was sigmoidal and significantly lower at each [Na⁺]o than values found using bilateral pH 6.0. Electrogenicity of JSO4 oi at pH 8.0 for both high and low [Na+]o was demonstrated by using a valinomycin-induced transmembrane electrical potential difference. A model is proposed for proton regulation of sodium sulphate cotransport where flux stoichiometry is controlled by [H⁺]i and sodium binding affinity is modified by [H⁺]o. Preliminary experiments with rabbit proximal tubular brush border membrane vesicles disclosed similar JSO₄ oi kinetic properties and a common transport mechanism may occur in both tissues

[en] Glycophorin A, a major glycoprotein of the erythrocyte membrane, has been incorporated into small unilamellar vesicles composed of a variety of pure and mixed phospholipids. Nuclear spin labels including 31P and 19F have been used at natural abundance or have been synthetically incorporated in lipids to act as probes of lipid-protein interaction. Interactions produce broadening of resonances in several cases and it can be used to demonstrate preferential interaction of certain lipids with glycophorin. 31P and 19F probes show a strong preferential interaction of glycophorin with phosphatidylserine over phosphatidylcholine. There is some evidence that interactions are more pronounced at the inner surface of the bilayer and these results are rationalized in terms of the asymmetric distribution of protein and lipid

[en] Purified rat peritoneal and pleural mast cells preincubated briefly with radioactively labeled fatty acid were treated with A23187, which bypasses primary receptors in stimulating exocytosis. An enhanced incorporation of fatty acid into phosphatidyl choline (PC) that occurred in parallel with histamine release at 24-25 degrees C was observed and was initially proportional to the total amount of histamine discharged. Enhanced PC labeling and histamine secretion were also proportional at temperatures ranging from 17-37 degrees C. Both radioactive linoleic and palmitic acids were incorporated selectively at the beta-position of the glycerol backbone of PC. PC labeling by [3H]choline was not detectably different in control and stimulated cells, and phosphatidic acid did not exhibit selectively enhanced beta-acylation. Thus, the stimulated labeling in A23187-treated cells may occur secondary to the action of a phospholipase A2 that favors PC as a substrate. Other peritoneal cell types exhibit a very similar A23187-stimulated selective labeling of PC. Therefore, autoradiography has been used to provide a direct demonstration that in purified preparations, mast cells are the principal cell type engaged in A23187-elicited incorporation of fatty acid into PC. The efficacy of this approach has relied on special procedures devised to obtain significantly different autoradiographic grain densities between control and stimulated preparations that can be attributed to differences in the level of [3H]palmitate-labeled PC. Preliminary tests using compound 48/80 as a secretory stimulus for mast cells have identified a similar selectively enhanced PC labeling. In either case, however, consideration of possible relationships between PC metabolism and the secretory process are premature since they have not been tested directly

[en] Stimulations or inhibitions by various agents of ⁴⁵Ca efflux from prelabeled cells or tissues display distinct and reproducible profile patterns when the results are plotted against time as fractional efflux ratios (FER). FER is the fractional efflux of ⁴⁵Ca from stimulated cells divided by the fractional efflux from a control unstimulated group. These profile patterns fall into three categories: peak patterns, exponential patterns, and mixed patterns. Each category can be positive (stimulation) or negative (inhibition). The interpretation of these profiles is difficult because ⁴⁵Ca efflux depends on three variables: the rate of calcium transport out of the cell, the specific activity of the cell compartment from which the calcium originates, and the concentration of free calcium in this compartment. A computer model based on data obtained by kinetic analyses of ⁴⁵Ca desaturation curves and consisting of two distinct intracellular pools was designed to follow the concentration of the traced substance (⁴⁰Ca), the tracer (⁴⁵Ca), and the specific activity of each compartment before, during, and after the stimulation or the inhibition of calcium fluxes at various pool boundaries. The computer model can reproduce all the FER profiles obtained experimentally and bring information which may be helpful to the interpretation of this type of data. Some predictions of the model were tested experimentally, and the results support the views that a peak pattern may reflect a sustained change in calcium transport across the plasma membrane, that an exponential pattern arises from calcium mobilization from an internal subcellular pool, and that a mixed pattern may be caused by a simultaneous change in calcium fluxes at both compartment boundaries

[en] Triphenylmethylphosphonium (TPMP+) partitions into the mitochondrial and cytosolic compartments in the rat white adipocyte in a potential-dependent fashion. The relationship between [³H]TPMP+ distribution, intracellular cAMP generation and lipolysis in response to hormones and cAMP-mimetic compounds was examined. Half-maximal [³H]TPMP+ efflux and glycerol release were produced by 15 and 9 nM adrenocorticotropin, 170 and 110 nM 1-epinephrine, 70 and 27 microM isobutylmethylxanthine and 800 and 750 microM dibutyryl cAMP, respectively. Hormone-stimulated cAMP generation was also correlated with [³H]TPMP+ efflux and lipolysis in terms of concentration dependency. In kinetic experiments, glycerol release and [³H]TPMP+ efflux in response to adrenocorticotropin or cholera toxin proceeded over a similar time course, whereas an earlier rise in cAMP generation was detected. The depolarizing effect of lipolytic compounds was localized to the mitochondrial compartment. When cells were incubated in elevated-[K+]0 buffer, the stimulatory effect of dibutyryl cAMP on [³H]TPMP+ efflux and lipolysis persisted, suggesting that maintenance of the plasma membrane potential is not critical for demonstration of these responses. When the extracellular concentration of serum albumin, which provides binding sites for free fatty acids, was increased from 1 to 3%, an increase in glycerol release and a decrease in [³H]TPMP+ efflux was observed. We suggest that intracellular free fatty acid accumulation in response to lipolytic agents causes dissipation of the mitochondrial membrane potential and efflux of [³H]TPMP+ from the organelle and cell

[en] According to the conventional carrier model, an inhibitor bound at the substrate transfer site inhibits competitively when on the same side of the membrane as the substrate, but noncompetitively when on the opposite side. This prediction was tested with the nonpenetrating choline analog dimethyl-n-pentyl (2-hydroxyethyl) ammonium ion. In zero trans entry and infinite trans entry experiments, where the labeled substrate and the inhibitor occupy the same compartment, the inhibition was competitive, but in zero trans exit it was noncompetitive, in accord with the model. Similar behavior was seen with dimethyl-n-decyl (2-hydroxyethyl) ammonium ion. With this property of the choline transport system established, it becomes possible to estimate the relative affinity inside and outside of inhibitors present on both sides of the membrane. The tertiary amine, dibutylaminoethanol, which enters the cell by simple diffusion, is such an inhibitor. Here the inhibition kinetics were the reverse of those for nonpenetrating inhibitors; zero trans and infinite trans exit was inhibited competitively, and zero trans entry noncompetitively. It follows that dibutylaminoethanol binds predominantly to the inner carrier form

[en] The transport of [³H] 1,L 5-formyltetrahydrofolate, [³H] folic acid, and [³H]methotrexate by L1210 cell plasma membrane vesicles exhibited multicompartmental behavior. Two separate vesicular compartments (parallel relationship) of approximately equal volume were revealed during measurements of influx and efflux. Flux in one compartment was rapid, saturable, highly temperature-sensitive, and inhibited by pCMBS. Flux in the other compartment exhibited all of the characteristics of passive diffusion. These results imply that our plasma membrane vesicle preparations consist of a mixture of two functional species. Transport of folate into one of these species occurs by passive diffusion alone, whereas transport into the other kind of vesicle occurs by both passive diffusion and carrier-facilitated transport