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Biomedical subjects

A Darszon

Publications and source records attributed to A Darszon.

At least 73 records · Page 4Linked to original sources
PubMed1989

Incorporation of ionic channels from yeast plasma membranes into black lipid membranes.

Recently, patch-clamping of yeast protoplasts has revealed the presence of plasma membrane K+ channels (Gustin, M. C., B. Martinac, Y. Saimi, M. R. Culberston, and C. Kung. 1986. Science (Wash. DC). 233:1195-1197). In this work we show that fusion of purified plasma membranes into planar bilayers allows the study of the yeast channels. The main cationic conductances detected were of 64 and 116 pS, however, larger and smaller conductances have been observed. The two main conductances were sensitive to the K+ channels blockers tetraethylammonium (TEA+) and Ba2+. Bionic experiments indicated that both conductances were K+ selective.

Cell Membrane↗
PubMed1988

Sea urchin sperm head plasma membranes: characteristics and egg jelly induced Ca2+ and Na+ uptake.

Sea urchin sperm respond to egg factors with changes in the ionic permeability of their plasma membrane. It has been previously shown that plasma membranes isolated preferentially from sea urchin sperm flagella respond to egg jelly increasing their Ca2+ and Na+ uptake (Darszon et al. (1984) Eur. J. Biochem. 144, 515-522). However, the egg jelly induced acrosome reaction occurs in the sperm head, and there is evidence for an heterogeneous distribution of plasma membrane components within the various regions of this cell. We here report a method for purifying sperm head membranes using positively charged beads according to Jacobson (1977) Biochim. Biophys. Acta 471, 331-335). Under the transmission electron microscope these membranes appeared homogeneous and apparently free of internal membranes. The yield of the preparation was 0.9% of the total protein in the sperm homogenate. The preparation contained less than 5% of the mitochondrial marker cytochrome oxidase, and 10% of the total DNA/mg protein. Surface labeling with 125I indicated a 2.5-3-fold enrichment in specific activity of the head membranes with respect to whole sperm. The SDS band pattern and the lipid composition of this preparation were different from those of isolated flagellar membranes. Phosphatidylcholine was higher in the head membranes, while phosphatidylserine and phosphatidylethanolamine were lower. The head membranes displayed a 1.7-2.3-fold higher Ca2+-ATPase activity and a 2.5-fold lower Na+/K+-ATPase activity, than the flagellar membranes. These results are consistent with a heterogeneous distribution of membrane components along the sea urchin sperm plasma membranes. Isolated head membranes sonicated in the presence of soybean phospholipid liposomes responded to egg jelly with a species-specific increase in Ca2+ and Na+ uptake. As in whole sperm, Ca2+ uptake was inhibited by the Ca2+ channel blocker nisoldipine. A close analog of this compound, [3H]nitrendipine, binds with high affinity to head membranes in a saturable, reversible manner, showing a Kd and Bmax of 31 nM and 5.3 pmol/mg protein, respectively.

Animals↗
PubMed1988

Transfer of spores, bacteria and yeast into toluene containing phospholipids and low amounts of water: preservation of the bacterial respiratory chain.

A method that allows the transfer of spores, bacteria and yeasts into a ternary system composed of toluene, phospholipids and low amounts of water is described. Initially an emulsion is formed by sonication of cells suspended in water in presence of toluene and phospholipids. The emulsion formed was subsequently clarified by blowing N2 on its surface and transparency was achieved when the water content of the system was reduced to 1-3 microliter/ml of organic solvent. The cells in ternary systems exhibit a reduction of cell volume, but the general structure is preserved. About 1/10,000 bacilli or yeast were viable, but spores were viable after 30 days in the ternary system. Yeast cells transferred to the ternary system, and back to an all water media failed to show oxygen uptake. In contrast bacilli that remained in the ternary system for 2 days respired to 80% of their maximal capacity when returned to an aqueous media.

Bacillus cereus↗
PubMed1987

Single-channel activity in sea urchin sperm revealed by the patch-clamp technique.

Ionic fluxes are deeply involved in the response of spermatozoa to the egg. Using the patch-clamp technique, we show for the first time single ion channel activity in sea urchin spermatozoa and spermatozoa heads. Due to their small size gigaseals were obtained in suspended cells by applying suction through the pipette. The rate of gigaseal formation was very low and improved to 6% (n = 1145) when flagella were detached from sperm. Current-voltage curves created from single-channel events showed conductances of approx. 65 and 170 pS, suggesting the presence of two types of channels. At least one appears to be a K+ channel.

Acrosome↗
PubMed1987

A fast transient hyperpolarization occurs during the sea urchin sperm acrosome reaction induced by egg jelly.

The egg jelly-induced sea urchin sperm depolarization, assayed with the membrane potential-sensitive dye diS-C3-(5), is preceded by a fast quenching, which was initially taken as an artifact. Here we show that part of this quenching results from a K+-dependent transient hyperpolarization (approximately 4 s). Seawater containing 25-35 mM KCl inhibits this hyperpolarization, the depolarization and the acrosome reaction induced by egg jelly, or by the acrosome reaction inducing factor purified therefrom. These results suggest that egg jelly induces a transient hyperpolarization mediated by an increase in K+ permeability, which may be involved in triggering the acrosome reaction.

Acrosome↗
PubMed1987

Internal pH can regulate Ca2+ uptake and the acrosome reaction in sea urchin sperm.

The egg jelly-induced acrosome reaction of sea urchin sperm is accompanied by intracellular alkalinization and Ca2+ entry. We have previously shown that in the absence of egg jelly, NH4Cl, which increases intracellular pH (pHi), induces Ca2+ uptake and the acrosome reaction in sperm of the sea urchin, Strongylocentrotus purpuratus. Here we show that at a constant concentration of NH4Cl (20 mM) in seawater, sperm react less as external pH is lowered from the normal 8 to 7.25. The pH dependence of the NH4Cl response is not very sensitive to temperatures between 12 and 17 degrees C. NH4Cl (15-50 mM) stimulates Ca2+ uptake and acrosome reactions in sperm suspended in Na+-free seawater, a condition known to inhibit the inductive effect of jelly. Jelly does not further stimulate Ca2+ uptake of sperm preincubated in NH4Cl, indicating that once the permeability to Ca2+ is increased by raising the pHi, the jelly has no further effect. We have used the membrane potential-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide to follow the membrane potential change that occurs when NH4Cl is added. Depolarization (25 mV) is associated with the acrosome reaction when either the natural inducer, egg jelly, or NH4Cl is added to sperm. Response to both inducers is inhibited under conditions known to abolish the acrosome reaction, i.e., low-pH seawater and nisoldipine. These results indicate that the NH4Cl-induced depolarization that accompanies the reaction is probably due to the opening of channels that allow Ca2+ to enter the cell and not to the depolarization by NH4+ ions. High-K+ seawater, which depolarizes sperm, and tetraethylammonium, a K+ channel blocker, inhibit the jelly-induced depolarization and the acrosome reaction, but do not inhibit NH4Cl-induced changes. It has already been shown that nigericin promotes Ca2+ entry and the acrosome reaction in sea urchin sperm. We found that the action of this ionophore depends on the pH of normal seawater. In the absence of external Na+ (replaced by choline), nigericin does not induce the reaction and does not stimulate Ca2+ uptake.

Acrosome↗
PubMed1986

Simultaneous synthesis and hydrolysis of ATP regulated by the inhibitor protein in submitochondrial particles.

Coupled submitochondrial particles from bovine heart with ATP synthases devoid of control by the inhibitor protein of Pullman and Monroy [J. Biol. Chem. 238, 3762-3769 (1963)] can be prepared by incubation of Mg-ATP particles in 50 mM phosphate, 250 mM sucrose, and greater than 95% D2O (pD 7.8) at 38 degrees C. As monitored with oxonol, the respiring particles build up and maintain a delta psi about 5-10% lower than that of the starting preparation. With oligomycin delta psi of the two preparations is the same. In the presence of an ATP trap (hexokinase and glucose), the two types of particles carry out oxidative phosphorylation at comparable rates. Low concentrations of oligomycin induce a small enhancement of the rate of ATP synthesis in non-controlled particles. In the absence of an ATP trap, net accumulation of ATP, as driven by electron transport in particles without control by the inhibitor protein, is low. Apparently this is due to lack of control by the inhibitor protein of ATP hydrolysis that occurs during oxidative phosphorylation.

ATP Synthetase Complexes↗
PubMed1986

Thermostability of membrane systems in organic solvents.

Two multisubunit enzymes of the inner mitochondrial membrane, cytochrome oxidase and the H+-ATPase may be transferred into highly apolar solvents as protein-lipid complexes. At 70 degrees C and an initial water concentration of 13 microliters per ml organic solvent (toluene), the half-life of the ATPase was approx. 11 h, whereas that of cytochrome oxidase was about 100 s. Thermostability of cytochrome oxidase could be increased more than 100-times by decreasing the water concentration to 3 microliters per ml toluene. At this latter concentration of water the half-life of the ATPase at 90, 80 and 70 degrees C was 5, 48 and 96 h, respectively.

Animals↗
PubMed1985

Extraction of mitochondrial membrane proteins into organic solvents in a functional state.

Protein-lipid complexes in organic solvents can be used as the starting material in the reassembly of functional planar and spherical bilayers (Montal, M., Darszon, A. and Schindler, H. (1981) Q. Rev. Biophys. 14, 1-79). The transfer of three enzymes of the inner mitochondrial membrane into organic solvents as protein-lipid complexes has been studied to understand better the extraction process. The enzymes studied were cytochrome c oxidase, ATPase and succinate dehydrogenase. These enzymes were transferred into hexane and diethyl ether in an active state, however, the activities extracted varied quantitatively, depending on the amount of protein of the starting preparation, the concentration of phospholipids and the cation employed. In all conditions cytochrome c oxidase was extracted with the highest yield and specific activity, and it was actually enriched in the organic extract. The values for succinate dehydrogenase and ATPase were lower, but their specific activities were similar to those of the starting material. This indicates that some membrane proteins are preferentially extracted into organic solvents in a functional state. The enzymes, as protein-lipid complexes, are fairly stable in organic solvents; in a month of storage at 4 degrees C in hexane some enzymes loose less than 50% of their activity.

Adenosine Triphosphatases↗
PubMed1985

High pH-induced acrosome reaction and Ca2+ uptake in sea urchin sperm suspended in Na+-free seawater.

The egg jelly-induced acrosome reaction of sea urchin sperm requires the presence of Ca2+ and Na+ in seawater at its normal pH 8. Sperm suspended in seawater at pH 9 undergo the acrosome reaction in the absence of jelly. We have attempted to understand the role of external Na+ in this reaction. Sperm were suspended in Na+-free seawater and the percentage of acrosome reaction and the amount of Ca2+ uptake were determined as a function of external pH. High pH (9.0) in Na+-free medium without jelly triggered a high percentage (above 65%) of sperm acrosome reactions and a two to fourfold increase in Ca2+ uptake. Both the percentage of acrosome reactions and the amount of Ca2+ uptake were similar to those induced by either jelly or pH 9 in Na+-containing seawater. On the other hand, the absence of Na+ in seawater inhibits jelly from inducing Ca2+ uptake and acrosome reactions at pH 8.0 and even at pH 8.5. These results indicate that the Na+ requirement for the acrosome reaction induced by jelly is lost when triggering is by high pH. In contrast, Ca2+ was strictly required since sperm did not react in Ca2+-free seawater at pH 9. We also found that like the jelly-induced acrosome reaction the high-pH-induced acrosome reaction and Ca2+ uptake in complete and Na+-free seawater were inhibited by D600. This finding suggests that the same transport system for Ca2+ uptake associated with the acrosome reaction operates at both triggering conditions, i.e., jelly or pH 9. Although D600 is not now considered a specific blocker, its effect has suggested the involvement of Ca2+ channels in the acrosome reaction. This proposal is supported by our results with nisoldipine, a highly specific inhibitor of calcium channels. The drug inhibited both the sperm acrosome reaction and Ca2+ uptake induced by jelly or pH 9 in complete seawater.

Acrosome↗
PubMed1985

Single-channel activity of bilayers derived from sea urchin sperm plasma membranes at the tip of a patch-clamp electrode.

Changes in the plasma membrane permeability of echinoderm sperm play a fundamental role in the acrosome reaction. During the reaction there is an increase in intracellular Ca2+ and Na+ and an efflux of H+ and K+. We have formed bilayers at the tip of patch pipets from a mixture of lipid vesicles and sea urchin sperm plasma membranes (12-50 microgram protein/ml). We observed three types of K+ channels (conductances: 22, 46, and 82 pS), two of which are partially blocked by TEA, and one Cl- channel (148 pS). The presence of K+ channels in sperm plasma membranes is consistent with the inhibition by TEA of the acrosome reaction in whole sperm and the membrane potential change that occurs during the reaction.

Acrosome↗
PubMed1984

Response of isolated sperm plasma membranes from sea urchin to egg jelly.

The acrosome reaction in sea urchin sperm is induced by a glycoprotein jelly surrounding the egg and is accompanied by changes in ion permeability of sperm plasma membrane. In an attempt to learn what membrane components are involved in the response to jelly, we have begun to reassemble sperm membrane components into artificial membranes and assay for permeability changes mimicking those that occur in sperm. Jelly in sea water at concentrations that induce the acrosome reaction did not significantly change 45Ca2+ uptake of sonicated unilamellar vesicles made with soybean lipid only (ratio jelly:control uptake = 1.08 +/- 0.36 SD, n = 21). Experiments with pure lipid planar bilayers made with soybean lipid or a lipid extract from sperm and held at various voltages, also did not reveal substantial permeability changes at comparable jelly concentrations. Thus, jelly by itself does not change the conductance of a pure lipid bilayer. In contrast, significant (P----0.0005, t test for two sample means) 45Ca2+ uptake was observed with vesicles made by cosonicating soybean phospholipids and Strongylocentrotus purpuratus sperm membranes isolated by the method of Cross, N. L. [1983, J. Cell Sci. 59, 13-25] (ratio jelly: control uptake = 1.51 +/- 0.75, n = 20, 16 positive out of 20 experiments). The calcium uptake response of the mixed vesicles was also species-specific: it did not occur with jelly from Arbacia punctulata (ratio Arbacia jelly: control = 1.18 +/- 0.51; ratio Strongylocentrotus jelly: control = 1.71 +/- 0.97, n = 10; P----0.025, paired t statistic). Vesicles made with soybean lipid and an octyl glucoside extract of sperm membranes also responded to jelly with increased 45Ca2+ uptake. Our results indicate that we have the starting conditions to isolate and characterize the sperm membrane components that participate in the egg jelly induced permeability changes.

Animals↗
PubMed1984

Release of the inhibitory action of the natural ATPase inhibitor protein on the mitochondrial ATPase.

The rate of ATP hydrolysis by submitochondrial particles prepared from bovine heart mitochondria in the presence of Mg2+ and ATP increases from a value of 0.4 mumol min-1 mg-1 to 6-7 mumol min-1 mg-1 upon incubation for 5-6 h at 38 degrees C. The increase in activity does not occur in particles that have been passed through a Sephadex column. The activation is prevented and partially reversed by ATP. This indicates that the increase in hydrolytic activity is due to abolition of the inhibitory action of the natural ATPase inhibitor protein of Pullman and Monroy [(1963) J. Biol. Chem. 238, 3762-3769]. At maximal activation approximately 50% of the inhibitor protein of the starting preparation remains in the particles as inferred from direct assay of inhibitor protein content and by its interaction with 125I-labeled antibodies directed against the inhibitor protein. The extent of the activation, which presumably is an index of the equilibrium between active and inactive enzymes, is strictly dependent on salts. The rate of the activation depends on the concentration of salts and is favored by alkaline pH. From results of experiments on the effect of temperature on the rate of activation of the ATPase, it was calculated that the activation energy, delta H not equal to and delta S not equal to of the process were 53.34 kJ/mol, 50.83 kJ/mol and -158.99 J mol-1 K-1, respectively. The data indicate that in its native inhibiting state, the interaction of the inhibitor protein with the enzyme involves electrostatic interactions. Also it is concluded that abolition of the inhibitory action of the protein on ATPase activity is not compulsorily linked to release of the protein into the water space.

Adenosine Triphosphate↗
PubMed1984

Oxidative phosphorylation and the Pi-ATP exchange reaction of submitochondrial particles under the influence of organic solvents.

The effect of some organic solvents, in particular dimethyl sulfoxide, on oxidative phosphorylation, the Pi-ATP exchange reaction, and ATP hydrolysis has been studied in submitochondrial particles from bovine heart. The three reactions are inhibited by dimethyl sulfoxide, but with a different sensitivity. Oxidative phosphorylation takes place at considerable rates in the presence of concentrations of dimethyl sulfoxide that completely inhibit the Pi-ATP exchange reaction. The sensitivity of the Pi-ITP exchange reaction to dimethyl sulfoxide measured in the presence of electron transport is also higher than that of oxidative phosphorylation. Dimethyl sulfoxide inhibits an electrochemical H+ gradient-dependent stimulating effect of inorganic phosphate on ATP hydrolysis which is linked to the Pi-ATP exchange reaction. In the absence of phosphate or in the presence of phosphate plus uncoupler, dimethyl sulfoxide does not inhibit hydrolysis. These findings, together with the observation that electron transport increases the rate of the Pi-ITP exchange reaction, suggest that electrochemical H+ gradients modify the kinetic characteristics of particulate Fi-ATPase. Glycerol and methanol, but not dimethylformamide, induce effects similar to those of dimethyl sulfoxide on oxidative phosphorylation and the Pi-ATP exchange reaction. This suggests that the described effects of solvents could be due to alterations of water structure.

Adenosine Triphosphate↗
PubMed1983

A small angle x-ray scattering study of a rhodopsin-lipid complex in hexane.

A small angle x-ray scattering study of a rhodopsin-lipid complex in hexane demonstrates the presence of two distinct particle populations with corresponding radii of gyration of approximately 22 and 160 A. A model based on the existence of inverted lipid micelles surrounding the protein polar moieties while the protein hydrophobic domains act as cross-links between the inverted micelles is presented. It accounts for the results herewith reported and explains most of the properties hitherto characterized for the rhodopsin-lipid complex in hexane.

Animals↗
PubMed1982

On the function of the natural ATPase inhibitor protein in intact mitochondria.

The recently described methodology to extract the mitochondrial ATPase along with other mitochondrial proteins into organic solvents, and their subsequent incorporation into liposomes [Eur. J. Biochem. (1982) 121, 427-433] has been employed to estimate the number of ATPases that contain the natural ATPase inhibitor protein in its inhibitory site in intact mitochondria incubated in various metabolic states. It was found that in the presence of electrochemical gradients about 50% of the ATPases are without inhibitor protein in its inhibitory site (active ATPases). In the transition from state 4 to state 3 the percentage of active ATPases diminishes from about 50% to approximately 20%. This indicates that during steady-state phosphorylation only a limited number of ATPases are in the active catalytic state, and that not only during active hydrolysis does the inhibitor protein interact with its inhibiting site; rather the inhibitor seems to interact with an intermediate state of the enzyme that appears either during the synthetic or hydrolytic reactions. In addition it was found that ATP, with or without uncoupler, induces the interaction of the inhibitor protein in more than 80% the ATPases through an oligomycin-sensitive process. Thus, notwithstanding other factors the interaction may account for the low hydrolytic activity of mitochondria.

Adenosine Triphosphatases↗