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Biomedical subjects

E Sigel

Publications and source records attributed to E Sigel.

68 records · Page 4Linked to original sources
PubMed1984

The purified GABA/benzodiazepine/barbiturate receptor complex: four types of ligand-binding sites, and the interactions between them, are preserved in a single isolated protein complex.

A GABA/benzodiazepine/barbiturate receptor complex has been purified from bovine cerebral cortex by affinity chromatography on a benzodiazepine column. Depending on the detergent present during the isolation of the receptor (deoxycholate/Triton X-100 or CHAPS/Asolectin), and during the binding assays (Triton X-100 or CHAPS), the receptor displays different binding properties for the GABAA agonist [3H]muscimol and for the chloride ion channel blocking agent [35S]t-butylbicyclophosphorothionate (TBPS), whereas the binding properties for the benzodiazepine [3H]flunitrazepam are independent of isolation and assay conditions. Both methods of isolation yield a protein complex consisting of the same two subunits of Mr 53 000 and Mr 57 000. Therefore the different binding properties reflect different conformations of the isolated receptor protein. [3H]flunitrazepam binding to the CHAPS-purified receptor is stimulated by GABA and the barbiturate pentobarbital in a dose-dependent manner. Photo-affinity labeling of the purified receptor with [3H]flunitrazepam leads to incorporation of radioactivity into both subunits, but predominantly into the Mr 53 000 band, as shown by fluorography. Proteolytic degradation by trypsin of the isolated photo-affinity labeled receptor in detergent solution proceeds via a labeled Mr 48 000 polypeptide. Proteolytic destruction of the reversible [3H]flunitrazepam and [3H]muscimol binding activities requires greater than 100 fold higher concentrations of trypsin than the decomposition of the receptor polypeptides into fragments less than Mr 10 000.

Animals↗
PubMed1983

A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex.

The gamma-aminobutyric acid/benzodiazepine receptor from bovine cerebral cortex was solubilized with sodium deoxycholate and purified by affinity chromatography on benzodiazepine-agarose and ion exchange chromatography. The benzodiazepine binding protein was enriched 1800-fold. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of two major bands of Mr = 57,000 and 53,000. [3H]Flunitrazepam, after UV irradiation, was incorporated irreversibly into both bands of the isolated protein. A high affinity binding site for gamma-aminobutyric acid was co-purified with the benzodiazepine binding site and the two sites were shown to reside on the same physical structure. The dissociation constants were 10 +/- 4 nM for [3H] flunitrazepam and 12 +/- 3 nM for the gamma-aminobutyric acid agonist [3H]muscimol. The maximum specific activity for [3H] muscimol binding was 4.3 nmol/mg of protein. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was between 3 and 4. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 7.3 +/- 0.5 nm and a sedimentation coefficient of 11.1 +/- 0.3 S, respectively. The purified complex had a pharmacological profile that corresponds to the receptor specificity found in membranes and crude soluble extracts.

Animals↗
PubMed1981

Rotation of cytochrome oxidase in phospholipid vesicles. Investigations of interactions between cytochrome oxidases and between cytochrome oxidase and cytochrome bc1 complex.

Cytochrome oxidase was incorporated into lipid vesicles composed of phosphatidylethanolamine-phosphatidylcholine-cardiolipin. Large proteoliposomes of 1,000-15,000 A diameter were prepared by calcium-induced fusion of small vesicles. Rotational diffusion of cytochrome oxidase was measured by detecting the decay of the absorption anisotropy, r(t), after photolysis of the heme a3.CO complex by a vertically polarized laser flash. Because of the large size of the proteoliposomes, there was no contribution of vesicle-tumbling to r(t) over the experimental time range of 5 ms for samples in 60% sucrose. Analysis of r(t) curves was based on a "rotation-about-membrane normal" model. The measurements were used to investigate intermolecular interactions between cytochrome oxidases and between cytochrome oxidase and cytochrome bc1 complex co-reconstituted in the above lipid vesicles. In vesicles of a high lipid to protein ratio (congruent to 27), nearly all cytochrome oxidase molecules are rotating with an approximate rotational relaxation time, phi 1, on the order of 500 microseconds. In contrast, about 20% of cytochrome oxidase is immobile in vesicles with a relatively low lipid to protein ratio (congruent to 5), although phi 1 of the mobile population remains about 500 microseconds. In contrast, about 20% of cytochrome oxidase is immobile in vesicles with a relatively low lipid to protein ratio (congruent to 5), although phi 1 of the mobile population remains about 500 microseconds. The immobilized fraction is presumably due to nonspecific self-aggregation of cytochrome oxidase. The presence of cytochrome bc1 complex does not change r(t) curves significantly, either in the presence or absence of cytochrome c. Previously, we have observed the co-existence of mobile and immobile populations of cytochrome oxidase in bovine heart and rat heart mitochondria (Kawato, S., Sigel, E., Carafoli, E., and Cherry, R. J. (1980) J. Biol. Chem. 255, 5508-5510). The present results suggest that the immobile population of about one-half of cytochrome oxidase could be simply due to nonspecific protein aggregation resulting from the high concentration of enzymes in the inner mitochondrial membrane (lipid to protein ratio, less than or equal to 0.5). We also conclude that there is no specific interaction between cytochrome oxidase and cytochrome bc1 complex in the above large lipid vesicles. A lateral collision-controlled model for electron transfer from cytochrome bc1 complex to cytochrome oxidase through cytochrome c is discussed based on the above results.

Animals↗
PubMed1980

Cytochrome oxidase rotates in the inner membrane of intact mitochondria and submitochondrial particles.

A transient dichroism is detected after photolysis by a linearly polarized laser flash of the cytochrome oxidaseCO complex in bovine heart mitochondria, rat heart mitochondria, and bovine heart submitochondrial particles. A decay in the absorption anisotropy is characterized by a time constant of about 300 to 400 mus in both mitochondria and submitochondrial particles. Since vesicle tumbling in the time range less than 5 ms can be excluded in these experiments, we conclude that cytochrome oxidase rotates in the mitochondrial membrane with a relaxation time of several hundred microseconds. However, it is likely that only about one-half of cytochrome oxidase contributes to the observed decay, the remainder being relatively immobile.

Animals↗
PubMed1980

Quantitative analysis of the proton and charge stoichiometry of cytochrome c oxidase from beef heart reconstituted into phospholipid vesicles.

The proton and charge stoichiometry of cytochrome c oxidase reconstituted into phospholipid vesicles has been analysed using a fast responding oxygen electrode and a system for the simultaneous measurement of H+, K+ or lipophilic cations, and O2. From initial rate measurements after addition of reductant (ascorbate /N,N,N',N'-tetramethyl-p-phenylenediamine) the following stoichiometries could be extrapolated: K+/e-, between 1.85 and 2.05, H+/e-, between 0.58 and 0.75. Lipophilic cations can replace valinomycin/potassium as the charge . compensating system. Net H+ extrusion was observed for up to 11 turnovers of the enzyme (11 O2/cytochrome aa3). The variation of the internal pH buffering capacity of the vesicles has an influence on the number of turnovers during which net proton translocation can be observed, but has no influence on the determined stoichiometries.

Animals↗
PubMed1979

The charge stoichiometry of cytochrome c oxidase in the reconstituted system.

Purified cytochrome c oxidase was reconstituted into phospholipid vesicles having high internal pH buffering capacity. In the presence of valinomycin, 2 K+ ions were taken up by the vesicles per electron transferred from cytochrome c to oxygen. The charge stoichiometry of 2 was obtained from simultaneous measurement of changes of K+, H+, and oxygen in the medium after addition of the reductant ascorbate/TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine). The changes in oxygen concentration were measured with a fast responding oxygen electrode (90% response time, 0.4 s). The existence of a proton pump in cytochrome c oxidase could thus be confirmed, and its charge stoichiometry measured, in a reconstituted system uncomplicated by other respiratory chain components.

Animals↗
PubMed1978

The proton pump of cytochrome c oxidase and its stoichiometry.

The operation of cytochrome c oxidase with ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine as substrate in antimycin-A-inhibited rat liver mitochondria is coupled to proton ejection. Measurements of the initial rate of valinomycin-dependent K+ uptake have shown that nearly 4 K+ are taken up as 2 electrons are transferred from cytochrome c to oxygen. This proves directly that a charge separation of nearly 4 occurs across the inner mitochondrial membrane each time 2 electrons are transferred to oxygen. Measurements of the initial rate of proton movement after addition of the reductant show that about 1.6 protons are released by the mitochondria as 2 electrons are transferred from cytochrome c to oxygen. The data support the suggestion of a proton pump coupled to the operation of cytochrome c oxidase [Wikström, M. F. K. (1977) Nature (Lond.) 266, 271--273].

Animals↗
PubMedDate not supplied

Functional modulation of ligand-gated GABAA and NMDA receptor channels by phosphorylation.

Ion channels are ubiquitous membrane proteins that may be organized into different families according to their predicted transmembrane topology. They are concerned with rapid signalling over plasma and intracellular membranes and are activated, depending on their type, by transmembrane voltage, intracellular second messengers or extracellular neurotransmitters. Intracellular activities of protein kinases and phosphatases act to modulate ion channel activity. The modulation of the function of ligand activated, neuronal ion channels, that are crucial for synaptic transmission, may be an important basis for a modulation of a synaptic efficiency. The following review concentrates, due to space limitations, on the postranslational modification, and on the modulation of the function by protein kinase C and protein kinase A, of ligand-gated GABAA channels and NMDA channels on a molecular level.

Animals↗