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K J Angelides

Publications and source records attributed to K J Angelides.

At least 55 records · Page 3Linked to original sources
PubMed1986

Hyperoxia reduces plasma membrane fluidity: a mechanism for endothelial cell dysfunction.

To evaluate the relative contributions of three possible mechanisms that can be advanced to explain the observation that hyperoxia decreases serotonin uptake by endothelial cells, we examined the effect of high O2 tensions on Na+-K+-ATPase activity, ATP content, and plasma membrane fluidity in cultured endothelial cells. Confluent monolayers of pulmonary artery and aortic endothelial cells were exposed to 95% O2 (hyperoxia) or 20% O2 (controls) in 5% CO2 at 1 ATA for 4-42 h. Exposure to high O2 tensions had no effect on Na+-K+-ATPase activity or ATP content in pulmonary artery or aortic endothelial cells in culture. However, hyperoxia decreased the fluidity of the plasma membrane of pulmonary artery and aortic endothelial cells in culture, and the time course for the decrease in fluidity parallels that of the hyperoxic inhibition of serotonin transport. These results indicate that hyperoxia decreases fluidity in the hydrophobic core of the plasma membranes of cultured endothelial cells. Such decreases in plasma membrane fluidity may be responsible for hyperoxia-induced alterations in membrane function including decreases in transmembrane transport of amines.

Adenosine Triphosphate↗
PubMed1986

Glucocorticoid-mediated alteration of fluidity of brush border membrane in rat small intestine.

The normal biophysical properties of the maturing mammalian enterocyte membrane are poorly understood. While the effects of glucocorticoids on maturation of intestinal enzyme function has been intensively investigated, their effects on membrane biophysical properties are not known. We used 1,6-diphenyl-1,3,5-hexatriene as a probe in fluorescence anisotropy studies to determine the fluidity of rat brush border membrane. Maturational changes and the effects of glucocorticoids administered antenatally or postnatally were determined. Fluorescence anisotropy values for 1,6-diphenyl-1,3,5-hexatriene in mature brush border membranes were higher than those values in membranes obtained from younger animals reflecting a less fluid membrane. Glucocorticoids administered to suckling rats increased the anisotropy values of 1,6-diphenyl-1,3,5-hexatriene in the membranes compared to saline-administered littermates. The anisotropy of intestinal brush border membrane was also increased in fetal rats whose mothers received dexamethasone. These alterations may relate to protein-binding properties and permeability characteristics of the enterocyte membrane.

Animals↗
PubMed1985

Physicochemical characterization of the alpha-peptide of the sodium channel from rat brain.

The alpha-peptide of the rat brain sodium channel of apparent molecular weight 260K has been purified to homogeneity in order to determine its structural and chemical properties. By negative-stain electron microscopy, the molecule morphology of the solubilized channel protein appears as a stack of disks or rouleaux whose dimensions are 40 A X 200 A. Measurement of the secondary structure by circular dichroism shows that the alpha-peptide is a conformationally flexible polypeptide that contains mostly beta-sheet and random-coil in mixed detergent-phospholipid micelles and folds into a conformation that has approximately 65% alpha-helix after reconstitution into phosphatidylcholine vesicles. Preparative polyacrylamide gel electrophoresis was used to obtain a chemically homogeneous peptide to analyze the amino acid and carbohydrate composition. The amino acid composition shows a reasonably high content of acidic amino acids with no striking excess of hydrophobic amino acids, while carbohydrate analyses show that carbohydrate is 31% by weight of the protein with sialic acid representing over 50% of the total carbohydrates. The high alpha-helical content, the amino acid composition, and the large carbohydrate mass are similar to those of the eel electroplax sodium channel and appear to be general features of the sodium channels which have been analyzed structurally and chemically to date.

Animals↗
PubMed1985

Characterization of mammalian neurofilament triplet proteins. Subunit stoichiometry and morphology of native and reconstituted filaments.

The three major proteins of mammalian neurofilaments of molecular weights 179,000 (NF1), 129,000 (NF2), and 66,500 (NF3) have been purified to homogeneity by multiple anion-exchange and hydroxylapatite absorption chromatography in 8 M urea. Silver staining of polyacrylamide gels of the purified proteins show single bands. In order to gain further insight into the molecular organization of the neurofilament triplet proteins, the molar stoichiometries and morphologies of native and reconstituted filaments and those isolated from developing brain were studied. Denaturing polyacrylamide gel electrophoresis followed by quantitative dye-binding analysis shows that the molar ratio of the three components in neurofilaments isolated from bovine spinal cord myelinated nerve is 4:2:1 (NF3:NF2:NF1). Comparison of the molar ratios of each component in neurofilaments isolated from rat, bovine, and human brain shows a variation in the ratio of each of these polypeptides and raises questions about the physiological uniqueness of the molar composition of the neurofilament triplet. Reconstitution of the three bovine polypeptides into 10-nm filaments was accomplished under conditions in which the NF3 protein was limiting. Reassembly of 10-nm filaments with varying amounts of NF2 and NF1 indicate that the NF3 homopolymer has a limiting capacity to bind NF2 and NF1 and is saturated at a molar ratio of 2:2:1 (NF3:NF2:NF1). Isolation of the neurofilament complex at various stages of rat brain maturation indicates that NF3 and NF2 are integrated into the neurofilament complex as early as embryonic day 17, while NF1 copurifies with these proteins at postnatal day 16, eventually reaching a molar stoichiometry of 2:2:1 in the adult rat. The molecular stoichiometry of the neurofilament proteins, the differential integration of these proteins during brain development, and the variation of the molar composition between mammalian species suggest accessory roles for the NF2 and NF1 proteins in the neurofilament complex.

Amino Acids↗
PubMed1985

Functional unit size of the neurotoxin receptors on the voltage-dependent sodium channel.

Radiation inactivation was used in situ to determine the functional unit sizes of the neurotoxin receptors of the voltage-dependent sodium channel from rat brain. Frozen or lyophilized synaptosomes were irradiated with high energy electrons generated by a linear accelerator and assayed for [3H]saxitoxin, 125I-Leiurus quinquestriatus quinquestriatus (alpha-scorpion toxin), 125I-Centruroides suffusus suffusus (beta-scorpion toxin), and batrachotoxinin-A 20 alpha-[3H]benzoate binding activity. The functional unit size of the neurotoxin receptors determined in situ by target analysis are 220,000 for saxitoxin, 263,000 for alpha-scorpion toxin, and 45,000 for beta-scorpion toxin. Analysis of the inactivation curve for batrachotoxinin-A 20 alpha-benzoate binding to the channel yields two target sizes of Mr approximately 287,000 (50%) and approximately 51,000 (50%). The results are independent of the purity of the membrane preparation. Comparison of the radiation inactivation data with the protein composition of the rat brain sodium channel indicates that there are at least two functional components.

Animals↗
PubMed1985

Left-handed DNA and the synaptic pairing reaction promoted by Ustilago rec1 protein.

Left-handed Z-DNA binds tightly to Ustilago rec1 protein. The binding reaction is strongly dependent on ATP, but complexes formed are rapidly dissociated by ADP. The parallel between the kinetics of Z-DNA binding and the synaptic pairing reaction leading to paranemic joint molecules suggests that formation of nascent heteroduplex structures in recombination is coupled with formation of left-handed Z-like DNA on the protein. Equilibrium and kinetic studies show that rec1 protein appears to have a strong Z-DNA binding site that binds Z-DNA 75 times tighter than the B form of the DNA. We propose that DNA with a structure approximated best by a left-handed Z-DNA conformation is a key intermediate in homologous pairing promoted by rec1 protein.

Animals↗
PubMed1984

Structural mapping of the voltage-dependent sodium channel. Distance between the tetrodotoxin and Centruroides suffusus suffusus II beta-scorpion toxin receptors.

A 7- dimethylaminocoumarin -4-acetate fluorescent derivative of toxin II from the venom of the scorpion Centruroides suffusus suffusus (Css II) has been prepared to study the structural, conformational, and cellular properties of the beta-neurotoxin receptor site on the voltage-dependent sodium channel. The derivative retains high affinity for its receptor site on the synaptosomal sodium channel with a KD of 7 nM and site capacity of 1.5 pmol/mg of synaptosomal protein. The fluorescent toxin is very environmentally sensitive and the fluorescence emission upon binding indicates that the Css II receptor is largely hydrophobic. Binding of tetrodotoxin or batrachotoxin does not alter the spectroscopic properties of bound Css II, whereas toxin V from Leiurus quinquestriatus effects a 10-nm blue shift to a more hydrophobic environment. This is the first direct indication of conformational coupling between these separate neurotoxin receptor sites. The distance between the tetrodotoxin and Css II scorpion toxin receptors on the sodium channel was measured by fluorescence resonance energy transfer. Efficiencies were measured by both donor quenching and acceptor-sensitized emission. The distance between these two neurotoxin sites is about 34 A. The implications of these receptor locations together with other known molecular distances are discussed in terms of a molecular structure of the voltage-dependent sodium channel.

Amino Acids↗
PubMed1984

Fluorescence resonance energy transfer on the voltage-dependent sodium channel. Spatial relationship and site coupling between the batrachotoxin and Leiurus quinquestriatus quinquestriatus alpha-scorpion toxin receptors.

A fluorescent N- methylanthraniloyl derivative of the potent depolarizing agent batrachotoxin has been used to probe the structural and conformational properties of the neurotoxin receptor site on the voltage-dependent sodium channel. Batrachotoxin A 20-alpha-N- methylanthranilate (BTX-NMA) retains high affinity for its receptor site on the synaptosomal sodium channel with a Kd between 78 and 91 nM and an average site capacity of 2 pmol/mg of synaptosomal protein in the presence of Leiurus quinquestriatus quinquestriatus alpha-scorpion toxin. The fluorescence emission of BTX-NMA upon binding to synaptosomes indicates a hydrophobic environment. Toxin V from L. quinquestriatus, an allosteric activator, effects a 20-nm red shift in the spectrum of bound BTX-NMA and a 4-fold enhancement in the fluorescence quantum yield disclosing a conformational change into a hydrophilic environment. Fluorescence resonance energy transfer measurements show that the distance separating the receptor sites is 37 +/- 10 A. Thus, the binding of alpha-scorpion toxin must involve conformational changes that extend over large distances from the batrachotoxin-binding locus. This information together with the distance measurements between the tetrodotoxin and alpha-scorpion toxin receptors and the conformational transition associated with this distance upon batrachotoxin addition indicate a conformationally flexible channel with coupling of sites through the polyatomic framework of individual subunits or through extensive alterations in subunit/subunit interactions.

Animals↗
PubMed1983

Preparation and characterization of fluorescent scorpion toxins from Leiurus quinquestriatus quinquestriatus as probes of the sodium channel of excitable cells.

Fluorescent derivatives of scorpion toxin V from Leiurus quinquestriatus quinquestriatus have been prepared so that the topographical, dynamic, and cellular properties of the neurotoxin receptor site on the voltage-dependent sodium channel could be studied. Four different modification strategies have been pursued in which acylated, amidinylated, thio-amidinylated, and reductively alkylated scorpion toxins were prepared. Acylation induces a loss of net positive charge on the toxin and these derivatives are purified by preparative isoelectric focusing and ion-exchange chromatography. Amidinylation and reductive alkylation preserve the protonation state of the toxin and maintain the native tertiary structure of the toxin. Because the native toxin does not contain cysteine, we have introduced new sulfhydryls through modification with the cyclic imidoester 2-iminothiolane which also preserves the net charge on the toxin. Novel purification methods with small amounts of toxin by immunoprecipitation using antibodies directed against the chromophores or through covalent thiol-disulfide exchange chromatography have been utilized. The biological activities, equilibrium binding, and spectroscopic properties indicate that these derivatives retain high affinity for the sodium channel and are as active or only 2-3 times less active than L. quinquestriatus V toxin itself. The spectroscopic properties of these fluorescent derivatives cover the absorption range from 290 to 470 nm, and fluorescence emissions range from 360 to 550 nm where suitable filters and spectral overlap with previously synthesized fluorescent tetrodotoxin can be found. The fluorescent properties in particular show excellent environmental sensitivity and are suitable for probing the molecular dynamics of the toxin receptor and for topographic mapping of the sodium channel by fluorescence resonance energy transfer measurements.

Action Potentials↗
PubMed1983

Mapping the molecular structure of the voltage-dependent sodium channel. Distances between the tetrodotoxin and Leiurus quinquestriatus quinquestriatus scorpion toxin receptors.

The Leiurus quinquestriatus quinquestriatus receptor site of the voltage-dependent sodium channel has been characterized using several fluorescent scorpion toxins. The derivatives show fluorescence enhancements upon binding to the receptor site on the channel together with blue shifts. The fluorescence properties of the bound probes indicate a conformationally flexible, hydrophobic site. Binding of tetrodotoxin has no effect on the fluorescence spectra of the bound derivatives, whereas binding of the allosteric activator batrachotoxin enhances the fluorescence about 2-fold and causes a red shift in the emission spectra, suggesting a batrachotoxin-induced conformational change in the scorpion toxin receptor. The distance between the tetrodotoxin receptor and the Leiurus scorpion toxin receptor on the channel was measured by fluorescence resonance energy transfer. Five different chromophoric scorpion toxin derivatives were used as energy transfer acceptors or donors with anthraniloyltetrodotoxin or N-methylanthraniloylglycine-tetrodotoxin as the energy donor or acceptor. Because of the presence of three tetrodotoxin receptors for each Leiurus receptor, the positions of the donors and acceptors were exchanged. Efficiencies of transfer were measured by both donor quenching and sensitized emission. The average distance of separation between these sites is 35 A. Upon batrachotoxin addition, this distance changes to 42 A indicating a conformational change in one subunit of the channel or a change in the interaction between two subunits coupled to the batrachotoxin-binding site. On the basis of these studies, we present a model suggesting that tetrodotoxin binds to a subunit/site which is extracellularly placed and is 35 A from the Leiurus subunit/site which is located in a protein cleft of the channel which extends partly into the membrane, and undergoes a neurotoxin and voltage-dependent conformational change.

Animals↗
PubMed1981

Application of photoactivatable fluorescent active-site directed probes to serine-containing enzymes.

A photoactivatable fluorescent anthraniloyl group has been directed to the active-site serine group of alpha-chymotrypsin and trypsin. The acylated derivatives are nonfluorescent until irradiated. When activated by light a highly reactive nitrene is generated which is capable of covalent insertion into the protein matrix. The resultant insertion product of this photolysis is a highly fluorescent reporter group which has little rotational mobility and is cross-linked through the serine to the protein matrix in the active site region of the protein. Because of the sensitivity to the polarity of the environment shown by the anthraniloyl chromophore, the dipolar relaxation characteristics of the cross-linked through the serine to the protein matrix in the active site region of the protein. Because of the sensitivity to the polarity of the environment shown by the anthraniloyl chromophore, the dipolar relaxation characteristics of the cross-linked enzyme and deacylated enzyme were determined. These measurements show that little relaxation occurs on the nanosecond time scale for the cross-linked enzyme, but upon deacylation of the serine increased dipolar relaxation of the protein with the attached reporter group is observed. The use of these active-site directed photoactivatable fluorescent probes can be extended to probe the active-site structure of complex enzymes and conformational dynamics of active-site regions in proteins and to serve as potential functional site labels in fluorescence resonance energy transfer measurements.

Affinity Labels↗
PubMed1981

Fluorescent and photoactivatable fluorescent derivatives of tetrodotoxin to probe the sodium channel of excitable membranes.

Fluorescent and photoactivatable fluorescent derivatives of tetrodotoxin (TTX) have been synthesized. N-Methylanthraniloylglycine hydrazide, anthraniloyl hydrazide, and 2-azidoanthraniloylglycine hydrazide were coupled to the carbonyl at C6 of oxidized tetrodotoxin to form stable fluorescent hydrazones. The C6 ketone can be reductively aminated with either ammonium or methylammonium acetate to form 6-amino- or 6-(methylamino)tetrodotoxin, which can then be acylated by a variety of fluorescent reagents. The biological activity, competitive binding with [3H]tetrodotoxin for the receptor on rat axonal membranes, and equilibrium binding isotherms obtained by fluorescence enhancement or anisotropy indicate that the derivatives are only about 2-5 times less active then tetrodotoxin itself. The 2-azidoanthraniloylglycine hydrazone of oxidized tetrodotoxin, when activated by light, generates a reactive nitrene which is capable of covalent insertion into the toxin receptor. The product of the photolysis is a highly fluorescent tetrodotoxin derivative which is irreversibly linked to the receptor site. The excitation and emission spectra of the fluorescent tetrodotoxin derivatives vary with solvent polarity, and this sensitivity has been used to determine the immediate environmental characteristics of the toxin binding site of the sodium channel. It is concluded that the toxin binding site is highly polar. Emission and excitation spectra reveal that radiationless energy is transferred from tryptophan residues of the receptor to the anthraniloyl group of the TTX derivatives.

Animals↗
PubMed1980

Transport of catecholamines by native and reconstituted rat heart synaptic vesicles.

Highly purified "heavy" synaptic vesicles were isolated from rat heart by differential centrifugation. Because of the high intravesicular concentrations of proteins, catecholamine, and ATP, resealed vesicle ghosts were prepared and used to study the detailed kinetics of catecholamine transport. ATP stimulated the uptake of l-norepinephrine and was saturable with a Km for l-norepinephrine at 3.3 microM and 1.8 mM for ATP. The ghosts also accumulated 5-hydroxytryptamine and l-epinephrine via an ATP-dependent mechanism. Uptake was stereospecific for the l-form. A functional catecholamine transporter could be solubilized by the detergent octyl-glucoside and incorporated into phospholipid vesicles, which, after detergent removal, generated proteoliposomes that accumulated l-norepinephrine. Reserpine- and l-propranolol-sensitive accumulation against a concentration gradient is achieved by artificially creating a pH gradient across the membrane, and lends further support to the idea that at least the initial phase of catecholamine transport is driven by the trans-membrane pH gradient created by the proton-translocating ATPase.

Adenosine Triphosphate↗