Isolation and characteristics of dihydropyridine calcium antagonist receptor from rabbit skeletal muscles

Up to 80% of the dihydropyridine receptor is solubilized from transverse tubules of rabbit skeletal muscle by 3-[(3-cholamidopropyl)-dimethylammonium]-2-oxy-1-propane sulfonate (CHAPSO). The DHP receptor is an oligomeric complex made up of two subunits with molecular masses of 160 and 53 kD as shown by DHP-Sepharose affinity chromatography and SDS gel electrophoresis of specifically eluted proteins. The reduction of disulfide bridges of the 160 kD subunit is accompanied by a decrease in its apparent molecular mass up to 125 kD. A method is proposed for preparative isolation of the DHP receptor which is based on ion-exchange chromatography and WGA-Sepharose chromatography. Individual subunits of DHP receptor were isolated by Sepharose 4B gel filtration in SDS; their amino acid composition was determined. Both the 160 and 53 kD subunits are N-glycosylated, and the oligosaccharide portions make up to 7.5% and 6.6%, respectively.     

The characteristics of the neuronal reactions of the cat somatosensory cortex to a heteromodal complex conditioned signal]

On alert cats the change was studied of the activity of the neurones of the sensorimotor cortical area at instrumental reaction to a simultaneous heteromodal complex stimulus. It was shown that in the projection of distal limb areas a group could be singled out of neurones, which changed their activity in one direction depending on the character of presented signals. In these cells an increase of discharges frequency was observed in response to complex stimulus, consisting of light and sound signals. After the extinction of the motor reaction both to the complex stimulus and to its components neuronal reactions of lesser intensity was recorded, what determined the absence of the motor reaction. This group of neurones had receptive fields localized on distal limb areas, it was activated at fulfillment of the movement of catching the reinforcement and belonged to neurones of the pyramidal tract. The neurones with receptive fields on the whole limb surface or changing their activity at the animal pose change, had variable reactions to positive and differentiation stimuli.     

Use of the fluorescent probe method to study the interaction of barbiturates with biological membranes]

The binding of the negatively charged fluorescence dye ANS and neutral dye NPN2 with lipid and erythrocyte membranes in the presence of barbiturates was studied. It was found that barbiturates decreased the amount of binding sites of ANS and NPN2 with membranes did not affect the quantum yield and the dissociation of the membrane-dye complex. It was shown that all barbiturates investigated were bound with the membranes in a neutral form.     

Modulation of the GABA-benzodiazepine receptor complex by taurine in rat brain membranes

The interactions of taurine and its precursor hypotaurine with the GABA-benzodiazepine receptor complex were studied by investigating their effects on GABA and flunitrazepam binding in rat brain membranes. Taurine, and to a lesser degree also hypotaurine, displaced the high- and low-affinity GABA binding. The maximal binding capacities of both sites were decreased in the presence of taurine, while the binding constants remained the same, suggesting noncompetitive interactions. Taurine and hypotaurine affected flunitrazepam binding only at a very high concentration (50 mmol/l), whereas GABA (within the concentration range of 0.1–100 mol/l) significantly enhanced the binding. Taurine inhibited the GABA-stimulated binding dose-dependently. These modulatory effects of taurine on the GABA-benzodiazepine receptor complex could result from interactions with the GABA recognition site but not from direct actions on the benzodiazepine site.     

Thermodynamic and spectral study of DNA-prospidin complex

By the methods of heat denaturation and luminescence the interaction between an antitumor drug prospidine and DNA in aqueous solutions at two ionic strengths (0.1 and 0.001 M NaCl) and at various prospidine concentrations was studied. For the first time it has been demonstrated that the interaction occurs at 0.1 M NaCl and therapeutic prospidine concentrations. In the framework of Frank-Kamenetsky's theory of melting of a polymer with stabilizing ligands the size of the binding site and binding constants (K) with the decrease of ionic strength, the lack of alterations in the DNA UV absorption spectrum on complex formation and the data on the competitive binding of ethydium bromide suggest that at the first stage of the reaction an external complex is formed due to electrostatic interactions between quaternary nitrogen atoms of prospidine and DNA phosphate groups. Incubation of the complex at 37 0 C leads to a decrease of the DNA melting temperature and hyperchromic effect. Presumably this is due to the relatively slow formation of chemical bonds between alkylating groups of prospidine and nucleophilic groups of DNA bases, which results in the destabilization and denaturation of DNA. It is concluded that the interaction between prospidine and DNA must be taken into consideration when studying the molecular mechanism of prospidine antitumour activity.     

Ouabain sensitivity of Na+,K+-ATPase in neuronal membranes]

Na+, K(+)-ATPase preparations of the rat and bovine brain and kidney were studied for ouabain sensitivity. Differences in apparent affinities to inhibitor of alpha(+)- and alpha-isozymes of Na+, K(+)-ATPase catalytic subunit were detected only in rat tissues but not in bovine ones. It is concluded that glycoside-sensitive and glycoside-resistant enzymic forms are not fully identical to alpha(+)- and alpha-subunit forms of Na+, K(+)-ATPase.     

3D structure of the skeletal muscle dihydropyridine receptor

The dihydropyridine receptors (DHPR) are L-type voltage-gated calcium channels that regulate the flux of calcium ions across the cell membrane. Here we present the three-dimensional (3D) structure at approximately 27A resolution of purified skeletal muscle DHPR, as determined by electron microscopy and single particle analysis. Here both biochemical and 3D structural data indicate that DHPR is dimeric. DHPR dimers are composed of two arch-shaped monomers approximately 210A across and approximately 75A thick, that interact very tightly at each end of the arch. The roughly toroidal structure of the two monomers encloses a cylindrical space of approximately 80A diameter, which is then closed on each side by two dome-shaped protein densities reaching over from each monomer arch. The dome-shaped domains have a length of approximately 80-90A and a maximum height of approximately 45A. Small orifices punctuate their exterior surface. The 3D structure disclosed here may have important implications for the understanding of DHPR Ca(2+) channel function. We also propose a model for its in vivo interactions with the calcium release channel at the junctional sarcoplasmic recticulum.     

Mass spectral analysis of complex lipids desorbed directly from lyophilized membranes and cells

Three desorption ionization techniques--laser desorption, plasma desorption and fast atom bombardment mass spectrometry--have been applied to lyophilized cells, membranes, lysed cells and various extracts. It has been shown that intact polar lipids are selectively desorbed from biological membranes by these methods and that their mass spectra provide "fingerprints" which reflect the unique biochemical composition of each class of cell or membrane.     

Rapid incorporation of the solubilized dihydropyridine receptor into phospholipid vesicles

We describe the rapid incorporation of the CHAPS solubilized dihydropyridine receptor into phospholipid vesicles. A series of sucrose gradient sedimentation experiments demonstrate that the (+)-[3H]PN200-110-labeled dihydropyridine receptor is associated with lipid vesicles following detergent removal by Extracti-gel chromatography. Solubilization of the receptor results in a loss of (+)-[3H]PN200-110 binding affinity relative to that observed in native membranes; the high affinity binding of (+)-[3H]PN200-110 can be restored upon reincorporation of the receptor into phospholipid vesicles. Similarly, the incorporation of the receptor restores its stability to incubation at 37 degrees C relative to that of the detergent solubilized receptor, thereby mimicking the properties of the membrane bound form of the receptor. The dissociation rate of (+)-[3H]PN200-110 from the reconstituted receptor is shown to be allosterically regulated by verapamil and diltiazem, indicating that the binding sites for these calcium antagonists have been inserted along with the dihydropyridine receptor into phospholipid vesicles. The results presented in this report, thus demonstrate the successful reconstitution of the dihydropyridine receptor into phospholipid vesicles by a variety of criteria. The reconstitution method described here is rapid and efficient, and should now facilitate structure-function studies of this receptor and its interrelationships with other regulatory components of the voltage-sensitive calcium channel system.     

Comparison of the energy characteristics of Acetabularia membranes in light and darkness]

Energetic parameters of the membrane of marine alga Acetabularia were compared at light and dark during the action potential (AP). Direct current resistance of the resting membrane at dark as well as at light is of the order 1000-5000 omega-cm2 without considerable difference. The maximum resistance of the excited Acetabularia membrane is somewhat increased at dark as compared to its value at light. The maximum power of the membrane system and that of its regulating mechanism along with the energy dissipating AP at light exceed the same values at dark. The dissipating energy and the work Acetabularia cell performs during the AP are also compared for light and dark conditions.     

Fluorescence study of lymphocyte plasma membranes]

ANS binding parameters--dissociation constant, number of binding sites, rotation freedom--are measured by fluorescence studies of a complex between ANS and lymph node cell plasma membranes. Divalent ions, Mg++ and Ca++, enhance the complex fluorescence intensity without shifting its maximum wavelength : this enhancement is induced by affinity and quantum yield increases, while the number of binding sites remains constant. The complex fluorescence quenching by ethacrynic acid shows the presence of free SH groups in the ANS binding site. An energy transfer takes place between membrane protein tryptophan residues and bound ANS ; the energy transfer yield is unaffected by Ca++ ions. A correlation of these results is postulated with the biological activity of the membrane.     

Use of model organisms for the study of neuronal ceroid lipofuscinosis

Neuronal ceroid lipofuscinoses are a group of fatal progressive neurodegenerative diseases predominantly affecting children. Identification of mutations that cause neuronal ceroid lipofuscinosis, and subsequent functional and pathological studies of the affected genes, underpins efforts to investigate disease mechanisms and identify and test potential therapeutic strategies. These functional studies and pre-clinical trials necessitate the use of model organisms in addition to cell and tissue culture models as they enable the study of protein function within a complex organ such as the brain and the testing of therapies on a whole organism. To this end, a large number of disease models and genetic tools have been identified or created in a variety of model organisms. In this review, we will discuss the ethical issues associated with experiments using model organisms, the factors underlying the choice of model organism, the disease models and genetic tools available, and the contributions of those disease models and tools to neuronal ceroid lipofuscinosis research. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.     

Further characterisation of the [35S]-TBPS binding site of the GABA receptor complex in locust (Schistocerca gregaria) ganglia membranes

1. Using homogenates of supraoesophageal ganglia from locust we observed specific binding of [35S]-TBPS which was linear with protein concentration up to 7 mg/ml, showed a pH optimum at pH 9.0 and was linear with NaCl concentration up to 350 mM. 2. Kinetic analysis of the binding showed positive cooperativity as a result of changes in on and off-rates with occupation of the binding site by the ligand. The apparent KD = 417 nM and Bmax = 1083 fmol/mg of membrane protein were calculated using a computer program for dose-response curve fitting. 3. The binding was enhanced by GABA, pentobarbital and benzodiazepines. Picrotoxinin had no effect on the binding at 0.1 mM. Only the cage convulsants TBPS and IBP were able to displace the binding. 4. Whilst the characteristics of the binding are similar to those reported for house fly thorax and abdomen preparations they are significantly different from those reported for house fly head, cockroach nerve cord and rat brain.     

The spectral correlation characteristics of the brain electrical activity in the rabbit during thirst]

Spectral-correlation analysis of the summate electrical activity of a number of subcortical structures of rabbit brain, having, by literature data, a relation to drinking behaviour showed that the increase of drinking excitability, induced by water deprivation was accompanied by definite reconstruction of biopotentials. In electrical activity of the studied structures, the spectrum power, as a rule, decreased (except in the paraventricular nucleus), and a definite structure of coherent connections between the subcortical and cortical potentials was established. Among the studied subcortical formations, structures (anterior hypothalamic area, lateral preoptic area, medial preoptic area, paraventricular nucleus) could be singled out where reconstructions of spectral characteristics of biopotentials took place most regularity, and the electrical processes in which were characterised by coherence index by an increase of spatial interconnection with the neocortex potentials, what allows to consider them as most significant for organization of drinking excitation.     

Comparative cytotoxic characteristics of the interferon-inducing yeast RNA-tilorone complex]

Cytotoxicity of the yeast RNA-tilorona molecular complex (MC) with interferonogenic properties and its influence on the DNA replicative synthesis were studied in experiments with human lymphocytes and 3 cell lines. It was shown that the MC doses of 25, 100 and 250 micrograms/ml were absolutely nontoxic for all the cell lines. The main parameters of the MC toxicity based on the cell viability were calculated. The parameters were found to correlate in the order of their magnitude with those relating to interferonogens of the polynucleotide nature. Within the dose ranges of 10 to 100 micrograms/ml the MC had a stimulating effect on replicative processes in the cells. It was concluded that the use of the MC as an inductor in large-scale manufacture of human and animal interferons of type 1 was promising.