Changes in the properties of brain opiate receptors during the development of morphine tolerance in rats

The effects of prolonged administration of morphine on the properties of opiate receptors of rat brain were studied. For this purpose the isotherms of binding of labeled mu-, delta-, and chi-ligands--morphine, D-Ala2, D-Leu5-enkephalin and ethylketocyclazocine--with brain membrane preparations of morphine-tolerant rats as well as those of control animals were analyzed. For quantitative determination of dissociation constants of the ligand-receptor complexes (K) and receptor concentrations ([Q]), the difference and simulation methods were used. It was shown that the values of K and [Q] vary within broad ranges in individual animals, whereas the individual variations of the [Q]/[K] ratios in controls or in morphine-tolerant rats are not so significant. This suggests [Q]/K to be one of the basic criteria for a comparison of properties of opiate receptors in different groups of animals. The use of this criterion and of the simulation method demonstrated that the development of tolerance causes changes in the properties of delta-receptors (the [Q]/K ratio decreases by greater than 50%). Unlike delta-receptors, the tolerance has no appreciable effect on the properties of mu- or chi-receptors or on the superhigh affinity binding sites of the ligands tested.     

Analysis of new opiate-like peptides using a radioreceptor method

A screening of new synthetic opioid-like peptides has been carried out by the radioreceptor assay using selective labeled ligands to mu-, delta- and gamma-opioid receptors of the rat brain membranes. With this aim peptides from sequences of the following proteins were used: kapporphin-Tyr-Ser-Phe-Gly-Gly and its analogues-Tyr-Ser-Phe-Gly-Gly-NH2, Tyr-D-Ser-Phe-Gly-Gly, Tyr-D-Ser-Phe-Gly-Gly-NH2, myelorphin-Phe-Gly-Tyr-Gly-Gly, interenkephalin B-Arg-Arg-Gln-Phe-Lys and chimeric peptide IEPhBin 1-Tyr-Gly-Gly-Phe-Leu-Arg-Pro-Tyr-Ile-Leu consisting of leu-enkephalin and pentaneurotensin. It has been found that myelorphin has a prevalent affinity to mu-receptor, while the kapporphin analogues both to mu- and delta-receptors. The presence of pentaneurotensin in chimeric peptide does not affect the specificity of binding to opioid receptors, but decreases affinity to mu- and delta-receptors approximately by an order as compared to leu-enkephalin. Kapprorphin and interenkephalin B displace neither of the selective labeled opioid ligands under study.     

Kinetic mechanisms of enzyme activity of the thromboxane synthetase system. Thromboxane synthetase of human platelets

Partially purified preparations of prostaglandin endoperoxide-synthetase (PGH-synthetase) and thromboxane synthetase (PGH-convertase) were obtained from human platelets by ion exchange chromatography. The kinetics of prostaglandin H2 enzymatic conversion was studied in the presence of thromboxane synthetase from human platelets. It was found that prostaglandin H2 conversion into both thromboxane A2 and malonic dialdehyde is a catalytic process, i. e., its rate increases as the protein concentration rises. The linear dependence of the enzymatic reaction velocity on substrate concentration at a prostaglandin H2 concentrations below 10-15 microM was demonstrated. PGH-synthetase and PGH-convertase from human platelets exhibit similar enzymatic activity dependence on pH and temperature, PGH-convertase being more thermostable.     

Kinetic model of a multienzyme system of blood prostanoid synthesis. I. Mechanism of stabilization of thromboxane and prostacyclin levels

A kinetic scheme of the prostacyclin-thromboxane system has been evolved on the basis of the authors experimental data and the results described elsewhere. The kinetic behavior of the model has been analysed with the aid of computer technology by varying the following parameters: phospholipase activities, free arachidonic acid exchange rates between platelets and endothelium, PGH-synthetase biosynthesis rates, velocities of arachidonic acid pathways other than the cyclooxygenase ones. It has been demonstrated that the biological system is capable of sustaining prostacyclin and thromboxane concentrations at steady fixed levels within a wide range of kinetic parameters.     

Prostaglandin H synthetase as a multisubstrate enzyme. Fluorimetric study of enzyme kinetics

The kinetics of a multisubstrate enzymatic reaction catalyzed by prostaglandin H synthase (PGH-synthase, EC 1.14.99.1) was studied, using homovanillic acid, a new electron donor for the given system. Homovanillic acid was shown to be a participant in a reaction with arachidonic acid/O2 stoichiometric ratios and is oxidized to a readily fluorescing product with an absorbance maximum (excitation) at 315 nm and fluorescence maximum at 425 nm. This allows for determination of the rate of enzymatic reaction with the sensitivity exceeding by one order of magnitude that of polarographic or spectrophotometric assays. Using fluorescent techniques, the dependence of the rate of PGH-synthase reaction on substrate (arachidonic acid, O2 and homovanillic acid) concentrations was studied, and the corresponding Km values were determined. The effect of Tween-20 and Lubrol PX concentrations on the reaction rate were examined. It was shown that with a decrease in the surfactant concentration the reaction rate increases.     

Inner and outer immobilization of chloroplasts]

The effects of glutaric aldehyde on pea leave chloroplasts and their inactivation kinetics were studied. Optimization of the chloroplasts fixation by glutaric aldehyde resulted in a 5-fold increase of stability of the chloroplasts. Immobilization of the chloroplasts in agar-agar gels was performed; the ability of chloroplasts for photooxidation of H2O was thereby retained. Immobilization did not actually affect the stability of chloroplasts. The inactivation kinetics of fixed and immobilized chloroplasts are in good agreement with the previously described model for inactivation of native chloroplasts.     

Isolation, purification and study of the stability of the soluble hydrogenase of Alcaligenes eutrophus Z-1]

Soluble hydrogenase was isolated from the hydrogen-oxidizing bacterium Alcaligenes eutrophus Z-1 and purified to electrophoretical homogeneity. The purification procedure included fractionation by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose and gelfiltration through Ultragel AcA-34. The resulting preparation had a specific activity of 25 mkmoles H2.min-1.mg of protein as measured by the rate of hydrogen evolution from sodium dithionite-reduced methyl viologen. The enzyme has a molecular weight of 200,000 and is made up of two subunits with mol. weights of 30,000 and two subunits with mol. weights of 65,000. The effects of pH, oxidants and reducers, as well as aerobic and anaerobic conditions on the hydrogenase preparations inactivation kinetics in intact cells and in a highly purified state were studied. The kinetic data suggest a possible existence of two enzyme forms differing in their activities and stabilities to denaturating influences.     

New possibilities for studying complex macromolecular structures of the cell by encasing cells in polyacrylamide microspheres]

A method is proposed for incorporation of mammalian cells into spheres permeable for protein molecules, the dimensions of these spheres not overcoming those of the cells. The method permits fractionation of the cells, preserving them as discrete units resistant to mechanical and hydrodynamic destruction in the course of routine laboratory manipulations, as well as using flow cytometry for their analysis. With this approach, the flow cytometric measurements were made of DNA being shielded in the chromatin complex which prevents from binding fluorescent dyes with low-molecular weights.     

Heterogeneity of mu- and delta-opioid active sites in the mouse cerebral cortex. Selective increase in the affinity of a superhigh affinity delta-site during long-term administration of morphine]

The interaction of the selective mu-ligand [D-Ala2, MePhe4, Glyol5]enkephalin (DAMGO) and the selective delta-ligand [D-Ser2, Leu5, Thr6]enkephalin (DSLET) with the binding sites in rat brain cortex membranes, has been studied. Analysis of the binding isotherms in the presence and absence of nonlabelled DAMGO and DSLET revealed that these ligands can interact with both superhigh affinity and high affinity binding sites that are distinct from one another. Prolonged administration of morphine and formation of physical dependence caused no appreciable changes in the parameters of either the superhigh affinity binding sites for the mu-ligands or the high affinity binding sites for mu- and delta-ligands. At the same time, the affinity of the superhigh affinity site for delta-ligands increased 3.5 times (tau 1/2 approximately equal to 17.5 h).