Ago Rinken

Kinetic evidence for isomerization of the dopamine receptor-raclopride complex.

The binding kinetics of the specific dopamine D2 antagonist [3H]raclopride to dopamine D2 receptors in rat neostriatum were studied. The pseudo-first-order rate constants of [3H]raclopride binding with these membranes revealed a hyperbolic dependence upon the antagonist concentration, indicating that the reaction had at least two consecutive and kinetically distinguishable steps. The first step was fast binding equilibrium, characterized by the dissociation constant KA = 12 +/- 3 nM. The following step corresponded to a slow isomerization of the receptor-antagonist complex, characterized by the isomerization equilibrium constant Ki = 0.11. The dissociation constant Kd = 1.3 nM, calculated from these kinetic data, was similar to Kd = 2.4 nM, determined from equilibrium binding isotherm for the radioligand. Implications of the complex reaction mechanism on dopamine D2 receptor assay by [3H]raclopride were discussed.

Solubilization of muscarinic receptor subtypes from baculovirus infected sf9 insect cells

Five different subtypes (human m1, m2, m5 and rat m3, m4) of muscarinic acetylcholine receptors (mAChR) were produced in insect Sf9 cells by infection with recombinant baculoviruses. N-[3H]methylscopolamine ([3H]NMS) has a similar affinity to each of these mAChR subtypes in cell membranes, while pirenzepine, 11-((2-[(diethylamino)methyl]-1-piperidinyl)acetyl)-5,11-dihydro-6H-pyri do- (2,3-b)(1,4)benzo-diazepin-6-on (AF-DX 116) and (+/-)-p-fluoro-hexahydrosiladifenidol (p-F-HHSiD) have a higher affinity for m1, m2 and m3, respectively, than for the other subtypes, indicating the maintenance of subtype specificity of mAChR in this system. Digitonin (1%, w/w) with sodium cholate (0.1%, w/w) solubilized 51% of m1, 36% of m2, 3% of m3, 28% of m4 and 17% of m5 mAChR from these cell membranes with retention of the [3H]NMS binding activity. Optimization of cholate concentrations resulted in solubilization of up to 50-60% for m1, m2 and m4, but up to 25% for m5 and 7% for m3. Optimal concentrations of cholate differed from one subtype to another. Sucrose monolaurate solubilized 21-43% of m1, m2 and m4, but only up to 12% for m5 and 2% for m3. 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS) was practically ineffective in mAChR solubilization from Sf9 cell membranes for all subtypes investigated. Solubilization with digitonin and cholate had little influence on [3H]NMS affinity for m2 and m4, but decreased m1 and m5 affinity by 10-fold and that of m3 by more than 50-fold. These results indicate that the solubility and stability of mAChR in detergents differ among the subtypes, in spite of their structural similarities. These differences should be taken into account when comparing the five subtypes, particularly when determining the proportion of each subtype in a given tissue by precipitating the solubilized mAChR with subtype-specific antibodies.

Regulation of dopamine D2 receptor affinity by cholecystokinin octapeptide in fibroblast cells cotransfected with human CCKB and D2L receptor cDNAs.

Alteration in dopamine (DA) and/or cholecystokinin (CCK) transmission in the CNS may be of relevance for schizophrenia. Previous findings in striatal membranes give indications of a modulation of DA D2 receptor affinity by CCKB receptor activation. In the present study receptor binding studies were performed in a mouse fibroblast cell line (L-hD2l/CCK), expressing both human D2 receptors (long form, D2L) and human CCKB receptors, and binding sites for [3H]CCK-8S (sulfated CCK octapeptide), the D2 agonist [3H]NPA and the D2 antagonist [3H]raclopride were found and characterized in saturation and competition experiments. 1 nM of CCK-8 caused a significant 38% increase in the KD value of the D2 agonist [3H]NPA binding sites in the L-hD2l/CCK cell membranes. This change was blocked by the CCKB receptor antagonist PD 134308 (50 nM). Furthermore, 1 nM of CCK-8 increased the KD value of the D2 antagonist [3H]raclopride binding sites by 34% (P < 0.05) in the L-hD2l/CCK cell membranes. Control cells (L-hD2l cells) expressing D2L receptors showed no specific [3H]CCK-8S binding sites and no modulation by CCK-8 of the D2L receptors. These findings indicate a modulation of the D2L receptor affinity by activation of the CCKB receptor also when they are coexpressed in a fibroblast cell line. One possible explanation of these data may include a receptor-receptor interaction between the CCKB and D2L receptors.

Serotonergic agonists behave as partial agonists at the dopamine D2 receptor

RAT dopamine D2short receptors expressed in CHO cells were characterized by activation of [35S]GTPgammaS binding. There were no significant differences between the maximal effects seen in activation of [35S]GTPgammaS binding caused by dopaminergic agonists, but the effects of 5-HT, 8OH-DPAT and 5-methoxytryptamine amounted to 47 +/- 7%, 43 +/- 5% and 70 +/- 7% of the dopamine effect, respectively. The dopaminergic antagonist (+)butaclamol inhibited activations of both types of ligands with equal potency (pA2 = 8.9 +/- 0.1), indicating that only one type of receptor is involved. In competition with [3H]raclopride binding, dopaminergic agonists showed 53 +/- 2% of the binding sites in the GTP-dependent high-affinity state, whereas 5-HT showed only 20 +/- 3%. Taken together, the results indicate that serotonergic agonists behave as typical partial agonists for D2 receptors with potential antiparkinsonian activity.

Striatal dopamine denervation decreases the GDP binding affinity in rat striatal membranes

The role of G-proteins in D2 receptor supersensitivity was studied in striatal membranes from rats with unilateral 6-hydroxydopamine (6-OHDA) induced lesions of the nigral dopamine (DA) system. Thirteen months after the lesion the number of [3H]raclopride binding sites was increased in the DA denervated striatum, but no changes in ligand binding affinities and in proportion of high-affinity agonist binding sites could be detected. The affinity of [35S]GTPγS binding was unaltered after the striatal DA denervation, whereas the binding affinity of GDP was decreased in the DA denervated as compared to the intact striatum. It is proposed that the decrease in GDP binding affinity to D2 DA receptor-coupled G proteins is an important factor in the D2 receptor super-sensitivity following degeneration of the striatal DA terminals.

Immobilisation and Kinetic Study of Tyrosinase for Biosensor Construction

ABSTRACT The catalytic properties and stability of soluble and immobilised in nylon-6,6 mesh tyrosinase were studied with the help of an oxygen sensor. A variety of methods were examined for the immobilisation of tyrosinase, although active immobilised enzyme was obtained only with the help of benzidine and dicyclohexylcarbodiimide. The immobilisation caused an increase in the Km value for catechol almost 2 times in comparison with that found for soluble enzyme (0.39 and 0.22 mM, respectively). The immobilised tyrosinase retained sufficient activity for several months. Due to its characteristic suicide inactivation induced by catechol, it is only of single use for analytical purposes. Obtained data were also used for evaluation of the model of oximeter-based biosensors. This model allows the calculation of steady-state parameters from transient state data, excluding the influence of accompanying side processes; for tyrosinase-bound biosensors it gave very reproducible results for automatic data processing.

A Model of Oximeter - Based Enzyme Electrode

A model of an oximeter-based two-substrate enzyme electrode is presented. The simplifications of the general model lead to a solution to differential equations describing the influence of the rate of the enzyme reaction in combination with the diffusion phenomena on measurable oxygen flux. Both transient phase and steady state conditions for experimental data can be considered within the frames of the model and provide possibilities for the calibration of the biosensor. The optimal parameters for the biosensor calibration can be used in practical design of enzyme electrodes.

N-alkylated dipeptide amides and related structures as imitations of the melanocortins’ active core

Thirty-three low molecular mass structures combining both peptide and peptoid features were prepared and tested on human melanocortin receptors MC1,3-5R. Most of them displayed low micromolar activity with preference for diamines, guanidino and 2-naphthyl derivatives compared to monoacetylated, amino and 3-indolyl counterparts. Some contained L- or D-histidine residues, but the change did not influence affinity. QSAR modelling yielded excellent models for the MC3-5 receptors explaining R2Y=0.89-0.91 and predicting Q2=0.77-0.80 of the affinity variations. One compound displayed MC1R selectivity (13-fold and more). An NMR study of showed that it exists as a mixture of four rotamers at its tertiary amide bonds. Comparisons with earlier data for melanocortin core tetrapeptide analogues indicate that the novel peptide-peptoids interact with the melanocortin receptors in a different way.

Mechanism of modulation of [3H]raclopride binding to dopaminergic receptors in rat striatal membranes by sodium ions

The mechanism of modulation of [3H]raclopride binding to dopaminergic receptors in rat brain striatal membranes by sodium ions was studied by means of equilibrium and kinetic measurements. Among different mono- and divalent cations studied, only sodium and lithium ions significantly enhanced [3H]raclopride binding to rat striatal membranes, but the effect of lithium was considerably smaller if compared with that of sodium. The equilibrium binding studies revealed that the increase in Na+ concentration from 0.5 to 150 mM increased both the radioligand affinity and the number of binding sites. The meaning of these changes was established by kinetic studies, which yielded hyperbolic plots of [3H]raclopride binding rate constants over the radioligand concentration. These plots correspond to the two-step ligand binding reaction mechanism, involving fast binding equilibrium followed by a slow isomerization of the receptor-antagonist complex. Sodium ions did not influence the antagonist affinity for the receptor sites in the first step of the binding process, nor the rate of isomerization of the receptor-ligand complex, but slowed down the rate of deisomerization. This led to a change in the value of the receptor-ligand dissociation constant Kd determined under equilibrium conditions. The same change in deisomerization rate was also sufficient to alter the receptor density (Bmax), measured by the conventional ligand binding procedure.