Kinetic evidence for different mechanisms of interaction of black mamba toxins MT alpha and MT beta with muscarinic receptors.

By studying the influence of two toxins from the black mamba Dendroaspis polylepis on the kinetics of [3H]-N-methylscopolamine binding to muscarinic acetylcholine receptors from rat cerebral cortex, it was revealed that these toxins, MT alpha and MT beta, interact with the receptors via kinetically distinct mechanisms. MT beta bound to receptors in a one-step, readily reversible process with the dissociation constant K(d)=5.3 microM. The binding mechanism of MTalpha was more complex, involving at least two consecutive steps. A fast receptor-toxin complex formation (K(T)=3.8 microM) was followed by a slow process of isomerisation of this complex (k(i)=1.8 x 10(-2) s(-1), half-time 39 s). A similar two-step interaction mechanism has been established for a related toxin, MT2 from the green mamba D. angusticeps (K(T)=1.4 microM, k(i)=8.3 x 10(-4) s(-1), half-time 840 s). The slow isomerisation process delays the effect of MT alpha and MT2, but increases their apparent potency compared to toxins unable to induce the isomerisation process.

Bi-substrate analogue ligands for affinity chromatography of protein kinases.

Novel affinity ligands, consisting of ATP-resembling part coupled with specificity determining peptide fragment, were proposed for purification of protein kinases. Following this approach affinity sorbents based on two closely similar ligands AdoC-Aoc-Arg4-Lys and AdoC-Aoc-Arg4-NH(CH2)6NH2, where AdoC stands for adenosine-5'-carboxylic acid and Aoc for amino-octanoic acid, were synthesized and tested for purification of recombinant protein kinase A catalytic subunit directly from crude cell extract. Elution of the enzyme with MgATP as well as L-arginine yielded homogeneous protein kinase A preparation in a single purification step. Also protein kinase A from pig heart homogenate was selectively isolated using MgATP as eluting agent. Protein kinase with acidic specificity determinant (CK2) as well as other proteins possessing nucleotide binding site (L-type pyruvate kinase) or sites for wide variety of different ligands (bovine serum albumin) did not bind to the column, pointing to high selectivity of the bi-functional binding mode of the affinity ligand.

Adenosine-derived non-phosphate antagonists for P2Y(1) purinoceptors.

Novel type antagonists for P2Y(1) adenine nucleotide receptors were synthesized by coupling of adenosine 5'-OH group with oligo-aspartate chain via a carbonyl linker. All these conjugates (AdoOC(O)Asp(n), n = 1-4) inhibited the 2MeSADP-stimulated synthesis of inositol phosphates in 1321N1 human astrocytoma cells stably expressing human P2Y(1) receptors. This inhibitory effect followed the rank order AdoOC(O)Asp(2)> AdoOC(O)Asp(3)> AdoOC(O)Asp(1)> AdoOC(O)Asp(4) with antagonistic constant pA(2) = 5.4 for AdoOC(O)Asp(2). Potency of this non-phosphate inhibitor was comparable with the previously known adenosine 3',5'- and 2', 5'-bisphosphates. Chemical and biological stabilities of these novel adenosine derived antagonists of the nucleotide receptor provide perspectives of their pharmacological implication.

Adenosine triphosphate is full antagonist at human P2Y(1) purinoceptors.

Both agonistic and antagonistic effects have been reported for ATP at P2Y(1) purinoceptors at micromolar ligand concentrations. These conflicting data hamper specification of the true pharmacological profile as well as structural requirements for antagonistic ligands of this receptor. In this report the type of ATP activity at human P2Y(1) receptors in hP2Y(1)-1321N1 cells was revisited. In parallel, kinetics of degradation of ATP in the assay mixture was analysed. It was found that transformation of this ligand to ADP was responsible for initiation of synthesis of inositol phosphates, observed in the presence of ATP in hP2Y(1)-1321N1 cells. This agonistic effect was abolished in the presence of the triphosphate regeneration system (CP/CPK). On the other hand, if the agonistic effect caused by degradation product of ATP was taken into consideration, this ligand behaved as a full antagonist at P2Y(1) receptors and was characterized by the apparent inhibitory constant 5 microM.

Peptide phosphorylation by calcium-dependent protein kinase from maize seedlings.

Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS15VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cbeta (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose-membrane-attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK-1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L-5X-4R-3X-2X-1SX+1R+2Z+3R+4, where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA.

Dual effect of nucleotides on P2Y receptors.

The interaction of ADP, 2MeSADP, and ADPbetaS with the adenine nucleotide receptor P2Y1 in the hP2Y1-1321N1 cell line and of UDP with a receptor or receptors recognizing pyrimidine nucleotides in NG108-15 cells was studied over a wide range ofligand concentrations. Bell-shaped dose-response curves for stimulation of phosphoinositide hydrolysis were obtained in these cells. This dual behavior of the agonists studied was characterized by two dissociation constants, K(agon) and K(antag), which quantify the agonistic and antagonistic activity of these ligands and can be compared with the conventional EC50 and IC50 values, respectively. The data revealed a common pattern of agonistic and antagonistic behavior of nucleoside diphosphates and their derivatives at these two types of P2Y receptors, pointing to some similar properties of their nucleotide binding sites.

Only pyrimidinoceptors are functionally expressed in mouse neuroblastoma cell lines.

The ability of UTP, UDP, ATP, and ADP to influence inositol phospholipid hydrolysis in neuroblastoma origin cell lines was assessed. The mouse neuroblastoma lines N1E 115, Neuro 2a, and NB4 1A3 and the rat glioma/mouse neuroblastoma hybrid line NG108-15 gave robust responses to both UTP and UDP, which were essentially equipotent. Thus a range of cell lines of mouse neuroblastoma origin express a pyrimidine-selective P2Y receptor. The NG108-15 cells were the only cell type tested at which ATP and ADP displayed activity with EC50 values of greater than 100 microM, compared with values of 0.58 and 1.25 microM for UTP and UDP, respectively. In contrast to the cell lines derived from mouse neuroblastoma, the human neuroblastoma lines SH-SY5Y and SK-N-SH did not respond to any nucleotides, although both responded well to carbachol.

Adenosine-5'-carboxylic acid peptidyl derivatives as inhibitors of protein kinases.

A new class of protein kinase bisubstrate-analog inhibitors was designed on the basis of adenosine-5'-carboxylic acid derivatives, where a short peptide was attached to the 5'-carbon atom of the adenosine sugar moiety via a linker chain. The potency and selectivity of these inhibitors were adjusted by relevant combination of these structural fragments, resembling the structure of the bisubstrate complex of the peptide phosphorylation reaction.

Influence of atropine on carbachol dual effect on Ca2+ mobilization in SH-SY5Y neuroblastoma cells.

The muscarinic receptor stimulated mobilisation of calcium ions in SH-SY5Y neuroblastoma cells was measured as a function on carbachol and atropine concentrations. The combined application of this pair of muscarinic agonist and antagonist yielded a set of bell-shaped dose-response curves. In the presence of atropine the cell responses were smaller and the up-going phase of these relationships was shifted towards higher agonist concentration, while the down-going phase of these curves was not influenced by the antagonist. These results pointed to a similar mechanism of the receptor inhibition at high carbachol (agonist) concentrations and by atropine (antagonist).

Pyrimidinoceptor potentiation by ATP in NG108-15 cells.

Regulation of inositol phospholipid hydrolysis by UTP and UDP in neuroblastoma x glioma hybrid cell line NG108-15 was potentiated in the presence of ATP. The effect of ATP was dose dependent and shifted the EC50 value for these uracil nucleotides up to three powers of magnitude, having no influence on the maximal value of the response. Adenine nucleotides (ADP, AMP, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), beta,gamma-methyleneadenosine 5'-triphosphate (betagammaMeATP), 3'-O-(4-benzoyl)benzoyl ATP (BzATP) and 3'-deoxyadenosine 5'-O-(1-thio)triphosphate (dATPalphaS)) as well as adenosine, had no influence on the pyrimidinoceptor response. The potentiation effect was abolished by excess of EDTA. The results were in agreement with the hypothesis of pyrimidinoceptor affinity regulation via extracellular phosphorylation of the receptor protein, initiated by ATP. This mechanism may have physiological implication for functioning of uracil nucleotides as endogenous signaling molecules.

Calibration of glucose biosensors by using pre-steady state kinetic data.

A new method for biosensor calibration and data processing, allowing the prediction of steady state parameters from the analysis of transient response curves (Rinken et al., 1996. Analytical Letters 29, 859), has been evaluated in the case of an oxygen sensor based two-substrate enzyme electrode for glucose determination. The electrochemical glucose biosensor was prepared by covering the surface of oxygen sensor with glucose oxidase (EC 1.1.3.4) immobilized in nylon mesh. This decreased the oxygen flow to the sensor in the presence of glucose and resulted in time-dependent decrease of the biosensor signal. Except the lag period of the response in the beginning of the assay, the oxygen consumption by the immobilized enzyme was described by an exponential function: [formula: see text] The parameter C, which corresponded to the steady-state output of the biosensor, was found to be the most suitable for glucose determination. The non-linear fitting for data of over 1000 independent experiments to the equation above always revealed correlation coefficients greater than 0.97. The calculation of the steady state parameter from the transient phase data makes the analysis fast and precise, especially for sensors with thick membranes, being convenient to use in the case of enzyme electrodes. The theoretical essence of the parameter C also gives valuable information for the optimal design of biosensors.

Activation of cAMP synthesis in rat brain cortical membranes by rubidium and cesium ions.

Rubidium and cesium chlorides accelerated cAMP synthesis in rat brain cortex membranes, while other alkali metal chlorides had no influence on the rate of this process. The effect was dose-dependent and yielded above 2-fold activation of adenylate cyclase. It has been shown that Rb+ and Cs+ influenced directly the catalytic subunit of the enzyme and did not substitute Mg2+ in formation of the metal-ATP complex in this reaction. The stimulatory effect of Rb+ was additive to the activation of adenylate cyclase by the half-maximal (0.3 microM) as well as by the saturating (10 microM) forskolin concentrations, pointing to the fact that these effectors activate different isoforms of the enzyme in rat brain cortex.

Statistical analysis of protein kinase specificity determinants.

The site and sequence specificity of protein kinases, as well as the role of the secondary structure and surface accessibility of the phosphorylation sites on substrate proteins, was statistically analyzed. The experimental data were collected from the literature and are available on the World Wide Web at http://www.cbs.dtu.dk/databases/PhosphoBase/. The set of data involved 1008 phosphorylatable sites in 406 proteins, which were phosphorylated by 58 protein kinases. It was found that there exists almost absolute Ser/Thr or Tyr specificity, with rare exceptions. The sequence specificity determinants were less strict and were located between positions -4 and +4 relative to the phosphorylation site. Secondary structure and surface accessibility predictions revealed that most of the phosphorylation sites were located on the surface of the target proteins.

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.

Modulation of [3H]quinpirole binding to dopaminergic receptors by adenosine A2A receptors.

Alteration of ligand binding to dopamine D2 receptors through activation of adenosine A2A receptors in rat striatal membranes has been studied by means of kinetic analysis. The binding of dopaminergic agonist [3H]quinpirole to rat striatal membranes was characterized by the constants Kd = 1.50+/-0.09 nM and Bmax = 115+/-2 fmol/mg of protein. The kinetic analyses revealed that the binding had at least two consecutive and kinetically distinguishable steps, the fast equilibrium of complex formation between receptor and agonist (KA = 5.9+/-1.7 nM), followed by a slow isomerization equilibrium (Ki = 0.06). Activation of adenosine A2A receptors by CGS 21680 caused enhancement of the rate [3H]quinpirole binding, altering mainly the formation of the receptor-ligand complexes (KA) as well as the isomerization rate of this complexes (ki), while the deisomerization rate (k[-i]) and the apparent dissociation rate remained unchanged.

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.

Dual effect of muscarinic receptor agonists on Ca2+ mobilization in SH-SY5Y neuroblastoma cells.

The muscarinic receptor-stimulated mobilisation of calcium ions in SH-SY5Y neuroblastoma cells was measured as function of the concentration of seven muscarinic receptor agonists and partial agonists: carbamoylcholine, acetylcholine, propionylcholine, butyrylcholine, acetylthiocholine, methylfurmethide and tetrametylammonium. The dose-response curves reached a clear maximum followed by a downturn of the curve. The concentration interval where the activatory and inhibitory effects occurred depended on the structure of the ligand. The bell-shaped dose-response curves were analysed assuming that the drugs interact with two sites, which are responsible for agonistic and antagonistic effects, on the muscarinic receptors. The results indicate that full vs. partial agonism is at least in part determined by relative affinities of these two sites.

A model of non-exclusive binding of agonist and antagonist on G-protein coupled receptors.

A modified concept of functioning of G-protein coupled receptors is presented, on the assumption that agonistic and antagonistic effects of drugs are related to their interaction with two separate receptor sites that exist simultaneously on a single receptor molecule and possess different ligand-specificity patterns. This proposal distinguishes between agonists and antagonists as binding at one of these sites triggers the receptor response, whereas the other site elicits another response leading to the receptor blockade. As these sites are simultaneously present on a single receptor molecule the formation of a ternary complex between agonist, antagonist and receptor is possible. Practical consequences of this concept are analysed with reference to experimental data on muscarinic acetylcholine receptors.