Substrate Report for: NBD-5-acylcholine

We have synthesized a homologous series of fluorescent analogs of acetylcholine, N-7-(4-nitrobenzo-2-oxa-1,3-diazolyl)-omega-amino-n-alkanoic acid beta (N,N,N-trialkylammonium) ethylesters (NBD-n-acylcholines) and report here on their physiological and biochemical properties. All NBD-n-acylcholines trimethylated at the cholinergic nitrogen are agonists of acetylcholine at the frog neuromuscular junction. Their potencies in depolarizing frog muscle cells decrease with decreasing chain length. The affinities of binding to the purified receptor from Electrophorus electricus also decrease with decreasing chain length with a large drop in affinity for the derivatives n = 4 and n = 3. The rate constants of association to acetylcholine receptor and to acetylcholine esterase are of the order of 10(8) M-1 S-1 and do not vary significantly with the chain length of the NBD-n-acylcholines. In contrast, the dissociation rate constants decrease with increasing chain length. The quenching of fluorescence of NBD-n-acylcholines accompanying binding to purified receptor and esterase from E. electricus appears to be due to the formation of a hydrogen bond between the omega-amino group as donor and an unidentified acceptor group in a hydrophobic pocket of the protein. With their advantageous fluorescence properties, their simple pharmacology, and their clear structure-function relationships, these compounds are useful tools for the study of cholinergic mechanisms.

We have studied the binding equilibria of the membrane-bound acetylcholine receptor from Torpedo marmorata with representative cholinergic ligands by means of two fluorescence and a rapid centrifugation assay. Based on the established mechanism of acetylcholine binding to the receptor (Fels, G., Wolff, E. K., and Maelicke, A. (1982) Eur. J. Biochem. 127, 31-38), the obtained binding and competition data were analyzed assuming two classes of interacting sites for all ligands studied. The experimental data were consistent with this assumption and, based on the obtained KD values, suggest weak positively cooperative interactions of binding sites when occupied by agonists but independent (or negatively cooperative interacting) sites when occupied by antagonists. Based on the fluorescence binding assay employed, agonists and antagonists induce different conformational states of the liganded receptor. These states seem to be similar for all antagonists tested but differ for the different agonists tested. The existence of ligand-specific conformational states suggests a close link of these states with receptor function.

We have developed a simple, direct and time-resolved method to monitor ligand-induced changes in agonist affinity of the membrane-bound acetylcholine receptor. The assay is based on the quenching of fluorescence of NBD-5-acylcholine observed upon binding of this cholinergic agonist to the receptor. Under conditions of partial saturation with the fluorescent agonist, agonists and local anesthetics but not antagonists can induce an increase in affinity of the receptor for NBD-5-acylcholine. The effect is not observed with receptor fully saturated with the fluorescent agonist. The half-life of the observed change in affinity is independent of the nature of the agonist or local anesthetic applied (t1/2 approximately 60 s at 22 degrees C). We conclude that the same state transition of the receptor can be induced by two groups of cholinergic ligands that are assumed to be non-competitive with each other and to have distinctly different modes of action. The time course of the transition is reminiscent of the slow process of desensitization observed in vivo.

We have synthesized a homologous series of fluorescent analogs of acetylcholine, N-7-(4-nitrobenzo-2-oxa-1,3-diazolyl)-omega-amino-n-alkanoic acid beta (N,N,N-trialkylammonium) ethylesters (NBD-n-acylcholines) and report here on their physiological and biochemical properties. All NBD-n-acylcholines trimethylated at the cholinergic nitrogen are agonists of acetylcholine at the frog neuromuscular junction. Their potencies in depolarizing frog muscle cells decrease with decreasing chain length. The affinities of binding to the purified receptor from Electrophorus electricus also decrease with decreasing chain length with a large drop in affinity for the derivatives n = 4 and n = 3. The rate constants of association to acetylcholine receptor and to acetylcholine esterase are of the order of 10(8) M-1 S-1 and do not vary significantly with the chain length of the NBD-n-acylcholines. In contrast, the dissociation rate constants decrease with increasing chain length. The quenching of fluorescence of NBD-n-acylcholines accompanying binding to purified receptor and esterase from E. electricus appears to be due to the formation of a hydrogen bond between the omega-amino group as donor and an unidentified acceptor group in a hydrophobic pocket of the protein. With their advantageous fluorescence properties, their simple pharmacology, and their clear structure-function relationships, these compounds are useful tools for the study of cholinergic mechanisms.

We have studied the binding equilibria of two fluorescent ligands and several nonfluorescent cholinergic ligands with the purified acetylcholine receptor from Electrophorus electricus. The assay was based on the specifically cholinergic and reversible quenching of fluorescence observed upon complex formation between the receptor protein and N-7-(4-nitrobenzo-2-oxa-1,3-diazole)-5-aminopentanoic acid beta-(N-trimethylammonium) ethyl ester. This way, a large body of accurate, true equilibrium data was obtained. We find 1) all ligands studied compete for the same number of binding sites at the receptor; 2) agonists compete for half of these sites with high affinity and for the other half of these sites with significantly lower affinity; 3) antagonists compete for all of these sites with one affinity; and 4) in the presence of disulfide reducing agents, the binding patterns of some agonists and antagonists are changed in accordance with the electrophysiological changes observed under the same conditions with Rana pipiens Sartorius muscle fibers. Our studies indicate that the mechanism of ligand recognition is still functional at equilibrium and is not subject to the presence of an intact membrane environment. Furthermore, the existence of two types of agonist sites at every receptor molecule excludes most of the presently discussed two-state models as the basis for a mechanism of receptor-ligand interaction. To explain sigmoidal dose-response curves, a two-site model is already sufficient.

We have studied the kinetics of interaction of N-7-(4-nitrobenzo-2-oxa-1,3,diazole)-5-aminopentanoic acid beta-(N-trimethylammonium) ethyl ester with the purified acetylcholine receptor from Electrophorus electricus. By employing a laser stopped flow fluorimeter, the full course of the kinetics was observed. The initial analysis of data yielded the following results. 1) Both association and dissociation kinetics are composed of several overlapping reaction steps. 2) Dissociation of monoliganded receptor is biphasic. This indicates the existence of two forms in equilibrium of the monoliganded receptor. 3) Displacement of fluorescent ligand by competing ligand follows a more complicated pattern. In particular, the late stages of the dissociation kinetics are increasingly slowed down the higher the concentration of competing ligand. This indicates a strictly ordered mode of association and dissociation, i.e. first on-last off for cholinergic ligands and the receptor. These and the previous finding of two agonist binding sites at the receptor (Prinz, H., and Maelicke, A. (1983) J. Biol. Chem. 258, 10263-10272) were employed to develop minimal reaction schemes that can account for the experimental data. They require two sites but four different ligand-receptor complexes and a strictly ordered mode of binding. The reaction schemes were used to simultaneously fit whole sets of association and dissociation kinetics. The fits reinforced the conclusions drawn by the initial analysis and also provided the rate constants for all reaction steps considered in the schemes. The following additional results were obtained. 4) One form of the monoliganded and diliganded receptor are formed with very large rate constants (greater than 2 X 10(8) M-1 s-1); the other complexes are formed more slowly. 5) The rapidly formed diliganded receptor (AR-A) has a rather short half-life while the other form of diliganded receptor (AR*A) develops more slowly and dominates at equilibrium. The obtained reaction schemes and rate constants invite comparison with physiological data. For example, the properties of the two forms of diliganded receptor correlate with those of the active and desensitized state of the endplate as defined by electrophysiological studies.

The binding of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to chicken liver fatty acid synthase has been studied by using both fluorescence titrations and the direct binding method of forced dialysis. Four apparently identical sites are found per enzyme molecule, with an intrinsic dissociation constant of 0.6 microM at pH 7.0, 23 degrees C. The acyl-binding sites on the enzyme have been studied with a fluorescent analogue of acetyl-CoA, beta-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)]alanyl coenzyme A (NBDA-CoA). The enzyme slowly transfers NBDA to acyl-binding sites. Analysis of the kinetics of binding and of the stability and hydroxylamine sensitivity of the acyl-enzyme at pH 7.5 suggests that binding occurs predominantly at two classes of sulfhydryl sites, with two sites of each class per enzyme molecule. Up to one NBDA per enzyme molecule is bound to a nonsulfhydryl site after overnight incubation of enzyme with NBDA-CoA. The acyl linkage at one class of sulfhydryl sites appears to be hydrolyzed by the thioesterase activity of the enzyme. This hydrolysis can be prevented by modifying the enzyme with tosyl fluoride. The binding of NBDA is inhibited by acetyl-CoA, malonyl-CoA, and NADPH. The NBDA-enzyme adduct is inactive, although activity can be partially restored by incubation at 35 degrees C. The binding of NADPH to the enzyme is not significantly altered by the binding of NBDA. Fluorescence resonance energy transfer between enzyme-bound NADPH and enzyme-bound NBDA suggests that the acyl-binding sites are 30-40 A from NADPH-binding sites. This distance can only be defined approximately because of the presence of multiple energy donors and acceptors and the uncertainty in the dipole-dipole orientations of the energy acceptors and donors.

We have studied the properties of N-7-(4-nitrobenzo-2-oxa-1,3-diazole)-omega-aminohexanoic acid beta-(N-trimethylammonium)ethyl ester, a fluorescent analog of acetylcholine at the cellular level by using pharmacological and electrophysiological techniques and at the molecular level by measuring the kinetics of interaction with solubilized acetylcholine receptor and with acetylcholine esterase (EC 3.1.1.7). The fluorescent drug is a powerful agonist of acetylcholine at the neuromuscular junction and also strongly desensitizes muscle fibers. Interaction with acetylcholine receptor is accompanied by large changes in the drug's fluorescence. From the kinetics of interaction studied by means of a stopped-flow fluorimeter with laser light source, we obtained a second-order forward rate constant in excess of 1 X 10(8) M-1 sec-1 and an initial dissociation rate constant (k1) of 0.5 sec-1 for receptor from Electrophorous electricus. Interaction of this analog with acetylcholine esterase from E. electricus is accompanied by a transient decrease in fluorescence followed by an increase leading to a stable plateau value at a level near the original one. The initial decrease in fluorescence followed second-order kinetics with k2 of the order of 10(9) M-1 sec-1. The slower consecutive reaction which could be blocked by phosphorylation of the esteratic site, was of first order with k1 = 0.05 sec-1.