Conformers of acetylcholinesterase: a mechanism of allosteric control.

Rate control in acetylcholinesterase (AChE) involves a single anionic site whose anionic center controls rate-related biochemical and conformational changes in the E (free enzyme) and EA (acylated enzyme) conformers. Change in conformer structure and biochemistry affect binding, acylation, and hydrolysis. It is significant that the anionic-esteratic intersite distance is not altered during conformer change as E is converted to EA. In this enzyme system, cationic acetylcholine and anionic AChE are true structural, functional, and biochemical counterparts. The anionic center in the E conformer lies at the bottom of a sterically restricted, hydrophobic cleft < 8 A wide at the top and > 3 A wide at the bottom, while the anionic center in the EA conformer is relatively open. It is characterized by a decrease in the relative binding of hydrophobic cations and by an ability to bind large organic cations. Binding of acetylcholine, H+, or organic cations at the anionic site controls k2(acylation) in the E conformer and k3(hydrolysis) in the EA conformer. Acetylcholine binding forms the ES complex in which the cation maximizes k2. In the EAS complex, the cation reduces k3 and provides allosteric control. Anionic site structure and biochemistry and the effect of pH on k2 and k3 differentiates AChE from butyrylcholinesterase. This comprehensive study of kinetic and thermodynamic processes in AChE was made possible by the synthesis and/or use of families of over 30 cationic and acylation probes of known stereochemistry. They act as rulers of the E and EA conformers of AChE and provide comparative data on kinetic-based and thermodynamic-based constants. Cationic inhibitors affect decarbamylation rates in AChE and provide an additional set of comparative data related to the mechanism of substrate hydrolysis by AChE. Acridine araphanes are unique neural receptor and cholinergic enzyme probes. Their parallel plane and coplanar conformations are related to bridge length. Two parallel plane acridine araphanes are pure uncompetitive inhibitors of AChE. Scatchard plots of the binding of methylacridinium and 9-aminoacridine with the E conformer and 9-aminoacridine with the EA conformer indicate binding at a single anionic site. No ternary complex (EII or EAII) from two-site binding was detected. In AChE, nonspecific, low-level binding at surface ionic and hydrophobic areas is ubiquitous. Binding affinity differences greater than two orders of magnitude distinguish binding at the anionic site from low level binding at surface moieties. Surface binding provides environmental and stability changes in the enzyme but does not modify the fundamental biochemistry of the E and EA conformers.

Affinity chromatography of acetylcholinesterase from Electrophorus electricus electroplax. Investigations on 9-aminoalkylacridine affinity ligands.

The neural enzyme 11-S acetylcholinesterase (E.C. 3.1.1.7) was purified by affinity chromatography from a trypsin digest of Electrophorus electricus electric organ. Unquaternized affinity ligands were reported which were comparable in efficacy to the routinely employed "quaternized acridine MAC ligand". A study was made of the quaternization reactions of various 9-aminoalkylacridines and 9-aminoacridine along with their relative binding affinities to acetylcholinesterase. Ease of synthesis in conjunction with the column performance of these unquaternized 9-aminoalkylacridine compounds made them the preferred affinity ligand in acetylcholinesterase chromatography. A new carbodiimide synthetic route for these unquaternized ligands was described.

Acridine araphanes: a new class of probe molecules for biological systems.

The bis-acridine ring system forms the basis for new biophysical probes of novel stereochemistry. Spectral data indicate that certain alkylene bridged bis-9-aminoacridines have a parallel plane conformation of predictable interplane distance. The parallel plane conformation is independent of solvent and thus is different from nucleic acid systems. This stable conformation allows these compounds to be used as sensitive "rulers" for describing binding site geometry in cholinergic enzymes and in the delineation of the mechanism of allosteric control in acetylcholinesterase.

Spruce budworm mortality as a function of aerial spray droplet size.

The size and number of aerial spray drops impinging on spruce budworm in its conifer forest habitat were determined by means of a new tracer method that uses fluorescent particles in a liquid spray. Examination of 1113 larvae affected by an experimental insecticide that had been applied to a 5000-acre (2024-ha) test area in Montana showed that 93 percent had not been contacted by any droplets larger than 50 micro in diameter. Small numbers of droplets 50 to 100 micro in diameter were found on 7 percent of the larvae, along with lethal numbers of smaller drops. No evidence was found that significant numbers of drops larger than 100 micro reached the target insects. Because about 95 percent of the spray applied to forests by current methods consist of droplets larger than 50 micro, the biologically effective portion of the drop spectrum is only a few percent. The data foreshadow a major potential reduction in insecticide requirements for the successful control of spruce budworm.