Wolfgang Herbert Hopff

«Affinity Labelling» des cholinergischen Rezeptors der motorischen Endplatte mit Diazo-Acetylcholin

Diazoacetylcholinebromide was used for affinity labelling of the cholinergic receptors of endplates in the mouse diaphragm. Under irradiation by light (Hg-high-pressure-lamp), irreversible depolarizing block of neuromuscular transmission developed. Acetylcholinesterase was influenced only by much higher concentrations, which demonstrates different localizations of the active enzyme center and the cholinergic receptor.

Blockade of acetylcholine synthesis in organophosphate poisoning

In spite of worldwide research efforts in the search for the treatment of organophosphate poisoning, the substances with practical antidotal capabilities remain to be discovered. This problem has generally been approached by attempting to reactivate the inhibited acetylcholinesterase. Our approach consisted of reducing the amount of the lethal agent acetylcholine by blocking its synthesizing enzyme cholineacetylase with methyl methane thiol sulfonate (MMTS). We have taken into consideration that we are dealing with acute toxicological problems. This applies for poisoning as well as for treatment, and therefore in the present stage we can only present minimal results. The time from sarin (2 mg/kg) injection to death in rats (controls) was 2:59 min. With a MMTS dosage of 133.5 mg/kg prior to sarin, it was prolonged to 20:55 min (p less than 0.01). With the same dosage of MMTS under identical conditions, the time from soman (2 mg/kg) injection to death was prolonged from 6:08 to 14:48 min (p less than 0.01). Although MMTS cannot be used as a therapeutic agent, our attempt has demonstrated a utility in treating organophosphate poisoning in mice and rats and points in a direction where further work might be fruitful.

Isolation and purification of acetylcholine receptor proteins by affinity chromatography.

In order to further molecular investigations on the binding capacity of acetylcholine receptors, a method was developed for the affinity chromatography of the nicotinic acetylcholine receptor. Reversibly binding cholinergic ligand groups were used as affinity ligands, instead of the well known snake venom alpha-toxins. These ligands are small in size, chemically well defined and fixed to long spacer chains (at least 40 nm). One ligand, with a pharmacologically stabilizing effect on the receptor, was a derivative of gallamine. Another, with a depolarizing effect, resembled carbamoylcholine and a third was a derivative of decamethonium. The receptor proteins were isolated from Torpedo marmorata electric organs. Preparation included solubilization with a non-ionic detergent, alkaline treatment to extract peripheral membrane proteins and affinity purification. The receptor proteins eluted from the three affinity resins were identical in their assembly of subunits (alpha, beta, gamma and delta) but of different purity. Receptor proteins were obtained on a large scale within a short time and under mild conditions for elution with the affinity ligands of the decamethonium or the gallamine type. This was a considerable advantage compared to the use of alpha-bungarotoxin.

Synthesis of specific cholinergic inhibitors for affinity chromatography

Our first aim was to simplify the spacer-synthesis for affinity-chromatography of cholinergic proteins. Further-more we synthesized 2 new inhibitors which proved to be useful for purification of acetylcholine-receptor protein.

Affinity Chromatography of Cholinergic Receptor Proteins

Cholinergic binding proteins were purified from torpedo electric organ. The preparation comprises: solubilization by non-ionic detergents followed by unspecific prepurification. For prepurification the double reversed technique proved to be very useful. Finally we applied affinity chromatography. For the affinity purification we used resins with chemically well defined small ligand groups from the depolarizing type (carbachol- and decamethonium-analogue), and from the stabilizing type (gallamine amide amine). The purified receptor proteins from all three resins showed different subunit compositions and different properties of alpha-bungarotoxin binding.

Cholinergic Receptor Isolation

The isolation of specific cholinergic bindin protein (acetylcholine receptor = AChR) has been achieved successfully by numerous research groups (1-4, 7-9, 11, 12). The ideal source for such preparations was the electric organ of several species of Torpedo. All successful preparations notwithstanding, there are still some serious problems waiting to be solved. The first problem is the stability of AChR. Compared to acetylcholinesterase (AChE) which can be kept at room temperature for several days without losing enzymatic activity, the AChR is less stable. Regarding stability as shown by immunological properties only, the AChR is remarkably stable. Even methods such as freeze drying of the electric organ or protein fractions during preparation, binding to animal toxins (mostly snake venoms) and application of drastic methods to release it from affinity columns do not interfere with the immunological properties. Regarding stability in terms of pharmacological properties, however, the AChR is very unstable and continuously loses its binding properties during isolation and over the course of time. It is still an open question whether a free isolated receptor in solution will ever behave like a receptor in its natural environment embedded in membranes. Some factors which might interact with this very sensitive protein are 1) ambient oxygen, 2) proteolytic enzymes (a number of proteolytic enzymes are liberated in the course of isolation), 3) microbes, 4) acetylcholine (ACh) (reasonable amounts of ACh are liberated during preparation), and 5) other agonist type molecules (small molecules with depolarizing ability have been used for affinity chromatography and/or elution from the affinity column.

Organophosphate Poisoning and its Treatment by Oximes

In our last report in Oglebay Park (Waser et al., 1983) we described the poisoning of animals with 14C-labeled sarin and its kinetics. We demonstrated the low antitoxic activity of pralidoxime and obidoxime injected 2–60 minutes after the poisoning, but causing a marked decrease of radioactivity when injected 20 minutes before 14C-sarin. We will report here on the kinetics and distribution of another organophosphate, 32P-diisopropyl-fluorophosphonate (DFP) (phosphofluoridic acid bis (1-methylethyl) ester) in mice. The interaction between sarin and obidoxime or pralidoxime (2-PAM) will be described, and possible mechanisms of reactivation of the phosphorylated enzyme, or of antagonizing the overflowing acetylcholine will be discussed.