Cholinergic Receptor Isolation

Abstract

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.