Rapid purification of a phospholipase-free alpha-bungarotoxin: maintenance of cation barriers of acetylcholine receptor membranes upon preincubation with purified toxin.

ABSTRACT

The purification of highly homogeneous, phospholipase-free alpha-bungarotoxin (alpha-Bgt) from the venom of the elapid Bungarus multicinctus or from commercial samples of alpha-Bgt is described. The method combines a conventional procedure for the purification of alpha-Bgt [D. Mebs, K. Narita, S. Iwanaga, Y. Samejima, and C. Y. Lee (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 243-262] with high-resolution gel-filtration and cation-exchange chromatography steps to remove membrane-damaging, contaminating phospholipase activity. The procedure also removes contaminating radioactive peptides from commercial preparations of 125I-alpha-Bgt. Apparent homogeneity of the purified alpha-Bgt (referred to as fraction D in the text), as well as the absence of contaminating phospholipase A2 activity, is assessed by (i) polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, (ii) gel-filtration and cation-exchange high-performance liquid chromatography, (iii) direct measurements of phospholipase A2 activity under conditions where very low enzymatic levels should be detected, (iv) lack of interference with the passive cation permeability properties of acetylcholine receptor membranes, (v) competitive inhibition of 125I-alpha-Bgt binding to the acetylcholine receptor membranes, and (vi) amino acid analysis and end-group (C- and N-terminus) determination. alpha-Bgt preparations subjected to these criteria do not exert the increase in membrane passive permeability to cations detected with other laboratory or commercial samples of alpha-Bgt. Availability of the new alpha-Bgt preparation allows for an assessment of the inertness of alpha-Bgt on lipid membrane properties while preventing cholinergic ligand binding to nicotinic acetylcholine receptor-rich membranes. These conditions are necessary for experiments requiring maintenance of the physical and phospholipid integrity of membranes.