HETEROGENEITY OF ACETYLCHOLINESTERASE IN NEUROBLASTOMA

Abstract

Abstract– Multiple forms of acetylcholine hydrolyzing activity have been observed in Triton X‐100 treated homogenates of mouse neuroblastoma cells. All these forms appear to be the true acetylcholinesterase, AChE (EC 3.1.17): they are substrate‐inhibited; hydrolyze acetylcholine > acetyl‐β‐methylcholine ≫ butrylcholine and are preferentially inhibited by specific AChE inhibitors. Almost all of the cell AChE activity is membrane associated, but readily ‘solubilized’ by Triton X‐100 and as such appears free of membrane contamination. With the aid of affinity chromatography the ‘solubilized’ neuroblastoma AChE has been partially purified (490‐fold) to a specific activity of 34,300 nmol/min/mg protein.Four active neuroblastoma AChE species appear upon acrylamide gel electrophoresis (with MWs of 64,000; 116,000; 186,000 and 284,000) while three species (4S, 6S and 9.6S) have been found upon sucrose gradient sedimentation analysis. We have determined that the 4S form migrates on acrylamide as the 116,000 MW species and the 9.6S form contains, in equal amounts, the 186,000 and 284,000 MW acrylamide species. Numerous active AChE forms are seen on Sepharose 6B chromatography. From comparing the crude, 4S, 9.6S and partially purified AChEs on acrylamide gels, sucrose gradients and Sepharose, mouse neuroblastoma appears to contain active AChE units which are capable of multiple types of dissociation and reassociation. An attempt is made to correlate all the observed AChE forms as well as relate this data to that known about AChE obtained from other sources.