Purification of acetylcholinesterase by tacrine affinity chromatography.
Acridine ligand affinity chromatography is an effective means of acetylcholinesterase (AChE) purification. However, the synthesis of these resins is laborious and expensive. We have developed an acridine ligand affinity resin that is easy to produce, inexpensive, and selective for AChE over butyrylcholinesterase. The resin is produced in a single synthetic step by attaching the aminoacridine tacrine to epoxy-activated Sepharose. AChE from bovine serum (59% yield), Torpedo electric organ (27-60% yield), and two commercial sources of eel AChE (> 92% yield) is purified using the affinity resin. One commercial source of eel AChE contains two proteins with molecular weights of 80 and 55 kDa upon purification, while two proteins with molecular weights of 55 and 25 kDa are isolated from the other commercial source, presumably representing degraded AChE. The degradation state of the commercially available eel AChE preparations did not influence their specific activities. The isolation of AChE from bovine serum results in a single 80-kDa protein. However, butyrylcholinesterase is not purified from the serum. Using the tacrine affinity resin, and 80-kDa AChE, solubilized from Torpedo electric organ membranes by protease digestion, can also be purified. Velocity sedimentation analysis of the Torpedo AChE reveals that the molecular forms isolated are either tetrameric or asymmetric when solubilized by collagenase or trypsin, respectively. Overall, the tacrine affinity resin which is simple and inexpensive to produce allows for the selective isolation of AChE from diverse biological matrices.
Neurodegenerative Diseases Group, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48106-1047, USA.
R T
RT
Carroll
J L
JL
Grimm
T W
TW
Hepburn
M R
MR
Emmerling
D000110:Acetylcholinesterase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D004524:Eels; D004557:Electric Organ / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D012117:Resins, Synthetic / Q000138:chemical synthesis; D013619:Tacrine / Q000138:chemical synthesis; D014101:Torpedo
D012117:Resins, Synthetic; D013619:Tacrine; D000110:Acetylcholinesterase