Purification and partial amino acid sequence analysis of human erythrocyte acetylcholinesterase

Abstract

A single step immunoaffinity purification procedure for human erythrocyte acetylcholinesterase is described which permitted the isolation of milligram quantities of enzyme from 10 U of erythrocytes, with 113 000-fold purification and a yield of about 22%. In SDS-PAGE analysis, the enzyme corresponds to a disulfide linked dimer of 140 kDa which is converted to a 70 kDa monomer upon disultide reduction. The tryptic peptides generated from purified enzyme were separated by reverse-phase HPLC. Five of these peptides were analysed to determine the amino acid sequences. The obtained sequences showed no homology to the already known amino acid sequences for human serum and brain butyrylcholinesterase and Torpedo californica acetylcholinesterase.

Complete amino acid sequence of fetal bovine serum acetylcholinesterase and its comparison in various regions with other cholinesterases

“…The partial amino acid sequences of human AChEs shown here were determined by Soreq and Prody (human brain) [14l, Chhajlani et al (human erythrocyte) [13], and Haas and Rosenberry (human erythrocyte) [12]. figure).…”

“…(C) The gapping introduced in (h) between sites 6 and 7 by Soreq and Prody [14] to align this region with serine hydrolases has been eliminated; the active site serine is marked with an asterisk. (D) Sequence for human erythrocyte AChE (i) is reported by Chhajlani et al [13]. (E,F) Xs denote unidentified amino acids; gapping is introduced into the Drosophila sequence for alignment in these regions.…”

“…(G) hHis-423 and His-438 regions are merged with gapping between amino acids 439 and 442 for alignment with FBS ACHE. Chhajlani et al [13] reported the amino acid sequences of 5 tryptic peptides of human erythrocyte ACHE, shown in Fig. 3D-H …”

“…Doctor [6][7][8][9][10][11], as have partial sequences of AChEs isolated from human erythrocytes [12,13], human brain [14], and bovine brain [15]. Pharmacological studies and enzyme kinetic studies with these enzymes, including multiple molecular forms, have demonstrated that they possess similar catalytic properties.…”

Comparative study of the inhibitory effect of antidepressants on cholinesterase activity inBungarus sindanus(krait) venom, human serum and rat striatum

“…The physiologic function of human BChE is still unknown; but it can take the place of AChE in acetylcholine (ACh) degradation when AChE is inhibited or absent [8]. AChE and BChE share 65% amino acid sequence homology and have a similar molecular form and their substrate hydrolysis is carried out by the "catalytic triad" of Ser, His and Glu in the active centre of both types of enzymes [9]. The 3-D structure of the enzyme showed the long and narrow active site gorge is about 20 A8 deep and includes two sites of ligand interaction: an acylation site at the base of the gorge with the catalytic triad and a peripheral site in its mouth.…”