Isolation of a galactose-free 20-kDa fragment exhibiting butyrylcholine esterase and aryl acylamidase activity from human serum butyrylcholine esterase by limited α-chymotrypsin digestion

Abstract

Purified human serum butyrylcholine esterase (~ 90-kDa subunit), which also exhibits aryl acylamidase activity, was subjected to limited α-chymotrypsin digestion. Three major protein fragments of ~ 50 kDa, ~ 21 kDa and ~ 20 kDa were found to be produced, as observed by SDS-gel electrophoresis of the chymotryptic digest. The purified butyrylcholine esterase could fully bind to a Ricinus-communis-agglutinin-Sepharose column but after chymotryptic digestion about 15-20% of the enzyme activity remained unbound and was recovered in the run-through fractions. Sephadex G-75 chromatography of the chymotryptic digest showed an enzymatically active fragment eluted at an approximate molecular mass of 20 kDa, apart from the undigested butyrylcholine esterase eluted at the void volume. The butyrylcholine esterase fragment that did not bind to Ricinus communis agglutinin also was eluted at an approximate molecular mass of 20 kDa from a Sephadex G-75 column. This enzymatically active low-molecular-mass fragment from Sephadex G-75 chromatography showed a single protein band of ~ 20 kDa on SDS-gel electrophoresis. Neutral sugar analysis of the ~ 20 kDa fragment showed the presence of mannose only, whereas the undigested butyrylcholine esterase showed the presence of both mannose and galactose. Amino-terminal-sequence analysis of the ~ 20 kDa fragment showed the sequence Arg-Val-Gly-Ala-Leu, which agrees with amino acid residues 147-151 reported for human serum butyrylcholine esterase [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. Both cholinesterase and aryl acylamidase activities were co-eluted in all chromatographic procedures. The results suggested that limited α-chymotrypsin digestion of human serum butyrylcholine esterase resulted in the formation of a ~ 20-kDa enzymatically active fragment with Arg¹⁴⁷ as its N-terminal residue and which was devoid of galactose.