Localization of the peptidase activity of human serum butyrylcholinesterase in a – 50-kDa fragment obtained by limited α-chymotrypsin digestion

Abstract

Purified human serum butyrylcholinesterase (∼90-kDa subunit) is known to exhibit aryl acylamidase and peptidase activity. Limited α-chymotrypsin digestion of the purified butyrylcholinesterase gave three major protein fragments of ∼ 50 kDa, ∼21 kDa and ∼ 20 kDa. In our earlier studies [Rao and Balasubramanian (1989) Eur. J. Biochem. 179, 639-644] we characterized the ∼ 20-kDa fragment and showed that it exhibited both butyrylcholinesterase and aryl acylamidase activities. In the present studies the ∼ 50-kDa fragment is characterized. This fragment, after isolation by Sephadex G-75 chromatography from a chymotryptic digest of purified butyrylcholinesterase, exhibited only peptidase activity and was devoid of cholinesterase and aryl acylamidase activities. It could bind to a column of Ricinus communis agglutinin bound to Sepharose, indicating its glycosylated nature and the presence of galactose. The peptidase activity in the ∼50-kDa fragment could be immunoprecipitated by a polyclonal antibody raised against purified butyrylcholinesterase. SDS-gel electrophoresis of this fragment isolated by R. communis agglutinin - Sepharose and Sephadex G-75 chromatography showed a protein band of ~ 50 kDa by silver staining. Amino-terminal sequence analysis of the ∼ 50-kDa fragment gave the sequence of Gly-Pro-Thr-Val-Asp which corresponded to amino acid residues 291-295 in the butyrylcholinesterase sequence [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. The combined results suggested that α -chymotrypsin digestion of human serum butyrylcholinesterase resulted in the formation of a ∼ 20-kDa fragment exhibiting both cholinesterase and aryl acylamidase activities and a ∼50-kDa fragment exhibiting only peptidase activity.