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

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.