Localization of the peptidase activity of human serum butyrylcholinesterase in a approximately 50-kDa fragment obtained by limited alpha-chymotrypsin digestion.
Purified human serum butyrylcholinesterase (approximately 90-kDa subunit) is known to exhibit aryl acylamidase and peptidase activity. Limited alpha-chymotrypsin digestion of the purified butyrylcholinesterase gave three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa. In our earlier studies [Rao and Balasubramanian (1989) Eur. J. Biochem. 179, 639-644] we characterized the approximately 20-kDa fragment and showed that it exhibited both butyrylcholinesterase and aryl acylamidase activities. In the present studies the approximately 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 approximately 50-kDa fragment could be immuno-precipitated 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 approximately 50 kDa by silver staining. Amino-terminal sequence analysis of the approximately 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 alpha-chymotrypsin digestion of human serum butyrylcholinesterase resulted in the formation of a approximately 20-kDa fragment exhibiting both cholinesterase and aryl acylamidase activities and a approximately 50-kDa fragment exhibiting only peptidase activity.
Rao
R V
RV
Department of Neurological Sciences, Christian Medical College and Hospital, Vellore, India.
Balasubramanian
A S
AS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002091:Butyrylcholinesterase / Q000097:blood* / Q000302:isolation & purification; D002802:Cholinesterases / Q000097:blood*; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D002918:Chymotrypsin; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification*; D011233:Precipitin Tests
D000596:Amino Acids; D010446:Peptide Fragments; D007531:Isoflurophate; D002091:Butyrylcholinesterase; D002802:Cholinesterases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D002918:Chymotrypsin