Abstract
Human serum cholinesterase was digested with pepsin under conditions which left disulfide bonds intact. Peptides were isolated by high pressure liquid chromatography, and those containing disulfide bonds were identified by a color assay. Peptides were characterized by amino acid sequencing and composition analysis. Human serum cholinesterase contains 8 half-cystines in each subunit of 574 amino acids. Six of these form three internal disulfide bridges: between Cys65-Cys92, Cys252-Cys263, and Cys400-Cys519. A disulfide bond with Cys65 rather than Cys66 was inferred by homology with Torpedo acetylcholinesterase. Cys571 forms a disulfide bridge with Cys571 of an identical subunit. This interchain disulfide bridge is four amino acids from the carboxyl terminus. A peptide containing the interchain disulfide is readily cleaved from cholinesterase by trypsin (Lockridge, O., and La Du, B. N. (1982) J. Biol. Chem. 257, 12012-12018), suggesting that the carboxyl terminus is near the surface of the globular tetrameric protein. The disulfide bridges in human cholinesterase have exactly the same location as in Torpedo californica acetylcholinesterase. There is one potential free sulfhydryl in human cholinesterase at Cys66, but this sulfhydryl could not be alkylated. Comparison of human cholinesterase, and Torpedo and Drosophila acetylcholinesterases to the serine proteases suggests that the cholinesterases constitute a separate family of serine esterases, distinct from the trypsin family and from subtilisin.
Highlights
Cys", Cy~~~"-Cys~a'n'd, C y ~ " ~ - C y s A~ ' d~i.sulfide bond with Cyse6 ratherthanCysee was inferredby homology with Torpedo acetylcholinesterase
Purification of Cholinesterase-Outdated human plasma was a gift from the Michigan Department of Public Health, Lansing, MI. 7.5 litera of plasma were used for each cholinesterase purification procedure, which consisted of three steps: ion-exchange chromatography a t pH 4.0, followed by affinity chromatography on procainamideSepharose 4B,and by ion-exchange chromatography a t pH 7.0 [3,11,12]
Our strategy for identifying disulfide-linked peptides was based on knowledge of the complete amino acid sequence of cholinesterase[7] and on our observation that the protein
Summary
Human serum cholinesterasewas digested with pep- by antibodiesaroused suspicionthat theamino acid sequences sin under conditions whichleft disulfide bonds intact. Human serum cholinesterase contains 8 half-cys- lengths are nearly the same at 574 and 575 amino acids/. This interchain disulfide bridgies four amino acids from the carboxyl termiAnupse.ptide containing the interchaindisulfideis readily cleavedfrom cholincholinesterase and compare the results with the disulfide bonds in Torpedo acetylcholinesterase[9]. We found that the disulfide bonds in human cholinesterase are in the same location as inTorpedo acetylcholinesteraseand have the same number of amino acids within each disulfide loop.This suggests that protein folding in thetwo enzymesis similar. The disulfide bridges in quence is known; the Torpedo acetylcholinesterase but not human cholinesterase have exactly the same location the Torpedo cholinesterase sequence is known. The PRPLOT program was used to plot the hydropat.hyindex
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