title: Pure human butyrylcholinesterase hydrolyzes octanoyl ghrelin to desacyl ghrelin.
abstract: The ghrelin hormone is a 28 amino acid peptide esterified on serine 3 with octanoic acid. Ghrelin is inactivated by hydrolysis of the ester bond. Previous studies have relied on inhibitors to identify human butyrylcholinesterase (BChE) as the hydrolase in human plasma that converts ghrelin to desacyl ghrelin. The reaction of BChE with ghrelin is unusual because the rate of hydrolysis is very slow and the substrate is ten times larger than standard BChE substrates. These unusual features prompted us to re-examine the reaction, using human BChE preparations that were more than 98% pure. Conversion of ghrelin to desacyl ghrelin was monitored by MALDI TOF mass spectrometry. It was found that 5 different preparations of pure human BChE all hydrolyzed ghrelin, including BChE purified from human plasma, from Cohn fraction IV-4, BChE immunopurified by binding to monoclonals mAb2 and B2 18-5, and recombinant human BChE purified from culture medium. We reasoned that it was unlikely that a common contaminant that could be responsible for ghrelin hydrolysis would appear in all of these preparations. km was <1 μM, and kcat was ~1.4 min(-1). A Michaelis-Menten analysis employing these kinetic values together with serum concentrations of ghrelin and BChE demonstrated that BChE could hydrolyze all of the ghrelin in serum in ~1 h. It was concluded that BChE is physiologically relevant for the hydrolysis of ghrelin.
authors.affiliation: Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950, USA. Electronic address: lmschopf@unmc.edu.
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Schopfer
authors.affiliation: Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950, USA. Electronic address: olockrid@unmc.edu.
authors.forename: Oksana
authors.initials: O
authors.lastname: Lockridge
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA. Electronic address: brimijoin@mayo.edu.
authors.forename: Stephen
authors.initials: S
authors.lastname: Brimijoin
mesh_terms: D002091:Butyrylcholinesterase / Q000378:metabolism*; D002853:Chromatography, Liquid; D054439:Ghrelin / Q000031:analogs & derivatives / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D053719:Tandem Mass Spectrometry
chemical_list: D054439:Ghrelin; C422650:ghrelin, des-n-octanoyl; D002091:Butyrylcholinesterase
keywords: Butyrylcholinesterase; Ghrelin; LC ESI MS; MALDI TOF; Monoclonal antibodies; SDS PAGE