Natural monomeric form of fetal bovine serum acetylcholinesterase lacks the C-terminal tetramerization domain.
Acetylcholinesterase isolated from fetal bovine serum (FBS AChE) was previously characterized as a globular tetrameric form. Analysis of purified preparations of FBS AChE by gel permeation chromatography revealed the presence of a stable, catalytically active, monomeric form of this enzyme. The two forms could be distinguished from each other based on their molecular weight, hydrodynamic properties, kinetic properties, thermal stability, and the type of glycans they carry. No differences between the two forms were observed for the binding of classical inhibitors such as edrophonium and propidium or inhibitors that are current or potential drugs for the treatment of Alzheimer's disease such as (-) huperzine A and E2020; tacrine inhibited the monomeric form 2-3-fold more potently than the tetrameric form. Sequencing of peptides obtained from an in-gel tryptic digest of the monomer and tetramer by tandem mass spectrometry indicated that the tetramer consists of 583 amino acid residues corresponding to the mature form of the enzyme, whereas the monomer consists of 543-547 amino acid residues. The subunit molecular weight of the protein component of the monomer (major species) was determined to be 59 414 Da and that of the tetramer as 64 239 Da. The N-terminal of the monomer and the tetramer was Glu, suggesting that the monomer is not a result of truncation at the N-terminal. The only differences detected were at the C-terminus. The tetramer yielded the expected C-terminus, CSDL, whereas the C-terminus of the monomer yielded a mixture of peptides, of which LLSATDTLD was the most abundant. These results suggest that monomeric FBS AChE is trimmed at the C-terminus, and the results are consistent with the involvement of C-terminal amino acids in the assembly of monomers into tetramers.
Saxena
Ashima
A
Division of Biochemistry, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910-7500, USA. ashima.saxena@na.amedd.army.mil
Hur
Regina S
RS
Luo
Chunyuan
C
Doctor
Bhupendra P
BP
D000110:Acetylcholinesterase / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D002417:Cattle; D002800:Cholinesterase Inhibitors / Q000737:chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004491:Edrophonium / Q000737:chemistry; D005312:Fetal Blood / Q000201:enzymology*; D006031:Glycosylation; D007527:Isoenzymes / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification; D010446:Peptide Fragments / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D011419:Propidium / Q000737:chemistry; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013816:Thermodynamics
D002800:Cholinesterase Inhibitors; D007527:Isoenzymes; D010446:Peptide Fragments; D011419:Propidium; D004491:Edrophonium; D000110:Acetylcholinesterase