Bovine brain acetylcholinesterase primary sequence involved in intersubunit disulfide linkages.

W L Roberts,B P Doctor,J D Foster,T L Rosenberry

doi:10.1016/s0021-9258(20)89471-x

W L Roberts, B P Doctor + Show 2 more

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https://doi.org/10.1016/s0021-9258(20)89471-x

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Abstract

Three distinct classes of membrane-bound acetylcholinesterases (AChEs) have been identified. A12 AChE is composed of 12 catalytic subunits that are linked to noncatalytic collagen-like subunits through intersubunit disulfide bonds. G2 AChE is localized in membranes by a glycoinositol phospholipid covalently linked to the C-terminal amino acid. Brain G4 AChE involves two catalytic subunits linked by a direct intersubunit disulfide bond while the other two are disulfide-linked to a membrane-binding 20-kDa noncatalytic subunit. Molecular cloning studies have so far failed to find evidence of more than one AChE gene in any organism although alternative splicing of torpedo AChE mRNA results in different C-terminal sequences for the A12 and G2 AChE forms. Support for a single bovine AChE gene is provided in this report by amino acid sequencing of the N-terminal domains from the G2 erythrocyte, G4 fetal serum, and G4 brain AChE. Comparison of the 38-amino acid sequences reveals virtually complete identity among the three AChE forms. Additional extensive identity between the fetal serum and brain AChEs was demonstrated by sequencing several brain AChE peptides isolated by high performance liquid chromatography after trypsin digestion of nitrocellulose blots of brain AChE catalytic subunits. Cysteines involved in intersubunit disulfide linkages in brain AChE were reduced selectively with dithiothreitol in the absence of denaturants and radioalkylated with iodoacetamide. The observed sequence of the major radiolabeled tryptic peptide was C*SDL, where C* was the radioalkylated cysteine residue. This sequence is precisely the same as that observed at the C terminus of fetal bovine serum AChE and shows close homology to the C-terminal sequence of torpedo A12 AChE. We conclude that the mammalian brain G4 AChEs utilize the same exon splicing pattern as the A12 AChEs and that factors other than the primary sequence of the AChE catalytic subunits dictate assembly with either the collagen-like or the 20-kDa noncatalytic subunits.

Highlights

From the Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106 and the SDiuision of Biochemistry, Walter Reed Army Institute of Research, Washington, D.C . 20307
AChE is composed of 12 catalytic subunits that are ences in the size of the oligomeric assemblies but linked to noncatalytic collagen-like subunits through in the subunits that attach the enzyme to membranes
We report here extensive identity in the primary amino sequences of bovine brain and erythrocyte AChEs with that published previously for fetal bovine serum AChE (Doctor et al, 1990)

Summary

RESULTS

Itol, and the mixtures were incubated for 30 min at the indicated Amino Acid Sequences of Intact Bovine AChEs-Bovine temperature. Amino acid analysis of each I4C-radiomethylated AChE demonstrated thatthe only significant N e methylated amino acid coeluted with a [3H]dimethylglutamic acid standard and that thirsadiomethylated amino acid gave a stoichiometry of nearly one per polypeptide in all three AChEs (Table I) Assignment of this labeled amino acid as N-terminal glutamic acid is consistent with the complete protein sequence shown in Fig. 5 below for the fetal bovine serum AChE catalytic subunit polypeptide (Doctor et al, 1990). The most prominent radioalkylated tryptic peptide eluted at 32 min, andits observed sequence was C*SDL, where C* wasthe radiolabeled cysteine residue This sequencecorresponds precisely to theC-terminal tetrapeptide in fetal bovine serum AChE (Fig. 5).

G L FAHVRL OSGAPNGPwgTVGV GRERA r

DISCUSSION

Findings

Methods