Molecular Forms of Acetylcholinesterase in Bovine Caudate Nucleus and Superior Cervical Ganglion: Solubility Properties and Hydrophobic Character

Abstract

In the present paper, we report an analysis of acetylcholinesterase molecular forms in the bovine caudate nucleus and superior cervical ganglion. We show that: (1) The superior cervical ganglion contains a significant proportion (∼ 15%) of collagen‐tailed forms (mostly A12 and A8), but these molecules are found only as traces (ca. 0.002%) in the caudate nucleus, even in favorable extraction conditions (i.e., in the presence of 1 m‐NaCl, 5 mm‐EDTA, 1% Triton X‐100). (2) The bulk of acetylcholinesterase corresponds to globular forms, mostly the tetrameric G4 and the monomeric G1 forms, with a smaller proportion of the dimeric G2 form. (3) The tetrameric enzyme exists as a minor soluble component (GS4) that does not interact with Triton X‐100, and a major hydrophobic component (GH4) that is partially solubilized in the absence of detergent in the caudate nucleus, but not in the superior cervical ganglion. (4) The monomeric G1 form presents a marked hydrophobic character, as indicated by its interaction with Triton X‐100, although it may be solubilized in large part in the absence of detergent in both tissues. (5) The detergentsolubilized forms aggregate upon removal of detergent. This property disappears after partial purification of G4) that does not interact with Triton X‐100, and a major hydrophobic component (GH4, but is restored upon addition of an inactivated crude extract, indicating that it is attributable to interactions with other hydrophobic components. (6) The proportions of molecular forms solubilized in detergent‐free buffers vary with the ionic composition of the medium. Repeated extractions of caudate nucleus in Tris‐HCl buffer produce a larger overall yield of G1 form (e.g., 40%) than appears in a single quantitative detergent solubilization (<15%). This G1 form apparently derives in part from a pool of GH4 form. (7) However, detergents that allow a quantitative solubilization of acetylcholinesterase yield the same proportions of forms (about 85% G4) independently of the ionic conditions. (8) Modifications of the molecular forms occur spontaneously during purification, or storage of the crude aqueous ex‐tracts, in a manner that depends on the ionic conditions. In Tris‐HCl buffer, G1 is converted into a well‐defined 7.5S form. In Ringer, polydisperse components are formed. The effects observed in Ringer cannot be reproduced by addition of 5 mm‐Ca2‐ to the Tris buffer either during or after extraction. (9) Proteases, such as pronase, convert the hydrophobic forms into molecules that do not appear to interact with Triton X‐100, and do not aggregate in its absence. These results raise fundamental questions regarding the status of acetylcholinesterase in situ, the structure and interactions of its molecular forms. They are discussed with reference to previous publications.

Protein CutA Undergoes an Unusual Transfer into the Secretory Pathway and Affects the Folding, Oligomerization, and Secretion of Acetylcholinesterase

“…All media were diluted 1 ⁄2. The fraction of the cellular content released per h was about 1.7% for CutA (1,24,44) , 0.5% for CutA (24) , and less than 0.1% for CutA (44) .…”

“…The shorter of the other two isoforms, which exist in both species, is contained in the longer one but starts at a downstream methionine encoded by a different exon (Fig. 1B); in the mouse, the corresponding methionines are Met 1 and Met 24 (the residues are numbered according to the longer protein) (Fig. 1C).…”

“…In some mutants, the first ATG was mutated to a stop codon, indicated as X, and the second and third ATGs were mutated to serine codons; the corresponding mutants are named according to the remaining methionines, e.g. CutA (1,24,44) for the wild type. Introduced peptidic motifs are indicated in brackets: a FLAG epitope (DYKDDDDK) at the N terminus (after Met 1 ) or at the C terminus, an HA epitope (YPYDVPDYA), and an endoplasmic reticulum retention motif (KDEL).…”

“…The first ATG was mutated to a stop codon, to avoid the possible use of an upstream initiation site, and the downstream ones to serine codons. The resulting mutants were designated according to presence or absence of ATG codons at each position: CutA (24,44) , CutA (1,44) , CutA (1,24) , CutA (1) , CutA (24) , CutA (44) , and CutA (-) . Note that CutA (24,44) corresponds to the splice variant 2 ( Fig.…”

“…The major molecular species of AChE 2 in mammalian brain consists of membrane-bound tetramers (1). In these hetero-oligomers, four catalytic subunits, corresponding to the AChE T variant that possesses a C-terminal t peptide (2), are associated with a hydrophobic 20-kDa protein (3,4).…”