Characterization of trimeric acetylcholinesterase from a legume plant, Macroptilium atropurpureum Urb.

Abstract

We recently identified plant acetylcholinesterases (E.C.3.1.1.7; AChEs) homologous to the AChE purified from a monocotyledon, maize, that are distinct from the animal AChE family. In this study, we purified, cloned and characterized an AChE from a dicotyledon, siratro. The full-length cDNA of siratro AChE is 1,441 nucleotides, encoding a 382-residue protein that includes a signal peptide. This AChE is a disulfide-linked 125-kDa homotrimer consisting of 41-42 kDa subunits, in contrast to the maize AChE, which exists as a mixture of disulfide and non-covalently linked 88-kDa homodimers. The plant AChEs apparently consist of various quaternary structures, depending on the plant species, similar to the animal AChEs. We compared the enzymatic properties of the dimeric maize and trimeric siratro AChEs. Similar to electric eel AChE, both plant AChEs hydrolyzed acetylthiocholine (or acetylcholine) and propionylthiocholine (or propionylcholine), but not butyrylthiocholine (or butyrylcholine), and their specificity constant was highest against acetylcholine. There was no significant difference between the enzymatic properties of trimeric and dimeric AChEs, although two plant AChEs had low substrate turnover numbers compared with electric eel AChE. The two plant AChE activities were not inhibited by excess substrate concentrations. Thus, similar to some plant AChEs, siratro and maize AChEs showed enzymatic properties of both animal AChE and animal BChE. On the other hand, both siratro and maize AChEs exhibited low sensitivity to the AChE-specific inhibitor neostigmine bromide, dissimilar to other plant AChEs. These differences in enzymatic properties of plant AChEs may reflect the phylogenetic evolution of AChEs.

Molecular cloning ofacetylcholinesterasegene fromSalicornia europaeaL.

“…The cultivation and treatment of Salicornia plants were performed with same method as Sagane et al 33 who used Residues not identical, but similar to the conserved one, are highlighted in gray. The blocks of sequences conserved in the lipase GDSL family enzymes and the putative catalytic triad Ser/Asp(Glu)/His residues in the lipase GDSL family enzymes, which were presumed previously, [32][33][34] are indicated by boxes and arrowheads, respectively. The accession numbers used in the analysis are as follows: siratro AChE, AB294246; maize AChE, AB093208.…”

“…The catalytic Ser/Asp(Glu)/His triads of the enzymes belonging to the lipase GDSL family were found in some of these proteins, and the sequences surrounding the consensus residues are well conserved. [29][30][31] Therefore, the putative catalytic triad for Salicornia AChE, Ser-45/Asp-184/ His-355 (indicated by this study), siratro AChE, Ser-41/Asp-180/ His-350, 32 and maize AChE, Ser-47/Asp-185/His-360, 33 can be deduced by superimposing the sequences on those of the lipase GDSL family enzymes (Fig. 4) (Fig.…”

“…The plants grown at 3% NaCl condition were used for molecular analysis. AChE activity for molecular analysis was measured by the DTNB method following precisely in the same way of Yamamoto et al 32 Plant materials were cut in 2 mm lengths and incubated at 30°C for 10 min in vials containing 300 μl of 100 mM sodium phosphate buffer (pH 7.0) with 24 μl of 100 mM neostigmine bromide or 24 μl of distilled water for a control. After 10 min incubation, 500 μl of 12.5 mM acetylthiocholine (ASCh) chloride as a substrate of AChE was added, and the solution was incubated at 30°C for 2 h. Following incubation, 300 μl of the solution was transferred to a vial, and 1,425 μl of 100 mM sodium phosphate buffer (pH 7.0) and 75 μl of 10 mM DTNB in 100 mM checked using the TargetP program 35 (http://www.cbs.dtu.dk/ services/TargetP/).…”

Subcellular localization of overexpressed maize AChE gene in rice plant

“…However, all these phenomena have been investigated using indirect techniques. Thus, to understand the plant ACh-mediated system, we purified AChEs and cloned the AChE genes from maize 14 and siratro 15 seedlings. The maize AChE was found to exist as two types of 88-kDa homodimers, which in turn consisted of disulfide-linked and noncovalently-linked 42-to 44-kDa subunits.…”

“…14 The siratro AChE might exist as a disulfide-linked 125-kDa homotrimer consisting of 41-or 42-kDa subunits. 15 The plant AChEs apparently from various quaternary structures, depending on the plant species, similar to animal AChEs. Furthermore, maize and siratro AChEs showed enzymatic properties of both animal AChE and animal butyrylcholinesterase, compared with electric eel AChE.…”