title: Zingipain, a ginger protease with acetylcholinesterase inhibitory activity.
abstract: In order to search for new acetylcholinesterase inhibitors (AChEIs), 15 Zingiberaceae plants were tested for AChEI activity in rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Zingiber officinale contained a significant AChEI activity. Eighty percent saturation ammonium sulfate precipitation and diethylaminoethyl cellulose ion exchange chromatography (unbound fraction) enriched the protein to a single band on nondenaturing and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (approximately 33.5 kDa). Gelatin-degrading zymography showed that the AChEI-containing band also contained cysteine protease activity. The AChEI activity was largely stable between -20 and 60 °C (at least over 120 min) and over a broad pH range (2-12). The AChEI activity was stimulated strongly by Mn(2+) and Cu(2+) at 1-10 mM and weakly by Ca(2+), Fe(2+), Mg(2+), and Zn(2+) at 1 mM, but was inhibited at 10 mM. In contrast, Hg(2+) and ethylenediaminetetraacetic acid were very and moderately strongly inhibitory, respectively. In-gel tryptic digestion with liquid chromatography-tandem mass spectroscopy resolution revealed two heterogeneous peptides, a 16-amino-acid-long fragment with 100 % similarity to zingipain-1, which is a cysteine protease from Z. officinale, and a 9-amino-acid-long fragment that was 100 % identical to actinidin Act 2a, suggesting that the preparation was heterogeneous. AChEI exhibited noncompetitive inhibition of AChE for the hydrolysis of acetylthiocholine iodide with a K(i) value of 9.31 mg/ml.
authors.affiliation: Program in Biotechnology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand.
authors.forename: Porlin
authors.initials: P
authors.lastname: Rungsaeng
authors.forename: Polkit
authors.initials: P
authors.lastname: Sangvanich
authors.forename: Aphichart
authors.initials: A
authors.lastname: Karnchanatat
mesh_terms: D000110:Acetylcholinesterase / Q000378:metabolism*; D000122:Acetylthiocholine / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D057056:Cysteine Proteases / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation; D057075:Enzyme Assays; D004795:Enzyme Stability; D020939:Zingiber officinale / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D010936:Plant Extracts / Q000378:metabolism; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D027343:Rhizome / Q000201:enzymology; D013696:Temperature
chemical_list: D002800:Cholinesterase Inhibitors; D010936:Plant Extracts; D010940:Plant Proteins; C543539:acetylthiocholine iodide; D000122:Acetylthiocholine; D003300:Copper; D000110:Acetylcholinesterase; D057056:Cysteine Proteases; C584512:zingipain, Zingiber officinale; D002118:Calcium