Acetylcholinesterase inhibitory activity of proteins from the rhizomes of zingiberaceae plants

Abstract

Acetylcholinesterase inhibitors (AChEIs) have been used as drugs for the symptomatic treatment of Alzheimer’s disease (AD). In order to search for new AChEIs, 15 Zingiberaceae plants were tested for AChEI activity in rhizome extracts. The crude homogenate and ammonium sulphate cut fraction of Zingiber officinale contained a significant AChEI activity. Eighty % saturation (NH4)2SO4 precipitation and DEAE-cellulose ion-exchange chromatography (unbound fraction) enriched the protein to a single band on non-denaturing and reducing SDS-PAGE (ca 33.5 kDa), but in-gel-tryptic digestion with LC-MS/MS resolution revealed two heterogenous peptides, a 16 amino acid fragment with 100% similarity to Zingipain-1, a cysteine protease from Zingiber officinale, suggesting the preparation was heterogeneous. Gelatin-degrading zymography showed that the AChEI containing band also contained protease activity. The AChEI activity was largely stable between -20 – 60 °C (at least over 120 mins), and over a broad pH range (2 - 12). The AChEI activity was stimulated strongly by Mn2+ and Cu2+ at 1 - 10 mM, and weakly by Ca2+, Fe2+, Mg2+ and Zn2+ at 1 – 5 mM but inhibited at 10 mM. In contrast, Hg2+ and EDTA were very and moderately strongly inhibitory. AChEI exhibited non-competitive inhibition of AChE for the hydrolysis of acetylthiocholine iodide with a Ki value of 9.31 mg/ml, a Vmax of 3.41 mM/min and a Km of 130.6 mM.