Zingipain, a Ginger Protease with Acetylcholinesterase Inhibitory Activity

Appl Biochem Biotechnol (2013) 170:934950

DOI 10.1007/s12010-013-0243-x

Porlin Rungsaeng & Polkit Sangvanich & Aphichart Karnchanatat

Received: 21 February 2012 /Accepted: 15 April 2013 /

Published online: 28 April 2013# Springer Science+Business Media New York 2013

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 (212). The

AChEI activity was stimulated strongly by Mn2+ and Cu2+ at 110 mM and weakly by Ca2+, Fe2+, Mg2+, and Zn2+ at 1 mM, but was inhibited at 10 mM. In contrast, Hg2+ and ethylenediaminetetraacetic acid were very and moderately strongly inhibitory, respectively. In-gel tryptic digestion with liquid chromatographytandem 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 Ki value of 9.31 mg/ml.

Keyword Acetylcholinesterase inhibitor . Ginger protease . Zingipain

P. Rungsaeng

Program in Biotechnology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand

P. Sangvanich

Department of Chemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand

A. Karnchanatat (*)

Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailande-mail: [email protected]

Zingipain, a Ginger Protease with Acetylcholinesterase Inhibitory Activity

Appl Biochem Biotechnol (2013) 170:934950 935

Introduction

Aging is a universal natural biological process that takes place in all organisms, leading to progressive and deleterious changes in the body. It is now widely accepted that aging is a multifarious event resulting from the collective effects of genetic variation, environmental risk factors, nutritional factors, and...