Planta Med 2004; 70(10): 942-947
DOI: 10.1055/s-2004-832621
Original Paper
Physiology, in vitro Biotechnology
© Georg Thieme Verlag KG Stuttgart · New York

Isolation and Characterisation of a Cysteine Protease (Phytolacain G), from Phytolacca americana Roots

Ursula Sußner1 , Gudrun Abel2 , Ralf Schulte2 , Wolfgang Kreis1
  • 1Lehrstuhl für Pharmazeutische Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
  • 2Bionorica AG, Neumarkt/Opf, Germany
Further Information

Publication History

Received: May 4, 2004

Accepted: July 7, 2004

Publication Date:
18 October 2004 (online)

Abstract

Protein extracts obtained from dried and fresh roots of Phytolacca americana L. (Phytolaccaceae) were examined in order to identify and characterise individual proteins. The extracts were compared with a commercial pokeweed mitogen standard using SDS polyacrylamide gel electrophoresis (SDS-PAGE). A dominant protein, present in both the extracts and the pokeweed mitogen standard, was isolated by subsequent ammonium sulphate fractionation, anion exchange chromatography, gel filtration and SDS-PAGE. In this way it was purified 140-fold with about 20 % yield and 70-fold with about 13 % yield from dried and fresh roots, respectively. Its molecular mass as determined by gel filtration and SDS-PAGE was estimated to be about 25 kDa. Subsequent isoelectric focussing revealed one single protein band at pH 6.0. LysC digestion of the 25 kDa protein yielded several peptides which were subjected to micro-sequencing. Comparison with published sequences revealed that the protein isolated was phytolacain G, a cysteine protease previously isolated from unripe fruits of P. americana L. The enzyme showed a high affinity towards the oxidised insulin B-chain and was completely inhibited by trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane (E64). The purified phytolacain G showed ”lectin-like” activities such as haemagglutination and mitogenic effects towards human lymphocytes.

Abbreviations

CP:cysteine protease (Phytolacain G)

Pa, PL:Phytolacca lectin

PHA:phytohaemagglutinin

PVPP:polyvinylpolypyrrolidone

PWM:pokeweed mitogen

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Prof. Dr. Wolfgang Kreis

Institute for Botany and Pharmaceutical Biology

Friedrich Alexander University Erlangen-Nürnberg

Staudtstr. 5

91058 Erlangen

Germany

Phone: +49-9131-852-8241

Fax: +49-9131-852-8243

Email: wkreis@biologie.uni-erlangen.de