Isolation and Characterisation of a Cysteine Protease (Phytolacain G), fromPhytolacca americanaRoots

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.

Mitogenic Activity of CEL-I, anN-Acetylgalactosamine (GalNAc)-Specific C-Type Lectin, Isolated from the Marine InvertebrateCucumaria echinata(Holothuroidea)

“…It is known that PWM is mitogenic to both T and B cells, but it is unlikely that commercially available PWM is a completely purified lectin, and it has been pointed out that commercially available PWM might be contaminated with large amounts of endotoxin and unidentified proteins. 31,32) According to a previous report, 33) PWM is composed of at least five isolectins (designated Pa-1 to Pa-5), and it has been reported that Pa-1 is mitogenic to both murine T and B cells, whereas Pa-2, Pa-4, and Pa-5 are T cell-specific mitogens. Probably, a comparative study of Pa-1 and CEL-I would provide useful information on the structure-activity relationships of mitogenic lectins.…”

Isolation and characterization of a dual function protein from Allium sativum bulbs which exhibits proteolytic and hemagglutinating activities

“…Phytolacain, a proteolytic enzyme isolated from P. americana roots exhibiting lectin-like activity and mitogenic properties was reported [7]. A protein with a dual behavior functioning as a lectin and as a trypsin inhibitor was isolated from Peltophorum dubium seeds [34].…”

“…CPs have been found in latex, fruits, and seeds [2e5], although a few reports on cysteine peptidases isolated from the underground parts of the plant have been published. Among them, a cysteine protease from Zingiber officinale Roscoe rhizomes [6] and from Phytolacca americana roots [7] has been studied. In addition, there were reports on the cysteine peptidase activity exhibited by a protein isolated from garlic bulbs [8e10].…”

“…Hemagglutinating activity is a well-known characteristic of lectins, although in the past it was reported that certain microbial and plant proteases have a red-cell agglutinating activity in addition to proteolytic activity [1,7,16].…”

Molecular and cytotoxicity investigations of Phytolacca americana (l.) root, leaf, and berry extracts

“…Lectin belongs to a class of proteins with high carbohydratebinding affinity. These proteins are known to cause red blood cells to clump together and to stimulate abnormal cell growth (Sussner et al 2004 Karadi et al (2011) investigated the antimicrobial activities in the extracts of two plant species, Musa paradisiaca and Cocos nucefera, on three bacterial strains and three fungal species. Their results indicated that the extracts of both plant species showed antibacterial and antifungal activities against the organisms tested, and found that gram positive bacteria are more susceptible than gram negative bacteria.…”