Purification and characterization of a stable cysteine protease ervatamin B, with two disulfide bridges, from the latex of Ervatamia coronaria.
Latex of the medicinal plant Ervatamia coronaria was found to contain at least three cysteine proteases with high proteolytic activity, called ervatamins. One of these proteases, named ervatamin B, has been purified to homogeneity using ion-exchange chromatography and crystallization. The molecular mass of the enzyme was estimated to be 26 000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon(1%)(280 nm)) of the enzyme was 20.5 with 7 tryptophan and 10 tyrosine residues per molecule. The enzyme hydrolyzed denatured natural substrates such as casein, azoalbumin, and azocasein with a high specific activity. In addition, it showed amidolytic activity toward N-succinyl-alanine-alanine-alanine-p-nitroanilide with an apparent K(m) and K(cat) of 6.6 +/- 0.5 mM and 1.87 x 10(2) s(-)(1), respectively. The pH optima was 6.0-6.5 with azocasein as substrate and 7.0-7.5 with azoalbumin as substrate. The temperature optimum was around 50-55 degrees C. The enzyme was basic with an isoelectric point of 9.35 and had no carbohydrate content. Both the proteolytic and amidolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. Interestingly, the enzyme had only two disulfide bridges versus three as in most plant cysteine proteases of the papain superfamily. The enzyme was relatively stable toward pH, denaturants, temperature, and organic solvents. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and typical color in ELISA. Other related proteases do not cross-react with the antisera to ervatamin B showing that the enzyme is immunologically distinct. The N-terminal sequence showed conserved amino acid residues and considerable similarity to typical plant cysteine proteases.
Kundu
S
S
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India.
Sundd
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M
Jagannadham
M V
MV
D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000302:isolation & purification*; D004220:Disulfides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007840:Latex / Q000737:chemistry; D008969:Molecular Sequence Data; D010946:Plants, Medicinal / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D004220:Disulfides; D007840:Latex; D003546:Cysteine Endopeptidases; C406685:ervatamin B