Purification and biochemical characterization of a highly active cysteine protease ervatamin A from the latex of Ervatamia coronaria.
Ervatamia coronaria, a flowering plant (family Apocynaceae) indigenous to India, has medicinally important applications. A search for biochemical constituents of the latex of the plant yielded at least three thiol proteases with distinctly different properties. One of them, a highly active protease (ervatamin A), was purified to homogeneity by ion exchange and gel filtration chromatography. The enzyme exhibited high proteolytic activity toward natural substrates and amidolytic activity toward synthetic substrates. The pH and temperature optima for proteolytic activity were 8-8.5 and 50-55 degrees C, respectively. Proteolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. The estimated molecular mass of the enzyme was 27.6 kDa. The extinction coefficient (epsilon(1%) 280) of the enzyme was estimated as 21.9, and the protein molecule consists of 8 tryptophan, 11 tyrosine and 7 cysteine residues. Isoelectric point of the purified enzyme was 8.37. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and a typical color in ELISA. The N-terminal sequence of the enzyme showed conserved amino acid residues to other plant cysteine proteases. Ervatamin A shows high activity in relation to the other thiol proteases isolated from the same source.
Nallamsetty
Sreedevi
S
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India.
Kundu
Suman
S
Jagannadham
Medicherla V
MV
D000595:Amino Acid Sequence; D029062:Apocynaceae / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D005779:Immunodiffusion; D007525:Isoelectric Focusing; D007840:Latex / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
D015853:Cysteine Proteinase Inhibitors; D007840:Latex; D003546:Cysteine Endopeptidases; C116225:ervatamin