title: Purification and characterization of a novel protease from the latex of Pedilanthus tithymaloids.
abstract: A novel protease was purified to homogeneity from the latex of Pedilanthus tithymaloids by a simple purification procedure involving ammonium sulfate precipitation and cation-exchange chromatography. The molecular weight of the protease was estimated to be approximately 63.1 kDa and the extinction coefficient (epsilon(1%)(280nm)) was 28.4. The enzyme hydrolyzes denatured natural substrates like casein, azoalbumin and azocasein with a high specific activity but little activity towards synthetic substrates. The pH and temperature optima were pH 8.0-9.5 and 65-70 degrees C, respectively. The proteolytic activity of the enzyme was inhibited by different protease-specific inhibitors (e.g., thiol, serine, metallo, etc.) up to a certain extent but not completely by any class of inhibitors. The enzyme was relatively stable towards pH change, temperature, denaturants and organic solvents. The enzyme consists of five disulfide bridges compared to three observed in most plant cysteine proteases. Overall, the striking features of this protease are its high molecular weight, high cysteine content and only partial inhibition of activity by different classes of protease inhibitors contrary to known proteases from other plant sources. The enzyme is named as pedilanthin as per the protease nomenclature.
authors.affiliation: Molecular Biology Unit Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India.
authors.forename: Reshma
authors.initials: R
authors.lastname: Bhowmick
authors.forename: N K Prasanna
authors.initials: NK
authors.lastname: Kumari
authors.forename: M V
authors.initials: MV
authors.lastname: Jagannadham
authors.forename: Arvind M
authors.initials: AM
authors.lastname: Kayastha
mesh_terms: D000645:Ammonium Sulfate; D002852:Chromatography, Ion Exchange; D004228:Dithionitrobenzoic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators; D019665:Euphorbiaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007840:Latex / Q000737:chemistry; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D055550:Protein Stability; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature; D014364:Tryptophan / Q000032:analysis; D014443:Tyrosine / Q000032:analysis
chemical_list: D020536:Enzyme Activators; D007840:Latex; D010940:Plant Proteins; D014443:Tyrosine; D014364:Tryptophan; D004228:Dithionitrobenzoic Acid; D010447:Peptide Hydrolases; D000645:Ammonium Sulfate