title: A high cysteine containing thiol proteinase from the latex of Ervatamia heyneana: purification and comparison with ervatamin B and C from Ervatamia coronaria.
abstract: A cysteine protease, with a high cysteine content and a high degree of amino terminal sequence homology with ervatamins B and C, has been purified from the latex of Ervatamia heyneana (Family Apocynaceae). The enzyme designated as heynein (M(r) = 23 kDa) has a comparatively high cysteine content (11), high isoelectric point (10.8), and high stability against pH (2.5-11.5), temperature (63 degrees C, 15 min), strong denaturants, and organic solvents. The enzyme has high specific activities for natural substrates such as casein and azoalbumin. The pH and temperature optima are pH 8.0-8.5 and 52 +/- 2 degrees C, respectively. Hydrolysis of synthetic substrates and digestion of bovine serum albumin confirm a distinct specificity of heynein as compared to ervatamins and papain. Also, heynein has distinct immunogenicity as monitored by enzyme-linked immunosorbent assay and Ouchterlony's double immunodiffusion. Strong enzyme activation by reducing agents such as beta-mercaptoethanol, dithiothreitol, and strong enzyme inhibition by thiol proteinase inhibitors such as E-64 and iodoacetic acid have evidenced heynein to be a cysteine protease. High stability, specific activity, and easy purification may make heynein a potential protease for food and biotechnology applications.
mesh_terms: D000595:Amino Acid Sequence; D029062:Apocynaceae / Q000201:enzymology*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003545:Cysteine / Q000032:analysis*; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D017385:Sequence Homology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D010446:Peptide Fragments; D011480:Protease Inhibitors; D003546:Cysteine Endopeptidases; C406685:ervatamin B; D003545:Cysteine