A Kunitz proteinase inhibitor from Archidendron ellipticum seeds: purification, characterization, and kinetic properties.
Leguminous plants in the tropical rainforests are a rich source of proteinase inhibitors and this work illustrates isolation of a serine proteinase inhibitor from the seeds of Archidendron ellipticum (AeTI), inhabiting Great Nicobar Island, India. AeTI was purified to homogeneity by acetone and ammonium sulfate fractionation, and ion exchange, size exclusion and reverse phase chromatography (HPLC). SDS-PAGE of AeTI revealed that it is constituted by two polypeptide chains (alpha-chain, M(r) 15,000 and beta-chain, M(r) 5000), the molecular weight being approximately 20 kDa. N-terminal sequence showed high homology with other serine proteinase inhibitors belonging to the Mimosoideae subfamily. Both Native-PAGE as well as isoelectric focussing showed four isoinhibitors (pI values of 4.1, 4.55, 5.27 and 5.65). Inhibitory activity of AeTI remained unchanged over a wide range of temperatures (0-60 degrees C) and pH (1-10). The protein inhibited trypsin in the stoichiometric ratio of 1:1, but lacked similar stoichiometry against chymotrypsin. Also, AeTI-trypsin complex was stable to SDS unlike the SDS unstable AeTI-chymotrypsin complex. AeTI, which possessed inhibition constants (K(i)) of 2.46 x 10(-10) and 0.5 x 10(-10)M against trypsin and chymotrypsin activity, respectively, retained over 70% of inhibitory activity after being stored at -20 degrees C for more than a year. Initial studies on the insecticidal properties of AeTI indicate it to be a very potent insect anti-feedant.
Bhattacharyya
Arindam
A
Centre for Environmental Management of Degraded Ecosystems, University of Delhi, Delhi 110007, India. ady1111@rediffmail.com
Mazumdar
Suman
S
Leighton
Sudeshna Mazumdar
SM
Babu
Cherukuri R
CR
D000595:Amino Acid Sequence; D000818:Animals; D007887:Fabaceae / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism; D007306:Insecticides / Q000378:metabolism; D007700:Kinetics; D007814:Larva / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D018411:Spodoptera / Q000201:enzymology
D007306:Insecticides; C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D010940:Plant Proteins; D012697:Serine Endopeptidases