title: Structural and functional characterization of proteinase inhibitors from seeds of Cajanus cajan (cv. ICP 7118).
abstract: Proteinase inhibitors (C11PI) from mature dry seeds of Cajanus cajan (cv. ICP 7118) were purified by chromatography which resulted in 87-fold purification and 7.9% yield. SDS-PAGE, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrum and two-dimensional (2-D) gel electrophoresis together resolved that C11PI possessed molecular mass of 8385.682 Da and existed as isoinhibitors. However, several of these isoinhibitors exhibited self association tendency to form small oligomers. All the isoinhibitors resolved in Native-PAGE and 2-D gel electrophoresis showed inhibitory activity against bovine pancreatic trypsin and chymotrypsin as well as Achaea janata midgut trypsin-like proteases (AjPs), a devastating pest of castor plant. Partial sequences of isoinhibitor (pI 6.0) obtained from MALDI-TOF/TOF analysis and N-terminal sequencing showed 100% homology to Bowman-Birk Inhibitors (BBIs) of leguminous plants. C11PI showed non-competitive inhibition against trypsin and chymotrypsin. A marginal loss (<15%) in C11PI activity against trypsin at 80 (°)C and basic pH (12.0) was associated with concurrent changes in its far-UV CD spectra. Further, in vitro assays demonstrated that C11PI possessed significant inhibitory potential (IC50 of 78 ng) against AjPs. On the other hand, in vivo leaf coating assays demonstrated that C11PI caused significant mortality rate with concomitant reduction in body weight of both larvae and pupae, prolonged the duration of transition from larva to pupa along with formation of abnormal larval-pupal and pupal-adult intermediates. Being smaller peptides, it is possible to express C11PI in castor to protect them against its devastating pest A. janata.
authors.affiliation: Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
authors.forename: Marri
authors.initials: M
authors.lastname: Swathi
authors.affiliation: Department of Biotechnology & Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
authors.forename: Vadthya
authors.initials: V
authors.lastname: Lokya
authors.affiliation: Department of Biotechnology & Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
authors.forename: Vanka
authors.initials: V
authors.lastname: Swaroop
authors.affiliation: Legumes Cell Biology, Grain Legumes Program, ICRISAT, Patancheru, Hyderabad 502324, India.
authors.forename: Nalini
authors.initials: N
authors.lastname: Mallikarjuna
authors.affiliation: Proteomics Facility, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
authors.forename: Monica
authors.initials: M
authors.lastname: Kannan
authors.affiliation: Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
authors.forename: Aparna
authors.initials: A
authors.lastname: Dutta-Gupta
authors.affiliation: Department of Biotechnology & Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India. Electronic address: kpssl@uohyd.ac.in.
authors.forename: Kollipara
authors.initials: K
authors.lastname: Padmasree
mesh_terms: D036561:Cajanus / Q000196:embryology*; D015180:Electrophoresis, Gel, Two-Dimensional; D063347:Native Polyacrylamide Gel Electrophoresis; D011480:Protease Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D012639:Seeds / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011480:Protease Inhibitors
keywords: Achaea janata; Activity staining; BBI; Castor; Isoinhibitors; MALDI-TOF/TOF; Two-dimensional (2-D) gel electrophoresis