title: A bifunctional α-amylase/trypsin inhibitor from pigeonpea seeds: Purification, biochemical characterization and its bio-efficacy against Helicoverpa armigera.
abstract: This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa armigera. A bifunctional α-amylase/trypsin inhibitor was purified from the seeds of pigeonpea by native liquid phase isoelectric focusing (N-LP-IEF), affinity chromatography and preparative electrophoresis. Its in-vivo and in-vitro interaction with midgut amylases of H. armigera was studied along with growth inhibitory activity. One and two dimensional (2D) zymographic analyses revealed that the purified inhibitor is dimeric glycoprotein (60.2kDa and 56kDa) exist in a multi-isomeric form with five pI variants (pI 5.5 to 6.3). It was found to be heat labile with complete inactivation up to 80°C and stable over a wide range of pH (4-11). The slow binding and competitive type of α-amylase inhibition was observed with 0.08μM of dissociation constant (Ki) for the enzyme-inhibitor complex (EI). The internal protein sequence of two subunits obtained by mass spectrometry matched with cereal-type α-AI, a conserved domain from AAI_LTSS superfamily and sialyltransferase-like protein respectively. In-vivo studies indicated up-regulation of total midgut α-amylase activity with negative effect on growth rate of H. armigera suggesting its suitability for pest control.
authors.affiliation: Department of Biochemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra 431004, India.
authors.forename: Prafull P
authors.initials: PP
authors.lastname: Gadge
authors.affiliation: Department of Biochemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra 431004, India.
authors.forename: Sandip K
authors.initials: SK
authors.lastname: Wagh
authors.affiliation: Department of Biochemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra 431004, India.
authors.forename: Faiyaz K
authors.initials: FK
authors.lastname: Shaikh
authors.affiliation: Dr. John Barnabas Post Graduate School for Biological Studies, B. P. H Education Society's Ahmednagar College, Station Road, Ahmednagar, Maharashtra 414001, India.
authors.forename: Rajesh D
authors.initials: RD
authors.lastname: Tak
authors.affiliation: Department of Biochemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra 431004, India. Electronic address: manoharpadul@yahoo.co.in.
authors.forename: Manohar V
authors.initials: MV
authors.lastname: Padul
authors.affiliation: Department of Biochemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra 431004, India.
authors.forename: Manvendra S
authors.initials: MS
authors.lastname: Kachole
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D036561:Cajanus / Q000737:chemistry* / Q000235:genetics; D019476:Insect Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012639:Seeds / Q000737:chemistry*; D016415:Sequence Alignment; D014357:Trypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D019476:Insect Proteins; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D000516:alpha-Amylases; D014357:Trypsin
keywords: Bifunctional α-amylase/trypsin inhibitor; H. armigera; Host plant resistance; Mass spectrometry; Pigeonpea; Reverse zymography