Bowman-Birk proteinase inhibitor from Cajanus cajan seeds: purification, characterization, and insecticidal properties.
A red gram proteinase inhibitor (RgPI) was purified from red gram ( Cajanus cajan ) seeds by using ammonium sulfate precipitation and ion-exchange, affinity, and gel filtration chromatography. SDS-PAGE under nonreducing condition revealed two protein bands with molecular masses of approximately 8.5 and approximately 16.5 kDa corresponding to monomeric and dimeric forms of RgPI, respectively. Similarly, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry also confirmed the presence of dimer as well as other oligomeric forms: trimer, tetramer, and pentamer. Reduction of RgPI with dithiothreitol (DTT) led to the dissociation of the dimeric and oligomeric forms. Native-PAGE and two-dimensional gel electrophoresis indicated the existence of isoinhibitors with pI values of 5.95, 6.25, 6.50, 6.90, and 7.15, respectively. The MALDI-TOF-TOF mass spectrum and N-terminal sequence 'DQHHSSKACC' suggested that the isolated RgPI is a member of the Bowman-Birk inhibitor family. RgPI exhibited noncompetitive type inhibitory activity against bovine pancreatic trypsin and chymotrypsin, with inhibition constants of 292 and 2265 nM, respectively. It was stable up to a temperature of 80 degrees C and was active over a wide pH range between 2 and 12. However, reduction with DTT or 2-mercaptoethanol resulted in loss of inhibitory activity against trypsin and chymotrypsin. It also decreased the activity of larval midgut trypsin-like proteinases in Manduca sexta . Its insecticidal property was further confirmed by reduction in the growth and development of these larvae, when supplemented in the diet.
Prasad
Elaprolu R
ER
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India.
Merzendorfer
H
H
Madhurarekha
C
C
Dutta-Gupta
A
A
Padmasree
K
K
D000595:Amino Acid Sequence; D036561:Cajanus / Q000196:embryology*; D004591:Electrophoresis, Polyacrylamide Gel; D007306:Insecticides / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D012639:Seeds / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
D007306:Insecticides; D014358:Trypsin Inhibitor, Bowman-Birk Soybean