Bowman-Birk proteinase inhibitor from Cajanus cajan seeds: purification, characterization, and insecticidal properties
| Autor: | H Merzendorfer, C Madhurarekha, Kollipara Padmasree, Elaprolu R Prasad, Aparna Dutta-Gupta |
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| Rok vydání: | 2010 |
| Předmět: |
Gel electrophoresis
Insecticides Chromatography Chymotrypsin Trypsin inhibitor General Chemistry Biology Trypsin Dithiothreitol chemistry.chemical_compound Biochemistry chemistry Cajanus Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Protein purification Seeds medicine biology.protein Electrophoresis Polyacrylamide Gel Amino Acid Sequence General Agricultural and Biological Sciences Polyacrylamide gel electrophoresis Ammonium sulfate precipitation medicine.drug Trypsin Inhibitor Bowman-Birk Soybean |
| Zdroj: | Journal of agricultural and food chemistry. 58(5) |
| ISSN: | 1520-5118 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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