RESUMEN
Bowman-Birk inhibitor (BBI ~10 kDa) and Kunitz inhibitor (KI ~20 kDa) are serine protease/proteinase inhibitor(s) [PI(s)] ubiquitously found in several Leguminous plant species with insecticidal and therapeutic properties. Due to narrow molecular mass differences, the separation of these inhibitors from a single seed variety is tedious. The present study is aimed to develop a rapid protocol (<24 h) for purifying BBI and KI from legume seeds using mild trichloroacetic acid (TCA) extraction followed by trypsin-affinity chromatography. The mature seeds of Vigna radiata and Cajanus platycarpus are used as a model to purify BBI and KI using this protocol. The BBI and KI purified from the seeds of V. radiata are labeled as VrBBI & VrKI, and C. platycarpus are labeled as CpBBI & CpKI, respectively. These PIs are confirmed by immunodetection and MALDI-TOF studies and further characterized for their structural (CD & fluorescence spectroscopy) and functional properties (temperature & DTT stability). BBI(s) purified using the above process are effective in the management of castor semi-looper 'Achaea janata', while KI(s) are effective in the management of pod borer 'Helicoverpa armigera'. Besides, both BBI(s) and KI(s) have significant potential in controlling the growth of methicillin-sensitive 'Staphylococcus aureus', a gram-positive pathogenic bacterium.
Asunto(s)
Antiinfecciosos , Fabaceae , Insecticidas , Mariposas Nocturnas , Animales , Fabaceae/química , Secuencia de Aminoácidos , Insecticidas/química , Verduras , Inhibidores de Serina Proteinasa , Semillas/química , Antiinfecciosos/análisis , Inhibidores de Tripsina/farmacología , Inhibidores de Tripsina/químicaRESUMEN
Rhynchosia sublobata, a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216â¯Da) and RsKI (19,412â¯Da) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Kiâ¯=â¯128.5⯱â¯4.5â¯nM) and chymotrypsin (Kiâ¯=â¯807.8⯱â¯23.7â¯nM) while RsKI (Kiâ¯=â¯172.0⯱â¯9.2â¯nM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100⯰C. But, RsBBI completely lost its TI and CI activities on reduction with 3â¯mM DTT. Conversely, RsKI lost its TI activity on heating at 100⯰C and retained >60% of its TI activity in presence of 3â¯mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coilsâ¯>â¯ß-sheets/ß-turnsâ¯>â¯α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75⯰C. Further, the significant inhibitory activity of RsBBI (IC50â¯=â¯24â¯ng) and RsKI (IC50â¯=â¯59â¯ng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively.
Asunto(s)
Cajanus/embriología , Fabaceae/embriología , Semillas/química , Inhibidor de la Tripsina de Soja de Bowman-Birk/química , Inhibidor de la Tripsina de Soja de Bowman-Birk/aislamiento & purificación , Inhibidor de la Tripsina de Soja de Kunitz/química , Inhibidor de la Tripsina de Soja de Kunitz/aislamiento & purificación , Secuencia de Aminoácidos , Cromatografía Liquida/métodos , Ditiotreitol/química , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Fabaceae/química , Calor , Cinética , Espectrometría de Masas/métodos , Estructura Secundaria de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Crude proteinase inhibitors (CPIs) extracted from the seeds of Rhynchosia sublobata, a wild relative of pigeon pea showed pronounced inhibitory activity on the larval gut trypsin-like proteases of lepidopteran insect pest - Achaea janata. Consequently, a full-length cDNA of Bowman-Birk inhibitor gene (RsBBI1) was cloned from the immature seeds of R. sublobata. It contained an ORF of 360 bp encoding a 119-amino acid polypeptide (13.3â¯kDa) chain with an N-terminus signal sequence comprising of 22 amino acids. The amino acid sequence and phylogenetic analysis together revealed that RsBBI1 exhibited a close relation with BBIs from soybean and Phaseolus spp. A cDNA sequence corresponding to RsBBI1 mature protein (89 amino acid stretch) was expressed in E. coli. The recombinant rRsBBI1 protein with a molecular mass of 9.97â¯kDa was purified using trypsin affinity chromatography. The purified rRsBBI1 exhibited non-competitive mode of inhibition of both bovine trypsin (Ki of 358⯱â¯11â¯nM) and chymotrypsin (Ki of 446⯱â¯9â¯nM). Its inhibitory activity against these proteases was stable at high temperatures (>95⯰C) and a wide pH range but sensitive to reduction with dithiothreitol (DTT), indicating the importance of disulphide bridges in exhibiting its activity. Also, rRsBBI1 showed significant inhibitory activity (IC50â¯=â¯70â¯ng) on A. janata larval gut trypsin-like proteases (AjGPs). Conversely, it showed <1% inhibitory activity (IC50â¯=â¯8⯵g) on H. armigera larval gut trypsin-like proteases (HaGPs) than it has against AjGPs. Besides, in vivo feeding experiments clearly indicated the deleterious effects of rRsBBI1 on larval growth and development in A. janata which suggests it can be further exploited for such properties.
Asunto(s)
Fabaceae/química , Péptido Hidrolasas/metabolismo , Semillas/química , Inhibidor de la Tripsina de Soja de Bowman-Birk/metabolismo , Inhibidores de Tripsina/farmacología , Animales , Bovinos , Mariposas Nocturnas , Inhibidor de la Tripsina de Soja de Bowman-Birk/química , Inhibidor de la Tripsina de Soja de Bowman-Birk/aislamiento & purificación , Inhibidores de Tripsina/química , Inhibidores de Tripsina/aislamiento & purificaciónRESUMEN
The pituitary function is regulated by a complex system involving the hypothalamus and biological networks within the pituitary. Although the hormones secreted from the pituitary have been well studied, comprehensive analyses of the pituitary proteome are limited. Pituitary proteomics is a field of postgenomic research that is crucial to understand human health and pituitary diseases. In this context, we report here a systematic proteomic profiling of human anterior pituitary gland (adenohypophysis) using high-resolution Fourier transform mass spectrometry. A total of 2164 proteins were identified in this study, of which 105 proteins were identified for the first time compared with high-throughput proteomic-based studies from human pituitary glands. In addition, we identified 480 proteins with secretory potential and 187 N-terminally acetylated proteins. These are the first region-specific data that could serve as a vital resource for further investigations on the physiological role of the human anterior pituitary glands and the proteins secreted by them. We anticipate that the identification of previously unknown proteins in the present study will accelerate biomedical research to decipher their role in functioning of the human anterior pituitary gland and associated human diseases.