ABSTRACT Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) b... more ABSTRACT Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a simple and sensitive gel X-ray film contact print technique. About 17 AlTIs were detected in the seed extracts of A. lebbeck. Two groups of AlTIs-1 major (10 AlTIs; slow migration on the gel) and 1 minor (7 AlTIs; fast migration on the gel) were identified. The former was specific only toward trypsin. However, the latter was specific toward both trypsin and Helicoverpa armigera gut proteinases (HaGPs). The most potent AlTI (AlTI13) was purified to assess its in vivo bioefficacy toward HaGPs. Purification was achieved using (NH4)(2)SO4 fractionation, Sephadex G-100 column chromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependent bioefficacies of AlTIs in the (NH4)(2)SO4 F-3 fractions (0.1%, 0.5%, and 1%) were approximately 79%, 83%, and 90%, respectively, resulting in reductions in the average larval weight of H. armigera. Artificial diet containing a single dose of AlTI13 (5 mu g/g diet) reduced the larval weight by about 76%, with 60% mortality. The half-maximal inhibitory concentrations (IC50) of AlTI13 for trypsin and HaGPs were 0.14 and 0.17 mu mol/ml, respectively. The optimum conditions for AlTI13 were pH 8 and temperatures ranging from 35 to 40 C. Reducing sodium dodecyl sulfate-PAGE analysis indicated that similar to 28 kDa Kunitz-like trypsin inhibitor was present. Thus, we showed that particularly, AlTI13 of A. lebbeck could be used as a transgene macromolecule to markedly increase insect resistance in genetically engineered plants.
This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa... more 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.
Trypsin protease inhibitor have been identified and purified from extracts of Mucuna Pruriens. Mu... more Trypsin protease inhibitor have been identified and purified from extracts of Mucuna Pruriens. Mucuna Pruriens seed sample were purified using ammonium sulphate precipitation, Saphadex G-75 gel filtration chromatography technique including analysis of trypsin inhibitors by dot-blot method, and electrophoretic analysis and visualization of protease inhibitor bands by gel X-ray film contact print method. Saphadex G-75 gel filtration chromatography identified a small molecular weight trypsin inhibitory fraction. In the present paper we described, a simple and inexpensive procedure to detect protease inhibitors of seed by the gel-x-ray film contact print technique.
The sample from seed of Peganum harmala plant tree was extracted in hexane, chloroform, acetone a... more The sample from seed of Peganum harmala plant tree was extracted in hexane, chloroform, acetone and methanol and separated by thin layer chromatography on silica gel plate by using chloroform : Methanol (85:15) solvent mixture and detected under the ultraviolet light. Maximum bands were detected on TLC of methanol extract. Those bands were scraped and the purification of the single band of interest molecule was done by preparative silica gel column chromatography. Antimicrobial activity of single band isolated was tested against standard ATCC (American Type Culture Collection) strain of E.coli ( 25922), Klebseilla , Staphylococcus aureus (25923), Pseudomonas aeruginosa (27853), S. pneumonia (6305) using Disc diffusion method and Punch Plate technique. The Purified fraction of alkaloid show zone of growth inhibition with 10mm on ATCC Staphylococcus aureus. The sensitivity test of water extract was showing effect mostly against Staphylococcus aureus and E.coli
In this report protein content and catalase (CAT) activity were used to study the effect of hydro... more In this report protein content and catalase (CAT) activity were used to study the effect of hydrogen peroxide (H2O2) on leaf senescence in detached Pigeon pea leaves. A decrease in protein content measured as an indicator of leaf senescence, and a drop in CAT activity was observed following treatment with H2O2 in Pigeon pea detached leaves compared with control leaves. However after longer incubations CAT activity significantly increased in comparison with day 1 treatment. Protein content and CAT activity were also studied in the leaves treated by 0.025, 0.05, 0.075 and 0.1 mM H2O2. The optimal concentration of H2O2 in reducing protein content seems to be 0.075 mM, whereas concentration of 0.025, 0.05, 0.075 mM increase the CAT activity while lower and higher concentrations shown the opposite effect. The observed changes revealed that H2O2 induces oxidative stress and oxidative damage thereby leaf senescence in the detached leaves of Pigeon pea.
The present study tries to examine the influence of natural and oxidative stress [hydrogen peroxi... more The present study tries to examine the influence of natural and oxidative stress [hydrogen peroxide (H2O2) treatment]-induced senescence on peroxidase (POD) activity in neem (Azadirachta indica A. juss) leaves. Data indicated that incubation of detached leaves in presence of H2O2 has induced POD(s), that enzyme activity is also enhanced in natural senescing leaves. Changes in POD activity and protein loss during H2O2-promoted senescence has shown similarity with natural senescence suggests that the underlying regulatory mechanisms might be the same in both stresses, at least in neem. Initial induction of POD activity under H2O2 stress suggests that PODs play a very important role during the early phases of leaf senescence. Reduction in POD activity along with the increase in protein loss at latter stages suggests that there was no correlation between PODs and senescence, at least in neem.
In this report peroxidase (POD) and polyphenol oxidase (PPO) activities were used to study the ef... more In this report peroxidase (POD) and polyphenol oxidase (PPO) activities were used to study the effect of hydrogen peroxide (H2O2) on leaf senescence in detached pigeonpea (Cajanus cajan [L.] Millsp.) leaves. The activities of POD and PPO were observed to be greater in H2O2-stressed pigeonpea leaves than in water treated control leaves. However, after longer incubations activities of these enzymes were markedly reduced. The observed changes revealed that exogenous H2O2 may induce oxidative stress tolerance by enhancing the activities of POD and PPOs. On the other hand, reduction found in H2O2-induced POD and PPO activities at later stages may be due to destruction of these proteins along with other proteins. This study will help to improve the tolerability of plants to environmental stresses by enhancing the expression of POD and PPOs.
This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa... more 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.
ABSTRACT Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) b... more ABSTRACT Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a simple and sensitive gel X-ray film contact print technique. About 17 AlTIs were detected in the seed extracts of A. lebbeck. Two groups of AlTIs-1 major (10 AlTIs; slow migration on the gel) and 1 minor (7 AlTIs; fast migration on the gel) were identified. The former was specific only toward trypsin. However, the latter was specific toward both trypsin and Helicoverpa armigera gut proteinases (HaGPs). The most potent AlTI (AlTI13) was purified to assess its in vivo bioefficacy toward HaGPs. Purification was achieved using (NH4)(2)SO4 fractionation, Sephadex G-100 column chromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependent bioefficacies of AlTIs in the (NH4)(2)SO4 F-3 fractions (0.1%, 0.5%, and 1%) were approximately 79%, 83%, and 90%, respectively, resulting in reductions in the average larval weight of H. armigera. Artificial diet containing a single dose of AlTI13 (5 mu g/g diet) reduced the larval weight by about 76%, with 60% mortality. The half-maximal inhibitory concentrations (IC50) of AlTI13 for trypsin and HaGPs were 0.14 and 0.17 mu mol/ml, respectively. The optimum conditions for AlTI13 were pH 8 and temperatures ranging from 35 to 40 C. Reducing sodium dodecyl sulfate-PAGE analysis indicated that similar to 28 kDa Kunitz-like trypsin inhibitor was present. Thus, we showed that particularly, AlTI13 of A. lebbeck could be used as a transgene macromolecule to markedly increase insect resistance in genetically engineered plants.
This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa... more 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.
Trypsin protease inhibitor have been identified and purified from extracts of Mucuna Pruriens. Mu... more Trypsin protease inhibitor have been identified and purified from extracts of Mucuna Pruriens. Mucuna Pruriens seed sample were purified using ammonium sulphate precipitation, Saphadex G-75 gel filtration chromatography technique including analysis of trypsin inhibitors by dot-blot method, and electrophoretic analysis and visualization of protease inhibitor bands by gel X-ray film contact print method. Saphadex G-75 gel filtration chromatography identified a small molecular weight trypsin inhibitory fraction. In the present paper we described, a simple and inexpensive procedure to detect protease inhibitors of seed by the gel-x-ray film contact print technique.
The sample from seed of Peganum harmala plant tree was extracted in hexane, chloroform, acetone a... more The sample from seed of Peganum harmala plant tree was extracted in hexane, chloroform, acetone and methanol and separated by thin layer chromatography on silica gel plate by using chloroform : Methanol (85:15) solvent mixture and detected under the ultraviolet light. Maximum bands were detected on TLC of methanol extract. Those bands were scraped and the purification of the single band of interest molecule was done by preparative silica gel column chromatography. Antimicrobial activity of single band isolated was tested against standard ATCC (American Type Culture Collection) strain of E.coli ( 25922), Klebseilla , Staphylococcus aureus (25923), Pseudomonas aeruginosa (27853), S. pneumonia (6305) using Disc diffusion method and Punch Plate technique. The Purified fraction of alkaloid show zone of growth inhibition with 10mm on ATCC Staphylococcus aureus. The sensitivity test of water extract was showing effect mostly against Staphylococcus aureus and E.coli
In this report protein content and catalase (CAT) activity were used to study the effect of hydro... more In this report protein content and catalase (CAT) activity were used to study the effect of hydrogen peroxide (H2O2) on leaf senescence in detached Pigeon pea leaves. A decrease in protein content measured as an indicator of leaf senescence, and a drop in CAT activity was observed following treatment with H2O2 in Pigeon pea detached leaves compared with control leaves. However after longer incubations CAT activity significantly increased in comparison with day 1 treatment. Protein content and CAT activity were also studied in the leaves treated by 0.025, 0.05, 0.075 and 0.1 mM H2O2. The optimal concentration of H2O2 in reducing protein content seems to be 0.075 mM, whereas concentration of 0.025, 0.05, 0.075 mM increase the CAT activity while lower and higher concentrations shown the opposite effect. The observed changes revealed that H2O2 induces oxidative stress and oxidative damage thereby leaf senescence in the detached leaves of Pigeon pea.
The present study tries to examine the influence of natural and oxidative stress [hydrogen peroxi... more The present study tries to examine the influence of natural and oxidative stress [hydrogen peroxide (H2O2) treatment]-induced senescence on peroxidase (POD) activity in neem (Azadirachta indica A. juss) leaves. Data indicated that incubation of detached leaves in presence of H2O2 has induced POD(s), that enzyme activity is also enhanced in natural senescing leaves. Changes in POD activity and protein loss during H2O2-promoted senescence has shown similarity with natural senescence suggests that the underlying regulatory mechanisms might be the same in both stresses, at least in neem. Initial induction of POD activity under H2O2 stress suggests that PODs play a very important role during the early phases of leaf senescence. Reduction in POD activity along with the increase in protein loss at latter stages suggests that there was no correlation between PODs and senescence, at least in neem.
In this report peroxidase (POD) and polyphenol oxidase (PPO) activities were used to study the ef... more In this report peroxidase (POD) and polyphenol oxidase (PPO) activities were used to study the effect of hydrogen peroxide (H2O2) on leaf senescence in detached pigeonpea (Cajanus cajan [L.] Millsp.) leaves. The activities of POD and PPO were observed to be greater in H2O2-stressed pigeonpea leaves than in water treated control leaves. However, after longer incubations activities of these enzymes were markedly reduced. The observed changes revealed that exogenous H2O2 may induce oxidative stress tolerance by enhancing the activities of POD and PPOs. On the other hand, reduction found in H2O2-induced POD and PPO activities at later stages may be due to destruction of these proteins along with other proteins. This study will help to improve the tolerability of plants to environmental stresses by enhancing the expression of POD and PPOs.
This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa... more 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.
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