Inhibitory Substances of a Tau-Type Pumpkin Glutathione S-Transferase: Their Existence and Chemical Properties

Hossain, Md. Daud,Suzuki, Toshisada,Fujita, Masayuki The Korean Society of Crop Science 2007 Journal of crop science and biotechnology Vol.10 No.2

Distributions of physiological inhibitors of a tau-type pumpkin glutathione S-transferase(CmGSTU3) have been investigated in different organs of pumpkin plants, including the onion bulb and water hyacinth root. Inhibitory effects were observed in alcoholic extracts of all plant parts, but the extracts prepared from the roots of either water hyacinth or pumpkin plant showed the highest effect on CmGSTU3 toward 1-chloro-2,4- dinitrobenzene(CDNB). Results of various chromatographies indicated that a number of inhibitory substances were present in the alcoholic extract of each plant organ. Some macromolecules in the plant extracts exhibited inhibitory effects; however, the extracts might contain a large number of unknown low-molecular-weight inhibitory substances. Some of the low-molecular-weight inhibitors in water hyacinth root extract showed characteristics fluoresce under UV light.

Suppressive Effects of Coumarins on Pumpkin Seedling Growth and Glutathione S-Transferase Activity

Md. Daud Hossain,Jing Li,Shirong Guo,Masayuki Fujita 한국작물학회 2008 Journal of crop science and biotechnology Vol.11 No.3

The effects of some coumarins (coumarin, 7-hydroxycoumarin, scopoletin and esculetin) were investigated on pumpkin (Cucurbita maxima Duch.) seedlings and on pumpkin glutathione S-transferases (GSTs). Coumarin and esculetin suppressed the growth of seedlings, especially the elongation of roots as well as hypocotyls. Among the compounds tested, only esculetin inhibited the activity of a particular pumpkin GST by 50%, CmGSTU3 toward 1-chloro-2, 4- dinitrobenzene (CDNB) and at a concentration of 22 μM. Both ethylacetae (EtOAc) and water fractions in pumpkin seedlings and different organs of one-month-old pumpkin plants contained esculetin or similar hydrophobic fluorescent substances as well as hydrophilic substances, which showed different degrees of inhibitory effects on CmGSTU3 activity. The effects of some coumarins (coumarin, 7-hydroxycoumarin, scopoletin and esculetin) were investigated on pumpkin (Cucurbita maxima Duch.) seedlings and on pumpkin glutathione S-transferases (GSTs). Coumarin and esculetin suppressed the growth of seedlings, especially the elongation of roots as well as hypocotyls. Among the compounds tested, only esculetin inhibited the activity of a particular pumpkin GST by 50%, CmGSTU3 toward 1-chloro-2, 4- dinitrobenzene (CDNB) and at a concentration of 22 μM. Both ethylacetae (EtOAc) and water fractions in pumpkin seedlings and different organs of one-month-old pumpkin plants contained esculetin or similar hydrophobic fluorescent substances as well as hydrophilic substances, which showed different degrees of inhibitory effects on CmGSTU3 activity.

Comparative Investigation of Glutathione S-Transferases, Glyoxalase-I and Alliinase Activities in Different Vegetable Crops

Hossain, Md Daud,Rohman, Md Motiar,Fujita, Masayuki The Korean Society of Crop Science 2007 Journal of crop science and biotechnology Vol.10 No.1

Glutathione S-transferases(GSTs, EC 2.5.1.18), glyoxalase-I(EC 4.4.1.5) and alliin lyase(alliinase, EC 4.4.1.4) are important enzyme systems in plant bodies. The first two are mainly detoxifying enzymes that utilize glutathione(GSH) in the defense mechanism, and the last one is mainly involved in secondary metabolism and relevant to sulfur compounds derived from GSH. The activities of the three enzymes have been investigated in soluble extracts of vegetable crops, including pumpkin, cabbage, broccoli, radish, carrot, potato, sweet potato, mungbean, and onion. GST activities were detected in all of the vegetables, and the extract of onion bulb exhibited the highest specific activity(648 nmol/min/mgP). The putative GSTs of most of the vegetables were found to be induced by ethanol. The activities of GSTs in onion bulb were found to be markedly inhibited by S-hexyl glutathione and were also inhibited by S-butyl glutathione and S-propyl glutathione. The anti-CmGSTF1 antiserum recognized a thick band for putative onion GST. The estimated glyoxalase-I activity level was also high in onion bulb(4540 nmol/min/mgP), indicating that the thick band detected by Western blot analysis might result from partial recognition of glyoxalase-I by the antiserum. The specific activities for glyoxalase-I were moderate in radish and carrot, and the extracts of other vegetables had rather low levels of activities. The extract of onion also showed the highest specific activity level for alliinase(2069nmol pyruvate/mgP). The extracts of other vegetables also had alliinase activities, although the estimated values were much lower than that of onion.

Suppressive Effects of Coumarins on Pumpkin Seedling Growth and Glutathione S-Transferase Activity

Hossain, Md. Daud,Li, Jing,Guo, Shirong,Fujita, Masayuki The Korean Society of Crop Science 2008 Journal of crop science and biotechnology Vol.11 No.3

The effects of some coumarins(coumarin, 7-hydroxycoumarin, scopoletin and esculetin) were investigated on pumpkin(Cucurbita maxima Duch.) seedlings and on pumpkin glutathione S-transferases(GSTs). Coumarin and esculetin suppressed the growth of seedlings, especially the elongation of roots as well as hypocotyls. Among the compounds tested, only esculetin inhibited the activity of a particular pumpkin GST by 50%, CmGSTU3 toward 1-chloro-2, 4- dinitrobenzene(CDNB) and at a concentration of 22 ${\mu}M$. Both ethylacetae(EtOAc) and water fractions in pumpkin seedlings and different organs of one-month-old pumpkin plants contained esculetin or similar hydrophobic fluorescent substances as well as hydrophilic substances, which showed different degrees of inhibitory effects on CmGSTU3 activity.

Enzymatic Formation of Guaiacylglycerol 8-O-4´- (Coniferyl Alcohol) Ether from Coniferyl Alcohol with Enzyme Preparations of Eucommia ulmoides

Md. Shameul Alam,Takeshi Katayama,Toshisada Suzuki,Deeder Sultana,Saima Sultana,Md. Daud Hossain 한국작물학회 2008 Journal of crop science and biotechnology Vol.11 No.1

Lignans and neolignans are optically active plant secondary metabolites. Research on biosynthesis of lignans has already been advanced especially for the formation of (+) pinoresinol but information on the biosynthesis of 8-O-4´- neolignans is still limited. Moreover, the chemical structure (position of substituents on aromatic rings) and stereochemistry of 8-O-4´ neolignans is not clear. Katayama and Kado discovered that incubation of cell-free extracts from E. ulmoides with coniferyl alcohol in the presence of hydrogen peroxide gave (+)-erythro- and (-)-threo- guaiacylglycerol 8-O-4´-(coniferyl alcohol) ether (GGCE) (diastereomeric ratio, 3:2) which is the first report on enzymatic formation of optically active -8-O-4´ neolignans from an achiral monolignol. In this aspect, enzymatic formation of guaiacyl 8-O-4´ neolignan is noteworthy to clarify its stereochemistry from incubation of coniferyl alcohol with enzyme prepared from Eucommia ulmoides. In this experiment, soluble and insoluble enzymes prepared from E. ulmoides were incubated with 30 mM coniferyl alcohol (CA) for 60 min. The enzyme catalyzed GGCE, dehydrodiconiferyl alcohol (DHCA), and pinoresinol identified by reversed phase HPLC. Consequently, diastereomeric compositions of GGCE were determined as erythro and threo isomer. Enantiomeric composition was determined by the chiral column HPLC. Both enzyme preparations enantioselectively formed (-)-erythro, (+)-erythro and (+)-threo, (-)-threo-GGCEs respectively. Lignans and neolignans are optically active plant secondary metabolites. Research on biosynthesis of lignans has already been advanced especially for the formation of (+) pinoresinol but information on the biosynthesis of 8-O-4´- neolignans is still limited. Moreover, the chemical structure (position of substituents on aromatic rings) and stereochemistry of 8-O-4´ neolignans is not clear. Katayama and Kado discovered that incubation of cell-free extracts from E. ulmoides with coniferyl alcohol in the presence of hydrogen peroxide gave (+)-erythro- and (-)-threo- guaiacylglycerol 8-O-4´-(coniferyl alcohol) ether (GGCE) (diastereomeric ratio, 3:2) which is the first report on enzymatic formation of optically active -8-O-4´ neolignans from an achiral monolignol. In this aspect, enzymatic formation of guaiacyl 8-O-4´ neolignan is noteworthy to clarify its stereochemistry from incubation of coniferyl alcohol with enzyme prepared from Eucommia ulmoides. In this experiment, soluble and insoluble enzymes prepared from E. ulmoides were incubated with 30 mM coniferyl alcohol (CA) for 60 min. The enzyme catalyzed GGCE, dehydrodiconiferyl alcohol (DHCA), and pinoresinol identified by reversed phase HPLC. Consequently, diastereomeric compositions of GGCE were determined as erythro and threo isomer. Enantiomeric composition was determined by the chiral column HPLC. Both enzyme preparations enantioselectively formed (-)-erythro, (+)-erythro and (+)-threo, (-)-threo-GGCEs respectively.

Enzymatic Formation of Guaiacylglycerol 8-O-4'-(Coniferyl Alcohol) Ether from Coniferyl Alcohol with Enzyme Preparations of Eucommia ulmoides

Alam, Md. Shameul,Katayama, Takeshi,Suzuki, Toshisada,Sultana, Deeder,Sultana, Saima,Hossain, Md. Daud The Korean Society of Crop Science 2008 Journal of crop science and biotechnology Vol.11 No.1

Lignans and neolignans are optically active plant secondary metabolites. Research on biosynthesis of lignans has already been advanced especially for the formation of (+) pinoresinol but information on the biosynthesis of 8-O-4'- neolignans is still limited. Moreover, the chemical structure(position of substituents on aromatic rings) and stereochemistry of 8-O-4' neolignans is not clear. Katayama and Kado discovered that incubation of cell-free extracts from E. ulmoides with coniferyl alcohol in the presence of hydrogen peroxide gave (+)-erythro- and (-)-threo- guaiacylglycerol 8-O-4'-(coniferyl alcohol) ether (GGCE)(diastereomeric ratio, 3:2) which is the first report on enzymatic formation of optically active -8-O-4' neolignans from an achiral monolignol. In this aspect, enzymatic formation of guaiacyl 8-O-4' neolignan is noteworthy to clarify its stereochemistry from incubation of coniferyl alcohol with enzyme prepared from Eucommia ulmoides. In this experiment, soluble and insoluble enzymes prepared from E. ulmoides were incubated with 30 mM coniferyl alcohol(CA) for 60 min. The enzyme catalyzed GGCE, dehydrodiconiferyl alcohol(DHCA), and pinoresinol identified by reversed phase HPLC. Consequently, diastereomeric compositions of GGCE were determined as erythro and threo isomer. Enantiomeric composition was determined by the chiral column HPLC. Both enzyme preparations enantioselectively formed (-)-erythro, (+)-erythro and (+)-threo, (-)-threo-GGCEs respectively.

Inhibitory Substances of a Tau-Type Pumpkin Glutathione S-Transferase: Their Existence and Chemical Properties

Masayuki Fujita,Md. Daud Hossain,Toshisada Suzuki 한국작물학회 2007 Journal of crop science and biotechnology Vol.10 No.2

Distributions of physiological inhibitors of a tau-type pumpkin glutathione S-transferase (CmGSTU3) have been investigated in different organs of pumpkin plants, as well as onion bulb and water hyacinth root. Inhibitory effects were observed in alcoholic extracts of all plant parts, but the extracts prepared from the roots of either water hyacinth or pumpkin plant showed the highest effect on CmGSTU3 toward 1-chloro-2,4- dinitrobenzene (CDNB). Results of various chromatographies indicated that a number of inhibitory substances were present in the alcoholic extract of each plant organ. Some macromolecules in the plant extracts exhibited inhibitory effects; however, the extracts might contain a large number of unknown, low-molecular-weight inhibitory substances. Some of the low-molecular-weight inhibitors in water hyacinth root extract showed characteristic fluorescence under UV light. Distributions of physiological inhibitors of a tau-type pumpkin glutathione S-transferase (CmGSTU3) have been investigated in different organs of pumpkin plants, as well as onion bulb and water hyacinth root. Inhibitory effects were observed in alcoholic extracts of all plant parts, but the extracts prepared from the roots of either water hyacinth or pumpkin plant showed the highest effect on CmGSTU3 toward 1-chloro-2,4- dinitrobenzene (CDNB). Results of various chromatographies indicated that a number of inhibitory substances were present in the alcoholic extract of each plant organ. Some macromolecules in the plant extracts exhibited inhibitory effects; however, the extracts might contain a large number of unknown, low-molecular-weight inhibitory substances. Some of the low-molecular-weight inhibitors in water hyacinth root extract showed characteristic fluorescence under UV light.

Comparative Investigation of Glutathione S-Transferases, Glyoxalase-I and Alliinase Activities in Different Vegetable Crops

Masayuki Fujita,Md Motiar Rohman,Md Daud Hossain 한국작물학회 2007 Journal of crop science and biotechnology Vol.10 No.1

Glutathione S-transferases (GSTs, EC 2.5.1.18), glyoxalase-I (EC 4.4.1.5) and alliin lyase (alliinase, EC 4.4.1.4) are important enzyme systems in plant bodies. The first two are mainly detoxifying enzymes that utilize glutathione (GSH) in the defense mechanism, and the last one is mainly involved in secondary metabolism and relevant to sulfur compounds derived from GSH. The activities of the three enzymes have been investigated in soluble extracts of vegetable crops, including pumpkin, cabbage, broccoli, radish, carrot, potato, sweet potato, mungbean, and onion. GST activities were detected in all of the vegetables, and the extract of onion bulb exhibited the highest specific activity (648 nmol/min/mgP). The putative GSTs of most of the vegetables were found to be induced by ethanol. The activities of GSTs in onion bulb were found to be markedly inhibited by S-hexyl glutathione and were also inhibited by S-butyl glutathione and S-propyl glutathione. The anti- CmGSTF1 antiserum recognized a thick band for putative onion GST. The estimated glyoxalase-I activity level was also high in onion bulb (4540 nmol/min/mgP), indicating that the thick band detected by Western blot analysis might result from partial recognition of glyoxalase-I by the antiserum. The specific activities for glyoxalase-I were moderate in radish and carrot, and the extracts of other vegetables had rather low levels of activities. The extract of onion also showed the highest specific activity level for alliinase (2069nmol pyruvate/mgP). The extracts of other vegetables also had alliinase activities, although the estimated values were much lower than that of onion. Glutathione S-transferases (GSTs, EC 2.5.1.18), glyoxalase-I (EC 4.4.1.5) and alliin lyase (alliinase, EC 4.4.1.4) are important enzyme systems in plant bodies. The first two are mainly detoxifying enzymes that utilize glutathione (GSH) in the defense mechanism, and the last one is mainly involved in secondary metabolism and relevant to sulfur compounds derived from GSH. The activities of the three enzymes have been investigated in soluble extracts of vegetable crops, including pumpkin, cabbage, broccoli, radish, carrot, potato, sweet potato, mungbean, and onion. GST activities were detected in all of the vegetables, and the extract of onion bulb exhibited the highest specific activity (648 nmol/min/mgP). The putative GSTs of most of the vegetables were found to be induced by ethanol. The activities of GSTs in onion bulb were found to be markedly inhibited by S-hexyl glutathione and were also inhibited by S-butyl glutathione and S-propyl glutathione. The anti- CmGSTF1 antiserum recognized a thick band for putative onion GST. The estimated glyoxalase-I activity level was also high in onion bulb (4540 nmol/min/mgP), indicating that the thick band detected by Western blot analysis might result from partial recognition of glyoxalase-I by the antiserum. The specific activities for glyoxalase-I were moderate in radish and carrot, and the extracts of other vegetables had rather low levels of activities. The extract of onion also showed the highest specific activity level for alliinase (2069nmol pyruvate/mgP). The extracts of other vegetables also had alliinase activities, although the estimated values were much lower than that of onion.