Glutamine synthetase （GS） is an important enzyme involved in nitrogen assimilation and metabolism in marine phytoplankton. However, little work has been done in situ due to the limitation of crude enzyme preparation...Glutamine synthetase （GS） is an important enzyme involved in nitrogen assimilation and metabolism in marine phytoplankton. However, little work has been done in situ due to the limitation of crude enzyme preparation methods. In this study, three enzyme preparation methods, high-speed centrifugation （HC, 〈10 000 g）, ultracentrifugation （UC, 70000 g）, and ultrafiltration （UF） with 100 kit molecular weight cutoff, were compared using two diatom species （Asterionellopsis glacialis and Thalassiosira weissflogii）, and two dinoflagellate species （Alexandriura catenella and Prorocentrum donghaiense） as experimental materials together with field samples collected from Xiamen Harbor, China. The results showed that HC is the best method to prepare crude enzymes for glutamine synthetase activity （GSA） in diatom species and diatom-dominant samples, while UF is the best method to extract GS from dinoflagellate species and dinoflagellate-dominant samples. For the HC method, the optimal centrifugal speed and time were 10 000 g and 35 min, respectively, and under these conditions, the highest GSA was obtained in all samples. This study indicates that both methods （HC and UF） overcome the limitation of centrifugal speed and could be applied to in situ GSA analysis, especially at sea.展开更多
Sulfotransferase (ST) is the first enzyme discovered in association with paralytic shellfish poisoning (PSP) toxin biosynthesis in toxic dinoflagellates. This study investigates the ST activity in crude enzyme extract...Sulfotransferase (ST) is the first enzyme discovered in association with paralytic shellfish poisoning (PSP) toxin biosynthesis in toxic dinoflagellates. This study investigates the ST activity in crude enzyme extraction of a toxic dinoflagellate species, Alexandrium tamarense CI01. The results show that crude enzyme can transfer a sulfate group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to N-21 in the carbamoyl group of gonyautoxin 2/3 (GTX2/3) to produce C1/C2, but is inactive toward STX to produce GTX5. The crude enzyme is optimally active at pH 6.0 and 15°C. The activity is enhanced by Co2+, Mg2+, Mn2+ and Ca2+ individually, but is inhibited by Cu2+. Moreover, the activity shows no difference when various sulfur compounds are used as sulfate donors. These results demonstrate that the ST specific to GTX2/3 is present in the cells of A. tamarense CI01 and is involved in PSP toxin biosynthesis. In addition, the ST from different dinoflagellates is species-specific, which explains well the various biosynthesis pathways of the PSP toxins in toxic dinoflagellates.展开更多
基金The National Natural Science Foundation of China under contract No 40331004the Ministry of Science and Technology of China under contract No 2005DFA20430
文摘Glutamine synthetase （GS） is an important enzyme involved in nitrogen assimilation and metabolism in marine phytoplankton. However, little work has been done in situ due to the limitation of crude enzyme preparation methods. In this study, three enzyme preparation methods, high-speed centrifugation （HC, 〈10 000 g）, ultracentrifugation （UC, 70000 g）, and ultrafiltration （UF） with 100 kit molecular weight cutoff, were compared using two diatom species （Asterionellopsis glacialis and Thalassiosira weissflogii）, and two dinoflagellate species （Alexandriura catenella and Prorocentrum donghaiense） as experimental materials together with field samples collected from Xiamen Harbor, China. The results showed that HC is the best method to prepare crude enzymes for glutamine synthetase activity （GSA） in diatom species and diatom-dominant samples, while UF is the best method to extract GS from dinoflagellate species and dinoflagellate-dominant samples. For the HC method, the optimal centrifugal speed and time were 10 000 g and 35 min, respectively, and under these conditions, the highest GSA was obtained in all samples. This study indicates that both methods （HC and UF） overcome the limitation of centrifugal speed and could be applied to in situ GSA analysis, especially at sea.
基金the National Natural Science Foundation of China (No.40376032)the Ministry of Science and Technology of the People’s Republic of China (No.2001CB409700)
文摘Sulfotransferase (ST) is the first enzyme discovered in association with paralytic shellfish poisoning (PSP) toxin biosynthesis in toxic dinoflagellates. This study investigates the ST activity in crude enzyme extraction of a toxic dinoflagellate species, Alexandrium tamarense CI01. The results show that crude enzyme can transfer a sulfate group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to N-21 in the carbamoyl group of gonyautoxin 2/3 (GTX2/3) to produce C1/C2, but is inactive toward STX to produce GTX5. The crude enzyme is optimally active at pH 6.0 and 15°C. The activity is enhanced by Co2+, Mg2+, Mn2+ and Ca2+ individually, but is inhibited by Cu2+. Moreover, the activity shows no difference when various sulfur compounds are used as sulfate donors. These results demonstrate that the ST specific to GTX2/3 is present in the cells of A. tamarense CI01 and is involved in PSP toxin biosynthesis. In addition, the ST from different dinoflagellates is species-specific, which explains well the various biosynthesis pathways of the PSP toxins in toxic dinoflagellates.
