Исследование каталитических свойств мультигемовой нитритредуктазы из галоалкалофильной бактерии Thioalkalivibrio nitratireducens
Пк1топоуа 1 М. а/, 2006]. Определение пространственной структуры ТуМК показало, что фермент существенно отличается от ранее описанных в литературе ША: мономер ТуМК представляет собой двухдоменный белок, содержащий восемь гемов с, пять из которых, включая каталитический, гомологичны пяти гемам известных ША, а дополнительные три гема расположены на отдельном домене. В кристалле ТуМК существует как… Читать ещё >
Содержание
- Литературный обзор
- 1. Биологический цикл азота
2. Восстановление нитрата и нитрита в бактериях. Транспорт через периплазматическую мембрану. з! Нитратредуктаза — ключевой фермент азотного метаболизма Нитритредуктаза — ключевой фермент последней стадии восстановления нитрата бактериями
3.1 Цитохром сс/? нитритредуктазы 15 !
3.2 Си — содержащие нитритредуктазы (Си-НиР)
3.3 Диссимиляторные цитохром с нитритредуктазы
3.3.1 Механизм респираторной аммонификации нитрита в бактериях
3.3.2 Структура цитохром с нитритредуктазного комплекса в протеобактериях
3.3.3 Строение и каталитические свойства цитохром с нитритредуктаз различных видов протеобактерий
3.3.4 Механизм реакции, катализируемой цитохром с нитритредуктазами
4.1 «» Исследование белков с помощью методов электрохимии. Исследование каталитических свойств нитритредуктаз с помощью методом вольтамперометрии.
5. Поиск возможного физиологического донора электронов «^N?[
6. Характеристика экстремофильной бактерии ТЫоа1ка1МЬпо пИгаИгебисепБ.
Материалы и методы
1. Реактивы, используемые в работе
2. Объект исследования и условия его культивирования
3. Выделение цитохром с нитритредуктазы методом ВЭЖХ
4. Определение субъединичного состава нитритредуктазы методом ВЭЖХ
5. Определение субъединичного состава нитритредуктазы методом малоуглового рассеяния.
6. Определение субъединичного состава нитритредуктазы методом аналитического ультрацентрифугирования.
7. Определение нитритредуктазной активности (по методу Грисса)
8. Определение активности нитритредуктазы методом циклической вольтамперометрии
9. Определение нитрит- и гидроксиламинредуктазных активностей по окислению метилвиологена
10. | Определение конечного продукта реакции
11. | Определение пероксидазной активности 12. | Фотовосстановление нитритредуктазы «NN?[4, 1 сенсибилизированное флавинами и птеринами 51 51 «
13. | Спектральные исследования
14. | Определение молекулярной массы и субъединичного состава 1 1 | белков методом электрофореза
15. Определения рН-оптимума нитритредуктазной и пероксидазной реакций
16. Масс-спектрометрический анализ
Результаты
1. Выделение, очистка и характеристика нитритредуктазы из клеток галоалкалофильной бактерии Гу.пИгаИгебисепэ.
2. Исследование олигомерного состава нитритредуктазы
3. Спектральные свойства ТуГ^Ш
4. 5″ 6. 7." Характеристика субстратной специфичности «П/Ы^ Зависимость активности нитритредуктазы от рН среды Определение кинетических параметров реакций, катализируемых Т/№ 1Ч Влияние различных ионов на активность «NN?[4 61 64 65~~ '
8. Изучение термостабильности и температурного оптимума нитритредуктазой реакции
9. Электрохимическое исследование кинетических параметров ' 69 | ТуГ^ I I
10. Определение пероксидазной активности Тх/Г^Ш
11. Фотовосстановление сенсибилизированное флавинами и птеринами
Выводы 98 |
Список публикаций
Список литературы 99 |
Список сокращений.
ТУГУ!" Цитохром с нитритредуктаза ТЫоа1ка1мЬп'о пИгаИгеёисепз
N1^ МВ АБТС цитохром с нитритредуктаза Метилвиологен 2,2'-азино-бис (3-этилбензтиазолин-6-сульфокислота) диаммонийная соль
НАДН НАД (Ф)Н Никотинамидадениндинуклеотид восстановленный Никотинамидадениндинуклеотидфосфат восстановленный
ФАД Флавинадениндинуклеотид
Ыа-ЭДТА Этилендиаминтетраацетат натрия
Рбфл РбфлНг ~ ЫЕЬА Рибофлавин Рибофлавин восстановленный N-(1 -нафтилэтилендиамин) дигидрохлорид
АТФ Аденозинтрифосфат
ФМН | Флавинмононуклеотид
ФМНН2 дт Флавинмононуклеотид восстановленный Дитионит натрия
ДСК Дифференциальная сканирующая калориметрия дмп 6,7-диметилптерин дмпн4 бДТ. в-тетрагидро-б.У-диметилптерин
Км Константа Михаэлиса-Ментен
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