Бесплатный автореферат и диссертация по биологии на тему

Катионные ингибиторы сериновых протеиназ из семян гречихи (Fagopyrum esculentum Moench. )

ВАК РФ 03.00.04, Биохимия

Содержание диссертации, кандидата биологических наук, Цыбина, Татьяна Александровна

Список сокращений.

ВВЕДЕНИЕ.

1. ОБЗОР ЛИТЕРАТУРЫ.

1.1. Классификация ингибиторов протеиназ растительного происхождения (введение).

1.1.1. Семейство соевого ингибитора трипсина Кунитца (STI).

1.1.2. Семейство ингибитора Баумана-Бирк.

1.1.3. Семейство ингибиторов I из картофеля.

1.1.4. Семейство ингибиторов II из картофеля.

1.1.5. Семейство тыквенных ингибиторов.

1.1.6. Семейство серпинов.

1.1.7. Семейство фитоцистатинов.

1.1.8. Семейство напинов.

1.1.9. Ингибиторы аспартильных протеиназ.

1.1.10. Семейство ингибиторов субтилизина/а-амилазы у злаков.

1.2. Другие ингибиторы протеиназ

1.3. Антимикробные пептиды и запасные белки, гомологичные ингибиторам протеиназ.

1.4. Физиологическое значение ингибиторов и возможности их применения.

2. ЭКСПЕРИМЕНТАЛЬНАЯ ЧАСТЬ.

2.1. Материалы и реактивы.

2.2. Методы исследования.

2.2.1. Определение активности ингибиторов.

2.2.2. Гидролиз ингибиторов пепсином.

2.2.3. Определение концентрации белка.

2.2.4. Нативный электрофорез в полиакриламидном геле.

2.2.5. Определение ИЭТ ингибиторов.

2.2.6. Определение аминокислот, входящих в реактивный центр ингибиторов.

2.2.7. Изучение рН-стабильности и термостабильности ингибиторов

2.2.8. Определение аминокислотного состава.

2.2.9. Восстановление и алкилирование белков.

2.2.10. Автоматическая деградация белков по Эдману.

2.2.11. Определение константы ингибирования (К,) протеиназ.

2.2.12. Определение молекулярной массы.

2.3. Результаты и их обсуждение.

2.3.1. Выделение и очистка катионных ингибиторов из покоящихся семян гречихи {Fagopyrum еэси1епШт МоепсИ.) сорта Шатиловская

2.3.2. Характеристика физико-химических свойств катионных ингибиторов из семян гречихи.

Заключение Диссертация по теме "Биохимия", Цыбина, Татьяна Александровна

ВЫВОДЫ:

1. Из покоящихся семян гречихи впервые выделены и охарактеризованы четыре новых ингибитора сериновых протеиназ: BWI-lc, BWI-2c, BWI-Зс и BWI-4c.

2. Молекулярные массы изученных ингибиторов лежат в пределах 5,27,76 кДа, а ИЭТ - в области рН 8,0-8,5. Все исследованные ингибиторы характеризуются высокой рН-стабильностью в широком диапазоне рН 2,0-12,0 и термостабильностью, особенно в кислой среде.

3. В положении Pj реактивных центров ингибиторов BWI-Зс и BWI-4c находится остаток лизина, а в реактивном центре BWI-2c - остаток аргинина.

4. Ингибиторы протеаз BWI-lc и BWI-2c способны инактивировать только трипсин, химотрипсин и трипсин-подобные протеиназы мицелиальных грибов, в то время как ингибиторы протеаз BWI-Зс и BWI-4c также эффективно подавляют активность субтилизинов бактерий и субтилаз грибов.

5. Катионные ингибиторы протеаз из семян гречихи в опытах in vivo подавляли прорастание спор и рост гиф фитопатогенных грибов.

6. На основании данных по N-концевым аминокислотным последовательностям в совокупности с физико-химическими свойствами ингибиторы протеаз BWI-Зс и BWI-4c из семян гречихи могут быть отнесены к семейству картофельного ингибитора протеиназ I. В то же время ингибиторы протеаз BWI-lc и BWI-2c по совокупности полученных данных не могут быть отнесены ни к одному из известных семейств ингибиторов протеолитических ферментов.

Библиография Диссертация по биологии, кандидата биологических наук, Цыбина, Татьяна Александровна, Москва

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