Биохимические свойства столбнячного токсина и механизмы его действия


Как показал еще Ehrlich, столбнячный токсин образующийся в культурах столбнячной палочки, состоит из двух различных и независимых веществ, оказывающих различный патогенный эффект: тетаноспазмина, вызывающего судорожный синдром, и тетанолизина, вызывающего гемолиз эритроцитов. Лизин более чувствителен к различным воздействиям, в частности к нагреванию, окислению и уменьшению рН среды. Он хорошо сорбируется эритроцитами. Все эти и другие свойства лизина используются для его выделения.

Лизин, по-видимому, не играет существенной роли в патогенезе столбняка. Некоторые авторы предполагают, что, помимо гемолитического действия, лизин может вызывать местный некроз тканей и разрушать фагоциты. Мы рассмотрим свойства главным образом тетаноспазмина, изучению которых и посвящена большая часть литературы по химии столбнячного токсина.

При электрофорезе неочищенный токсин разделяется на две фракции: одна перемещается к аноду, она высокоактивна, другая - к катоду, она нетоксична. Очищенный токсин соответствует в основном фракции Бета-глобулинов, которые ведут себя при рН крови как электроотрицательные белки. А. Ю. Иллютович показал, что чем сильнее токсин, тем более преобладает в нем количество электроотрицательных компонентов. Фракции неочищенного токсина, соответствующие по своей электрофоретической подвижности Альфа-глобулинам и альбуминам, нетоксичны.
 
Благодаря работам Pillemer и сотрудников, получивших очищенный кристаллический токсин, а также исследованиям Largier, Turpin и Raynaud и др., выделивших высокоочищенный столбнячный токсин, физико-химические свойства последнего относительно хорошо изучены. Кристаллы столбнячного токсина бесцветны, 1% раствор его имеет бледно-желтый оттенок.

Молекулярный вес токсина составляет 67 000-67 286. Константа седиментации токсина, по Pillemer, составляет 4,5 единицы Сведберга, по Largier - 3,9. Очищенный, особенно кристаллический, токсин в обычном растворе нестоек, его устойчивость повышается в присутствии биполярных ионов при рН 5-6. Падение патогенности токсина при его стоянии сопровождается изменением растворимости и возрастанием константы седиментации до 7,0 или 7,6. Этот факт позволил указанным авторам сделать заключение, что токсин в растворе димеризуется и превращается в нетоксичный токсоид. Однако, по данным Turpin и Raynaud, константа седиментации активного токсина может достигать 6,3. Raynaud, Turpin и Bizzini, исследуя свойства очищенного столбнячного токсина, обладающего высокой патогенной активностью, нашли, что последний может находиться в нескольких агрегатных состояниях, причем полимеризованный токсин может иметь константу седиментации 7,0. Деполимеризация большинства агрегатных форм осуществляется путем разрыва дисульфидных мостиков.

Как показали Pillemer и сотрудники, кристаллический столбнячный токсин представляет собой белок, содержащий 15,7% азота, около 1% (1,04%) серы и 0,1% (0,065%) фосфора. Токсин не содержит углеводов. В его состав входит 13 аминокислот, их соотношение представляет известный интерес. В отличие от других белков в столбнячном токсине содержится относительно большое количество аспарагиновой кислоты, изолейцина и лизина. С этим свойством токсина связывается его высокая поверхностная активность и способность сорбироваться различными веществами (в том числе и на стенке посуды). В столбнячном токсине аспарагина содержится в 1,5 раза больше, чем глютамина, и больше изолейцина, чем лейцина; аналогичные отношения имеются и в ботулиническом токсине в отличие от других белков.

Уже давно было отмечено многими исследователями значение качества различных токсинов в определении их патогенной активности. В работах разных авторов было показано, что разные токсины, полученные от разных штаммов, обладают разной токсичностью, причем особенно вариабельными являются отношения в степени токсичности для разных животных. Эти отношения могут колебаться в десятки и даже сотни раз. При этом антигенные свойства токсинов, их способность связываться противостолбнячной сывороткой in vitro могут быть одинаковыми. Относительно причин отмеченных различий в патогенных свойствах токсинов был высказан ряд предположений. Friedemann и Hollander выдвинули гипотезу о различных качественных свойствах разных молекул токсина; Smith о возможном наличии двух или более токсических субстанций, к которым по-разному чувствительны различные животные и которые в разных отношениях находятся в разных токсинах; А. Д. Сперанский, В. М. Аристовский, Friedemann и Hollander высказали предположение о роли балластных веществ в определении патогенного эффекта столбнячного токсина. Хотя трактовка механизма действия балластных веществ может быть различной (А. Д. Сперанский и др. делали акцент на значении добавочных воздействий на организм), это предположение получило фактическое подтверждение в работе Pillemer и сотр., показавших, что четыре кристаллических препарата, полученные из токсинов разных серий, по своей патогенной силе были одинаковы: они содержали одинаковое количество DLM в 1 мг азота. Важное значение в определении степени патогенности неочищенных токсинов может иметь и наличие в их препаратах того или иного количества анатоксина, способного интерферировать с действием токсина, а также связываться, как и токсин, ганглиозидами.

Различные вещества, добавляемые к токсину, могут усиливать его патогенное действие. К ним относятся сыворотка крови, вытяжки из органов, мясной бульон, яичный белок, гемоглобин, ликвор, пептон и др. По данным Marie, яичный желток благодаря лецитину может повысить токсичность токсина в 105 раз. Сообщение об этом необычном результате осталось в литературе одиноким, а по данным других авторов, лецитин вообще не оказывает потенцирующего влияния на столбнячный токсин.

Исходя из того что белковые вещества усиливают действие столбнячного токсина, Condrea и Poenaru выдвинули гипотезу, согласно которой столбнячная палочка продуцирует не активный токсин, а протоксин, который под влиянием белковых веществ, в частности пептона, превращается в собственно токсин. Пептон в опытах этих авторов мог повышать токсичность токсина на 600%. Однако другие исследователи показали, что действие пептона, как и других усиливающих веществ, проявляется не в истинном активировании токсина, а в предохранении последнего от разрушения. Zuger и соавторы установили, что потенцирующее действие экстракта из мышечной ткани не связано с увеличением количества токсина ("размножением" его молекул), так как для нейтрализации одинакового количества "активированного" и исходного токсина необходимо одинаковое количество антитоксина.

Содержащиеся в нативных токсинах балластные белковые вещества играют роль стабилизаторов или консервантов токсина. Роль последних могут играть и небелковые вещества, например глицерин. Вместе с тем балластные вещества, находящиеся в нативных токсинах, оказывают и детоксицирующее действие. Последнее связано главным образом с наличием в фильтрате культуры протеаз, которые образуются при развитии столбнячной палочки и разрушают токсин. Консервация токсина пептоном и другими белковыми веществами в значительной мере определяется тем, что эти вещества являются ингибиторами протеолитической активности нативного токсина.

Свежеприготовленные очищенные препараты токсина обладают очень высокой токсичностью.

Высокая биологическая активность токсина давно уже служит основанием для предположения, что столбнячный токсин является ферментом. Еще Tizzoni и Cattani высказали предположение о ферментативной природе токсина. Lenormant выдвинул гипотезу, согласно которой столбнячный токсин состоит из носителя, связанного с антигенной структурой, и фиксируется на чувствительном субстрате, и активной группировки, обладающей свойствами диастазы. Отношение между этими частями токсина аналогично отношениям в системе кодиастазы и анадиастазы. Это предположение в дальнейшем не было проверено. Petrie показал, что токсин содержит протеазу и пептидазу, которые определяют его реакции с различными протеинами; с этими свойствами, по-видимому, связана и чувствительность токсина к свободным SH-группам. Schultze также пришел к заключению о наличии в токсине протеазы и пептидазы, которые связаны с тетаноспазмином. Наличие протеолитических ферментов в столбнячном токсине описано и другими авторами. Остается, однако, неясным, присущи ли они самому токсину и имеют ли они значение в его патогенном действии. По данным Pelloja и Campani, токсин не обладает рибонуклеазной активностью, а по данным Zacks и Sheff токсин, лишенный оксидазной активности, не теряет своих патогенных свойств.

Pappenheimer выдвинул в общей форме гипотезу, согласно которой столбнячный токсин действует не как фермент и не как ингибитор ферментативной активности, а как вещество, которое нарушает образование каких-то ферментных систем, необходимых для осуществления нервных процессов. Feigen, Peterson и др. высказали предположение, что процесс активирования спонтанного выброса передатчика в нервно-мышечном аппарате под влиянием столбнячного токсина (или его фракции) имеет в своей основе какую-то ферментативную природу. Возможно, что нарушение тормозных механизмов в центральной нервной системе при столбнячной интоксикации также связано с каталитическим действием токсина.

Женский журнал www.BlackPantera.ru:  Георгий Крыжановский

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