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Regulación de la actividad de la ARN polimerasa II en las células híbridas CHO resistentes a la alfa-amanitina

A Guialis, B G Beatty, C J Ingles

|January 1, 1977

Resumen

Las células híbridas de ovario de hámster chino (CHO) demuestran un mecanismo regulador para la ARN polimerasa II. La exposición a la alfa-amanitina conduce a la inactivación de la ARN polimerasa II sensible, con formas resistentes que aumentan para mantener una actividad total constante.

Área de la Ciencia:

Biología Molecular Biología Molecular
Biología celular Biología celular.
La bioquímica es la bioquímica.

Sus antecedentes:

Las líneas celulares híbridas de ovario de hámster chino (CHO) se crearon mediante la fusión de células de tipo salvaje sensibles a la alfa-amanitina con células mutantes que poseen ARN polimerasa II resistente a la alfa-amanitina.
Estas células híbridas contienen formas sensibles y resistentes de ARN polimerasa II, lo que permite el estudio de la regulación de la actividad enzimática diferencial.

Objetivo del estudio:

Investigar los mecanismos reguladores que controlan los niveles intracelulares de ARN polimerasa II en respuesta a la alfa-amanitina.
Para determinar si los cambios en la actividad enzimática se correlacionan con los cambios en la masa enzimática total.

Principales métodos:

Cultivo de células híbridas CHO en medios que contienen alfa-amanitina para inducir la actividad enzimática diferencial.
Utilizando un radioinmunoensayo sensible para cuantificar la masa total de la enzima ARN polimerasa II.
Utilizando ensayos de unión a 3H-amanitina para evaluar la actividad y capacidad de unión de la ARN polimerasa II inactivada con alfa-amanitina.

Principales resultados:

Cuando se cultivaba en alfa-amanitina, la ARN polimerasa II sensible se inactivaba, mientras que la actividad de la ARN polimerasa II resistente aumentaba proporcionalmente, manteniendo una actividad total constante.
Un aumento de 2-3 veces en la actividad enzimática resistente en las células tratadas con alfa-amanitina no mostró cambios significativos en la masa enzimática total a través del radioinmunoensayo.
La cuantificación de la polimerasa II sensible inactivada por unión a 3H-amanitina reveló una pérdida de capacidad de unión correlacionada con la pérdida de actividad enzimática.

Conclusiones:

Existe un mecanismo regulador para controlar la concentración intracelular de la ARN polimerasa II.
Esta regulación implica ajustes en la concentración de la enzima, en lugar de sólo la modificación post-traducional o la modulación de la actividad.
La regulación observada es independiente de la actividad de la ARN polimerasa I.

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