Tubulin carboxypeptidase assay based on the action of the enzyme on [14C]tyrosinated tubulin bound to nitrocellulose membrane.
Anal Biochem
; 279(1): 9-17, 2000 Mar 01.
Article
en En
| MEDLINE
| ID: mdl-10683225
We have developed a method for the determination of tubulin carboxypeptidase activity which is based on the action of the enzyme on the substrate, [14C]tyrosinated tubulin, previously adsorbed on nitrocellulose membrane. In addition to being two to three times more sensitive than previous carboxypeptidase assays, this method allows the determination of dilute enzyme preparations even containing high salt (inhibitory) concentrations. This is a valuable property specially under circumstances in which numerous high salt-containing fractions with scarce activity should be analyzed (for example after certain chromatographic stages during enzyme purification). Our method is simpler, less time-consuming, and suitable for multiple, simultaneous determinations and the substrate bound to nitrocellulose can be stored for several months without significant alteration of its properties. Peptidases other than tubulin carboxypeptidase can act on [14C]tyrosinated tubulin bound to nitrocellulose, solubilizing radioactive compounds, suggesting the eventual applicability of this method to assay proteases in general. Other features and advantages of the assay as well as its limitations are discussed.
Buscar en Google
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Tubulina (Proteína)
/
Carboxipeptidasas
Tipo de estudio:
Diagnostic_studies
/
Evaluation_studies
Límite:
Animals
Idioma:
En
Revista:
Anal Biochem
Año:
2000
Tipo del documento:
Article
País de afiliación:
Argentina
Pais de publicación:
Estados Unidos