Tubulin carboxypeptidase assay based on the action of the enzyme on [14C]tyrosinated tubulin bound to nitrocellulose membrane.
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.
Sironi
J J
JJ
Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Universidad Nacional de Córdoba, Córdoba, 5000, Argentina.
Barra
H S
HS
Arce
C A
CA
D000818:Animals; D002250:Carbon Radioisotopes; D002268:Carboxypeptidases / Q000032:analysis* / Q000378:metabolism*; D002417:Cattle; D002847:Chromatography, Agarose / Q000379:methods; D003101:Collodion; D005069:Evaluation Studies as Topic; D066298:In Vitro Techniques; D008567:Membranes, Artificial; D010447:Peptide Hydrolases / Q000032:analysis; D051381:Rats; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000031:analogs & derivatives; D013379:Substrate Specificity; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry
D002250:Carbon Radioisotopes; D008567:Membranes, Artificial; D014404:Tubulin; C091883:phenyl-superose; D014443:Tyrosine; D003101:Collodion; D012685:Sepharose; D002268:Carboxypeptidases; D010447:Peptide Hydrolases; C023212:tyrosyltubulin carboxypeptidase