[22] Tyrosine incorporation in tubulin

Publisher Summary This chapter illustrates tyrosine incorporation in tubulin. Tubulin tyrosinolation refers to the tRNA-independent addition of tyrosine to the C-terminal glutamate of its α chain. It was first characterized in Caputto's laboratory, as the fortuitous result of a control experiment carried out while studying developmental fluctuations in tRNAs from brain. The assay method for tubulin-tyrosine ligase is based on the rate at which labeled tyrosine is fixed into a trichloroacetic acid-insoluble form in the presence of purified microtubule protein. A unit of enzyme is the amount transferring 1 nmol of tyrosine to tubulin in 1 min, under the assay conditions. Specific activity is defined as units per milligram of protein. All purification procedures are carried out at 0-4°. The pH optima are observed at 8 and 6.5, and 8.5 μM ATP or 30 μM L-tyrosine give half maximal activity. Tubulin dimer appears to be the only substrate. The enzyme is homogeneous by SDS-PAGE, and the native molecular weight appears to be 37,000 by HPLC gel filtration.