Inhibition of plant cell proteolytic activities that degrade tubulin

Abstract

The requirement for proteinase inhibitors during the chromatographic isolation of tubulin from cultured cells of rose ( Rosa sp. cv. Paul's scarlet) was examined by NadodecylSO 4-polyacrylamide gel electrophoresis, electron microscopy and immunoblotting. Tubulin fractions isolated in the absence of proteinase inhibitors showed substoichiometric ratios of α-subunit to β-subunit, and low molecular weight polypeptides, one (~32 Kd) of which coassembled with polymers. Electron microscopy revealed polymorphic structures, including C- and S-shaped ribbons and free protofilaments. Immunoblotting experiments with IgGs to the individual α- and β-subunits showed that some of the low molecular weight polypeptides were fragments of proteolytically degraded subunits. The use of low micromolar concentrations of the synthetic proteinase inhibitors leupeptin hemisulfate and pepstatin A protected tubulin from endogenous proteolytic activities during the isolation procedure and resulted in increased tubulin purity.