PURIFICATION AND CHARACTERIZATION OF 20-S PROTEASOME FROM WHEAT LEAVES

The 20 S proteasome (multicatalytic proteinase) from wheat leaves was purified to apparent homogeneity by successive chromatographies steps. The molecular mass of the purified proteasome complex was estimated t o be 580 kDa by gel filtration. Polyacrylamide gel electrophoresis of the proteasome gave a single protein band under non-denaturing conditi ons and at least ten bands in the range 20-32 kDa in the presence of s odium dodecyl sulfate. Two dimensional electrophoresis revealed the pr esence of more than 20 polypeptides. By electron microscopy after nega tive staining with sodium phosphotungstate the proteasome preparation appeared as symmetrical ring-shaped particles. The enzyme exhibited ch ymotrypsin-like, trypsin-like and peptidyl-glutamyl-peptide hydrolase activities. In addition, the differential sensitivity of these activit ies to several compounds (aldehyde peptides, sodium dodecyl sulfate an d polylysine) strongly support the multicatalytic nature of this enzym e. The structural and biochemical data obtained indicated that the 20 S proteasome isolated from wheat leaves exhibits the canonical charact eristics of this conserved particle. (C) 1997 Elsevier Science Ireland Ltd.