PURIFICATION AND CHARACTERIZATION OF PROTEASOME FROM OSTRICH LIVER

The proteasome (EC 3.4.99.46) is a high molecular mass (similar to 700 kDa) multisubunit enzyme complex which is the focus of worldwide rese arch in order to identify the structure, mechanism of action and speci ficity of the complex, The purpose of the present study was to investi gate the tryptic, chymotryptic and peptidylglutamyl-peptide hydrolysin g (PGPH) activities of ostrich liver proteasome. The proteasome was pu rified from ostrich Liver by employing ammonium sulphate fractionation , followed by three sequential chromatographic steps on Toyopearl Supe r Q-650 S, Sephadex G-150 and phenyl-Toyopearl columns, Temperature an d pH optima were examined and the effect of inhibitors, detergents, fa tty acids and cations on the peptidase activities was determined, Ostr ich proteasome exhibited a relative M-r of similar to 665 000 using no n-denaturing gradient PAGE and dissociated into the characteristic ''l adder'' associated with the proteasome subunits during SDS-PAGE, The p H optima for the peptidase activities mere found to be slightly alkali ne (tryptic activity) and neutral (chymotryptic-like and PGPH activiti es), Ostrich liver proteasome was found to be activated in terms of th e PGPH activity by fatty acids and SDS, whereas the chymotryptic and t ryptic-like activities were differentially inhibited, Ostrich proteaso me, in its inhibition by monovalent cations, was similar to the protea somes extracted from other sources. The specificity of the proteasome appears to be very broad, although it lacks aminopeptidase activity. T he yield compared favourably with similar extraction procedures which have been reported. On the basis of the physicochemical and kinetic pr operties which ostrich liver proteasome exhibited, it can be safely co ncluded that it corresponds well with the proteasomes isolated from ma ny other sources. (C) 1997 Elsevier Science Ltd.