AFFINITY PURIFICATION AND CHARACTERIZATION OF PROTEIN GENE-PRODUCT-9.5 (PGP9.5) FROM RETINA

Protein gene product 9.5 (PGP9.5) is a cytosolic protein that is highl y expressed in vertebrate neurons, which is now included in the ubiqui tin C-terminal hydrolase subclass (UCH) on the basis of primary-struct ure homology and hydrolytic activity on the synthetic substrate ubiqui tin ethyl ester (UbOEt). Some UCHs show affinity for immobilized ubiqu itin, a property exploited to purify them. In this study we show that this property can also be applied to PGP9.5, since a protein has been purified to homogeneity from bovine retina by affinity chromatography on a ubiquitin-Sepharose column that can be identified with: (a) PCP9. 5 with respect to molecular mass, primary structure and immunological reactivity; (b) the known UCHs with respect to some catalytic properti es, such as hydrolytic activity on UbOEt, (which also characterizes PG P9.5), K-m value and reactivity with cysteine and histidine-specific r eagents. However, it differs with respect to other properties, e.g. in hibition by UbOEt and a wider pH range of activity.