Figure 1

Purification of p48 The p48-containing fraction was purified as described in Materials and Methods. A, Q-Sepharose column chromatography: 35–80 % ammonium sulfate fraction was applied to a Q-Sepharose column (2.6 × 10 cm). Proteins were eluted with a linear gradient of NaCl and the 0.3–0.5 M eluate was collected in 10 ml fractions. B, SP-Sepharose column chromatography: p48-containing fractions from a Q-Sepharose column were pooled and applied to a SP-Sepharose column (1.6 × 10 cm). Proteins were eluted with a linear gradient of NaCl and the 0–0.25 M eluate was collected in 10 ml fractions. Fractions were assessed by immunoblotting using anti-Xenopus 20S proteasome. Protein bands of p48 and subunits of 20S proteasome are indicated by arrows and square brackets, respectively. Proteasome activity of the fractions toward a fluorogenic peptide substrate (Suc-Leu-Leu-Val-Tyr-MCA) was determined in the presence (●) and absence (○) of 0.05% SDS as described (21). Elution profile was monitored by absorbance at 280 nm (—).