FIGURE 1.

³⁵S-Labeled catalase tetramerizes in vitro. A, human catalase was synthesized in vitro in a rabbit reticulocyte lysate for 90 min at 30 °C in the absence or presence of 1 μm human PEX5, as indicated, and analyzed by native-PAGE/autoradiography. B, ³⁵S-labeled catalase was synthesized for 55 min. After adding cycloheximide, an aliquot was removed and frozen in liquid N₂ (lane 55′). The remainder of the reaction was then incubated at 30 °C, and aliquots were removed and frozen at the indicated time points. The samples were subjected to native-PAGE/autoradiography. The protein bands labeled with mCat and tCat correspond to the monomeric and tetrameric forms of catalase; the band labeled with an asterisk probably represents dimeric catalase (see text for details). C, catalase and a mutant version of it possessing two acidic amino acid residues at the C terminus (CatED) were synthesized in vitro for 55 min and supplemented with cycloheximide (lanes 1 and 2, respectively). Aliquots of each reaction were then combined and incubated for 4 h at 30 °C (lane 4) or incubated individually under the same conditions (lanes 3 and 5 for catalase and CatED, respectively), and subjected to native PAGE/autoradiography. Note that this gel was run for 2.5 h to improve separation of tetramers. Longer electrophoretic runs also result in more diffuse bands. The dots in lane 4 indicate the three expected heterotetramers. mCatED and tCatED indicate the monomeric and tetrameric forms of CatED, respectively.