FIGURE 1.

Purity and properties of purified hCtf4 and its derivatives. A, structure and domains of full-length (FL) hCtf4. See text for details. B, SDS-PAGE analysis of hCtf4 derivatives. Various hCtf4 derivatives (0.5 μg), isolated as described under “Experimental Procedures,” were subjected to SDS-10% PAGE followed by Coomassie staining. The specific proteins loaded (indicated above each lane) were as follows: lane 1, full-length hCtf4; lane 2, WD domain; lane 3, SepB domain; lane 4, HMG domain; lane 5, WD + SepB; lane 6, SepB + HMG domain. C, elution profile of hCtf4 from a Superose 6 column. hCtf4 (6 μg) was loaded onto a Superose 6 PC 3.2/30 column equilibrated with 25 mm Tris-HCl, pH 7.5, 10% glycerol, 0.2 m NaCl, and 1 mm EDTA. The column was developed at a rate of 30 μl/min at 4 °C and fractions (50 μl) collected. Aliquots (20 μl) were then subjected to SDS-PAGE and Coomassie staining. D, glycerol gradient sedimentation of hCtf4. SP-Sepharose-isolated hCtf4 (70 μg) was sedimented through a 5-ml 15–40% glycerol gradient for 18 h at 250,000 × g at 4 °C. Fractions were subjected to 10% SDS-PAGE and Coomassie staining. E, characterization of the hydrodynamic properties of hCtf4 and its derivatives. The hydrodynamic properties of each isolated protein were, including Stokes radii, s values, apparent molecular weight, and predicted molecular weight of hCtf4 derivatives. The results presented were derived as previously described (27).