Novel isolation method and structural stability of a eukaryotic chaperonin: The TCP‐1 ring complex from rabbit reticulocytes

In the course of removing a contaminant from preparations of aminoacyl‐tRNA synthetase complexes, a novel purification method has been developed for the eukaryotic cytoplasmic chaperonin known as TRiC or CCT. This method uses only three steps: ammonium sulfate precipitation, pelleting into a sucrose cushion, and heparin‐agarose chromatography. As judged by electrophoresis, sedimentation, and electron microscopy, the preparations are homogeneous. The particle is identified as a chaperonin from electrophoretic polypeptide pattern, electron microscopic images, direct mass measurement by sedimentation velocity analysis, amino‐terminal sequencing, and ATP‐dependent refolding of rhodanese and actin. Further investigation of the biochemical and physical properties of the particle demonstrates that its constituent polypeptides are not glycosylated. The particle as a whole binds strongly to polyanionic matrices. Of particular note is that negatively stained images of chaperonin adsorbed to a single carbon layer are distinctly different from those where it is sandwiched between two layers. In the former, the “characteristic” ring and four‐stripe barrel predominate. In the latter, most images are round with a highly reticulated surface, the average particle diameter increases from 15 to 18 nm, and additional side, end, and substrate‐containing views are observed. The particle structure is strikingly resistant to physical forces (long‐term storage, repeated cycles of freezing and thawing, sedimentation), detergents (Triton, deoxycholate), salts (molar levels of KC1 or LiCl), and pH changes (9‐6). Only a strongly chaotropic salt (NaSCN) and extremely acidic conditions (pH 4.5) cause aggregation and dissociation of TRiC, respectively. However, treatment with KC1 or deoxycholate reduces TRiC folding activity.