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 KCl 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 KCl or deoxycholate reduces TRiC folding activity.
Norcum
M T
MT
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505, USA. mnorcum@fiona.umsmed.edu
D000595:Amino Acid Sequence; D000818:Animals; D018833:Chaperonins / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011817:Rabbits; D012156:Reticulocytes / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
D018833:Chaperonins