The work presented in this thesis involves the isolation and characterization chaperonin 60 (cpn60) proteins from Rhodobactar sphaaroidas and the cytosol of Pisum sativum. Non-denaturing-PAGE analysis of R. sphaaroidas cell-free extracts fractionated in sucrose density gradients revealed the presence of a Mr 670,000 protein that was considered to be a cpn60 candidate protein. This protein was purified by a combination of ion exchange and size exclusion chromatography. The Mr 670,000 multimer was shown to consist of a single polypeptide of Mr 58,000. Anti-sera raised against the Escharichia coli cpn60 (GroEL) and the pea plastid cpn60 did not cross-react with this protein. However, anti-sera raised against the R. sphaaroidas protein was shown to cross-react with the E coli GroEL protein. Subsequent N- terminal amino acid sequence analysis demonstrated that the R. sphaaroidas polypeptide was identical to GroEL at 15 of the 19 residues determined and showed significant similarity to other known cpn60 sequences. Like other chaperonins the native protein tended to dissociate in the presence of MgATP, in this case into what appears to be a dimeric form. Examination of the R. sphaaroidas protein in the electron microscope revealed 'top' views with seven-fold symmetry and rectangular 'side' views with four equally spaced, approximately equally dense, transverse striations. The protein appeared to be a cylinder of diameter 12 nm and height 9 nm. This structure is characteristic of the majority of the cpn60s examined so far. Rotational symmetry analysis revealed structural details extending beyond (20 A)-|. The image of the chromosome-encoded R. sphaaroidas chaperonin (Rs cpn60) differed from similarly generated images of plasmid-encoded GroEL. The possible significance of these differences is discussed. The effects of various environmental stresses on protein synthesis in R. sphaaroidas was investigated by pulse-labelling cells. Particular attention was paid to the expression of Rs cpn60. Overall the stress responses observed were similar to those reported for other prokaryotes. Of particular note are the quite different views of the stress responses presented by non-denaturing- and SDS-PAGE analyses. Attempts were made to determine the range of polypeptides that might be bound to Rs cpn60 before and after heat shock. These studies possibly indicate that there is a size constraint on polypeptides that interact with Rs cpn60 and that the flux of polypeptides through Rs cpn60 alters during heat shock. The second part of this thesis concerns attempts to identify a cytosolic chaperonin in P. sativum Until very recently no chaperonins had been identified in the cytosol of eukaryotes. Sequence analysis had raised the possibility that the TCP-1 protein from mice might be a cytosolic chaperonin (van der Vies, 1989). Monoclonal antibodies raised against the mouse TCP-1 protein cross-reacted with a Mr 57,000 polypeptide with a possible cytosolic location in P. sativum leaf extracts. Subsequent attempts to purify and determine the sub-cellular distribution of this polypeptide were unsuccessful. However, the sedimentation behaviour of the native protein indicated that the polypeptide was part of a multimeric complex of similar size to other chaperonins. The behaviour on ion exchange columns was also similar to other known chaperonins. The main obstacles to purification and localization of the native protein were: a) the monoclonal antibody used did not recognise the native protein and b) the multimeric complex appeared to dissociate during purification.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:333997 |
| Date | January 1992 |
| Creators | Watson, Gregory Michael Francis |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/110796/ |
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