Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.795801
Title: Immunology and biosynthesis of the mammalian pyruvate dehydrogenase complex
Author: Marcucci, Olga G. Lambertus de
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1985
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Abstract:
The mammalian pyruvate dehydrogenase complex, located in the inner membrane-matrix compartment of mitochondria, is a large multi-molecular aggregate, Mr 8.5 x 10e6, containing multiple copies of its three constituent enzymes, pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2) and lipoamide dehydrogenase (E3). High-titre, monospecific, polyclonal antibodies against the native complex and its individual components are characterised and employed to study the events involved in the biosynthesis and import of the components of this complex into mitochondria. Antisera are examined with respect to their ability to inhibit the overall activity of the complex and of the intrinsic protein kinase. Monospecificity of all antisera is demonstrated when challenged with crude cell extracts and subsequent immunoblotting analysis. The antiserum to native PDC exhibits high reactivity against all components of the complex, except E3, which cannot be explained by a low content of E3 in the purified antigen or by its inaccessibility to the immune system. The significance of the low immunogenicity of E3 is probably related to the conservation of its primary sequence and tertiary structure during evolution. A major observation from this study is that ox heart PDC contains an additional polypeptide, M 51,000+/-1,000 of unknown function. This protein is called component X and constitutes approx. 6% of the total complex. Detailed immunological studies suggest that component X is a normal cellular component, located in the mitochondrial compartment and does not represent a fragment of subunit E2 or the intrinsic protein kinase of the complex. Additional studies on the subcellular localisation of PDC suggest that this enzyme is associated with the inner membrane of rat liver and ox heart mitochondria. These and other studies also indicate that component X is an integral component of the complex and not a membrane protein, which becomes associated with the PDC during its isolation. The individuality of protein X is also demonstrated by comparison of the peptide maps of the 125 I and 14C-labelled subunits E2 and X obtained with several proteases. Protein X seems to be tightly-associated with the E2 core of the complex and is at least partially exposed on the surface of the native assembly, as it is accessible to proteases and to antibodies directed against it. Further studies reveal that, after incubation of the complex in the presence of [2-14C] pyruvate, 14C-label, probably in the form of acetyl groups is incorporated rapidly into both E2 and component X. Phosphorylation of the complex causes a parallel decrease in the acetylation of both proteins, indicating the involvement of the E1 component in the acetylation of these groups. Similarly, the transfer of acetyl groups from E2 and X onto CoA is observed to occur in a parallel fashion from both proteins. Studies on the effects of NEM on the incorporation of 14C-labelled acetyl groups also reveal interesting features of the acetylation reaction, which suggest the existence of secondary NEM-sensitive acetylation sites on the complex. Additional studies on the nature of the 'acetylatable' group in protein X and the physiological acceptor of acetyl groups are required to clarify the function of this component in the acetylation reactions of the complex. Studies on cultured bovine kidney, rat liver and pig kidney cells, 35 incubated with [35S] methionine in the presence of uncouplers of oxidative phosphorylation, demonstrate the accumulation of larger M precursor polypeptides to the subunits E2, E3, E1 alpha and E1 beta of the complex. Precursor forms for the individual subunits of the PDC are identified by immunoprecipitation techniques using antisera against the native complex and its SDS-denatured subunits followed by fluorographic analysis. These precursors, possessing M values 2,000-8,000 larger than their mature counterparts in the mitochondria, are relatively stable in the cytoplasm of the cells when monolayers are incubated in the presence of uncouplers for several hours. Removal of the uncoupler and subsequent chase shows that these precursors are processed into their mature forms with a similar lag time. Complete processing is observed within 30 min.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.795801  DOI: Not available
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