Structural and functional studies on two mitochondrial proteins: The pyruvate dehydrogenase complex and the phosphate carrier

Phelps, Anne (1987) Structural and functional studies on two mitochondrial proteins: The pyruvate dehydrogenase complex and the phosphate carrier. PhD thesis, University of Glasgow.

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Abstract

(A) Binding of bovine heart pyruvate dehydrogenase complex (PDC) to the inner mitochondrial membrane. The interaction of bovine heart PDC with the inner mitochondrial membrane was investigated using a combined enzymological and immunological approach. Employment of high-titre monospecific antisera directed against intact PDC and its constituent subunits allowed the investigation of this association under conditions which inactivate the enzyme. Initial studies demonstrated that the bulk of PDC activity (70-80%) remains bound to the membrane while soluble control enzymes (citrate synthase and fumarase) are readily released into the supernatant fraction, with only minimal activity remaining membrane associated. Preferential association of PDC with the inner membrane was further investigated using the immune-replica technique. (B) Topographical and biosynthetic studies on phosphate transport protein (PTP). For these studies high-titre monospecific antiserum was produced against the purified phosphate transport protein from rat liver. This antiserum was shown to elicit a strong reaction against the parent antigen? i. e. the rat liver enzyme and also the corresponding protein from bovine heart when mitochondrial extracts were immunoblotted and challenged with anti-PTP-serum. The 34 000 Mr polypeptide was detected in crude extracts of the cell lines chosen for the biosynthetic studies on this protein i. e. Buffalo rat liver (BRL), rat kidney (NBL) and pig kidney (PK 15) cells. Initial studies to elucidate the mode of synthesis of PTP were carried out by labelling BRL and PK 15 cells overnight in the presence of [35s] -methionine. Immunoprecipitation with anti-PTP-serum and subsequent detection of radiolabelled polypeptides by fluorography failed to identify the parent antigen, although several other mitochondrial proteins and their precursors were detected in this manner. However, immunoblotting of a non-radiolabelled immunoprecipitate from bovine heart mitochondria successfully demonstrated the 34 000 Mr polypeptide. Topographical studies on the final membrane organisation of PTP were initiated by proteolytic digestion of bovine heart mitoplasts and sonicated particles. After resolving the protease-resistant membrane-bound polypeptides by SDS-PAGE, they were transferred to nitrocellulose and analysed by immunoblotting with anti-PTP-serum. It was found that PTP contains no protease-sensitive domains exposed at either the matrix or cytoplasmic surface of the inner mitochondrial membrane. Solubilisation of the protecting membrane by either Triton X-100 or SDS demonstrated that the protein was not inherently resistant to protease digestion while parallel incubations and subsequent analysis of the membranes using anti-SDH-serum provided an excellent control system for these studies. Results from these studies, native M determination and crosslinking data have allowed the presentation of a model for the molecular arrangement of PTP within the inner mitochondrial membrane of bovine heart.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry
Date of Award: 1987
Depositing User: Enlighten Team
Unique ID: glathesis:1987-76668
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 13:56
Last Modified: 19 Nov 2019 13:56
URI: http://theses.gla.ac.uk/id/eprint/76668

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