Immunological and biosynthetic studies on two mitrochondrial dehydrogenases

Clarkson, George H. D (1987) Immunological and biosynthetic studies on two mitrochondrial dehydrogenases. PhD thesis, University of Glasgow.

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Monospecific, polyclonal antisera were raised to purified bovine heart succinate dehydrogenase (SDH), and to the individual large (Mr = 70000) and small (Mr = 27000) subunits of this enzyme. The specificities of the antisera were determined by immune blotting. These antisera exhibited cross-reactivity with the corresponding polypeptides in Buffalo rat liver (BRL), pig kidney (PK-15) and bovine kidney (NBL-1) cell lines, and were employed to investigate some of the events involved in the biogenesis of succinate dehydrogenase in the BRL and PK-15 cell lines. Newly-synthesised forms of the large and small subunits of SDH were detected in cultured PK-15 and BRL cells labelled with 35 [35S]methionine in the presence of uncouplers of oxidative phosphorylation. In both cell lines, the precursor forms of the large and small subunits exhibit M values approx. 1-2000 and 4-5000 greater than the corresponding mature forms. When the uncoupler is removed in pulse-chase experiments, complete conversion of the precursor to the mature forms occurs within 45 min. Studies on the kinetics of processing of the large subunit precursor revealed that reversal of precursor accumulation is rapid, with processing occurring with a half-time of 5-7.5 min. The accumulated precursor exhibits long term stability when PK-15 cells are maintained in the presence of DNP. The arrangement of the large and small subunits of succinate dehydrogenase on the mitochondrial inner membrane was investigated by immune blot analysis of protease-treated bovine heart mitochondria (right side-out; outer membrane removed) or submitochondrial particles (inside-out). Both subunits were found to be absent from the cytoplasmic surface of the inner membrane. The bulk of the small subunit appears to protrude into the matrix compartment, since this subunit is degraded during protease treatment of submitochondrial particles, without the appearance of a membrane-associated fragment. The data obtained in this study suggests that the large subunit may interact with the matrix side of the inner membrane via two distinct regions; these are detected as membrane-associated fragments of 32000 and 27000 after treatment of submitochondrial particles with papain or protease K. An analogous biosynthetic study was performed on the mammalian branched chain 2-oxoacid dehydrogenase (BCOAD) multienzyme complex, a high-Mr assembly composed of multiple copies of a branched chain 2-oxoacid dehydrogenase (El), dihydrolipoamide acyltransferase (E2) and lipoamide dehydrogenase (E3). This was achieved using antisera raised to (a) the purified complex from bovine kidney (anti-BCOAD serum); (b) the purified El subcomplex and (c) the E2 subunit isolated by preparative SDS/polyacrylamide gel electrophoresis. Both anti-BCOAD and anti-El sera exhibited a relatively low titre of antibodies to the E1 B polypeptide. An unexpected finding of this study was that the lipoamide dehydrogenase (E3) component was present in immunoprecipitates of the PK-15 BCOAD complex which were obtained using an antiserum which lacked antibodies against E3. This finding suggests that the absence of E3 from most preparations of purified BCOAD complex does not reflect the physiological situation but is a result of the isolation procedure. The precursor form of the BCOAD complex E1 subunit was detected in the pig kidney and bovine kidney cell lines. This species exhibited a value which was approx. 3000 greater than the mature-sized polypeptide. In contrast, the newly-synthesised E1B subunit evaded detection, largely due to the low immunogenicity of this polypeptide. The precursor form of the BCOAD complex E2 subunit was detected as a 56000-Mr species and was therefore similar in size to the previously identified pre-E3 polypeptide. However, the separate identity of the 56000 species and the E3 precursor was confirmed in immunocompetition experiments using unlabelled purified pig heart E3. The presequence of the BCOAD complex E2 precursor is markedly smaller than those of the E2 precursors of the analogous 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes, which both have values of approx. 8000.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry
Date of Award: 1987
Depositing User: Enlighten Team
Unique ID: glathesis:1987-76630
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 14:01
Last Modified: 19 Nov 2019 14:01

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