The Enzymatic and Immunological Detection of the Plant 2-Oxoacid Dehydrogenase Complexes

Carmichael, Ailsa (1994) The Enzymatic and Immunological Detection of the Plant 2-Oxoacid Dehydrogenase Complexes. PhD thesis, University of Glasgow.

Abstract

Specific properties, including subcellular localisation, kinetic parameters and subunit composition, of the plant 2-oxoacid dehydrogenase complexes (pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chain 2-oxoacid dehydrogenase complexes) were investigated by applying enzymatic and immunological techniques. The intracellular distribution of this family of multienzyme complexes within pea root and leaf tissue confirmed the presence of a mitochondrial and plastid pyruvate dehydrogenase complex (PDC). The first extensive screening of plant organelles for the presence of PDCs sister complexes, 2-oxoglutarate dehydrogenase (OGDC) and branched-chain 2-oxoacid dehydrogenase (BCDC) complexes, was performed and indicated that these multienzyme complexes are confined to mitochondria. This distribution was based on enzymatic detection and addressed numerous potential limiting factors such as low in vivo concentrations of an active complex and the presence of NADH oxidases masking activity. The activities recorded for mitochondrial and plastid PDCs were consistent with previously reported values, OGDC activity was approx. 20% of mitochondrial PDC activity and BCDC-catalysed reaction displayed a very low reaction rate resulting from low concentrations of an active complex in the mitochondrial extracts. Initial velocity data for potato mitochondrial PDC and OGDC and pea chloroplast PDC-catalysed reactions were collected and the apparent Michaelis constants agreed with previously reported values. This family of multienzyme complexes were shown to be inhibited by the products of the reaction sequence, NADH and acyl CoA, displaying greatest sensitivity to the NAD+ : NADH ratio. The 2-oxoacid dehydrogenase- catalysed reactions have previously been shown to function by a ping-pong mechanism and the inhibition patterns observed in this investigation were consistent with such a mechanism; acyl CoA and NADH were competitive inhibitors versus their steady state precursors whilst product inhibition versus the 2-oxoacid substrate was uncompetitive. The availability of high-titre polyclonal antisera to the 2-oxoacid dehydrogenase complexes and to specific subunits isolated from bovine heart facilitated the immunological probing of plant mitochondrial and plastid extracts. Immuno-reactive plant polypeptides, which were shown to be associated with a high Mr aggregate i.e. a multienzyme complex, were detected and their Mr values compared to the analogous mammalian enzymes. Immunological investigations suggests that the E2 subunit of pea and potato mitochondria and pea chloroplast PDC carry one lipoyl domain paralleling the situation in the analogous yeast enzyme and contrasting the two lipoyl bearing domains which are carried by the mammalian subunit. Mr comparisons also indicate that the E2 subunit of plant OGDC and BCDC, like their mammalian and prokaryotic counterparts contain one lipoyl domain. No equivalent antigenic species were identified in chloroplast extracts supporting an exclusive mitochondrial location of OGDC and BCDC as indicated by enzymatic investigations. Immunological detection of the E3 subunit, a common enzyme to each 2-oxoacid dehydrogenase complex and the glycine decarboxylase complex (GDC), identified distinct mitochondrial and plastidic forms of the plant enzyme indicating that organelle-specific forms are expressed in the plant system. Separation of solubilised, heat-treated potato tuber mitochondrial protein by anion exchange chromatography resolved three distinct E3 activities. These activities appear to represent distinct forms of E3 arranged as alpha2 and beta2 homodimers and an alphabeta heterodimer (Mr, alpha, 59,000 and Mr beta, 56000) . Comparison of mitochondrial E3 profiles from photosynthetic and non-photosynthetic tissue suggest that the potential isoforms are differentially expressed in different tissues. Such observations indicated the three forms of the E3 display complex selectivity. Consequently, potato mitochondrial E3 isoforms may exhibit specificity at tissue, organelle and complex levels.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Gordon Lindsay
• Keywords: Biochemistry, Plant sciences
• Date of Award: 1994
• Depositing User: Enlighten Team
• Unique ID: glathesis:1994-75467
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 20:02
• Last Modified: 19 Nov 2019 20:02
• URI: https://theses.gla.ac.uk/id/eprint/75467