Isolation and characterisation of a novel dihydrolipoamide dehydrogenase from pea chloroplasts

Conner, Mark Joseph Alexander (1997) Isolation and characterisation of a novel dihydrolipoamide dehydrogenase from pea chloroplasts. PhD thesis, University of Glasgow.

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Dihydrolipoamide dehydrogenase (E3) is a common component of the three ?-ketoacid dehydrogenase complexes oxidising pyruvate, 2-oxoglutarate, and the branched chain 2-oxoacids. It is also a component in the glycine cleavage system. E3 belongs to the family of pyridine nucleotide-disulphide oxidoreductases, catalysing electron transfer between pyridine nucleotides and disulphide compounds. It exists as a homodimer, with one flavin adenine dinucleotide (FAD) per subunit and a redox-active disulphide. There is increasing evidence for the existence of multiple isoforms of E3 in mitochondria from bacterial and plant sources. Moreover, preliminary immunological analyses of pea mitochondria and chloroplasts have provided the first evidence of organelle-specific isoforms of dihydrolipoamide dehydrogenase existing in plants. Utilising anion exchange chromatography, two distinct dihydrolipoamide dehydrogenases were isolated from a crade extract of pea leaves and eluted from the anion exchange column by differing concentrations of potassium phosphate. Isolation and purification of pea leaf chloroplasts and mitochondria revealed the location of the differing dihydrolipoamide dehydrogenases, with the major isoform located in mitochondria and the second, minor isoform located in the chloroplasts. The chloroplast-specific dihydrolipoamide dehydrogenase was found to be a homodimer, with an apparent subunit molecular mass of 52 kDa, 4 kDa smaller than the apparent mass of pea mitochondrial E3. This polypeptide was not recognised by antiserum raised to the pea leaf mitochondrial isoform and antiserum against the chloroplastic dihydrolipoamide dehydrogenase failed to cross-react with the pea mitochondrial E3. Further analysis revealed that the N-terminal sequence was distinct from and did not correspond to any part of the mitochondrial sequence. Kinetic studies demonstrated that the chloroplastic enzyme behaved as a conventional dihydrolipoamide dehydrogenase and contained a redox-active disulphide at its active site. However, the Km for NAD+ and DHL and the Ki for NADH of the chloroplastic isoenzyme was found to be significantly different from the mitochondrial E3. Moreover, the specific activity of the chloroplastic enzyme was found to be significantly lower than its mitochondrial counterpart. Structural differences were revealed when guanidine hydrochloride, sodium chloride and elevated temperatures were used as denaturing agents. The chloroplastic dihydrolipoamide dehydrogenase was found to be more resistant to loss of activity in the presence of these agents than the mitochondrial isoform. Chromatographic and immunological analyses were performed on leaf extracts of barley, spinach and oil seed rape and in each, an equivalent polypeptide was found to cross-react with antiseram raised to the pea chloroplastic isoenzyme. An attempt was made to clone the cDNA corresponding to the chloroplastic dihydrolipoamide dehydrogenase protein using the available N-terminal sequence. This, however, proved difficult resulting in the amplification of a sequence which displayed only limited similarity to the N-terminal sequence of the chloroplastic enzyme outside the primer sequences. The protein-biochemical data reveal the existence of a pea chloroplastic dihydrolipoamide dehydrogenase which is distinct from the mitochondrial isoform, and this enzyme may be the first component of a chloroplast-specific pyruvate dehydrogenase complex to be isolated and characterised to date.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Plant sciences.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Supervisor, not known
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-71322
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 25 Aug 2022 09:19
Thesis DOI: 10.5525/gla.thesis.71322

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