title: Folding and assembly studies on the components of mammalian PDC and OGDC
Creators: name: McCartney, Richard Graham
Publisher: name: University of Glasgow
Year: 1998
Subjects: subject: Molecular biology, biophysics.

description: Component enzyme purification and reconstitution studies have been conducted on the dihydrolipoamide acetyltransferase-protein X core subcomplex (E2/X) of bovine heart pyruvate dehydrogenase complex (PDC). Gel permeation chromatography, in the presence of IM NaCl, produced an E2/X core and E1/E3 fraction. Subsequent, anion exchange chromatography successfully separated the El and E3 components into homogeneous fractions. Electrospray mass spectrometry (ES/MS) determined the Mr- of bovine E2 and protein X. GdnHCl induced dissociation of the E2/X core disclosed that it was an ordered co-operative event involving formation of specific lower Mr intermediates corresponding to dihydrolipoamide acetyltransferase trimers and monomers. Modulation of refolding conditions allowed for the reassembly of native E2 cores devoid or partially depleted of the protein X component, as determined by immunological analysis. Reconstitution studies, with stoichiometric levels of the E1/E3 components, were unable to sustain overall complex activity with the E2 core devoid of protein X. In contrast 30-35% recovery of PDC activity could be obtained, under the same conditions, with the E2 core depleted of protein X, as compared to the native E2/X core assembly. Further reconstitution studies with excess (up to 100 fold) E3 were able, in both instances, to promote significant additional stimulation of PDC activity (25-30%). This effect was dependent on the source of E3 used and was optimal with parent bovine E3. These studies, using the refolded E2 cores, demonstrated the low affinity, but specific, binding of the dihydrolipoamide dehydrogenase (E3) component to sites on E2. This provided in vitro evidence to support the low levels of PDC activity observed in cell lines derived from patients who do not express protein X. A protocol has been devised which facilitated the dissociation of the 2- oxoglutarate decarboxylase (El) and E3 components from the 24meric dihydrolipoamide succinyltransferase (E2) core of the mammalian 2-oxoglutarate dehydrogenase complex (OGDC). By mixing the components in stoichiometric quantities, it was possible to reconstitute significant levels of OGDC activity. Gel permeation analysis of the E1/E3 fraction, performed under associative conditions, indicated the ability of the two homodimeric components to interact and form a stable subcomplex, comprising single copies of the two components. N-terminal sequence analysis identified SP-22, a thiol specific antioxidant protein, which appears to associate with the E3 component of mammalian PDC. Using specific primers, the polymerase chain reaction (PCR) was employed to obtain double stranded SP-22 DNA from bovine brain cDNA. Following purification, the DNA was cloned, in a blunt ended fashion, into the pCR-script vector. Both single and double restriction enzyme analyses were performed to screen the clones obtained for the presence of the appropriate inset. Subsequent in vitro transcription /translation analysis of the clones succeeded in identifying one capable of synthesising a protein product of the predicted Mr.