Immunological and biosynthetic studies on the mammalian 2‐oxoglutarate dehydrogenase multienzyme complex

Hunter, Anne; Lindsay, J. Gordon

doi:10.1111/j.1432-1033.1986.tb09464.x

Cited by 41 publications

(29 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…5). The requirement for a membrane potential for uptake (hence processing) of cytosolic precursors of mitochondrial polypeptides is well established and optimal conditions for detection of precursors in these cell lines have been determined previously [14,151. Unambiguous identification of the E l r precursor (pre-Ela) is made in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For raising a subunit-specific anti-E2 serum, preparative SDS/polyacrylamide gel electrophoresis was employed to resolve milligram quantities of purified BCOADC. After staining with Coomassie blue, the band corresponding to E2 was excised and employed as antigen as described in an earlier paper [14].…”

Section: Production Oj Antibodiesmentioning

confidence: 99%

“…targeted to the organelle from their site of synthesis on cytoplasmic ribosomes. Earlier studies in this laboratory [14,151 have revealed that all the individual polypeptides of PDC and OGDC are synthesised initially as higher M , precursors including the Ela and Elfl subunits of pyruvate dehydrogenase. Moreover, there is evidence that the E2 precursors (pre-E2) of OGDC and PDC contain extended presequences in the M, range 6000 -8000.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Immunology, biosynthesis and in vivo assembly of the branched‐chain 2‐oxoacid dehydrogenase complex from bovine kidney

Clarkson

Lindsay

1991

European Journal of Biochemistry

View full text Add to dashboard Cite

Specific, polyclonal antisera have been raised to the native branched-chain 2-oxoacid dehydrogenase complex (BCOADC) from bovine kidney and each of its three constituent enzymes: El, the substrate-specific 2-oxoacid dehydrogenase; E2, the multimeric dihydrolipoamide acyltransferase 'core' enzyme and E3, dihydrolipoamide dehydrogenase. Purified BCOADC, isolated by selective poly(ethyleneglyco1) precipitation and hydroxyapatite chromatography, contains only traces of endogenous E3 as detected by a requirement for this enzyme in assaying overall complex activity and by immunoblotting criteria. A weak antibody response was elicited by the E 1 j subunit relative to the E2 and Elcr polypeptides employing either purified El or BCOADC as antigens.Anti-BCOADC serum showed no cross-reaction with high levels of pig heart E3 indicating the absence of antibody directed against this component. However, immunoprecipitates of mature BCOADC from detergent extracts of NBL-1 (bovine kidney) or PK-15 (porcine kidney) cell lines incubated for 3-4 h in the presence of [35S]methionine contained an additional 55000-Mr species which was identified as E3 on the basis of immunocompetition studies.Accumulation of newly synthesised [35S]methionine-labelled precursors for E2, Ela and E3 was achieved by incubation of PK-15 cells for 4 h in the presence of uncouplers of oxidative phosphorylation. Pre-E2 exhibited an apparent M , value of 56500, pre-Ela, 49000 and pre-E3, 57000 compared to subunit M , values of 50000,46000 and 55 000, respectively, for the mature polypeptides. Thus, like the equivalent lipoate acyltransferases of the mammalian pyruvate 'dehydrogenase (PDC) and 2-oxoglutarate dehydrogenase (OGDC) complexes, pre-E2 of BCOADC characteristically contains an extended presequence.In NBL-1 cells, pre-E2 was found to be unstable since no cytoplasmic pool of this precursor could be detected; moreover, processed Ela was not assembled into intact BCOADC as evidenced by the absence of E2 or E3 in immunoprecipitates with anti-(BCOADC) serum after a 45-min 'chase' period in the absence of uncoupler. Dihydrolipoamide dehydrogenase (E3), in its precursor state, was not present in immune complexes with anti-(BCOADC) serum, indicating that its co-precipitation with mature complex is by virtue of its high affinity for assembled complex in vivo whereas no equivalent interaction of pre-E3 with its companion precursors occurs prior to mitochondrial import.The mitochondrial branched-chain 2-oxoacid dehydrogenase complex (BCOADC) catalyses an irreversible step in the oxidation of the branched-chain amino acids leucine, isoleucine and valine (see [I, 21 for reviews). It may also be involved in the catabolism of threonine and methionine since 2-oxobutyrate and 4-methylthio-2-oxobutyrate can also act as substrates for this complex [3j.In common with the structurally and functionally analogous pyruvate dehydrogenase (PDC) and 2-oxoglutarate dehydrogenase (OGDC) complexes, BCOADC can be isolated as a functional high-Mr assembly containing multiple copies o...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Production Oj Antibodiesmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Immunology, biosynthesis and in vivo assembly of the branched‐chain 2‐oxoacid dehydrogenase complex from bovine kidney

Clarkson

Lindsay

1991

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Anti-Elk antisera were raised against sodium dodecyl sulfate (SDS) gel-purified Elk protein that was resolved from KGDHC purified from bovine kidney (Lai et al, 1988). Anti-KGDHC, -E2k, and -E3 antisera were prepared as described previously (Hunter and Lindsay, 1986). The specificities of these antisera were described previously .…”

Section: Antiseramentioning

confidence: 99%

Immunochemical Characterization of the Deficiency of the α‐Ketoglutarate Dehydrogenase Complex in Thiamine‐Deficient Rat Brain

Sheu

Calingasan

Lindsay

et al. 1998

Journal of Neurochemistry

View full text Add to dashboard Cite

Abstract:Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. The metabolic encephalopathy caused by thiamine deficiency (TD) is a classic example in which an impairment of cerebral oxidative metabolism leads to selective cell death. In experimental TD in rodents, a reduction in the activity of the thiamine diphosphate-dependent, mitochondrial enzyme a-ketoglutarate dehydrogenase complex (KGDHC) occurs before the onset of pathologic lesions and is among the earliest biochemical deficits found. To understand the molecular basis and the significance of the deficiency of KGDHC in TD-induced brain damage, the enzyme activity and protein levels of KGDHC were analyzed. The effect of TD on the subregional/cellular distribution of KGDHC and the anatomic relation of KGDHC with selective cell death were also tested by immunocytochemistry. Consistent with several previous studies, TD dramatically reduced KGDHC activity in both anatomically damaged (thalamus and inferior colliculus) and spared (cerebral cortex) regions. Immunocytochemistry revealed no apparent correlation of regional KGDHC immunoreactivity or its response to TD with affected regions in TD. The basis of the enzymatic and immunocytochemical behavior of KGDHC was further assessed by quantitative immunoblots, using antibodies specific for each of the three KGDHC components. Despite the marked decrease of KGDHC activity in TD, no reduction of any of the three KGDHC protein levels was found. Thus, TD impairs the efficacy of the KGDHC catalytic machinery, whereas the concentration of protein molecules persists. The generalized decline of KGDHC activity with no apparent anatomic selectivity is consistent with the notion that the compromised mitochondrial oxidation sensitizes the brain cells to various other insults that precipitate the cell death. The current TD model provides a relevant experimental system to understand the molecular basis of many neurodegenerative conditions in which mitochondrial dysfunction and KGDHC deficiency are prominent features. Key Words: Alzheimer's disease-et-Ketoglutarate dehydrogenase-2-Oxoglutarate dehydrogenaseOxidative stress-Thiamine deficiency-Thiamine diphosphate-dependent enzymes-Wernicke-Korsakoff's syndrome.

show abstract

“…[3,14,15]. 14C-labelled peptides were detected by fluorography following the method of Chamberlain [16].…”

Section: Introductionmentioning

confidence: 99%

Component X of mammalian pyruvate dehydrogenase complex: Structural and functional relationship to the lipoate acetyltransferase (E2) component

et al. 1989

View full text Add to dashboard Cite

The lipoate acetyltransferase (E2, Mr 70 000) and protein X (Mr 51 000) subunits of the bovine pyruvate dehydrogenase multienzyme complex (PDC) core assembly are antigenically distinct polypeptides. However comparison of the N-terminal amino acid sequence of the E2 and X polypeptides reveals significant homology between the two components. Selective tryptic release of the 14C-labelled acetylated lipoyl domains of E2 and protein X from native PDC generates stable, radiolabelled 34 and 15 kDa fragments, respectively. Thus, in contrast to E2 which contains two tandemly-arranged lipoyl domains, protein X appears to contain only a single lipoyl domain located at its N-terminus.Pyruvate dehydrogenase complex; Component X; Lipoyl domain; Homology; Component E2; (Bovine heart)

show abstract

Immunological and biosynthetic studies on the mammalian 2‐oxoglutarate dehydrogenase multienzyme complex

Cited by 41 publications

References 17 publications

Immunology, biosynthesis and in vivo assembly of the branched‐chain 2‐oxoacid dehydrogenase complex from bovine kidney

Immunology, biosynthesis and in vivo assembly of the branched‐chain 2‐oxoacid dehydrogenase complex from bovine kidney

Immunochemical Characterization of the Deficiency of the α‐Ketoglutarate Dehydrogenase Complex in Thiamine‐Deficient Rat Brain

Component X of mammalian pyruvate dehydrogenase complex: Structural and functional relationship to the lipoate acetyltransferase (E2) component

Contact Info

Product

Resources

About