Rapid purification of bovine kidney branched‐chain 2‐oxoacid dehydrogenase complex containing endogenous kinase activity

Lawson, R. A. S.; Cook, Kenneth G.; Yeaman, Stephen J.

doi:10.1016/0014-5793(83)81115-6

Cited by 51 publications

(22 citation statements)

References 18 publications

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“…PDC and OGDC were purified from bovine heart as in refs. 25 and 26, whereas BCOADC was purified from bovine kidney (27). The E2 (+X) component of PDC and E2 of BCOADC were obtained by resolution of the respective complexes, using gel filtration on Superose 6 (Pharmacia) in the presence of 1 M NaCI (28), as was an E1-E2 subcomplex of OGDC.…”

Section: Methodsmentioning

confidence: 99%

Identification and analysis of the major M2 autoantigens in primary biliary cirrhosis.

Fussey

Guest

James

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

245

102

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Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial antibodies in the serum. It is possible that the PBC-specific immunoreactive trypsin-sensitive antigens on the inner mitochondrial membrane, termed M2, are important in the pathogenesis of this autoimmune disease. We have previously shown that a major M2"a" antigen is the E2 component of the pyruvate dehydrogenase multienzyme complex located within mitochondria. Analysis of the primary structure of the E2 components of all three 2-oxo acid dehydrogenase complexes reveals a high degree of homology with a similar highly segmented structure including lipoyl domains, E3-binding domains, C-terminal catalytic domains, and interdomain linker sequences. Immunoblotting of PBC patients' sera against purified E2 protein from 2-oxoglutarate dehydrogenase complex and branched-chain 2-oxo acid dehydrogenase complex reveals that these polypeptides are also autoantigens in this disease. Sera from 29 of 40 (72.5%) PBC patients gave a positive response against bovine 2-oxoglutarate dehydrogenase complex E2 and from 25 of 40 (62.5%) PBC patients gave a positive response against bovine branched-chain 2-oxo acid dehydrogenase complex E2. All 40 PBC patients (100%) have autoantibodies directed against at least one of the E2 components of the family of 2-oxo acid dehydrogenase complexes.

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Section: Methodsmentioning

confidence: 99%

Identification and analysis of the major M2 autoantigens in primary biliary cirrhosis.

Fussey

Guest

James

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

245

102

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“…Complex was phosphorylated as described in Lawson et al (1983) (Cook et al, 1984) to be present on the a-subunit (results not shown).…”

Section: Methodsmentioning

confidence: 99%

“…Branched-chain 2-oxo acid dehydrogenase was purified and assayed as described by Lawson et al (1983). Complex was phosphorylated as described in Lawson et al (1983) (Cook et al, 1984) to be present on the a-subunit (results not shown).…”

Section: Methodsmentioning

confidence: 99%

“…As with the pyruvate dehydrogenase complex, the E I component of the branched-chain 2-oxo acid dehydrogenase complex consists of two polypeptides, termed a and fi. The activity of the complex can be controlled by phosphorylation/dephosphorylation of the a-subunit of the El component by a kinase intrinsic to the complex (Fatania et al, 1981;Odessey, 1982;Lawson et al, 1983). The kinase phosphorylates two sites closely grouped on the asubunit, and the amino acid sequence surrounding these sites has been determined, inactivation correlating closely with phosphorylation of one site (Cook et al, 1984).…”

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confidence: 99%

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Resolution and reconstitution of bovine kidney branched-chain 2-oxo acid dehydrogenase complex

Cook¹,

Bradford²,

Yeaman³

1985

Biochemical Journal

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Branched-chain 2-oxo acid dehydrogenase complex was resolved into component E 1 and E2-kinase subcomplex by gel filtration in the presence of 1 M-NaCl. Essentially all the original activity of the complex can be regained after reconstitution of the component enzymes, reassociation being a rapid process. The specific activities of El and E2 were 25.1 and 19.0 units/mg respectively. Non-phosphorylated active El has an approx. 6-fold higher affinity for E2 than does phosphorylated El. The components of the branched-chain 2-oxo acid dehydrogenase complex do not crossreact with the respective components from the pyruvate dehydrogenase complex. The significance of these results and of the tight association of the kinase with E2 are discussed.Branched-chain 2-oxo acid dehydrogenase complex is a multienzyme complex catalysing the committed step in the breakdown of the essential amino acids leucine, isoleucine and valine. This complex is analogous to the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, and comprises an acyltransferase core (E2) around which are arranged the 2-oxo acid dehydrogenase (El) and dihydrolipoamide dehydrogenase (E3) components. In each complex the enzymes act in sequence to complete the oxidative decarboxylation of their respective substrates. As with the pyruvate dehydrogenase complex, the E I component of the branched-chain 2-oxo acid dehydrogenase complex consists of two polypeptides, termed a and fi. The activity of the complex can be controlled by phosphorylation/dephosphorylation of the a-subunit of the El component by a kinase intrinsic to the complex (Fatania et al., 1981;Odessey, 1982;Lawson et al., 1983). The kinase phosphorylates two sites closely grouped on the asubunit, and the amino acid sequence surrounding these sites has been determined, inactivation correlating closely with phosphorylation of one site (Cook et al., 1984). The amino acid sequence surrounding this site shows a distinct homology with Abbreviation used: SDS, sodium dodecyl sulphate.

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“…6) In the early 1980 s, several reports regarding BCKDC purification from various animal tissues were published. [7][8][9][10] In addition, nearly two decades ago, we too reported a novel procedure for the purification of BCKDC from frozen rat liver. 3,11) In those reports, we described a phenyl-Sepharose-based hydrophobic interaction chromatography method that yielded highly purified BCKDC.…”

mentioning

confidence: 99%

Modified Method for Purifying Rat Liver Branched-Chain α-Ketoacid Dehydrogenase Complex

Fujimura

Muramatsu

Akita

et al. 2009

Bioscience, Biotechnology, and Biochemistry

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Rapid purification of bovine kidney branched‐chain 2‐oxoacid dehydrogenase complex containing endogenous kinase activity

Cited by 51 publications

References 18 publications

Identification and analysis of the major M2 autoantigens in primary biliary cirrhosis.

Identification and analysis of the major M2 autoantigens in primary biliary cirrhosis.

Resolution and reconstitution of bovine kidney branched-chain 2-oxo acid dehydrogenase complex

Modified Method for Purifying Rat Liver Branched-Chain α-Ketoacid Dehydrogenase Complex

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