Mammalian lipoate acetyltransferase: molecular weight determination by gel filtration in the presence of guanidinium chloride

Kresze, Georg-B.; Dietl, Brigitte; Ronft, Hedwig

doi:10.1016/0014-5793(80)80124-4

Cited by 16 publications

(4 citation statements)

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“…An additional band of Mr 50000, migrating below the E3 component, has been consistently found in our purified preparations of the complex when the electrophoretic analysis is performed with the Laemmli buffer system. The presence of this protein in preparations of the pyruvate dehydrogenase complex has been noted previously by Stanley & Perham (1980) and Kresze et al (1980 Fig. 1.…”

Section: Resultssupporting

confidence: 77%

“…E2 consists of a single type of polypeptide chain whose M, has been estimated by means of SDS/polyacrylamide-gel electrophoresis to be between 70000 and 74000 (Barrera et al, 1972;Hamada et al, 1975;Machicao & Wieland, 1980). This estimation is at variance with the value of 52000 reported by Barrera et al (1972) on the basis of electron microscopy, symmetry considerations, sedimentation-equilibrium analysis and gel filtration in 6M-guanidinium chloride (Kresze et al, 1980). Enzyme E3 is composed of two identical subunits.…”

contrasting

confidence: 59%

“…Each one contains a molecule of FAD and has an Mr value of 55000 [for reviews see and Wieland (1983)]. An additional component of Mr 50000-52000 has been observed by SDS/polyacrylamide-gel electrophoresis of the purified pyruvate dehydrogenase complex in the Laemmli system (Stanley & Perham, 1980;Kresze & Steber, 1979;Kresze et al, 1980). The subunit composition of the 2-oxoglutarate dehydrogenase complex from ox heart has also been established (Koike & Koike, 1976).…”

mentioning

confidence: 99%

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Low immunogenicity of the common lipoamide dehydrogenase subunit (E3) of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes

Marcucci¹,

Hunter²,

Lindsay³

1985

Biochemical Journal

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The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart is described. The specificity of these antisera and their precise reactivities with the individual components of the complexes were examined by immunoblotting techniques. All the subunits of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes were strongly antigenic, with the exception of the common lipoamide dehydrogenase component (E3). The titre of antibodies raised against E3 was, in both cases, less than 2% of that of the other subunits. Specific immunoprecipitation of the dissociated N-[3H]ethylmaleimide-labelled enzymes also revealed that E3 alone was absent from the final immune complexes. Strong cross-reactivity with the enzyme present in rat liver (BRL) and ox kidney (NBL-1) cell lines was observed when the antibody against ox heart pyruvate dehydrogenase was utilized to challenge crude subcellular extracts. The immunoblotting patterns again lacked the lipoamide dehydrogenase band, also revealing differences in the apparent Mr of the lipoate acetyltransferase subunit (E2) from ox kidney and rat liver. The additional 50 000-Mr polypeptide, previously found to be associated with the pyruvate dehydrogenase complex, was apparently not a proteolytic fragment of E2 or E3, since it could be detected as a normal component in boiled sodium dodecyl sulphate extracts of whole cells. The low immunogenicity of the lipoamide dehydrogenase polypeptide may be attributed to a high degree of conservation of its primary sequence and hence tertiary structure during evolution.

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Section: Resultssupporting

confidence: 77%

contrasting

confidence: 59%

mentioning

confidence: 99%

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Low immunogenicity of the common lipoamide dehydrogenase subunit (E3) of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes

Marcucci¹,

Hunter²,

Lindsay³

1985

Biochemical Journal

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“…In this study we demonstrate that kidney pyruvate dehydrogenase complex is disassembled when treated with papain due to limited proteolysis of E2, and that nicked E2 as well as intact E l and E3 can be isolated readily from papain-treated enzyme complex. Parts of our results have been presented [19].…”

Section: 2)mentioning

confidence: 99%

Bovine Kidney Pyruvate Dehydrogenase Complex

Kresze

Ronft

Dietl

1980

European Journal of Biochemistry

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Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated rapidly by papain. However, none of the component activities of the complex is destroyed during inactivation of the overall reaction. The core component, lipoate acetyltransferase, is cleaved by papain to give principal fragments with Mr 26500 and 26000 (as determined by dodecylsulfate gel electrophoresis). Much more slowly, the α chain of the pyruvate dehydrogenase component is attacked. Fragmented lipoate acetyltransferase retains its complete enzymatic activity and remains of high molecular weight. It is unable, however, to bind the other component enzymes, pyruvate dehydrogenase and lipoamide dehydrogenase. Therefore, the multienzyme complex is disassembled when treated with papain. A method is described which allows the rapid and convenient isolation of nicked lipoate acetyltransferase as well as unfragmented pyruvate dehydrogenase and lipoamide dehydrogenase from papain‐treated complex under very mild conditions. The two uncleaved component enzymes have identical properties and similar specific activities as enzyme preparations obtained by other, more laborious procedures.

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Dihydrolipoamide S-acetyltransferase

Springer Handbook of Enzymes

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Mammalian lipoate acetyltransferase: molecular weight determination by gel filtration in the presence of guanidinium chloride

Cited by 16 publications

References 17 publications

Low immunogenicity of the common lipoamide dehydrogenase subunit (E3) of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes

Low immunogenicity of the common lipoamide dehydrogenase subunit (E3) of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes

Bovine Kidney Pyruvate Dehydrogenase Complex

Dihydrolipoamide S-acetyltransferase

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