Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Mapping of the lipoyl-bearing domain by limited proteolysis.
1986; Elsevier BV; Volume: 261; Issue: 1 Linguagem: Inglês
10.1016/s0021-9258(17)42477-x
ISSN1083-351X
AutoresC W Hu, T A Griffin, Kim S. Lau, R. P. Cox, David Chuang,
Tópico(s)Muscle metabolism and nutrition
ResumoTo characterize the lipoyl-bearing domain of the dihydrolipoyl transacylase (Ez) component, purified branched-chain a-keto acid dehydrogenase complex from bovine liver was reductively acylated with [U-14C]a-ketoisovalerate in the presence of thiamin pyrophosphate and N-ethylmaleimide.Digestion of the modified complex with increasing concentrations of trypsin sequentially cleaved the E2 polypeptide chain (Mr = 52,000) into five radiolabeled lipoyl-containing fragments in the order of L1 (Mr = 28,000), LZ (Mr = = 14,000) as determined by the autoradiography of sodium dodecyl sulfate-polyacrylamide gel.In addition, a lipoate-free inner E2 core consisting of fragment A (Mr = 26,000) and fragment B (Mr = 22,000) was produced.Fragment A contains the active site for transacylation reaction and fragment B is the subunitbinding domain.Fragment L6 and fragment B were stable and resistant to further tryptic digestion.Mouse antiserum against E2 reacted only with fragments L1, Lz, and Ls, and did not bind fragments L4, Ls, A, and ' The abbreviations used are: El, branched-chain cu-keto acid decarboxylase; E*, dihydrolipoyl transacylase; E3, dihydrolipoyl dehydrogenase; TPP, thiamin pyrophosphate; TLCK, Ne-p-tosyl-L-lysine chlorornethyl ketone; SDS, sodium dodecyl sulfate.
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