Branched-chain alpha-keto acid dehydrogenase complex from bovine kidney: radial distribution of mass determined from dark-field electron micrographs.
Scanning transmission electron microscopy (STEM) was used to determine the radial distribution of mass within the bovine kidney branched-chain alpha-keto acid dehydrogenase complex (E1-E2) and its core enzyme, dihydrolipoamide acyltransferase (E2). The particle mass of E2 measured by STEM is (1.19 +/- 0.02) x 10(6). Assuming 24 subunits per E2 core, this value corresponds to a subunit molecular weight of (4.96 +/- 0.08) x 10(4), which agrees well with the subunit molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 5.2 x 10(4) (Pettit et al., 1978) and that deduced from the gene sequence, 46,518 (Griffin et al., 1988). Thus, the STEM data reaffirms the 24-subunit model for this E2. Previous studies indicated that the E2 subunits contain an extended, outer lipoyl-bearing domain connected by a trypsin-sensitive segment to a compact, inner catalytic domain. The assemblage of 24 inner domains comprises a cubelike inner core. The quantity and spatial distribution of mass determined from STEM images for the E2 inner core are consistent with this model. The lipoyl-bearing domains are shown to occupy a zone defined by radii of 80-130 A over which the lipoyl moiety may range. This zone overlaps the positions of the 24 branched-chain alpha-keto acid dehydrogenase (E1) molecules, which apparently are located on the of the cubelike inner core.
Hackert
M L
ML
Clayton Foundation Biochemical Institute, University of Texas, Austin 78712.
Xu
W X
WX
Oliver
R M
RM
Wall
J S
JS
Hainfeld
J F
JF
Mullinax
T R
TR
Reed
L J
LJ
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000818:Animals; D002417:Cattle; D007658:Ketone Oxidoreductases / Q000302:isolation & purification; D007668:Kidney / Q000201:enzymology*; D046911:Macromolecular Substances; D008854:Microscopy, Electron / Q000379:methods; D008855:Microscopy, Electron, Scanning / Q000379:methods; D008961:Models, Structural; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification; D011487:Protein Conformation
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)