1. Eur. J. Biochem. 196,95-100 (1991)
0FEBS 1991
001429569100117H
Immunology, biosynthesis and in vivo assembly
of the branched-chain 2-oxoacid dehydrogenasecomplex from bovine kidney
George H. D. CLARKSON and J. Gordon LINDSAY
Department of Biochemistry, University of Glasgow, Scotland, UK
(Received January 29/August 30, 1990) - EJB 90 0088
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 E1j
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-labelledprecursors 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 55000, 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
assembledcomplex in vivo whereas no equivalent interaction of pre-E3 with its companion precursors occurs prior
to mitochondrial import.
The mitochondrial branched-chain 2-oxoacid dehydro-
genase complex (BCOADC) catalyses an irreversible step in
the oxidation of the branched-chain amino acids leucine, iso-
leucine 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 anal-
ogous pyruvate dehydrogenase (PDC) and 2-oxoglutarate de-
hydrogenase (OGDC) complexes, BCOADC can be isolated
as a functional high-Mrassemblycontaining multiple copiesof
its three major component enzymes; El, a substrate-specific,
thiamin-diphosphate-requiring 2-oxoacid dehydrogenase
containing two types of subunits, CI and p; a distinct dihydro-
Correspondence to J. G. Lindsay, Departmcnt of Biochemistry,
University of Glasgow, Glasgow, GI2 SQQ, Scotland
Ahbreviations. PDC, pyruvate dehydrogenase complex; OGDC.
2-oxoglutarate dehydrogenase complex; BCOADC, branched-chain
2-oxoacid dehydrogenase complex; FCCP, carbonyl cyanide p-
trifluoromethoxyphenylhydrazone.
Enzymes. Branched-chain 2-oxoacid dehydrogenase, El (EC
1.2.4.4); dihydrolipoamide acyltransferase, E2 (EC 2.3.1.12); dihy-
drolipoamide dehydrogenase, E3 (EC 1.8.1.4).
lipoamide acyltransferase (E2) containing covalently bound
lipoic acid and dihydrolipoamide dehydrogenase (E3), an
FAD-linked enzyme which is common to all three
multienzymecomplexes. BCOADC activity, like that of PDC,
is regulated in vivo by the phosphorylation state of the Elcr
subunit of its substrate-specific dehydrogenase [4]with phos-
phorylation causing inactivation. Covalent modification of
branched-chain El is mediated by a specific, tightly bound
kinase which is not fully characterised as yet [5]and a loosely
associated phosphatase [6].
BCOADC was purified originally from bovine kidney [7]
and found to consist of a 24-subunit octahedral E2 core struc-
ture (subunit M, 52000) to which was attached copies of the
El enzyme composed of two types of polypeptide termed Ela,
subunit M, 46000 and ElP, subunit M , 36000. Subsequently
BCOADC has been purified from bovine liver [8, 91, rabbit
liver [lo], rat kidney 1111 and Pseudomonas putidu [12]. The
preparations of bovine kidney BCOADC describedby Danner
et al. [8] and Heffelfinger et al. [9] are distinct in that they
contain endogenous E3.
Since the dozen polypeptides which are products of the
mitochondrial genome have now been identified [13], the con-
stituent proteins of BCOADC must be nuclear-coded and

2. 96
Fig. 1. Immune replica analysis of mammalian PDC, OGDC, BCOADC using anti-E3 serum. Samples of purified pig heart E3, bovine heart
PDC and OGDC and bovine kidney BCOADC wcrc electrophoresed on a 10% (niass/vol.) SDS/polyacrylamide slab gcl. One section of the
gel (A) was stained with Coomassie blue; the remaining section (B) was processed for detection of immunoreactive polypeptides using a 1 :50
dilution of anti-E3serum. (A) Lane 1, low-M, markers; lane 2, pig heart E3 (3 pg); lane 3, purified OGDC (10 pg); lane 4, purified BCOADC
(10 pg); lane 5, PDC (15 pg). (B) Lane 1, '251-labelledlow-M, markers; lane 2, pig heart E3 (0.5 pg); lane 3, OGDC (2 pg); lane 4, BCOADC
(2 pg); lane 5, BCOADC (15 pg); lane 6, PDC (2 pg)
targeted to the organelle from their site of synthesis on cyto-
plasmic 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 dehydroge-
nase. Moreover, there is evidence that the E2 precursors (pre-
E2) of OGDC and PDC contain extended presequences in the
M, range 6000-8000.
These earlier in vivo precursor studies have now received
definitive support from cloning and sequence analysis of sev-
eral genes for human, rat and bovine PDC and BCOADC
subunits. In particular, it has been established conclusively
that the E2genesof these two complexeshave leader sequences
approximately 60 amino acids in length [I6- 191.Correspond-
ing genesfor Ela, E1P and E3 polypeptides generally code for
typical (25-35 amino acid) N-terminal extension sequences,
although Ela of BCOADC also appears to be an exception
in this regard [24, 251.
Here we report on (a) immunological studies on the
HCOADC; (b) the detection, apparent M , values and stabili-
ties of the E2 and Ela and E3 precursors in differing cell lines
and (c) the association of E3 with the mature BCOADC in
vivo.
MATERIALS AND METHODS
NBL-1 (bovine kidney) and PK-15 (pig kidney) cell lines
were obtained from Flow Laboratories (Irvine, Scotland)
while Glasgow-modified minimal essential Eagle's medium
(G-MEM), either standard or methionine-free was purchased
from Gibco-BRL (Paisley, Scotland). Pansorbin, a 10%
(mass/vol.) suspension of Staphylococcusaureus cells (Cowan
1 strain), was the product of Calbiochem-Behring. L-[~~S]-
Methionine (> 1100Ci/mol), Na'251 (carrier-free) and N-
ethyl[2,3-'4C]maleimide (10.1 Ci/mol) were obtained from
Amersham International (Aylesbury, Bucks) and X-ray film,
X-Omat S or XAR 5 was obtained from Kodak (UK) Ltd.
Low-Mimarkers calibration kit was the product of
Pharmacia and nitrocellulose paper (0.45-pm pore size) was
purchased from Schleicher & Schiill (Dassel, FRG). Phenyl-
methanesulphonyl fluoride, p-aminobenzamidine . HCl, 2,4-
dinitrophenol and protein A were obtained from Sigma (GB)
Ltd. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone
(FCCP) was bought from Aldrich Chemical Co. Iodogen, was
the product of Pierce and Warriner, UK. All other reagents
were of the highest grades available commercially.
Purijkation of BCOADC and constituent enzymes
BCOADC complex was purified from bovine kidney by
the method of Lawson et al. [26].Dihydrolipoamide dehydro-
genase (E3) from pig heart was purchased from Calbiochem-
Behring. Purified El subcomplex was the generous gift of Dr
Stephen Yeaman (Department of Biochemistry, University of
Newcastle-on-Tyne).
Production oj antibodies
Antibodies to native BCOADC and purified El and E3
were raised using a previously described immunisation regime
[27].For raising a subunit-specific anti-E2 serum, preparative
SDS/polyacrylamide gel electrophoresis was employed to re-
solve milligram quantities of purified BCOADC. After stain-
ing with Coomassie blue, the band corresponding to E2 was
excised and employed as antigen as described in an earlier
paper [14].
Generalprocedures
Detailed methods for the maintenance and metabolic
labelling of cultured cells with [35S]methionine,SDS/poly-
acrylamide gel electrophoresis, immunoblotting and immuno-
precipitation have been reported previously [28] (and refer-
ences therein).
Fluorography was performed by the method of Chamber-
lain [29]. Protein concentrations were determined by a modi-
fied Lowry procedure using bovine serum albumin as standard
[30].1251-labelledprotein and low-M, markers were prepared

3. 97
Fig. 2. Reactivity of anti-BCOADC and anti-E2 sera with purified bovine kidney BCOADC complex. Varying amounts of purified BCOADC
were subjected to electrophoresis on a 12.5% (massivol.) SDS/polyacrylamide gel. One portion of the gel (A) was stained with Coomassie
blue. Polypeptides on duplicate portions of the gel were transrerred electrophoretically onto nitrocellulose paper for incubation with anti-
BCOADC serum (B) or anti-E2 serum (C). Immune complexes were detected by autoradiography following incubation with '251-labelled
protein A. (A) Lanes I and 6, low-M, markers; lanes 2- 5 contain 25, 10, 5 and 2.5 pg enzyme, respectively.(B, C) Lanes 1-4 contain 5, 2,
1 and 0.5 pg enzyme, respectively
as described in earlier publications [27, 311 with minor alter-
ations. Denatured BCOADC was modified with N-ethy1[2,3-
14C]inaleimideby the method of Hodgson et al. [32].
RESULTS
BCOADC was purified from bovine kidney cortex to a
specificactivity of 35-45 nkat/mg protein in good agreement
with previously reported values [26]. The purity of BCOADC
was estimated at 96% from densitometric scanning of the
Coomassie-blue-stained SDS/polyacrylamide gel (Fig. 1A,
lane 4). A comparison was made with the subunit profiles of
purified PDC (lane 5) and OGDC (lane 3) to obtain accurate
M , values for component polypeptides of BCOADC in the
Laemmli gel system. Purified BCOADC contains three major
polypeptides corresponding to the E2 (Mr 50000), Ela ( M ,
46000) and E1P (Mr37000) subunits. No band of associated
E3 (Mr 55000) is evident in the purified BCOADC prep-
aration. Thevirtual absence of E3 in this complex isconfirmed
by immune replica analysis of the three complexes with anti-
E3 serum (Fig. 1B). No E3 could be detected on probing
versus 2 pg BCOADC (lane 4) while a strongly positive
immunoblot was obtained with 1 pg OGDC (lane 3) or 2 pg
PDC (lane6).A small amount of intrinsic E3could be detected
on analysing 15 pg BCOADC (lane 5). Fig. 2 illustrates the
immunological properties of antisera raised to intact
BCOADC (B) and the purified E2 subunit (C) when tested
against varying amounts of purified complex (A). The low
antigenicity of E3 and the presence of only trace amounts in
this complex means that no observable titre to this component
is present in antiserum to intact BCOADC (Fig. 2B). Anti-
body was also raised to intact El (a generous gift from Dr
S.J. Yeaman) and with both these antisera only a very weak
antibody response was elicited against the Elfi subunit
(Fig. 2B). Subunit-specific antisera to E2 (Fig. 2C) and Ela
(not shown) were also prepared by excision of the individual
bands from SDS/polyacrylamide gels using protocols de-
scribedpreviously [14,151.In each case, the monospecificity of
the resulting antiserum was also checked by immunoblotting
against BCOADC or mitochondria1extracts of bovine kidney
Fig. 3. Immunoprecipitation of subunits of BCOADCfrom PK-I5 cells
labelled with (3 5S]methionine.PK-15 cells were incubated overnight
with ["S]methionine (150 pCi/dish). After preparation of radio-
labelled cellextracts, immunoprecipitations wereperformed with vari-
ous antisera in conjunction with formalinised S. uureus cells to absorb
immune complexes. The resulting immunoprecipitates were resolved
on a 10% (mass/vol.) SDS/polyacrylamide slab gel and visualised by
fluorography (see Materials and Methods for details).
Immunoprecipitates were obtained as follows: lane 1, with pre-im-
mune serum; lanes 2 and 3, with anti-El serum; lane 4,with anti-E2
serum; lanes 5 and 6, with anti-BCOADC serum; lanes 7 and 8, with
anti-E3 serum: lane 9, with anti-PDC Elcc serum;lane 10,N-ethy1[2,3-
''C]maleimide-modified BCOADC as marker. Lane 9 was included
as a positive control
(NBL-1) or pig kidney (PK-15) .cell lines employed in these
studies.
Several of these antisera were examined for their ability
to iminunoprecipitate mature subunits of BCOADC from
detergent extracts of PK-15 cells labelled for 4 h in the pres-
ence of [35S]methionine (Fig. 3). Pure N-ethy1[2,3-14C]-
maleimide-labelled BCOADC was run as a marker (lane 10)
and all radiolabelled polypeptides visualised by fluorography

4. Fig. 4. Ident@lfi'cntiono j the 55000-M, species present in immunopre-
ripitales of PK-15 cells using anti-BCOADC serum. Immunopreci-
pitations were performed on detergent extracts of PK-15 cells which
had been previously incubated overnight with [3sS]methionine.In
some cases, small amounts of the extract were mixed with 10 pg pig
heart E3 or heat-treated at 60°C for 5 min before addition of anti-
serum. Products of immunoprecipitationwere isolated using S.uureus
cells, rcsolvcd by electrophoresis on a 10% (massivol.) SDS/
polyacrylamide gel, and the radiolabelled polypeptides visualised by
fluorography (as described earlier). Lane 1, '2s1-labelled low-M,
markers; lane 2, N-ethy1[2,3-14C]maleimide-labelledBCOADC;lanes
3-8, immunoprecipitates obtained with the following antisera: lane
3, non-immune serum; lane 4, anti-BCOADCserum; lane 5, as lane
4 using heat-treated extract; lane 6 as 4, plus 10 pg E3; lanc 7, anti-
E3 serum; lane 8, as 7, plus 10 pg E3
after resolution on 10% (mass/vol.) SDS/polyacrylamide gels.
tmmunoprecipitates with anti-BCOADC serum (lanes 5 and
6) contain the E2 and Ela subunits and a third polypeptide,
M, 55000, i.e. with the mobility of E3 (lanes 7 and 8). Only a
very weak band representing E1P was observed, presumably
reflecting the low titre of antibody to this component observed
on immunoblotting. A similar response was also indicated
with antibody directed against native El of BCOADC (lanes
2 and 3) although, surprisingly, in addition to Ela, anti-El
serum also precipitated an unidentified polypeptide of M ,
60000. The presence of this component could not be detected
in El preparations evenin trace amountsasjudged by immune
replica analysis. Subunit-specific immunoprecipitations with
anti-E2 (BCOADC) serum (lane 4), anti-E3 serum (lanes 7
and 8) and anti-Ela (PDC) serum (lane 9) are also shown for
comparison.
The identification of the Mr-55000 species which co-pre-
cipitates with BCOADC was confirmed as E3 (Fig. 4, lane 6)
by immunocompetition analysis in which immunoprecipita-
tion of [3'S]methionine-labelled subunits was performed in
the presence of excess, non-radiolabelled pig heart E3. Heat
treatment of the detergent extracts of PK-15 cells at 60°C for
5 min failed to release E3 from the complex (lane 5). It should
be noted also that N-ethylmaleimide modification of the com-
plex (lane 2) for use as a radiolabelled marker caused signifi-
cant decreases in the mobility of all the subunits.
Accumulation of precursor forms of BCOADC was at-
tempted in NBL-1 (bovine kidney) cell cultures by incubation
of cells for 4 h with [35S]methionineand uncouplers of oxida-
tive phosphorylation, namely 2,4-dinitrophenol or carbonyl
cyanide p-trifluoromethoxyphenylhydrazone (FCCP)
(Fig. 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 celllineshave been determined
previously [14, 151. Unambiguous identification of the Elr
precursor (pre-Ela) is made in Fig. 5, lanes 11 and 13, in
which subunits were synthesized in the presence of 2 mM
dinitrophenol (lane 11) or 10 pM FCCP (lane 13) and
immunoprecipitated with anti-Ela specific serum. After a
45-min 'chase' on removal of uncoupler, conversion to the
mature form is readily observed (Fig. 5, lanes 12and 14). Pre-
Ela has an estimated subunit M , value of 49000 compared
to 46000 for the mature species. The unidentified 60000-M,
component is also present in these immunoprecipitates. As it
is not formed as a larger precursor, it is unlikely that this
polypeptide is of mitochondrial origin.
Surprisingly, a similar pattern of immunoprecipitated
products was found when these identical samples were probed
with anti-BCOADC serum. In both the uncoupler-treated
(Fig. 5, lanes 5 and 7) and 'chase' samples (lanes 6 and S),
again the only major antigen in the immune complexes is the
pre-Ela and Ela subunit. An important feature (lanes 6 and
8) is that after the 'chase' period the processed Ela subunit is
not assimilated into mature, intact complex as indicated by
the lack of E2 or associated E3. In contrast, immune
complexes with anti-BCOADC serum from NBL-1 cells,
labelled for 4 h or 24 h with [3sS]methioninein the absence of
uncoupler (lanes 3 and 4) reveal the expected pattern of bands,
i.e. E2 and Ela with a weak E1B component and also E3.
The inability to detect the precursor state of E2 in NBL-1
cells prompted us to attempt a similar set of experiments in
the PK-15 (pig kidney) cell line(Fig. 6). As shown in this figure
(lanes 4 and 5), immunoprecipitation of mature complex with
anti-BCOADC serum yields the E2, Ela and associated E3
polypeptides. The identity of E3 is again confirmed by
immunocompetition with excess unlabelled E3 (lane 5). In
the presence of dinitrophenol, three radiolabelled bands were
visualised with M , values 56500, 49000 and 46000 (lane 6).
The middle (49000-M,) species,with an M , value between the
mature E2 and Ela polypeptides, corresponded to pre-Ela
identified in the previous figure. The 56500-M, component,
synthesised in the presence of uncoupler, was of similar mo-
bility to mature E3 but was no longer competed out by the
presence of pure, non-radioactive lipoamide dehydrogenase
(lane 7). The possibility that this band now represents pre-E2
is confirmed in lanes 8 and 9 which show specific immuno-
precipitation of pre-E2, M,56 500 with subunit-specific anti-
body. Specific detection of pre-E3, 57000-M,, with anti-E3
specificserum, is also shown for comparison (lanes 9 and 10).
The identity of the 46000-M, species in immunoprecipitated
products from uncoupler-treated PK-15 cells is still unknown.
Its possible origins are considered in the next section.
In Table I an up-to-date summary is presented of the M ,
value of the precursor and mature states of the component
polypeptides of all three mammalian mitochondrial 2-oxoacid
dehydrogenase multienzyme complexes, including the sizes of
their respective extension sequences as estimated by SDS/
polyacrylamide gelelectrophoresis. These estimated values are
in close agreement with the predicted sizes of the human,
rat or bovine presequences determined directly from recently
published nucleotide sequence data [I6-231. BCOADC con-
forms to the general pattern, observed previously for PDC
and OGDC, in that pre-E2 contains an extended 'signal' se-
quencein the M , range 5000-7000.In contrast, the equivalent
E2 gene for PDC from S. cerevisiae contains a standard mito-
chondrial presequence of 28 amino acids in length [33].

5. 99
Fig. 5. Dnmunoprecipitation of BCOADC subunits.from NBL-I cells labelled with (35S]methionine in the presence or absence of uncouplers of
oxidative phasphovylation. Bovinc kidney (NBL-1) cells were incubated either overnight or for 4 h with [35S]methionine(150-200 pCi/dish)
in the presence or absence of uncouplers. In some cases, cells maintained for 4 h plus uncouplers were subsequently incubated for 45 min after
removal of uncoupler in fresh medium containing non-radioactive methionine. lmmunoprecipitates were isolated and analysed on 10% (mass/
vol.) SDS/polyacrylamide gels as described previously. Lane S, '251-labelled low-M, markers; lane 1, N-ethy1[2,3-'4C]maleimide-modified
BCOADC; lane 2, control immunoprecipitate with non-immune serum; immunoprecipitates obtained with either anti-BCOADC serum (3-
8) or anti-Elx serum (9-14) from the following extracts: lanes 3 and 9, overnight label; lanes 4 and 10, 4-h label; lanes 5 and 11, 4-h
label +2 mM dinitrophenol; lanes 6 and 12, as 5 and 11 with 45-min 'chase' period; lanes 7 and 13, 4-h label +10 FM FCCP; lanes 8 and
14, as 7 and I 3 with 45-min 'chase' period
Table 1. Subunit M, values of the mature and precursor states of the
constituent polypeptides of PDC, OGDC and BCOADC as estimated
by their mobilities on SDS/polyacrylamide gel electrophoresis
All values are the average of three separate determinations in bovine
or pig kidney cells by 1000 or less. In rat liver cells, these values
correspond to 68000 and 75000 f 1000 for the mature and precursor
forms, respectively; n. d., not determined
Fig. 6. Identification of the precursor states ofthe E2 and E3 subunits
of BCOADC in PK-I5 c~lls.lmmunoprecipitates were obtained from
[35S]methionine-labelledextracts of PK-15cells which had been pulse-
labelled (4 h) in the presence or absence of 2 mM dinitrophenol. In
some cases, antiserum addition was preccded by addition of purc,
unlabelled pig heart E3 (10 pg). Lane 1, low-M, markers; lane 2, N -
ethy1[2,3-'4C]maleimide-modifiedBCOADC; lane 3, control immu-
noprecipitates with non-immune serum; lanes 4- 7, immune
complexes obtained with anti-BCOADC serum from 4-h pulse (lane
4); lane 5, as 4, plus 10 pg E3; lane 6, 4-h pulse plus 2 mM
dinitrophenol; lane 7, as 6 plus 10 pg E3; lanes 8 and 9,
immunoprecipitates with anti-E2 serum from 4-h pulse plus 2 m M
dinitrophenol; lancs 10 and 11, immunoprecipitates with anti-E3
serum from 4-h pulse plus 2 mM dinitrophenol
DISCUSSION
In this paper, evidenceispresented on the followingpoints:
(a) the tight association of lipoamide dehydrogenase (E3) with
BCOADC in vivo, despite its absence from most preparations
of purified complex; (b) the detection of the E2, E3 and Elcl
precursors in cultured NBL-1 and PK-15 cells,confirming the
presence of a long 'signal' sequence on E2 in vivo, and (c)
evidence for markedly differing stabilities of the E2 precursor
in these two cell lines.
~ ~~
Polypeptide Apparent M , value of
mature form precursor form
PDC Elcl
PDC E1/3
OGDC El
BCOADC Elx
BCOADC EIP
PDC E2
OGDC E2
BCOADC E2
E3
PDC-component X
42000
36000
96000
46000
37000
70000
48000
50000
55000
51000
44500
39000
98000
49000
n.d.
77000
56000
56 500
57000
n.d.
Association of E3 with BCOADC
BCOADC preparations from several laboratories contain
variable amounts of lipoamide dehydrogenase with most con-
taining only traces of this enzyme (see Fig. I), hence requiring
addition of exogenous E3 for measurement of overall complex
activity. However, the co-precipitation of E3 with anti-
BCOADC serum from [3 SS]methionine-labelledcellular ex-
tracts suggests a tight interaction with the intact complex in
vivo (Figs 3 and 4).
Variability in the E3 content of BCOADC appears to
reflect the differing protocols employed in its preparation. In
particular, the inclusion of a hydroxyapatite chromatography
step seems to be responsible for removing the bulk of
dihydrolipoamide dehydrogenase activity.BCOADC is eluted
from hydroxyapatite by 0.35 M potassium phosphate whereas
E3 binds tightly to this matrix and is only eluted at much
higher salt concentrations. Thus E3 is stripped from the E2
core structure at this stage in the purification. As expected,
BCOADC containing high levels of E3 is obtained by em-

6. ploying purification schemes which do not include a
hydroxyapatide step [8, 91. Interestingly, we have shown pre-
viously that E3 appears to exhibit a variable affinity for PDC
in differing cell lines. In NBL-1 cells it co-precipitates with the
complex by virtue of its physical association with the core
assembly, whereas it is absent from immune complexes of
PDC from PK-15 cells [15].
REFERENCES
Precursor states and stability of BCOADC polypeptides
Accumulation of E2, E3 and Elcc precursors of this
multienzyme complex has been observed in these cell lines.
Failure to detect a higher-Mr form of the E1P subunit may
reflect the low titre of antibody to the mature polypeptide but
may also indicate a low methionine content or rapid turnover
of the precursor or mature states in vivo. There is considerable
variation in the stability of pre-E2 of BCOADC in PK-15 cells
and NBL-1 cells as significant pools of this precursor can be
accumulated only in the pig kidney cell line. This result is not
consistent with the presence of a conformationally distinct
state of the precursor in NBL-1 cells which is not recognized
by antibody to native E2, as is the case for the equivalent pre-
E2 of OGDC [14]. Consequently, it was not possible (a) to
observe the appearance of the mature form of E2 following
reversal of uncoupler action or (b) to follow the incorporation
of processed E l or E3 into newly assembled complex. In
contrast, pre-E2 is accumulated readily in PK-15 cells and,
despite its similar M , value to pre-E3, this precursor can
be assigned unambiguously from immunocompetition studies
and employment of subunit-specific anti-E2 serum. In any
case pre-E3 should not be precipitated with anti-BCOADC
serum since its presence in immune complexes of intact
BCOADC is a consequence of its high affinity for the as-
sembled complex. Earlier studies on PDC have provided evi-
dence against any strong interaction of pre-E3 with other
cytosolic precursors of this complex prior to their uptake and
assembly [15].
An unidentified 45000-Mr species was also observed in
immunoprecipitates of BCOADC precursors from PIC-15
cells. Its identity was not investigated further but it may rep-
resent actin which is often reported to be a contaminant of
immune complexes [34].
The precursor form of E2 of BCOADC exhibits an appar-
ent M , value 6000 41000 greater than mature E2, in agree-
ment with previous evidence from this laboratory that pre-E2
of PDC and OGDC also have long presequences. In the case
of BCOADC and PDC, direct confirmation of the presence
of extended N-terminal signal sequences [16-191 has been
achieved by cloning and sequencing of full-length copies of
the appropriate bovine and human dihydrolipoamide acyl-
transferase genes.
Since extended leader sequences are usually located only
on the dihydrolipoamide acyltransferase (E2) enzymes, it is
possible that they are involved in mediating the assembly of
their characteristic oligoineric core structure. However, in S.
cerevisiae the corresponding E2 gene of PDC contains only a
28-amino-acid N-terminal presequence. The availability of
cloned genes for mammalian E2 enzymes will permit a direct
investigation of possible secondary functions of these se-
quences in addition to mitochondria1 targeting.
G.H.D.C.wasthe recipient of apostgraduatestudentshipfunded
by the Science and Engineering Research Council (UKL to whom~I
J:G. L. is also grateful fnr contiued financialsupport. JLJ.
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