山陽学園短期大学紀要

論第40巻１-８（2009）

文アフリカツメガエル Xenopus laevis
分岐鎖α- ケト酸脱水素酵素複合体のデカルボキシラーゼ成分の性質
Properties of the Decarboxylase Component of Xenopus laevis
Branched-chain α -Keto Acid Dehydrogenase Complex

小

野

和

夫

Kazuo Ono
キーワード：アフリカツメガエル、ミトコンドリア、分岐鎖α - ケト酸脱水素酵素複合体、デカルボキ
シラーゼ、サブユニット
Keywords : X enopus laevis, mitochondria, branched-chain α-keto acid dehydrogenase complex,
decarboxylase component, subunit
SUMMARY
Branched-chain α-keto acid dehydrogenase complex（ BCKADC）
contains decarboxylase（ E1）,
dihydrolipoyl transacylase, and dihydrolipoyl dehydrogenase as catalytic components. To characterize
BCKADC ofamphibian, mitochondrial extract of Xenopus laevis liver was subjected toimmunoblotting analysis using rat anti E1 antibodies. As a result, three immunoreactive proteins
were detected; the molecular mass of proteins was estimated to be 36, 37 and 46 kDa. Incubation ofliver mitochondria from Xenopus laevis prior to solubilization of E1 resulted in no significant change
in their appearance and amount. These proteins were also detected in heart of Xenopus laevis as wellas liver of the amphibian. Sucrose density gradient centrifugation under the condition, which brings
dissociation of BCKADC to each component enzyme, revealed that the three proteins of Xenopus
laevis moved together through the gradient. The density that three proteins settled corresponded
well with that of rat E1 did. Present results obtained here showed that E1 of Xenopus laevis contains
three proteins at least, and the 47 kDa protein corresponds to E1α subunit in comparison withmolecular mass of E1α of other species.

INTRODUCTION
Branched-chain α-keto acid dehydrogenase complex（BCKADC）
functions as a rate limiting enzyme
of branched-chain amino acids such as leucine, isoleucine and valine1,2,3,4）. Mammalian BCKADC isconstituted by three catalytic components of decarboxylase（E1）, dihydrolipoylacyl-transferase（E2）
山陽学園短期大学

食物栄養学科：Department of Food and Nutrition , Sanyo Gakuen College--山陽学園短期大学紀要

第40巻（2009）

4.5,6）and dihydrolipoamide dehydrogenase（ E3）. The multi enzyme complex is composed of 12

molecules of E1, 6

molecules of E3 and E2 core, which consists of 24-mer of E2 molecules. The E1

and E3 are bound noncovalently to the E2 core5,6,7）. The E2 subunit of mammals contains threestructural domains, a lipoyl- bearing domain in the amino-terminal portion, an E1/E3-binding domain
and an inner-core domain in the carboxyterminal portion, linked by two interdomain segments
6,7）（linkers）. In addition, mammalian BCKADH contains regulatory enzymes, E1 specific kinase andphosphorylated E1 phosphatase3,8,9）.
BCKADC is also found in non-mammalian vertebrates such as avian 10）and fish11,12,13）. It had been
already reported that the similarity in the primary structure of chicken E1α and E1β to human are
93.3 % and 95.9 % , respectively10）. E1 from these species worked interchangeably with rat E1 orbovine E1 as decarboxylase component of BCKADC10,13）.
We had also reported that E2 subunits of chicken and rainbow trout are comparable to mammalian
E2 in regard to the primary structure and function10,13）. However additional residues in the linkers of
E2 leading to reduced E1 binding capacity and reduced activity of the overall reaction catalyzed bychicken and rainbow trout BCKADC 10,13）. As to E3, its amino acid sequence and function areconserved very well among animal species14,15）
These studies suggest that both structure and functions of E1, E2 and E3 are conserved wellamong animal species. On the other hand, we had detected unexpectedly three proteins byimmunoblotting with mitochondrial extract of an amphibian, Xenopus laevis, liver using anti rat E1
IgG. E1 from mammals, chicken and rainbow trout consist of α and β subunit10,13）in tetramer of
α2β216,17）.
In this study, the relationship between E1 molecule and three antigens to the anti rat E1 wasinvestigated to assign three proteins to exact subunit（s）of E1 of Xenopus laevis. Interaction between
these three proteins and E2 core was also examined using sucrose density gradient centrifugation.

MATERIALS AND METHODS
Materials
Male Xenopus laevies was obtained from a domestic farm. Aprotinin and leupeptin were from
Boehringer（ Mannheim, Germany）and Peptide institute（ Osaka, Japan）, respectively. All otherchemicals were the highest grade commercially available. Rat E1, E2, E3, and chicken E2 wereprepared as described previously10, 18）. Polyclonal anti-rat E1 and rat E2 antibodies were raised in
rabbits with purified rat E1 and rat E2, respectively, as described19）.
Preparation of mitochondria and mitochondrial extract
Xenopus laevis liver mitochondria were prepared basically according to the method of Roche andCate20）. The mitochondria were suspended in a minimum volume of 20 mM potassium phosphatebuffer（KPB）（pH6.5）containing 2µg/ml of leupeptin, 2µg/ml of aprotinin, 0.1 mM phenylmethylsulfonyl
fluoride and 0.1 mM benzamidine. The suspension was frozen in an Erlenmeyer flask in dry ice/
isopropanol bath and stored at –80ºC until use. The frozen mitochondria were thawed in cold runningwater, diluted three times the original volume with 20 mM KPB（pH 6.5）
and homogenized with amotor-driven Potter-Elvehjem homogenizer. The suspension was made up in 50 mM NaCl, 0.1 mM--小野和夫：アフリカツメガエル Xenopus laevis 分岐鎖α - ケト酸脱水素酵素複合体のデカルボキシラーゼ成分の性質

phenylmethylsulfonyl fluoride, 0.5 mM benzamidine, 1µg/ml leupeptin and 1µg/ml aprotinin, followedby centrifugation at 32,000 g for 30 min at 4ºC. The supernatant was diluted with 20 mM KPB（pH7.5）to make a concentration of 9 mg protein per ml, and made up in 0.2 mM thiamine pyrophosphate（TPP）, 1%（w/v）bovine serum albumin（BSA）and 10 mM MgCl2. The solution was brought to pH 7.5
by adding 10 M KOH drop by drop with stirring.
SDS-PAGE and immunobloting
SDS-PAGE was performed according to Laemmli21）with 10% SDS-polyacrylamide gel, and proteins
were electro blotted onto polyvinylidene difluoride membrane（Nihon Millipore Ltd, Yonezawa, Japan）.
Western blot analysis was performed as described by Harlow and Lane22）, using goat anti-rabbit IgG
conjugated alkaline phosphatase（ZYMED, San Francisco, CA, USA）.
Sucrose density gradient centrifugation
The mitochondrial extract was incubated for 30minutes at 4℃ in the presence of 1M NaCl followed
by layering onto a ultracentrifugation tube（1.5 cm in diameter x 9.6 cm long）
containing 11.5 ml of alinear sucrose density gradient from 0 to 20%（w/v）
in KPB（pH7.5）
containing 5mM DTT and 1M
NaCl10,18）. The centrifugation was performed at 110,000 g in RPS40T rotor by Hitachi SCP 85H
centrifuge for 17 hours. After the centrifugation, the gradient was divided into 23 fractions in equalvolume（0.5ml）from the top. E1 in each fraction was detected by immunoblotting. E2 in the extractfrom rat liver mitochondria was detected by anti-rat E2 IgG．
Protein determination
The amount of protein was measured bybicinchoninic acid method 23）, using BCA
Protein Assay Reagent Kit（PIERCE, Rockford,
IL, USA）with BSA as a standard.

RESULTS
Immunoblotting of BCKADC E1 ofmitochondria from Xenopus laevis
liver
To evaluate whether the primary structureof Xenopus laevis E1 is distinguishedimmunologically from other vertebrates,
immunoblotting of the E1 was conductedusing anti rat E1 IgG. For comparison of thecomposition and molecular mass of E1 subunit,
liver mitochondria extracts of Xenopus laevis,
rat, chicken and rainbow trout were subjectedto the analysis. The result was shown in Fig.
1. Except Xenopus laevis, clearly two

Fig.1. Immunoblotting analysis of E1 component of
Xenopus Laevis.
Mitochondrial extract of liver from Xenopus laevis was
prepared as described in the section of “Materials amd
Methods. The extract was electrophorased on 10%
polyacrylamide gel, followed by electro-transferring topolyvinylidene difluoride membranes. The resolved
subunits were probed with anti-rat E1 antibodies. Bound
antibodies were detected with alkaline phosphataseconjugated goat anti-rabbit IgG. Lanes of R, C and T are
purified rat BCKADH（2 µg）, paritial purified chiken
BCKADH（14 µg）and purified rainbow trout BCKADH（3.6µg）, respectively. Lanes of XL1, XL1/2 and XL1/4 are 125,62.5 and 31.3 µg protein of mitochondrial extract fromXenopus laevis liver, respectively.

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第40巻（2009）

immunoreactive bands of 46 kDa and 36 kDa were detected.
In the mitochondrial extract of liver from Xenopus laevis, one more immunoreactive smear band was
detected in addition of 46 kDa and 36 kDa
proteins（Fig. 1）. The molecular mass of theprotein on the blot was estimated to beapproximately 37kDa by comparison ofrelative mobility of the α（46 kDa）and β（36kDa）subunits of rat E14,10,13）.
Effect of the incubation of Xenopus
laevis mitochondria on the pattern of
immunoblotting
E1 molecule has been reported so far
consists of α and β subunits in α2β216,17）. If
it is true of Xenopus laevis, it is uncertain how
three proteins with molecular mass of 36, 37
and 46 kDa correspond to the α and β
subunits of Xenopus laevis E1. One possible
explanation is that proteolytic cleavage of
E1α of 46 kDa by mitochondrial protease24）
produced 37 kDa protein.

Therefore, toascertain whether endogenous mitochondrialFig.2. Effect of incubation of mitochondria from liver of
Xenopus laevis on immunoblotting pattern with anti rat E1
antibodies.
Mitochondria from Xenopus laevis liver were incubatedat 25℃ for 2hours. For immunoblotting analysis, an aliquotsof mitochondria were taken at intervals as indicated, andthen the sample was solubilized in the presence of thefollowing protease inhibitors: 0.1mM PMSF, 0.1mM
benzamidine, 0.3mM aprotinin, and leupeptin 1mg/ml.
Immunoblotting of the extract conducted as described in“Materials and Methods” .

protease action involves or not, mitochondria
of liver from Xenopus laevis were incubated at
25℃ for 2 hours. The result indicates clearly
that the incubation caused no significant
change of the apparent molecular mass andquantitative ratio of the three immunoreactive
proteins as shown in Fig. 2.
Immunoblotting analysis of kidneyand heart of Xenopus laevis
Relatively high BCKADC activity was foundin ki dn ey a n d he ar t o f s ev e ra l an im a lspecies4,10,13）. Thus, immunoblotting analysis
was also conducted using kidney and heart of
Xenopus laevis to confirm the profile ofimmunoblot obtained with liver is applicableto these organs. As to kidney and heart ofrat, two major immunoreactive proteins withmolecular mass of 46 kDa and 36 kDa weredetected by anti rat E1 IgG（Fig. 3）. In case

Fig.3. Immunoblotting analysis of E1 component of
BCKADC from kidney and heart from Xenopus laevis.
Homogenate from liver, heart and kidney（250 µg protein
e a c h ）w e r e s u b j e c t e d t o S D S - P A G E w i t h 1 0 %polyacrylamide gel, followed by electro-transferring topolyvinylidene difluoride membranes. The resolved subunits
were probed with anti-rat E1 antibodies. Bound antibodies
were detected with alkaline phosphatase-conjugated goatanti-rabbit IgG as described in “Materials and Methods”.
H , K and L indicate heart , kidney and liver , respectively.

--小野和夫：アフリカツメガエル Xenopus laevis 分岐鎖α - ケト酸脱水素酵素複合体のデカルボキシラーゼ成分の性質

of heart from Xenopus laevis, it contained three immnoreactive proteins of 36, 37 and 46 kDa as wellas liver. Kidney showed very weak signal corresponding to 37 kDa and 46 kDa. These observation
indicate that there are at last three proteins, which are recognized as antigens to rat E1 antibodies, inliver and heart from Xenopus laevis .
Sucrose density gradient centrifugation of mitochondrial extract of liver fromXenopus laevis
It is still unclear which of the three immunoreactive proteins function as E1 subunit（s）
of E1?
Sucrose density gradient centrifugation was carried out to answer this query. E1 component of
BCKADH binds noncovalently to E1 binding domain on E2 molecule through E1β subunit 25）,
accordingly if the three proteins, which were detected by anti-rat E1 IgG, correspond to subunits of
E1 of Xenopus laevis, the immunoreactive proteins should move together through the gradient
independent on individual molecular mass under the experimental condition. Sucrose density gradientcentrifugation was conducted after incubation of the extract of liver mitochondria with 1M NaCl for
dissociation of BCKADC18）. Molecular mass of Rat E1 and E2 core are estimated to be 160 kDa and
2,000 kDa based on composition and molecular mass of subunits, respectively4）. As expected from theestimation, rat E1 was detected in fractions containing lower concentration of sucrose than that offractions E2 moved（Fig. 4）. Under the same experimental conditions, the three immunoreactive
proteins of liver of Xenopus laevis were detected in the fractions corresponding to the fractions inwhich rat E1α and E1β were detected（Fig. 4）.

Fig.4. Sucrose density gradient centrifugation under the condition of resolution of E1 from E2 core.
Mitochondrial extract（3 mg of protein）from liver of Xenopus laevis was incubated at 4 ℃ for 30
min with 1M NaCl. The sample was subjected to sucrose gradient centrifugation, followed byfractionation in order from the top, as described in Materials and Methods. The numbers in eachpanel correspond to the fraction number.

--山陽学園短期大学紀要

第40巻（2009）DISCUSSION
Three proteins with molecular mass of 36, 37 and 46 kDa were detected by immunoblotting of
mitochondrial extract of Xenopus laevis liver using rat anti E1 antibodies（Fig. 1）. The relative
amount of three proteins were not influenced by incubation of mitochondria at 25℃ for 2 hours before
extraction from mitochondria（Fig. 2）. These results demonstrate that the appearance of threeproteins on the blot was not caused by proteolysis of intact E1 subunit during solubilization frommitochondria. This thought is also supported by the result of which three proteins were detected inheart of Xenopus laevis（Fig. 3）.
Sucrose density gradient centrifugation showed that there was no significant difference on thesedimentation profile among three proteins of 36, 37 and 46 kDa in the presence of 1M NaCl
（Fig. 4）.
Moreover the ratios in amount of 37 kDa to 46 kDa and 36 kDa to 46 kDa were constant through theabove mentioned fractions（Fig. 4）. It is noteworthy that fractions to which these proteins moved
correspond to fractions rat E1 moved. These results suggest that all the imunoreactive proteins of
36, 37, 46 kDa in Xenopus laevis behaved together, not individually. During the centrifugation, eachspecies of macromolecule in the original solution moves through the gradient at a rate that is mainly
dictated by its mass or size10,18）. Since molecular mass of rat E1 was estimated to be 160 kDa
approximately4,10）, the above observation may provide a possible explanation of three proteinsassociating each other, and constituting E1 molecule of Xenopus laevis.
Taking into consideration obtained results from present study, it is concluded that Xenopus laevis
has three proteins, which react with anti rat E1 IgG. Based on the limited information on primarystructure of E1 of nonmammalian vertebrates 10,11,13）, it can be speculated that the protein withmolecular mass of 46 kDa is almost likely to be E1α subunit. As to E1β subunit, it is hard to
distinguish between 36 and 37 kDa proteins except the difference in the molecular mass. Which ofproteins correspond to functional E1β subunit? To clarify these queriess, cDNA cloning of E1
subunit（s）of Xenopus laevis is essential.

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