THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 249, No. 12, Issue of June 25, pp. 3336-3842, 1974

Printed in U.S.A.

Properties and Subunit Composition of the Pig Heart Z-Oxoglutarate Dehydrogenase”

(Received for publication, November 21, 1973)

KICHIKO KOIKE,~ MINORU HAMADA, NOBUYUKI TANAKA, KIN-ICHI OTSUKA, KYOKO OGASAHARA, AND

MASAHIKO KOIKE

From the Department of Pathological Biochemistry, Atomic Disease Institute, Nagasaki University School of Medicine, Salcamoto-cho, Nagasaki-shi, 852, Japan

SUMMARY

The 2-oxoglutarate dehydrogenase, one of the component enzymes of the 2-oxoglutarate dehydrogenase complex, has been highly purified. The enzyme has a sedimentation coefficient (s&~) of 10.3 S and diffusion coefficient (&o,~) of 3.92 X lo-’ cm2 s-l. The weight average molecular weight was estimated to be about 216,000 from the sedimentation equilibrium data. The content of right-handed LY helix in the enzyme molecule was estimated to be about 34% by both optical rotatory dispersion and circular dichroism. The en- zyme was found to contain 1 molecule of protein-bound thiamine-PP per mole; some other kinetic and protein chemi- cal properties are also reported.

In 6 M guanidine hydrochloride (pH 8.0), the enzyme was dissociated into its subunits and the molecular weight of sub- unit was estimated to be 97,000 from the sedimentation equi- librium data. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate the enzyme was dissociated into its subunits with an estimated molecular weight of 113,000. The results indicate that 1 molecule of 2-oxoglutarate dehy- drogenase consists of two similar subunits, which contains NH&erminal alanine.

The multi-enzyme complex which catalyzes a coenzyme A- and nicotinamide adenine dinucleotide-linked and lipoic acid- mediated oxidative decarboxylation of 2-oxoglutarate (Equation 1) in pig heart muscle has been isolated and highly purified.

2-Oxoglutarate + CoA-SH + N.4D’ -+ Succinyl-S-CoA + CO, + NADH + H+ (1)

The isolated complex has a molecular weight of 2.7 million (1,

* This investigation was supported in part by grants from the Ministry of Education of Japan, the Vitamin B Research Com- mittee,-and the U.S. Army -Research and Development Group (Far East). This is the ninth naner of the mammalian a-keto acid dehydrogenase complexes series.

$ Part of this work is taken from a thesis to be submitted by Kichiko Koike to Nagasaki University Postgraduate School of Medicine, in partial fulfillment of the requirements for the degree of Doctor of Medical Science.

2). The 2-oxoglutarate dehydrogenase complex has been fur-

ther separated into three component enzymes, 2-oxoglutarate

dehydrogenase, lipoate succinylt.ransferase, and lipoamide de- hydrogenase, and reconstituted from these enzymes (2).

The 2-oxoglutarate dehydrogenase (Et) is an essential com- ponent of the multi-enzyme complex, and apparently catalyzes a decarboxylation of 2-oxoglutarate (Equation 2) and the sub- sequent succinylation of the lipoyl moiety (Equation 3) which is bound covalently to the lipoate succinyltransferase (&) (3).

2-Oxoglutarate + [TPP]l-E, + [succinic semialdehyde-TPP]-E1+CO, (2)

[Succinic semialdehyde-TPP]-El + [Lips,]-& ---t [succinyl-S-Lip-SH]-& + [TPP]-El (3)

This paper reports the enzymic, physicochemical, and chemical properties of 2-oxoglutarate dehydrogenase, and the subunit composition of the enzyme.

EXPERIMENTAL PROCEDURE

Materials

2-Oxonlutaric acid was obtained from Kyowa Hakko Kogyo, Tokyo. -Takadiastase was a gift from Doctbrs G. Sunagawaand Y. Yusa, Sankyo Company, Tokyo. Permutit (50 to 80 mesh) for thiamine assay was obtained from the Japan Vitamin Society. B-Galactosidase, phosphorylase a, catalase, &albumin, aldolase, chymotrypsinogen, an> cytochrome c were obtained from Boehrin- eer Mannheim. Mannheim: bovine serum albumin and diisopropyl- iuorophosphate were obtained from Sigma, St. Louis; and &r- bonic anhydrase was from Worthington Biochemical Corporation, Freehold, N. J. Lipoamide dehydrogenase was prepared from the 2-oxoglutarate dehydrogenase complex as described by Tanaka et al. (2). The sources of all other chemicals are as described previously (l-3).

Methods

Protein was determined by the phenol method (4) with bovine serum albumin as the standard. All other methods were carried out as described in previous papers (l-3).

Enzyme Assays-A ferricyanide-linked assay of 2-oxoglutarate dehydrogenase, based on Equation 4, was carried out at 25” spec- trophotometrically as described by Massey (5).

2-Oxoglutarate + 2 Fe(CN)8 + H20 + Succinate + CO2 + 2 Fe(CN)G’- + 2 H+ (4)

The reaction mixture (2 ml) contained 1.34 rmoles of potassium ferricyanide, 100 pmoles of potassium phosphate buffer (pH 6.5),

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0.1 rmole of thiamine-PP, 1 rmole of calcium chloride, 6.7 rmoles of potassium 2-oxoglutarate (pH 6.5), and 1.34 mg of bovine serum albumin and enzyme. Reaction was begun by the addition of 2-oxoglutarate (6.7 rmoles/O.l ml) and followed at 420 run by a recording spectrophotometer. Ferricyanide-linked 2-oxogluta- rate dehydrogenase activity was also determined by the modified method (6) of Hager (7) for pyruvate dehydrogenase, using potas- sium 2-oxoglutarate instead of potassium pyruvate. Specific activity is expressed as one-half of the total micromoles of ferro- cyanide formed per hour per mg of protein. Lipoate succinyl- transferase and lipoamide dehydrogenase assays were carried out as described in previous papers (l-3).

Ultracentrifugal Anal~/ses-All sedimentation experiments were performed in a Beckman model E analytical ultracentrifuge eQUlDDed with the RTIC unit and schlieren and Ravleieh inter- - ** ference optical systems. Photographic plates we;e measured with a Mann type 829D comparator and a Nikon model 6CT2 microcomparator. Sedimentat’ ion coefficients were corrected to standard condition and extrapolated to infinite dilution as de- scribed by Schachman (8). Diffusion coefficient measurements were performed at 5227 rpm and 20” in an An-J rotor with a capil- lary type synthetic boundary cell, as described by Chervenka (9). The molecular weights of the native enzyme and its subunit were determined by the meniscus depletion sedimentation equilibrium method of Yphantis (10). Equilibrium runs of the native enzyme were carried out at 12,590 rpm and 20” in an An-D rotor with 12.mm double sector cells equipped with either a six-channel Kel-F or an aluminum-filled Epon centerpiece, sapphire windows, and interference masks. For determination of the subunit molecular weight, the protein samples previously dialyzed against deionized and redistilled water were lyophilized, dissolved in 6 M guanidine HCl (pH 8.0) containing 0.1 M 2-mercaptoethanol, and allowed to stand at 4” for 24 to 48 hours. Equilibrium runs were carried out at 21,740 rpm and 20” in an An-D rotor with a 12-mm double sector cell with a relatively long solution column (4 mm). Weight average molecular weights were represented as the average of those obtained from each of three black fringes. The partial specific volume (8) of the native enzyme was calculated from the amino acid composition (8). This partial specific volume for the native enzyme and a reduced value in guanidine (11) were used for the calculation of the subunit molecular weight. The value of density of aqueous solution of 6 M guanidine HCl containing 0.1 M 2-mercaptoethanol was taken from the data of Kawahara and Tanford (12).

Gel Electrophoresis-Acrylamide-agarose composite disc gel electrophoresis was performed with 2% polyacrylamide and 1% agarose as described by Peacock and Dingman (13).

Polyacrylamide gel electrophoresis in sodium dodecyl sulfate was performed by the method of Weber and Osborn (14) in 570 polyacrylamide gels, and used for monitoring stages of the enzyme purification and for determining subunit molecular weight. All protein samples were reduced and alkylated with iodoacetic acid before subjection to gel electrophoresis. About 0.1 mg of protein sample was dialyzed against deionized and redistilled water at 4” for 24 hours, transferred into a small test tube, and lyophilized. The dried sample was dissolved in 0.1 ml of 0.1 M Tris-HCl buffer (pH 8.5) containing 6 M guanidine HCl and 0.1 M 2-mercapto- ethanol, followed by addition of 1 ~1 of 0.1 M diisopropylfluoro- phosphate in I-propanol to avoid proteolysis (15). After 24-hour standing at room temperature under nitrogen, the protein was carboxymethylated essentially as described by Gibbons and Perham (16), dialyzed against deionized and redistilled water, and lyophilized. Carboxymethylated protein samples were either incubated at 37” for 2 hours or were heated at 100” for 5 min in 0.01 M sodium phosphate buffer (pH 7.0) containing 1% sodium dodecyl sulfate and 1% 2-mercaptoethanol. Similar results were obtained by both procedures. The gels were stained with 0.25% Coomassie brilliant blue for 40 min at room temperature and

are expressed in terms of mean residue rotation, [m’], and circular dichroism results in terms of molecular ellipticity, [e]. Both parameters have units of degrees.cmz per dmole. The mean residue weight in all calculations is taken to be 112, which was calculated from amino acid composition. The content of right- handed 01 helix was calculated from both [m’]233 as described by Blout et al. (19) and [f?]224 by Holzwarth and Doty (20).

Thiamirke-PP DeterminaliorL-Thiamine-PP content was deter- mined by the method of Kaziro (21) with yeast pyruvate decarbox- ylase (2-oxo-acid carboxylase, EC 4.1.1.1)) which was prepared by the modified procedure (22) of Green et al. (23). Thiamine-PP was also assayed spectrofluorometrically by the thiochrome method of Fujiwara and Matsui (24) after conversion to free thiamine by takadiastase.

Thiol Determination-The content of thiol groups was deter- mined spectrophotometrically with 5,5’-dithiobis(2-nitrobenzoic acid) in the presence of 6 M guanidine HCl in 0.04 M potassium phosphate buffer (pH 8.0) (25), and with p-chloromercuribenzoate in the absence (26) and presence (27) of 8 M urea in 0.05 M potassium phosphate bulfer (pH 7.0).

Ami,~o Acid Analyses-Amino acid analyses were performed by the method of Spackman et al. (28). Lyophilized protein (1 to 2 mg) was hydrolyzed with 1 ml of 5.9 N constant boiling HCl in uacuo at 110” for 24 to 72 hours (29), and the hydrolysate was analyzed on a Beckman model 116 amino acid analyzer. Serine and threonine contents were calculated by extrapolation to zero time. Half-cystine was determined as cysteic acid after performic acid oxidation (30). Tryptophan and tyrosine were estimated spectrophotometrically by the method of Goodwin and Morton (31).

NHz-terminal Group Analyses-NH*-terminal groups were determined by the method of Edman as modified by Eriksson and Sjoquist (32). Phenylthiohydantoins, separated by thin layer chromatography on Silica Gel Ft5a plates (from E. Merck) devel- oped in chloroform. were scraoed off and eluted with 95% ethanol. The amount of phenylthiohydantoin was estimated. from the absorption of the extract at 269 nm. NH*-terminal groups were also identified by the dansylr method of Hartley (33) on a poly- amide sheet.

Tryptic Digestion and Peptide Mapping-Peptide mapping of tryptic digest was performed on Whatman No. 3MM filter paper as described by Katz et al. (34). Performic acid-oxidized protein was digested for 10 hours at 37” with trypsin (substrate to enzyme ratio of 5O:l) in 0.2 M ammonium bicarbonate (pH 8.3). The peptides (2 to 2.5 mg) were separated by a combination of paper chromatography and electrophoresis. Chromatography was performedwi‘th”l-butanol-pyridine-acetic acid-water-(15:10:3:12) followed bv electroohoresis in nvridine-acetic acid-water (1~10: 289)) pH 3.6 at 3000’volts for l& “min. The air-dried papers were stained wit,h 0.257, ninhydrin in acetone. Arginine-containing peptides were detected on a separate paper with the modified Sakaguchi spray as described by Irreverre (35).

Preparation of 2.Oxoglutarate Dehydroge,nase-Pig heart 2-0x0- glutarate dehydrogenase, which was isolated from the 2-0x0- glutarate dehydrogenase complex as previously reported (2), was further purified by ammonium sulfate fractionation between 0.23 and 0.36 saturation. The purified enzyme had a specific activity of about 440 in the ferricyanide-linked 2-oxoglutarate dehydro- genase assay, and did not show any lipoate succinyltransferase or lipoamide dehydrogenase activity. The ammonium sulfate step increased the specific activity by 10 to 15%. The purified enzyme has been stored at - 18” in 0.05 M potassium phosphate buffer (pH 7.0) containing 0.5 mM EDTA for over 6 months without significant loss of activity. This preparation lost less than 10% of its activity by freezing at -18” and thawing once.

RESULTS

destained by diffusion in methanol-acetic acid (14): Optical Rotatory Dispersion and Circular Dichroism-Optical

Physicochemical Properties-The purity of the purified enzyme

rotatory dispersion and circular dichroism were recorded at 25” was examined by acrylamide-agarose composite gel electropho-

on a JASCO model ORD/UVd spectropolarimeter equipped with resis. The enzyme protein migrated as a single band. The

a circular dichroism attachment. Optical rotatory dispersion and circular dichroism intensities were, respectively, calibrated with 1 The abbreviations used are: [TPP], enzyme-bound thiamine- an aqueous solution of sucrose to give an [o~]~s~ of 66.5 (17) and PP; [Lip&], enzyme-bound lipoic acid; [succinyl-S-Lip-SHI, with an aaueous solution of d-lo-camnhorsulfonic acid at 290 nm enzyme-bound S-succinyl dihydrolipoic acid; dansyl, 5-dimethyl- t,o give an ;L - eR of 2.20 (18). Optical rotatory dispersion results aminonaphthalene-l-sulfonyl.

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z -

50.0 50.5 51.0 51.5 52.0

Y”

FIG. 1 (left). Sedimentation pattern of 2-oxoglutarate dehy- drogenase. The photograph was taken after 50 min at 59,780 rpm. Protein Concentration was 0.435 g/100 ml of 0.05 M pOtM3SiUIU

phosphate buffer (pH 7.0) containing 0.5 mM EDTA. FIG. 2 (right). Sedimentation equilibrium centrifugation of

2-oxoglutarate dehydrogenase. The initial protein concentration was 0.04 g/100 ml. The plot was obtained from the interference photograph of the equilibrium distribution in a 12-mm double sector cell with a 4 mm solution column at 12,590 rpm and 20” in an An-D rotor for 45 hours. Units of r are radial distance in centimeters, and units of c are fringe displacement in millimeters.

TABLE I Weight average molecular weight of 2-oxoglutarate dehydrogenase

by sedimentation equilibrium ultracentrifugation The solvent contained 0.05 M potassium phosphate buffer

(pH 7.0) and 0.5 mM EDTA. The meniscus depletion sedimenta- tion equilibrium runs were carried out at 12,590 rpm and 20” for 45 hours in an An-D rotor with double sector cells equipped with a g-channel Kel-F centerpiece.

Initial concentration

g/100 ml

0.02 0.04 0.06

Molecular weight

220,000 217,000 212,000

enzyme showed only one band (cf. Fig. 5B) on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, indicating the homogeneity of the preparation.

The sedimentation velocity pattern of the enzyme showed a single major component and one minor component, as shown in Fig. 1. The reciprocal plot of ~20,~ against protein concentra- tion was virtually linear, and s%,~ was estimated as 10.3 S. The diffusion coefficient also showed a slight concentration de- pendence and the value extrapolated to infinite dilution (DHo,2Lo was 3.92 X lo+ cm2 s-l. From these values of s and D the molecular weight was calculated as 236,000, using for the partial specific volume a value of 0.732 ml per g, calculated from the amino acid composition.

In the meniscus depletion sedimentation runs, the plots of the logarithm of the vertical displacement (c) of a single fringe versus the square of the radial distance (~2) were virtually linear, indicating homogeneity of the protein, as shown in Fig. 2. Values of the weight average molecular weight of the purified preparation of 2-oxoglutarate dehydrogenase at different initial protein concentration are summarized in Table I. At the initial concentration of 0.04 g/100 ml, values for several different prep-

LL 210 230 250 200 220 240

WAVELENGTH (nm)

FIG. 3. The optical rotatory dispersion curve (A) and circular dichroism spectrum (B) of 2-oxoglutarate dehydrogenase.

arations of the enzyme lay in the range of 214,000 to 218,000. The value of the weight average molecular weight of the enzyme was taken to be 216,000 f 4,000.

Optical Properties-The absorption spectrum showed a maxi- mum at 278 nm and a minimum at 250 nm. The absorbance ratio at 280 nm and 260 was 1.52. There was no absorption above 320 nm.

The optical rotatory dispersion curve of the enzyme at pH 7.0 showed a negative Cotton effect with a trough at 234.5 nm,,a crossover point at 222 nm, and a shoulder at 215 nm, as shown in Fig. 3A, and is characteristic of proteins having a right-handed Q! helical structure (19). The reduced mean residue rotation at 234 nm, [m’]234, was found to be - 6440 deg + cm2 per dmole, and from this the content of right-handed a! helix in the enzyme molecule was estimated to be 34%.

The circular dichroism spectrum of the enzyme showed two troughs at 218.5 nm and 210 nm and a crossover point at 202 nm, as shown in Fig. 3B. This, again, is characteristic of pro- teins containing LY helical structure (20). The molecular ellip- ticity at 219 nm, [0j219, was -13,520 deg.cm2 per dmole, from which the a helical content of the enzyme was calculated as 340/,, agreeing closely with the value obtained from the optical rota- tory curve. The circular dichroism spectrum in the near ultra- violet region (400 to 250 nm) showed negative maxima at 292, 285,279, and 272 nm, and a broad positive band around 263 nm. Further analyses of these circular dichroism bands will be pub- lished elsewhere.

Thiamine-PP Content-The isolated 2-oxoglutarate dehydro- genase contained all the protein-bound thiamine-PP in the 2- oxoglutarate dehydrogenase complex, as reported previously (2). The 2-oxoglutaratc dehydrogenase activity of the purified en- zyme was not stimulated by exogenous thiamine-PP even in the presence of Ca2+. This observation suggests that thiamine-PP was not dissociated from the enzyme during isolation. The thiamine-PP content of the enzyme was estimated to be about 1 mole per mole of the enzyme (mol wt 216,000) by both the enzymatic assay of thiamine-PP and the chemical analysis of total thiamine.

Thiol Content-The content of thiol groups and half-cystine residues is summarized in Table II. Approximately 32 moles of each were dktected per mole of the enzyme (mol wt 216,000), indicating the absence of disulfide bridges.

Kinetic Properties-As shown in Table III, the ferricyanide- linked oxidation of 2-oxoglutarate catalyzed by 2-oxoglutarate

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TABLE II

Content of total thiol groups and half-cystine in d-ozoglutarate dehydrogenase

Methods Thiol group 01 half-cyst&

DTNB* in 6 M guanidine HCl (pH 8.2). CMBc in 0.05 M potassium phosphate buffer

(pH 7.0). CMBc in 8 M urea in 0.05 M potassium phos-

phate buffer (pH 7.0). Total half-cystined as cysteic acid.

34

32

31 32

a Average molecular weight of 216,000. * 5,5’-Dithiobis(2-nitrobenzoic acid) (25). c p-Chloromercuribenzoate (26, 27). d Determined by amino acid analysis after performic acid oxida-

tion (30).

TABLE III

Effect of phosphate concentralion on %-oxoglutarale dehydrogenase activity

Each reaction mixture contained the standard 2-oxoglutarate dehydrogenase assay components, 100 pmoles of histidine buffer (pH 6.5) in place of potassium phosphate buffer, the indicated concentration of potassium phosphate buffer (pH 6.5), and 20 Mg of 2-oxoglutarate dehydrogenase in a final volume of 2 ml. Reac- tion was begun by the addition of 2-oxoglutarate and followed at 420 nm.

Amount of potassium phosphate added Specific activity

/A?noles ~molcs/hr/mg )rolein

5 176 10 242 20 282 40 339 60 392 80 418

100 440 200 497 400 515 800 497

dehydrogenase required the presence of dibasic phosphate ion (potassium salt). Therefore, the standard assay system con- tained 100 pmoles of potassium phosphate buffer (pH 6.5) in a final volume of 2 ml to give nearly full activity.

When the ferricyanide-linked 2-oxoglutarate dehydrogenase assay was carried out in potassium phosphate buffer the optimal pH was found to be approximately 6.5, as shown in Fig. 4. The optimal temperature at this pH was determined by the modified method (6) of Hager (7) and found to be near 36”. The activa- tion energy of the 2-oxoglutarate dehydrogenase reaction was calculated to be 1.75 x lo4 cal per mole of the enzyme. The heat stability of the enzyme was measured by estimating the activity remaining when samples of the enzyme in 0.05 M potas- sium phosphate buffer (pH 7.0) containing 0.5 mM EDTA were heated for 5 min at different temperatures. Loss of activity was negligible up to 25”, but rapid beyond 30”.

Substrate specificities are shown in Table IV. The results indicate that 2-oxoglutarate dehydrogenase is very specific for the oxidation of 2-oxoglutarate, but does show some activity

PH

FIG. 4. The pH activity curve for the 2-oxoglutarate dehy- drogenase reaction. The activity was measured by the standard assay procedure, as described under “Methods.”

TABLE IV

Substrate specificities of %oxoglutarale dehydrogenase

Enzyme activity was measured by the standard 2-oxoglutarate dehydrogenase assay, as described under “Methods.” Various a-keto acids (potassium salt) were used as substrate instead of 2-oxoglutarate.

Substrate Relative activity

% 2-Oxoglutarate............................. 100 a-Ketoadipate. .._....._....._..__....._. 18 a-Ketopimelate............................ 7 a-Ketobutyrate............................ 0 a-Ketovalerate.. 0 Oxalate.................................... 0 Pyruvate.................................. 0

obtained with the 2-oxoglutarate dehydrogenase complex (l), which contains 2-oxoglutarate dehydrogenase bound nonco- valently to lipoate succinyltransferase (2). The K, (apparent) value for 2-oxoglutarate under the assay condition is 0.038 mM.

2-Oxoglutarate dehydrogenase contained about 32 moles of thiol group per mole of the enzyme and its dehydrogenase ac- tivity was completely inhibited only by high concentration of p-chloromercuribenzoate (enzyme to p-chloromercuribenzoate molar ratio of 1:200). However, the role of thiol group is being investigated further.

Amino Acid Composition-The results of amino acid analyses of the enzyme are summarized in Table V. The final recovery of all amino acids was calculated to be 98%. The partial spe- cific volume (B) of the enzyme calculated from the amino acid composition (8) was 0.732 ml per g.

NHz-terminal Residue-The Edman method revealed alanine as the NHz-terminal residue, and the amount present was esti- mated as 2 moles per mole of the enzyme (mol wt 216,000). The result indicates that 2-oxoglutarate dehydrogenase probably consists of 2 polypeptide chains. Dansyl-alanine was likewise the only derivative revealed by the dansyl method.

Peptide Mapping-The peptide map of the tryptic digest of performic acid-oxidized enzyme showed a total of 66 ninhydrin- positive spots, 32 of which contained arginine. The amino acid analysis showed the presence of 46 lysine residues and 45 argi-

with cu-ketoadipate and cY-ketopimelate. Similar results were nine residues per subunit (mol wt 97,000). These data suggest

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that the two subunits of 2oxoglutarate dehydrogenase are probably similar, if not identical.

Polyacrylumide Gel Electrophoresis in Sodium Dodecyl Sulfate- The 2-oxoglutarate dehydrogenase, which was treated with 1% sodium dodecyl sulfate containing 1 y0 2-mercaptoethanol after reduction and alkylation in 6 M guanidine HCl (pH 8.5) and 0.1 M 2-mercaptoethanol, showed a single band (Fig. 5B) with estimated molecular weight 113,000 (Fig. 6), on electrophoresis in 5% polyacrylamide gel in 0.1% sodium dodecyl sulfate. The

TABLE V

Amino acid composition of d-oxoglutarate dehydrogenase

Amino acid I

Total residues

Aspartic acid. Threonine . Serine Glutamic acid. Proline. . Glycine. . . . Alanine........ Half-cystine Valine . Methionine . . . Isoleucine . . Leucine. . Tyrosine. . Phenylalanine Lysine. Histidine . Arginine. Tryptophan.. .

. . .

.

. .

?&es/n& enzymea

190 98

102 226 119 125 149 32b

148 48 91

158 55c 82

102 62

101 23~

a Average molecular weight of 216,000. b Determined as cysteic acid after performic acid oxidation (30). c Estimated spectrally according to the procedure of Goodwin

and Morton (31).

A B C D

pattern was not altered if the sample was carboxymethylated after denaturation with 6 M guanidine HCl or 8 M urea in the absence of diisopropylfluorophosphate. When subjected to similar gel electrophoresis the 2-oxoglutarate dehydrogenase complex gave three bands (Fig. 5A) with estimated molecular weights of 113,000, 55,000, and 48,000, respectively. The band of molecular weight 113,000 corresponds to the dissociated subunit of 2-oxoglutarate dehydrogenase (Fig. 5B), and the other two bands with molecular weights of 55,000 and 48,000 correspond, respectively, to lipoamide dehydrogenase (Fig. 5C) and lipoate succinyltransferase (Fig. 50). The data indicate that the complexed and uncomplexed 2-oxoglutarate dehydro- genase can be dissociated into a single subunit with a molecular weight of 113,000 in sodium dodecyl sulfate gels.

Sedimentation Equilibrium Analysis in 6 M Guanidine Hy- drochloride--The molecular weight of the dissociated subunit of 2-oxoglutarate dehydrogenase was measured by the meniscus depletion sedimentation equilibrium method (10). The sam-

ples, which were previously dialyzed against water and lyophilized, were dissolved in 6 M guanidine HCl (pH 8.0) con- taining 0.1 M 2-mercaptoethanol and allowed to stand under nitrogen at 4” for 24 to 48 hours. The plots of the logarithm of the vertical displacement of a single fringe versus the square of the radial distance were virtually linear, as shown in Fig. 7. The weight average molecular weight of the dissociated subunit of the enzyme for two values of D, the value for the native enzyme (0.732 ml per g) and a reduced value in guanidine (0.722 ml per g) (1 l), are given in Table VI. Using the value of 0.732 ml per g, the molecular weights obtained for several different prep- arations of the enzyme lay in the range of 96,000 to 99,500 at an initial concentration of 0.04 g/100 ml. The value of the weight average molecular weight of the subunit of the enzyme was taken to be 97,000 f 4,000. This indicates two subunits per mole of 2-oxoglutarate dehydrogenase with a molecular weight of 216,000. On the other hand, the enzyme was dissociated into a single subunit with a molecular weight of 113,000 by gel

1

Z2[ , ( , , j 0.2 0.4 0.6 0.8 1.0

FIG. 5 (left). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the 2-oxoglutarate dehydrogenase complex and its component enzymes. Each protein was treated with 1% SO- dium dodecyl sulfate containing 1% 2-mercaptoethanol for 2 hours at 37” and subjected to electrophoresis as described under “Methods.” A, 2-oxoglutarate dehydrogenase complex; B, 2-oxoglutarate dehydrogenase; C, lipoamide dehydrogenase; D, lipoate succinyltransferase. The bands denoted by arrows indi- cate the subunits corresponding to: 1,2-oxoglutarate dehydrogen- ase; d, lipoamide dehydrogenase; S, lipoate succinyltransferase.

FIG. 6 (right). Determination of the apparent molecular

MOBILITY weights of the dissociated subunits of 2-oxoglutarate dehydrogen- ase by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Standard protein samples and 2-oxoglutarate dehydro- genase itself were subjected to electrophoresis exactly as described under “Methods.” Mobility relative to the tracking dye was plotted against the known subunit molecular weight of each protein as described by Weber and Osborn (14). 1, bovine serum albumin (dimer) ; 9, (3-galactosidase; 3, 2-oxoglutarate dehydro- genase (arrow); 4, phosphorylase a; 6, bovine serum albumin (monomer); 6, catalase; 7, lipoamide dehydrogenase; 8, ovalbu- min; 9, aldolase; 10, carbonic anhydrase; 11, chymotrypsinogen.

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m / I + I /” I “1.5-

F - i

l.O- I 1

50.5 I

51.0 51.5 52.0 r-2

FIG. 7. Sedimentation equilibrium centrifugation of 2-0x0- glutarate dehydrogenase in 6 M guanidine HCl and 0.1 M 2-mer- captoethanol. The initial protein concentration was 0.02 g/100 ml. The centrifugation was carried out at 21,740 rpm and 20” for 48 hours in an An-D rotor with a 12-mm double sector cell equipped with sapphire windows and with a 4 mm solution column, after denaturation of the protein as described under “Methods.”

TABLE VI

Weight average molecular weight of dissociated subunit of 8-oxoglutarate dehydrogenase

The solvent contained 6 M guanidine HCl (pH 8.0) and 0.1 M 2-mercaptoethanol. The meniscus depletion sedimentation equilibrium runs were carried out at 21,740 rpm at 20” for 48 hours in an An-D rotor with double sector cells equipped with an alumi- num-filled Epon centerpiece.

Molecular weight Initial concentration

i = 0.73Za i = o.m*

g/100 ml

0.02 99,700 93,000 0.04 97,900 91,400 0.06 95,900 89,800

(L Value of 5 for the native enzyme (8). * Assumed value of 5 in guanidine HCl (11).

electrophoresis in sodium dodecyl sulfate. From the amino

acid analyses and peptide maps it is possible to conclude that

these two subunits are similar, if not identical. Given the content of thiamine-PP (1 mole per mole of the

enzyme) it appears that a pair of subunits with NHz-terminal alanine bind one thiamine-PP as coenzyme.

DISCUSSIOX

As reported in a recent paper of this series, the mammalian 2-oxoglutarate dehydrogenase complex has been isolated from the Keilin-Hartree particle preparation from pig heart muscle (l), further separated into three component enzymes, and recon- stituted from the isolated component enzymes (2). One of the component enzymes of this multi-enzyme complex, 2-0x0- glutarate dehydrogenase bound to the core enzyme, lipoate succinyltransferase, catalyzes the first step of the oxidative decarboxylation of 2-oxoglutarate. Various properties of the enzyme have now been described in detail in this paper.

The pig heart 2-oxoglutarate dehydrogenase contains 1 mole- cule of thiamine-PP tightly bound, in contrast to pyruvate dehydrogenase, which is free of thiamine-PP (6). Attempts to dissociate thiamine-PP resulted in complete loss of activity.

The 2-oxoglutarate dehydrogenase apparently consists of two similar subunits with an estimated molecular weight of 113,000

1.

2.

3.

4. 5. 6.

7. 8. 9.

10. 11.

12.

13.

14.

15.

16.

3841

by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. By the sedimentation equilibrium ultracentrifugation in 6 M

guanidine HCl, the molecular weight of the subunit was esti- mated to be 97,000 + 4,000 from the data calculated with two values of D, namely the value for the native enzyme and the value in guanidine WC1 (11). The amino acid analysis showed the presence of 46 lysine residues and 45 arginine residues per subunit with a molecular weight of 97,000. A total of 66 nin- hydrin-positive spots and 32 of arginine-containing spots were found on the peptide map of the native enzyme. The dis- crepancy between the ratios expected for lysine to arginine peptides per subunit predicted from the amino acid composition for an (~2 structure and those found in peptide map remains to be studied. These results, however, indicate that 2-oxoglutarate dehydrogenase consists of two similar, if not identical, subunits, and that either one of the two subunits or two subunits together contains 1 molecule of thiamine-PP. Recently, Pettit et al. (36) reported that Escherichia coli 2-oxoglutarate dehydrogenase with a molecular weight of 190,000 was a dimer of identical polypeptide chains. The subunit molecular weight was esti- mated to be about 94,000 by sedimentation equilibrium ultra- centrifugation in guanidine HCl and by gel electrophoresis in sodium dodecyl sulfate.

Studies of the binding sites for the core enzyme (lipoate suc- cinyltransferase), the mode of association of this enzyme with the core enzyme, and the binding of thiamine-PP to 2-0x0- glutarate dehydrogenase are still in progress.

Acknowledgments--We wish to thank Dr. K. Kawahara and Miss K. Ota, Department of Physical Chemistry, School of Pharmaceutical Sciences, Nagasaki University, for their advice in ultracentrifugal analyses. We are indebted to Miss M. Yoshida for her assistance in the preparation of the manuscript and to Mrs. S. Nakao for her technical assistance. We also thank Dr. Jean 0. Thomas, Department of Biochemistry, University of Cambridge, for her criticisms and kind help in the preparation of the manuscript.

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and Masahiko KoikeKichiko Koike, Minoru Hamada, Nobuyuki Tanaka, Kin-Ichi Otsuka, Kyoko Ogasahara

DehydrogenaseProperties and Subunit Composition of the Pig Heart 2-Oxoglutarate

1974, 249:3836-3842.J. Biol. Chem. 

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