mammalian a-keto acid dehydrogenase complexes · 2.5% sodium or potassium phosphotungstate (ph 7.4)...

THE JOURNAL OP BIOLOGICAL CHEMISTRY Vol. 244, No. 13, Issue of July 10, PP. 3660-3670, 1969

Ptinted in U.S.A.

Mammalian a-Keto Acid Dehydrogenase Complexes

V. RESOLUTION AND RECONSTITUTION STUDIES OF THE PIG HEART PYRUVATE DEHYDROGENASE COMPLEX*

(Received for publication, January 3, 1969)

TARO HAYAKAWA, TAMOTSU KANZAKI, TSUTOMU KITAMURA, YUKIKO FUKUYOSHI, YUKIHIKO SAKURAI,

KICHIKO KOIKE, TADASHI SUEMATSU, AND MASAHIKO KOIKE

From the Department of Pathological Biochemistry, Atomic Disease Institute, and Electron Microscope Center, Nagasaki University School of Medicine, Sakamoto-cho, Nagasaki-shi 852, Japan

SUMMARY

1. The pig heart pyruvate dehydrogenase complex was separated into lipoamide hydrogenase (&I,~ = 5.55 S) and a colorless fraction by the fractionation on a calcium phosphate gel-cellulose column in the presence of 4 M urea. Subse- quently, in the presence of 0.3 M potassium iodide, the colorless fraction was fractionated into two additional components, pyruvate dehydrogenase (7.45 S) and partially resolved lipoate acetyltransferase, with ammonium sulfate. Highly purified lipoate acetyltransferase (28.2 S) was isolated by the resolution of the yellow fraction with 4 M urea, which was separated from the complex in the presence of 0.02 M ethanolamine (pH 9.4) on the calcium phosphate gel-cellulose column.

2. The molecular weights of the original complex, pyruvate dehydrogenase, lipoate acetyltransferase, lipoamide dehydrogenase, and the reconstituted complex were approximately 7.4 million, 110,000, 150,000, 2.0 million, and 7.5 million, respectively.

3. All three isolated constituent enzymes were required to reconstitute coenzyme A- and nicotinamide adenine dinucleotide-linked oxidative decarboxylation of pyruvate. Re- constitution of these constituent enzymes to produce a large unit resembling the original complex in composition, hydrodynamic parameters, and enzymic activities was achieved. Lipoate acetyltransferase appears to possess specific binding sites for pyruvate dehydrogenase and lipoamide dehydrogenase.

4. Morphological resolution and reconstitution of the complex through electron microscopic data have been shown. The macromolecular organization of the complex is discussed.

* This investigation was supported in part by Grant 91431 from the Scientific Research Fund of the Ministry of Education of Japan, by the sponsorship of the United States Army Research and Development Group (Far East), Department of the Army under Grant DA-CRD-AFE-S92-5446%Gll3, and by Grant AM-10765-02 from the National Institutes of Health, United States Public Health Service. A part of this work was presented at the Seventh International Congress of Biochemistry, Tokyo, 1967.

The enzyme system which catalyzes a coenzyme A- and nicotinamide adenine dinucleotide-linked oxidative decarboxylation of pyruvate (Reaction 6) has been isolated from pig heart muscle as a multienzyme complex (1, 2). The complex catalyzes a co- ordinated sequence of reactions that may be represented by Equation 1 through 5 (the brackets indicate enzyme-bound intermediates).

Pyruvate + [thiamine-PP]-Er *

[acetaldehyde-thiamine-PP]-El + CO%

[Acetaldehyde-thiamine-PP]-Er + [LipS&& +

[acetyl-S-Lip-SH]-Ez + [thiamine-PP]-Er

[Acetyl-S-Lip-SH]-Et + CoA-SH --f

acetyl-S-CoA + [Lip(SH)z]-&

[Lip(SH)2]-Es + [FAD]-Es +

[Lip&]-E, + [reduced-FAD]-Es [Reduced-FAD]-E8 + NAD+ + [FAD]-Es + NADH + H+ Sum:

Pyruvate + CoA-SH + NAD+ --f

acetyl-X-CoA + CO2 + NADH + Hf

(4)

(5) -

(‘3)

where Lip& is lipoic acid, acetyl-X-Lip-SH is S-acetyl dihydrolipoic acid, Lip(SH)z is dihydrolipoic acid.

(1)

(2)

(3)

The Escherichia coli pyruvate dehydrogenase complex has been separated into three constituent enzymes, pyruvate dehydrogenase (Er), lipoate acetyltransferase (Et), and lipoamide dehydrogenase (ES). The complex has been reconstituted from the isolated enzymes (3). The pyruvate dehydrogenase complex isolated from pigeon breast muscle has been also separated into three constituent enzymes, and complete restoration of the over- all activity (Reaction 6) was achieved by combining all three isolated constituent enzymes, but there was a failure to produce a large enzyme aggregate (4). The highly purified preparation of the mammalian pyruvate dehydrogenase complex contains protein-bound lipoic acid and FAD, but it is free from thiamine-PP (1, 2). Previous attempts to separate the individual enzymes containing each coenzyme and to confirm the proposed reaction sequence had met with limited success, because each enzyme could not retain its activity during the course of the separation.

3660

by guest on October 13, 2019

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Issue of July 10, 1969 Hayakawa et al. 3661

This paper describes the separation of the pig heart pyruvate dehydrogenase complex into three constituent enzymes, pyruvate dehydroganase (catalyzed Reaction l), lipoate acetyltransferase containing protein-bound lipoic acid (catalyzed Reactions 2 and 3), and a flavoprotein, lipoamide dehydrogenase (catalyzed Reactions 4 and 5). Reconstitution of these constituent enzymes to produce a large unit resembling the original complex in composition, enzymatic activities, hydrodynamic parameters, and macromolecular organization is reported. Some of this work has already been reported briefly (5, 6).

EXPERIMENTAL PROCEDURE

Materials and Methods-The pyruvate dehydrogenase complex was isolated from pig heart muscle particles essentially as described in previous papers (1, 2). Dried Clostridium kluyveri cells were obtained from Nutritional Biochemicals. Ultra pure grade urea, purchased from Wako Pure Chemical Industries and Mann, was recrystallized from deionized water or 95% ethanol, and ethanolamine was redistilled just before use. Solutions of analytical grade phosphotungstic acid, E. Merck (Darmstadt), were adjusted to pH 7.2 to 7.4 with dilute sodium or potassium hydroxide. Butvar 98 and 72A were generously furnished by Plastic Products and Resins Division, Monsanto Company, Springfield, Massachusetts. Perforated product grids (400 mesh copper) were obtained from Ernest F. Fullam, Inc. (Schenec- tady, New York). The sources of other materials are described in a previous paper (2). All solutions were prepared with dis- tilled and deionized water, and the potassium phosphate buffer used for the dialyses of enzyme preparations contained 5 X 10h4 M EDTA. The ethanolamine-phosphate buffer was prepared by mixing 0.48 ml of ethanolamine, 240 ml of water, and 160 ml of 0.05 M potassium phosphate buffer (pH 7.0). The pH was adjusted to 9.4 with approximately 3 ml of 1 M potassium phosphate buffer (pH 6.0).

Assay Procedure-Pyruvate dismutation, lipoate acetyltransferase, lipoamide dehydrogenase, and pyruvate dehydrogenase activities were determined as described in a previous paper of this series (2). Protein was determined by the biuret method and the phenol method with crystalline bovine serum albumin as the standard (7). The lipoic acid content (calculated as (+)-lipoic acid) of enzyme preparations was determined after alkaline hydrolysis (2), with lipoic acid-deficient Streptococcus faecalis lOC1 cells (8). The flavin content of protein fractions was determined according to the method of Beinert and Page (9) by measuring the absorbance of the neutralized trichloracetic acid extract at 450 rnh before and after reduction with dithionite. Calcium phosphate gel suspended on Whatman cellulose powder (CF-11) was prepared as described by Price and Greenfield (lo), and 15 to 20 g of the same powder per batch were added to obtain the appropriate flow rate of the column.

Ultracentrifugal Analyses-All sedimentation experiments were-performed~~at 3-50 in a Beckmanmodel Eultracentrifuge equipped with the RTIC unit and the schlieren and Rayleigh optical systems. Photographic plates were measured with a Mann type 829D comparator. Sedimentation coefficients were corrected to standard condition (water at 20’) and extrapolated to infinite dilution. The molecular weights of the native complex, its constituent enzymes, and the reconstituted complex were determined by the method of sedimentation equilibrium according to the meniscus depletion technique as described by

Yphantis (11). The An-D rotor was used at the higher speeds, while at speeds below 12,000 rpm the heavy An-J rotor was used. For the equilibrium runs, a 12-mm six-channel Kel-F centerpiece purchased from Beckman was used. Molecular weights were usually determined as the average of those obtained from each of three black fringes. Errors were indicated as standard devia- tions from the mean of these molecular weights. In the calcu- lations of sedimentation coefficients and molecular weights, the partial specific volume (7) was assumed to be 0.73 ml per g. Details are given under “Results.”

Electron Microscopy-The complexes and their constituent enzymes were negatively stained and embedded by the microdroplet cross-spraying technique with a special multiple spraying apparatus provided with suitably arranged separate capillaries for protein and staining reagent, devised by Fernandes-Moran (12, 13). This technique gave a brief controlled interaction of microdroplets which collided and interacted very rapidly before hitting the specimen grid. All reagents were filtered through sintered glass filter. Enzyme proteins were examined at a final concentration of 125 to 135 pg per ml in 0.0015% sucrose in 0.00125 M potassium phosphate buffer (pH 7.0). An aliquot of 2.5% sodium or potassium phosphotungstate (pH 7.4) was diluted with water to give a final concentration of 0.5%. Ultra thin Butvar 98 film, supported on a carbon-stabilized net of Butvar 72A mounted on a copper grid, was moistened with water for 5 to 10 min and water droplets were absorbed by filter paper. Previously cooled 0.4 ml of enzyme solution and 0.5% sodium phosphotungstate solution were sprayed on the moistened grid in the lucite cylinder (S-cm in diameter) by the multiple spraying apparatus. The distance from the top of the capillaries to the surface of the grid was about 40 cm. After 2 to 5 min the specimen was examined in a JEM model 7A-3 electron microscope equipped with an anticontamination trap, operating at 80 kv. The micrographs were taken at an electron optical magnifi- cation of 100,000 times.

RESULTS

Resolution of Pyruvate Dehydrogenase Complex with Urea- The pyruvate dehydrogenase complex was separated into two components by fractionation on a calcium phosphate gel-cellulose column in the presence of 4 M urea as described in a previous paper (3) with some modifications. A typical resolution was performed as follows. A solution of the complex containing 30 mg of protein in 1.4 ml of 0.05 M potassium phosphate buffer (pH 7.0) was applied to a calcium phosphate gel-cellulose column (3 cm in diameter x 4.5 cm in height) which had been previously washed with a solution of 1 To ammonium sulfate in 0.1 M potassium phosphate buffer (pH 7.5). After adsorption of the protein, the column was washed with 18 ml (0.7 volume of the packed gel) of a solution of 4 M urea and 1% ammonium sulfate in 0.1 M potassium phosphate buffer (pH 7.5). The column was then washed with about 45 ml of a solution of 1% ammonium sulfate-in&i ‘M potassium phosphate-buffer--(pH 75) : f? colorless protein was eluted, leaving a broad yellow fluorescent band on the column. Immediately after elution, the colorless fraction, comprising 26.8 mg of protein in a volume of 21 ml, was fractionated with solid ammonium sulfate. The precipitate obtained between 0 and 0.5 saturation was collected by centrifugation for 30 min at 16,000 x g and dissolved in a minimum volume of 0.05 M potassium phosphate buffer (pH 7.0). The solution was dialyzed against the same buffer. The yellow band was then


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3662 Mammalian a-Keto Acid Dehydrogenase Complexes. V Vol. 244, No. 13

eluted from the column with a solution of 6% ammonium sulfate in 0.1 M potassium phosphate buffer (pH 7.5). The eluate, comprising 1.7 mg of protein in a volume of 7 ml, was fractionated with solid ammonium sulfate. The precipitate obt,ained between 0.50 and 0.90 saturation was collected by centrifugation and dissolved in a minimum volume of 0.05 M potassium phosphate buffer (pH 7.0). The solution was dialyzed overnight against the same buffer.

The composition and enzymatic activities of both the colorless fraction and lipoamide dehydrogenase are shown in Table I. The colorless fraction contained all of the protein-bound lipoic acid of the pyruvate dehydrogenase complex and exhibited both pyruvate dehydrogenase and lipoate acetyltransferase activities. Lipoamide dehydrogeanse contained all of the flavin in the complex, and its lipoamide dehydrogenase activity per mole of flavin was essentially the same as that found with the original complex.

The sedimentation pattern of the colorless fraction showed a major component (s~0,~ = 56 S) (Fig. 1A). The sedimentation coefficients of different preparations of the colorless fraction varied between approximately 53 and 58 S. This variation is

TABLE I

Composition and activities of pyruvate dehydrogenase complex and its components from urea resolution

I I I Specific activities

I / I

m#moles/mg protein ~moles/hrlmg protein

Pyruvate dehydrogenase complex. . . . . 3.3 1.57 100 6.66 107 372

Colorless fraction.. . . 4.0 0.08 13 7.40 120 18

Lipoamide dehydrogenase. 0 18.3 0 0 0 4260

lI i ;-^!. B

B FIG. 1. Sedimentation patterns obtained with the components

from resolution of the pyruvate dehydrogenase complex with urea. Pattemz A, the colorless fraction after 30 min at 35,606 rpm; bar angle, 60”; B, lipoamide dehydrogenase after 97 min at 59,730 rpm; bar angle, 55’. Protein concentrations were, respectively, 0.48 and 0.49 g/160 ml of 0.05 M potassium phosphate buffer (pH 7.0).

8- -A

5- I I I (

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

CONCENTRATION (g/lOOml)

FIG. 2. Concentration dependence of the sedimentation coefficients of lipoamide dehydrogenase (Curve A), lipoate acetyltransferase (Curve B), and pyruvate dehydrogenase (Curve C) isolated from the pyruvate dehydrogenase complex.

TABLE II Weight average molecular weights of lipoamide dehydrogenase

Experimental conditions: 0.05 M potassium phosphate buffer (pH 7.0); 3”, 23 hours at 21,740 rpm.

Initial concentration Molecular weights

g/l00 ml

0.132 106,700 f 700 0.666 103,566 f 766 0.013 111,300 f 200

due to differences in the contents of pyruvate dehydrogenase and lipoamide dehydrogenase in the preparations, which, in turn, are due to losses of pyruvate dehydrogenase during the purification of the native complex and its resolution on a calcium phosphate gel-cellulose column with 4 M urea or due to a remaining of lipoamide dehydrogenase. The sedimentation pattern of lipoamide dehydrogenase (Fig. 1B) showed a single component and represented slight concentration dependence of sedimentation coefficients as shown in Fig. 2 (Curve A). The value extrapolated to infinite dilution (&,,,J is 5.55 S. Plots of the logarithm of the vertical displacement of a single fringe against the square of the distance from the center of rotation were virtually linear and showed that the protein was homogeneous. Weight average molecular weight calculated from these data was 108,800 f 600 (Table II). Preparation of lipoamide dehydrogenase showed a FAD content of 18.3 mpmoles per mg of protein. It is thus apparent that lipoamide dehydrogenase contains 2 moles of FAD per mole of enzyme, corresponding to a hypothetical minimum molecular weight of 54,400.

Resolution of Pyruvate Dehydrogenase Complex at pH Q.&-The pyruvate dehydrogenase complex was allowed to stand in 0.02 M ethanolamine-phosphate buffers with pH ranging from 7.5 to 9.5. Aliquots of each mixture were removed at various time intervals (2,4, and 6 hours) and tested in the pyruvate dehydrogenase assay system after dialysis against 0.05 M potassium phosphate buffer (pH 7.0) for 3 hours. Pyruvate dehydrogenase activity rapidly decreased at pH ranging between 8.5 and 9.0 regardless of the incubation times. Ninety-three per cent of the original activity remained at pH 8.5, 59% at pH 9.5. Ex-


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


amination of the complex at pH 8.5 in an analytical ultracentrifuge revealed no detectable dissociation.

The complex was separated into pyruvate dehydrogenase and a yellow fraction by chromatography on a calcium phosphate gel- cellulose column as described in a previous paper (3) with some minor modifications. A typical resolution was performed as follows. A column (3.5 X 1.8 cm) of calcium phosphate gel- cellulose was washed with the 0.02 M ethanolamine-phosphate buffer (pH 9.4) until the pH of the effluent became approximately 9.4. A mixture of 1.7 ml (32 mg protein) of the pyruvate dehydrogenase complex and an equal volume of 0.04 M ethanolamine solution was applied to the column. The column was then washed with approximately 130 ml of 0.02 M ethanolamine-phosphate buffer (pH 9.4). A colorless fraction comprising 17 mg of protein was eluted, leaving a broad yellow fluorescent band on the column. Ten-milliliter fractions were collected and their protein contents were estimated by ultraviolet absorption at 280 rnp and 260 mp (7). Those fractions with a protein concentration of more than 0.1 mg per ml were combined and fractionated with solid ammonium sulfate between 0.29 and 0.55 saturation. The precipitate was collected by centrifugation and dissolved in a minimum volume of 0.05 M potassium phosphate buffer (PH 7.0). The solution was dialyzed against the same buffer. After dialysis for 2 to 3 hours, the most active preparation of pyruvate dehydrogenase was obtained by the refractionation with solid ammonium sulfate between 0.42 and 0.55 saturation.

The yellow band was eluted from the column with a solution of 4% ammonium sulfate in 0.1 M potassium phosphate btier (pH 7.5), and the yellow fraction, comprising 11.7 mg of protein in a volume of 20 ml, was precipitated with ammonium sulfate below 0.29 saturation and dialyzed against 0.05 M potassium phosphate buffer (pH 7.0).

Even during the operation, which lasted 1 hour, the activity of the refractionated pyruvate dehydrogenase had diminished by more than 40 to 60% at pH 9.4. However, both lipoate acetyltransferase and lipoamide dehydrogenase activities were unaf- fected. The composition and enzymatic activities of the two fractions are shown in Table III.

The yellow fraction contained protein-bound lipoic acid and flavin, and exhibited lipoate acetyltransferase and lipoamide dehydrogenase activities. The sedimentation pattern of the yellow fraction showed a major component (SZO,~ = 33.2 S) with which the boundary of the yellow color was associated (Fig. 3A).

In contrast to the E. coli complex the resolution of the mam-

TABLE III

Composition and activities of components from resolution of pyruvate dehydrogenase complex at pH 9.4

-

D

1

Pyruvate dehydrogenase. . 0

Refractionated pyruvate dehydrogenase (0.42-0.55). . . . 0

Yellow fraction. . 6.6

0

0 3.04

Specific activities

0 0.64

0 7.70 7.6 0.17

0

0 255

0

0 700~

A

A B FIG. 3. Sedimentation patterns of the yellow fraction (Pattern

A) after 36 min and lipoate ecetyltransferase (Pattern B) after 45 min at 42,040 rpm; bar angle, 55”. Protein concentrations were, respectively, 0.57 and 0.48 g/100 ml of 0.05 M potassium phosphate buffer (pH 7.0).

TABLE IV Composition and activities of components from urea resolution of

yellow fraction

Component

Lipoate ace- tyltransfer- ase . _ . . . . .

Lipoamide dehydrogenase. . .

I I Specific activities

11.0 0.13 2.3 0.36 282 46

0 18.1 0 0 0 4630

malian pyruvate dehydrogenase complex at pH 9.4 was only done to obtain the yellow fraction because of the loss of the pyruvate dehydrogeanse activity. Pyruvate dehydrogenase was obtained from the colorless fraction by potassium iodide resolution as described in the following paragraph.

Resolution of Yellow Fraction with Urea-The yellow fraction obtained by the resolution of the complex at pH 9.4 was separated into two additional components, a colorless fraction and lipoamide dehydrogenase, by fractionation on a calcium phosphate gel-cellulose column in the presence of 4 M urea, essentially the same as described above. With the same sized column, 5.4 mg of the colorless fraction and 1.7 mg of lipoamide dehydrogenase were obtained from 9.5 mg of the yellow fraction.

The colorless fraction contained all of the protein-bound lipoic acid and exhibited mainly lipoate acetyltransferase activity. However, it contained a trace of flavin and exhibited slight activities in dismutation, pyruvate dehydrogenase, and lipoamide dehydrogenase assays (Table IV). These results indicated that, very small amounts of pyruvate dehydrogenase and lipoamide dehydrogenase were still attached to lipoate acetyltransferase. The sedimentation pattern of lipoate acetyltransferase is shown in Fig, 3B. Lipoate acetyltransferase represented distinct concentration dependence of the sedimentation coefficients (Fig. 2,


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3664 Mammalian a-Keto Acid Dehydrogenase Complexes. V Vol. 244, No. 13

TABLE V Weight average molecular weights of lipoate acetyltransferase Experimental conditions: 0.05 M potassium phosphate buffer

(pH 7.0); 4”, 55 hours at 4327 rpm.


g/100 ml x 10’

0.119 1.96 & 0.01 0.074 1.93 f 0.03 0.01% 2.04 f 0.03

Q Different preparation, 5”, 52 hours at 4327 rpm.

Curve B), and the value extrapolated to infinite dilution (s:~,~) is 28.2 S. Plots of the logarithm of the vertical displacement of a single fringe against the square of the distance from the center of rotation were curved and the values of au(r) increased with concentration, indicating the polydispersity of the preparation. This polydispersity is mainly due to differences in the attach- ments of p.yruvate dehydrogenase and lipoamide dehydrogenase, as mentioned above (cf. Table IV). Weight average molecular weight calculated from these data was 1.98 & 0.02 million (Table V). Purified preparation of lipoate acetyltransferase contained 11 mpmoles of lipoic acid per mg of protein. It is apparent that a minimum molecular weight per mole of lipoic acid would be approximately 91,000.

The sedimentation pattern of lipoamide dehydrogenase showed a single component. Its flavin content and lipoamide dehydrogenase activity (Table IV), together with its sedimentation characteristics, indicated that it is identical with lipoamide dehydrogenase obtained by the resolution of the native complex with urea.

Resolution of Colorless Fraction with Potassium Iodide-In the presence of 0.3 M potassium iodide, the colorless fraction obtained in the course of the purification of the complex or by the resolution of the complex with urea was dissociated into two additional components.l Examination of the mixture of the colorless fraction and potassium iodide in an analytical ultracentrifuge revealed the presence of two components (Fig. 4A). A typical resolution was performed as follows. With continuous stirring, 4 g of solid potassium iodide were dissolved into 74 ml of ice-cold 0.05 M potassium phosphate buffer (pH 7.0). Then, a solution of the colorless fraction containing 144 mg of protein in 6 ml of 0.05 M potassium phosphate buffer (pH 7.0) was added. After stirring for 5 min, 14 g of solid ammonium sulfate (0.25 saturation) were added and after an additional 5-min stirring the mixture was centrifuged at 25,000 X g, for 10 min. To clear supernatant fluid obtained, 14 g of ammonium sulfate (0.50 saturation) were added and after 5-min stirring the mixture was centrifuged. The precipitate was dissolved in an appropriate volume of 0.05 M potassium phosphate buffer (pH 7.0) and the solution was dialyzed with stirring against two 500-ml portions of the same buffer as soon as possible after the precipitation. This operation should be completed within 1 hour to minimize the loss of the pyruvate dehydrogenase activity. The first fraction, precipitated between 0 and 0.25 saturation of ammonium sulfate

1 Potassium iodide is not a specific dissociating reagent for the separation of pyruvate dehydrogenase from the complex because it also dissociates the flavoprotein from the complex. Then, the complex itself is not a suitable source to obtain the pure preparation of pyruvate dehydrogenase from potassium iodide resolution.

and consisting of the colorless fraction partially resolved, was dissolved in 80 ml of ice-cold 0.05 M potassium phosphate buffer (pH 7.0) with the use of a small Teflon pestle homogenizer. With continuous stirring, 4 g of solid potassium iodide were added to it and the mixture was refractionated with solid ammonium sulfate as mentioned above. After dialysis, the two second fractions precipitated between 0.25 and 0.50 saturation were combined and diluted with 0.05 M potassium phosphate buffer (pH 7.0) to make a final volume of 25 ml. The fraction was refractionated with solid ammonium sulfate. The most active preparation of pyruvate dehydrogenase precipitated between 0.42 and 0.55 saturation. The composition and enzymatic activities of pyruvate dehydrogenase and the colorless fraction partially resolved are shown in Table VI.

The sedimentation pattern of pyruvate dehydrogenase showed a single component (Fig. 4B) and represented a slight concentration dependence of sedimentation coefficients as shown in Fig. 2 (Curve C). The value extrapolated to infinite dilution (s:~,~) is 7.45 S. Plots of the logarithm of the vertical displacement of a single fringe against the square of the distance from the center of rotation were virtually linear and showed that the protein was homogeneous. Weight average molecular weight calculated from these data was 153,300 f 800 (Table VII).

Reconstitution of Pyruvate Dehydrogenase Complex from Three Isolated Constituent Enzymes-All three enzymes, pyruvate dehydrogenase, lipoate acetyltransferase, and lipoamide dehydrogenase, were required to reconstitute CoA- and NAD-linked oxidative decarboxylation of pyruvate (Table VIII). Direct evi- dence that the three isolated constituent enzymes combined to produce a large unit resembling the original complex was obtained as described below. The mixture contained 4.5 mg (52.6%) of pyruvate dehydrogenase, 3.0 mg (35.5%) of lipoate acetyltransferase, and 1.0 mg (11.9%) of lipoamide dehydrogenase in a total volume of 1.6 ml of 0.05 M potassium phosphate

B

A B FIG. 4. Sedimentation patterns obtained with the components

from resolution of the colorless fraction with potassium iodide. Pattern A, the colorless fraction in 0.3 M potassium iodide in 0.05 M potassium phosphate buffer (pH 7.0), after 29 min at 50,740 rpm; bar angle, 60”. The colorless fraction of 0.4 ml (5.7 mg of protein) was mixed with the same volume of 0.6 M potassium iodide in 0.05 M potassium phosphate buffer (pH 7.0) and approximately after 30 min the run was started. Pattern B, pyruvate dehydrogenase after 75 min at 59,780 rpm; bar angle, 60”; protein concentration, 0.59 g/100 ml of 0.05 M potassium phosphate buffer (pH 7.0).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


buffer (pH 7.0). It was centrifuged for 2 hours at 198,000 x g in the No. 50 rotor of a Beckman model L-2 ultracentrifuge. The yellow pellet was dissolved in an appropriate volume of 0.05 M potassium phosphate buffer (pH 7.0). The sedimentation pattern of the reconstituted complex was similar to that of the original complex and showed a major peak with which the boundary of the yellow color of the flavoprotein was associated (Fig. 5, A and B). The sedimentation coefficient (~0,~) of the major component was 72.1 S. The composition and enzymatic activities of the yellow pellet (Experiment 1) were in agreement with the values of the original complex (Table IX).

In a second experiment with a different weight ratio of the three isolated constituent enzymes, the mixture contained 6.4 mg (63.8%) of pyruvate dehydrogenase, 2.6 mg (26.3%) of lipoate acetyltransferase, and 1 .O mg (9.9 %) of lipoamide dehydrogenase in a total volume of 1.6 ml of 0.05 M potassium phosphate buffer (pH 7.0). It was centrifuged for 2 hours at 198,000 x g. The yellow pellet was dissolved in 0.05 M potassium phosphate buffer (pH 7.0). Its sedimentation pattern was very similar to that shown in Fig. 5 (Pattern B), with the exception of the existence of a minor component with the same sedimentation coefficient (sz~,~) found with isolated pyruvate dehydrogenase. The sedlmentatlon coefficient (sz,,,~ ) of the major component was 76.0 S. The composition and enzymatic activities of the pellet (Experiment 2) are shown in Table IX.

Other sedimentation studies indicated that pyruvate dehydrogenase and lipoamide dehydrogenase did not combine with each other, but that each of these components did combine with

TABLE VI

Composition and activities of components from resolution of colorless fraction with 0.3 M potassium iodide

Pyruvat,e dehydrogenase

Ammonium sulfate fraction (O&0.42)..

Second ammonium&fate fraction (0.42- 0.55). .

Colorless fraction Partially re-

solved. .

-

-

Total protein

34.2

18.1

71.0

7

0

0

6.6 -

Specific activities

Pyruvate Lipoate dehydro- acetyl- genase transferase

5.34 0

12.8 0

1.14 237

TABLE VII

Weight average molecular weights of pyruvate dehydrogenase

Experimental conditions: 0.05 M potassium phosphate buffer (pH 7.0); 5’, 29 hours at 15,220 rpm.

Initial concentration

g/100 ml

0.117 0.059 0.023

Molecular weights

154,300 f 1,300 153,600 f 600 152,000 f 300

TABLE VIII

Reconstitution of pyruvate dismutation activity

The amounts of protein in the dismutation assay were: pyruvate dehydrogenase, 9.6 pg; lipoate acetyltransferase, 7.1 rg; lipoamide dehydrogenase, 1.9 pg. Other components and conditions were as described previously (2).

Fraction AcetyfJpt&Thate

wnoles/hr

Pyruvate dehydrogenase. 0 Lipoate acetyltransferase. <O.l Lipoamide dehydrogenase. 0 Pyruvate dehydrogenase + lipoate acetyltrans-

ferase........................................ 0.26 Pyruvate dehydrogenase + lipoamide dehydro-

genase........................................ 0 Lipoate acetyltransferase + lipoamide dehydro-

genase........................................ <O.l Pyruvate dehydrogenase + lipoate acetyltrans-

ferase + lipoamide dehydrogenase. 1.68

A

Y B

B FIG. 5. Sedimentation patterns of the native complex (Pattern

A) after 28 min and the reconstituted complex (Pattern B) after 21 min at 35,600 rpm; bar angle, 65” and 6OO”, respectively. Protein concentrations were, respectively, 0.47 and 0.45 g/100 ml of 0.05 M potassium phosphate buffer (pH 7.0).

lipoate acetyltransferase. Moreover, the specific activities and sedimentation coefficient of the reconstituted complex were not significantly affected by the order of mixing of the three components. These observations indicated that pyruvate dehydrogenase and lipoamide dehydrogenase combined independently of each other with specific binding sites of lipoate acetyltransferase.

Interchangeability of Flavoprokin of Pyruvate Dehydrogenase Complex with Three Other Flavoproteins-Two components, colorless fraction and lipoamide dehydrogenase, from urea resolution of the pyruvate dehydrogenase complex were required to reconstitute the CoA- and NAD-linked oxidation of pyruvate and they reassociated simultaneously when mixed to produce a large unit resembling the original complex (Table X). The flavoprotein of the pyruvate dehydrogenase complex was interchange- able with lipoamide dehydrogenase prepared from the following three different sources: (a) free lipoamide dehydrogenase prepared from the protamine sulfate supernatant fluid of amber- color extract (from which both the pyruvate and the 2-oxoglu-


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3666 Mammalian a-Keto Acid Dehydrogenase Complexes. I’ Vol. 244, No. 13

tarate dehydrogenase complexes were excluded) by the method described by Massey (14) with some modification; (b) lipoamide dehydrogenase separated from the 2-oxoglutarate dehydrogenase complex by chromatography on a gel-cellulose column in the presence of 4 M urea; and (c) human liver lipoamide dehydrogenase prepared by the method described in a previous paper (15). As summarized in Table X, the three flavoproteins were almost equal in their capacity to reconstitute the complex in respect to both ultracentrifugal analyses and the activity of over- all oxidation of pyruvate, as was, it is interesting to note, human liver lipoamide dehydrogenase in contrast to the E. coli lipoamide dehydrogenase (3). However, with the results of the immunochemical studies of lipoamide dehydrogenases, human liver lipoamide dehydrogenase was completely differentiated from three pig heart lipoamide dehydrogenases in respect to the Ouchterlony double-diffusion test and the titration studies of the activities

TABLE IX Composition and activities of native complex and reconstituted

complex from three isolated constituent enzymes

I I I Specific activities

I I I m)m&%/mg protein Irmoles/hr/mg firotei~

Native complex 3.2 1.53 93.5 6.52 122 378 Reconstitmed

complex Experiment 1 3.3 1.63 87.8 7.76 125 462 Experiment 2 2.4 1.56 82.2 8.90 99 397

TABLE X

Interchangeability of flavoprotein of pyruvate dehydrogenase complex in ultracentrifugal and enzymatic analyses

Ultracentrifugal analyses were performed as follows: the colorless fraction from the pyruvate dehydrogenase complex (3.1 mg of protein) and 750 rg of each flavoprotein were mixed in 0.8 ml of 0.05 M potassium phosphate buffer (pH 7.0), and approximately after 50 min the runs were started at 36,500 rpm. Over-all oxidation of pyruvate was performed as follows: 20 fig of the colorless fraction from the pyruvate dehydrogenase complex and 4 pg of each flavoprotein were previously incubated at 0” for 10 min and then the pyruvate dismutation assay was followed.

Fraction

Colorless fraction.. . . . . _. . . Colorless fraction + free lipoamide dehy-

drogenase.............................. Colorless fraction + lipoamide dehydro-

genase from 2-oxoglutarate dehydrogenase complex. . . . . . . . . . . .

Colorless fraction + lipoamide dehydrogenase from pyruvate dehydrogenase complex...............................

Colorless fraction + lipoamide dehydrogenase from human liver.

Colorless fraction + lipoamide dehydrogenase from E. coli.. . L!-

53.3

57.8

57.0

59.0

57.9

53.4

cetyl phos- late formed

pmoles/hr

0.36

1.2

1.2

1.5

1.3

0.36

TABLE XI

Weight average molecular weights of native complex

Experimental conditions: 0.05 M potassium phosphate buffer (pH 7.0); 4”, 70 hours at 2378 rpm.


E/loo ml x 10’

0.057” 7.31 f 0.1 0.042 7.57 f 0.03 0.014 7.42 f 0.04

a Different preparation, 4”, 72 hours at 2378 rpm

I I I

3.0

v 2.5 I n

v I 49 49.5 50

X2 (cmZ)

FIG. 6. Sedimentation equilibrium of the reconstituted complex at an initial concentration of 0.047 g/100 ml. The data were from outer channel of a 12-mm six-channel Kel-F centerpiece after 72 hours at 2378 rpm; temperature, 4”. LOG C represents fringe displacement and X represents the distance in centimeters from the axis of rotation.

TABLE XII

Weight average molecular weights of reconstituted complex

Experimental conditions: 0.05 M potassium phosphate buffer (pH 7.0); 4”, 72 hours at 2378 rpm.


g/loo ml x 100

0.070 7.61 f 0.09 0.047 7.24 f 0.09 0.019 7.50 f 0.10

with the immune globulin, as already reported in our previous paper (16).

Molecular Weights of Native and Reconstituted Complexes-As already reported in our previous paper (2), the highly purified complex showed a single component in the ultracentrifugal analysis (Fig. 5A), and the sedimentation coefficient to infinite dilution (s&J is 67.5 S.

Plots of the logarithm of the vertical displacement of a single fringe against the square of the distance from the center of rotation were virtually linear and showed that the protein was homogeneous. Weight average molecular weight calculated from these data was 7.43 f 0.06 million (Table XI).

As already mentioned, the sedimentation coefficients of different preparations of the reconstituted complex varied between 65 and 76 S. This variation might be due to a small difference in the contents of two constituent enzymes, pyruvate dehydro-


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Issue of July 10, 1969 Hayakawa et al.

FIG. 7. Electron micrographs of the native and the reconstituted pyruvate dehydrogenase complexes and their constituent enzymes negatively stained with sodium phosphotungstate (pH 7.4). A, the native pyruvate dehydrogenase complex; B, the reconstituted pyruvate dehydrogenase complex; C, lipoate acetyltransferase; D, pyruvate dehydrogenase; and E, lipoamide dehydrogenase. X 300,m.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3668 Mammalian cr-Keto Acid Dehydrogenase Complexes. ‘V Vol. 244, No. 13

FIG. 8. Electron micrographs of the colorless fraction (pyruvate dehydrogenase-lipoate acetyltransferase subcomplex) (A) and the yellow fraction (lipoamide dehydrogenase-lipoate acetyl transferase subcomplex) (B). X 300,000.

genase and lipoamide dehydrogenase. The molecular weight of nique showed images similar to those obtained with lipoate the reconstituted complex was estimated by the sedimentation acetyltransferase and much smaller particles in the background a equilibrium analysis with the same conditions as used for the little. This suggests that the reconstituted complex tends to native complex. The results are shown in detail in Fig. 6, in- dissociate during negative staining with sodium phospho- dicating the homogeneity of the reconstituted complex and the tungstate. When the native complex was mixed with 0.25% lack of dissociation under the conditions used. Weight average sodium phosphotungstate before spraying, it was observed that molecular weight calculated from these data was 7.45 f 0.09 the complex also tended to dissociate. Electron micrographs of million (Table XII). the native complex shadow-casted with platinum-carbon showed

Electron Microscopic Structures of Pyruvate Dehydrogenase a globular structure with a diameter of about 284 A and a height Complex, Its Constituent Enzymes, and Reconstituted Complex- of about 260 A.2 The appearances of both the colorless fraction The resolution of the pyruvate dehydrogenase complex into its (pyruvate dehydrogenase-lipoate acetyltransferase subcomplex) constituent enzymes and the reconstitution of the complex from and the yellow fraction (lipoamide dehydrogenase-lipoate acetyl- them have been visualized by the correlative electron micro- transfera,se subcomplex) are very similar as shown in Fig. 8, A scope studies. An electron micrograph (Fig. 7C) of lipoate and B, respectively. The images of both subcomplexes showed acetyltransferase negatively stained with 0.25% sodium phos- the various aspects of a polyhedron structure similar to that of photungstate showed the appearance of a polyhedron structure lipoate acetyltransferase. with a gross diameter of about 170 A. The various orienta- These electron microscopic observations suggest that the mole- tions of this enzyme molecule are visible; however, it is very cules of pyruvate dehydrogenase and lipoamide dehydrogenase complicated to define the internal structure with a molecular are apparently bound to the lipoate acetyltransferase molecule weight of 1.98 million. The images of pyruvate dehydrogenase and that they also distribute symmetrically on the lipoate acetyl- (Fig. 70) and lipoamide dehydrogenase (Fig. 7E) are globular transferase molecule to permit efficient interaction of three co- structures with a gross diameter of about 72 A and 70 A, respectively. The appearance of the native and the reconstituted

enzymes, thiamine-PP, lipoyl moiety, and FAD, which bound to

pyruvate dehydrogenase complexes is shown in Fig. 7, A and B, each constituent enzyme, respectively. Before the presentation

respectively. The comparison of these micrographs reveals of a model of the complex based on the correlative electron micros-

close similarity between these two complexes. The images of copy, it is necessary to define further the orientation of each

these complexes showed the various aspects of polyhedron with a constituent enzyme and the complex, and their structural ar-

gross diameter of about 210 to 250 A with no clearly determined rangement

internal structure. Electron micrograph of the reconstituted complex stained even by the microdroplet cross-spraying tech-

2 Masahiko Koike, Kichiko Koike, and Tadashi Suematsu, un- published data.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


DISCUSSION

Enzyme systems which catalyze Reaction 6 have been isolated as multienzyme complexes with molecular weights of several million from pigeon breast muscle (17, 18) and E. co& (19) in addition to pig heart muscle and bovine kidney (20). The E. coli pyruvate dehydrogenase complex is the first multienzyme complex which was separated into three constituent enzymes- pyruvate dehydrogenase, lipoate acetyltransferase, and lipoamide dehydrogenase-by the combination of its fractionations in the presence of 0.02 M ethanolamine (pH 9.5) and subsequently 4 M

urea on the calcium phosphate gel-cellulose column (3). The three isolated enzymes combined simultaneously to produce a large unit resembling the native complex. In the preliminary report of Mukherjee et al. (21), the E. coli 2-oxoglutarate dehydrogenase complex was also separated into three enzymes, 2- oxoglutarate dehydrogenase, lipoate transsuccinylase, and lipoamide dehydrogenase, by the combination of its fractionation on a calcium phosphate gel-cellulose column in the presence of 1 M sodium chloride in 0.05 M ethanolamine (PH 10.0) and 4 M urea and the complex was then reconstituted from these enzymes.

The results described in this report indicate that pig heart pyruvate dehydrogenase complex is the first organized multienzyme in mammalian tissue which has been separated into three constituent enzymes containing at least three coenzymes without any loss of each enzyme activity. They were reassociated simultaneously to produce a large enzyme aggregate resembling the original complex. It is apparent from the result that the pig heart pyruvate dehydrogenase complex is composed of at least three constituent enzymes containing three coenzymes: (a) thiamine-PPdependent pyruvate dehydrogenase; (b) lipoate acetyltransferase containing all of protein-bound lipoic acid; and (c) a flavoprotein, lipoamide dehydrogenase. It is not yet clear whether lipoate acetyltransferase is a single enzyme with two active sites which catalyzed Reactions 2 and 3 or a multienzyme of two separate enzymes which catalyzed Reactions 2 and 3, respectively. From the hydrodynamic parameters and molecular weight of lipoate acetyltransferase it is obvious that it is a large aggregate. It might be possible to separate lipoate acetyltransferase into a small subunit. If it is assumed that there is 1 molecule of protein-bound lipoic acid per subunit, the minimum molecular weight of the hypothetical subunit of this enzyme would be approximately 91,000. From the ultracentrifugal analyses and molecular weights of the native complex and its constituent enzymes, the number of molecules of each constituent enzyme per mole of the complex (mol wt 7.43 x 106) has been calculated approximately as follows: for the lipoate acetyltransferase (mol wt 1.98 x 106), 1; for the pyruvate dehydrogenase (mol wt 153,300), approximately 30; and for the lipoamide dehydrogenase (mol wt 108,800), approximately 6.

The mammalian pyruvate dehydrogenase activity rapidly decreased at pH ranging from 8.5 to 9.5 in contrast to the E. coli enzyme, so that previous attempts have met with limited success. This observation showed the conformational complexity of the mammalian pyruvate dehydrogenase molecule. As illustrated in the text, the sedimentation coefficient (szo,d of the reconstituted complex became greater than that of the original complex, and it reached to a value of 76.0 S by increasing of pyruvate dehydrogenase concentration (Fig. 9). The enzymatic activities, especially the pyruvate dehydrogenase activity of the reconstituted complex, also increased by about 30% of that of

76 76

0 74 ; 72

w$ 70 66

66 64 ,f--i

1.5 2.0 2.6

mgPYRUVATE CEHYDROCSENASE

mgLlPOATE ACETYLTRANSFERASE

FIG. 9. Effect of the concentration of pyruvate dehydrogenase on the sedimentation coefficient of the reconstituted complex. In the mixtures of three constituent enzymes, the concentration ratios of pyruvate dehydrogenase to lipoate acetyltransferase were changed and centrifuged for 2 hours at 198,999 X (7. Other conditions were as illustrated in the text. All of the runs were performed at the protein concentration of 0.45 g/199 ml of 0.05 M potassium phosphate buffer (pH 7.0).

the original complex, as well. This finding might suggest that the native complex lost pyruvate dehydrogenase during purification. The reason is not known yet. Similar results were also obtained with the native complex.

Further experiments to visualize the structure of the complex and its constituent enzymes are still in progress. Investigation of the characteristics of these constituent enzymes is also still in progress in respect to enzymatic, physicochemical, protein- chemical, and immunochemical properties. The results will appear elsewhere.

Acknourwen&We wish to thank Dr. W. R. Carroll, National Institutes of Health, Dr. C. R. Wilhns, Southwest Texas State College, and Dr. S. A. Kuby, University of Utah College of Medicine, for their discussion in molecular weight determination and Dr. H. Fernandez-Moran, The University of Chicago, Dr. L. J. Reed and Mr. R. M. Oliver, The Uni- versity of Texas at Austin, for their helpful advice in electron microscopy. We also wish to thank for their assistance in the preparation of the manuscript Miss M. Yoshida, Miss Beverley Carpenter, and Mr. Barrie Ward, and for their technical assistance Miss J. Tominaga, Miss S. Tokunaga, and Mrs. S. Nakao.

1.

2.

3.

4.

5.

;:

8.

9. 10.

11.

REFERENCES

HAYAKAWA, T., MUTA, H., HIBASHIMA, M., IDE, S., OKABE, K., AND KOIKE, M., Biochem. Biophys. Res. Commun., 17, 51 (1964).

HAYAKAWA, T., HIRASHIMA, M., IDE, S., HAMADA, M., OKABE, K., AND KOIKE, M., J. Bzol. Chem., 241,4694 (1966).

KOIKE, M., REED, L. J., AND CARROLL, W. R., J. Biol. Chem., 238, 30 (1963).

GLEMJA, A. A., ZILRER, L. S., AND SEVERIN, S. E., Biokhimiua, 31, 1033 (1966).

_

HAYAKAWA, T., AND KOIKE, M., J. Biol. Chem., 242, 1356 (1967 1.

K&KE,.M., J. Tritaminol. (Osaku), 14, 86 (1968). LAYNE, E., in S. P. COLOWICK AND N. 0. KAPLAN (Editors),

Methods in enzymolog2/, Vol. III, Academic Press, New York, 1957, p. 447.

GUNSALUS, I. C., DOLIN, M. I., AND STXUGLIA, L., J. Biol. Chem., 194, 849 (1952).

BEINERT, H., AND PAGE, E., J. Biol. Chem., 226, 479 (1957). PRICE, V. E., AND GREENFIELD, R. E., J. Biol. Chem., 209,

363 (1954). YPHANTIS, D. A., Biochemistry, 3, 297 (1964).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Mammalian a-Keto Acid Dehydrogenase Complexes. V Vol. 244, No. 13

12. FERN~NDEZ-MORAN, H., Res. Publ. Ass. New. Ment. LXs., 40, 235 (1962).

17. JAGANNATHAN, V., AND SCHWEET, R. S., J. Biol. Chem., 196, 551 (1952).

13. FERN~NDEZ-MORAN, H., in R. HARRIS (Editor), The interpre- 18. SCHWEET, R. S., KATCHMAN, B., BOCK, R. M., AND JAGANNA- tation of ultrastructure, Academic Press, New York, 1962, p. THAN, V., J. Biol. Chem., 196, 563 (1952). 411.

14. MASSEY, V., Biochim. Biophys. Acta, 37, 310 (1960). 19. KOIKE, M., REED, L. J., AND CARROLL, W. R., J. Biol. Chem.,

236, 1924 (1960). 15. IDE, S., HAYAKAWA, T., OKABE, K., AND KOIKE, M., J. Biol. 20. ISHIKAWA, E., OLIVER, R. M., AND REED, L. J., Proc. Nat.

Chem., 242, 54 (1967). Acad. Sci. U. 8. A., 66, 534 (1966). 16-i &&AKAWA; T;, i%IXAWA; T:, thmu; 2, AIii‘KGTkT; MY, 21; MOiJ~3 BY, Malmxm; J;, HoR-N-ET;-D;L., AN-l-Rh%m;

J. Biochem. (Tokyo), 62, 396 (1967). L. J., J. Biol. Chem., 240, PC2268 (1965).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Yukihiko Sakurai, Kichiko Koike, Tadashi Suematsu and Masahiko KoikeTaro Hayakawa, Tamotsu Kanzaki, Tsutomu Kitamura, Yukiko Fukuyoshi,

DEHYDROGENASE COMPLEXRECONSTITUTION STUDIES OF THE PIG HEART PYRUVATE

-Keto Acid Dehydrogenase Complexes: V. RESOLUTION ANDαMammalian

1969, 244:3660-3670.J. Biol. Chem.

http://www.jbc.org/content/244/13/3660Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/244/13/3660.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/content/244/13/3660

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;244/13/3660&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/244/13/3660

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=244/13/3660&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/244/13/3660

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/244/13/3660.full.html#ref-list-1

http://www.jbc.org/

mammalian a-keto acid dehydrogenase complexes · 2.5% sodium or potassium phosphotungstate (ph 7.4)...

Documents