Isolation and properties of pyruvate and α-ketoglutarate dehydrogenation complexes from pig heart muscle

BIOCHEMICAL

Vol. 17, No. 1, 1964

ISOLATION

AND BIOPHYSICAL

AND PROPERTIES

RESEARCH COMMUNICATIONS

OF PYRUVATE AND a-KETGGLUTARATE

DEHYDROGENATION COMPLEXES FROM PIG HEART MUSCLE

Tsro Hayakawa, Hiroo Satoshi Ide, Keiichiro

Muta, Masahiro Hirashima, Okabe and Masahiko Koike

Department of Pathological Biochemistry Atomic Disease Institute Nagasaki University School of Medicine Nagasaki-shi, Japan ReceivedJune24,

1964

Enzyme systems ation

of a-keto

(Korkes,

e

Gunsalus,

which

acids

al.,

1951;

CoA-SH a-ketoglutarate

form

pig

from

1960).

in

heart

In their

the presence

previous

into

(Crookes three

containing acetylase These

the original

et al.,

1952;

found

that

one of the authors

a crude

reported

dehydrogenation (Koike,

complexes etg.,

components:

and (c) were

complex

also

1952;

1960a, (a)

lipoic

pyruvic acid

a flavoprotein, reassociated

in composition

fraction sulfate

complex from

Reaction

1

been repoFted.

of studies

In

on the pyruvate

(PDC and KGDC) in Escherichia b and c)~

The PDC was separated

carboxylase,

and exhibiting

(b)

a large activities

a component

dihydrolipoic

dihydrolipoic

and enzymatic

51

purified

1953 and Massey,

catalyzed

a series

(1)

precipitated

has not

to produce

1963).

et al.,

H+

in highly

dehydrogenation

purification

activity,

sources

et al.,

was isolated

pyruvate

further

the p,rotein-bound

decarboxyl-

various

+ DPNH +

Kaufman,

of pyruvate;

essential

Schweet,

CO,

complex

of mammalian (1952)

+

ammonium

strain)

components

RCO-S-CoA

saturated

papers,

from

1952;

0.4

and a-ketoglutarate coli

been obtained

, et al.,

-

(Sanadi,

with

oxidative

1959).

DPN

studies

extract

1) have

dehydrogenase

and co-workers

heart

a CoA- and DPN-linked

Jagannawr

+

Mammalian

pig

(Reaction

1954 and Goldman,

RCOCOsH +

Korkes

catalyze

trans-

dehydrogenase. unit

resembling

(Koike,

et al.,

Vol. 17, No. 1, 1964

BIOCHEMICAL

In the present complexes

from

(PDG) and 3.3 bound

lipoic

heart

communication

pig

heart

1.

particles

are

For

purification

were

prepared

The resultant

adjusted

pH to 6 with by addition

1 N acetic

solution

phosphate

buffer,

pH 7, dialyzed

overnight

and centrifuged. eluates,

by centrifugation dissolved

on a calcium washed

in 0.05

0.05

on columns,

M phosphate

washed

ammonium sulfate band at the

top

with

acetylase

activities

pH 7.5.

The yellow sulfate

colorless

eluates

colorless

fractions

KGDG was precipitated

Tables

were

were were

and recovery

data

0.24

pH 7 protamine

were

The resultant

collected

yellow

pH 7, and purified (Price,

M

pellets

by chromatography

et al.,

1954)

The complexes

were

previously adsorbed and 1%

buffer,

leaving

a bright

yellow

pH 7.5,

carboxylase

with

the 0.05

with

solid

between and 0.29

of enzymatic

to the column

colorless

and dihydrolipoic M and 0.1

was then buffer,

precipitated

between

other,

0.1

pH 7.5

M phosphate

fractionated

buffer,

designated each

X g.

with

buffer,

band on each column in 0.1

from

precip-

0.1 M phosphate

pyruvic

removed

was

of ?$

was eluted

When PDC! was applied

both

et al.,

volume

M phosphate

pH 7.5.

M phosphate

exhibited

free

buffer,

M then

Pig

per ml,

to 0.03

fluids,

column

of each column.

which

4$ ammonium

0.05

in 0.1

fractions

0.05

at 144,000

buffer,

catalysis

was followed.

6 mg protein

and 0.015

almost

gel-cellulose

for

of Sanadi,

Each precipitate

M phosphate

phosphate

with

purification

hours

the protein-

procedure

The supernatant

2.5

for

contained coenzymes

of 10

and KGDG and PDC respectively

against

were

Both

extract,

5)'

which

weights

to the method

volume

(pli

multienzyme

molecular

essential

acid;

of 0 to 0.015

of both

with

original

amber-colored

sulfate

were

discussed.

according

protamine

precipitate

units

our

RESEARCH COMMUNICATIONS

and study

FAD and thiamine-IPas

(1952).

itated

isolation

as structural

(KGDG) million acid,

of Reaction

AND BIOPHYSICAL

trans-

M phosphate

buffer,

eluted

with

a solution

of

pH 7.5.

Both

the

and

ammonium

sulfate.

0.29

and 0.36

saturation. activities

yellow

saturation

A summary is

PDC and the

presented

and of typical in

I and II. Electrophoretic

analysis

the purified

52

PDC and KGDG gave homogenous

bands

Vol. 17, No. 1, 1964

BIOCHEMICAL

AND BIOPHYSICAL

Table Purification

of Pyruvate

I Dehydrogenation

Specific Fraction

Homogenate Particle

Total protein g 5,800

RESEARCH COMMUNICATIONS

activities

Dismutation

Yield total mmoles

0.23

1,330

Complex (pmoles/hr./mg

Carboxylase

protein)

Lipoic transac.

0.03

0.26

330

1.3

429

0.13

1.3

37

3.6

133

0.4

3.1

Amber-color extract

Lipoic DeH. -

66

Protamine ppt. eluate

2.44

13

32

0.82

15

36

Pellet

0.7

41

28

3.0

62

112

AmSO ppt. (0.29-0.36)

0.15

64

16

4.3

81

210

All assays were carried out as described previously (Koike, --et al., 1960a), except incubation at 37'C in the presence of crystalline bovine serum albumin. Lipoic transacetylase assay was carried out with 10 bmoles of dihydrolipoamide in 0.05 ml of 95$ ethanol. Lipoic dehydrogenase assay was carried out as described by Massey (1960) with some modification in ' the presence of lipoamide at room temperature.

Table Purification

II

of a-Ketoglutarate

Dehydrogenation

Specific Fraction

Homogenate Particle Amber-color extract

Total protein g 5,800

activities

Carboxylase

Yield total mmoles

0.19

1,100

Complex

(pmoles/hr./mg

DPN Reduction

Lipoic DeH.

protein) COs Evolution

-

0.48 1.7

330

1.0

330

-

37

2.7

100

25

66

6.0 8.6

46

64

119

10.4

27

82

216

16.7

12

178

373

24.5

Protamine ppt. eluate

7.7

Pellet

3.1

Amso ppt. (O-24-0.29)

0.7

16.3

4.2

c+Ketoglutarate carboxylase assay was carried out as described by Koike, et al., (1960a). DPN reduction and lipoic dehydrogenase assay were carried out as described by Massey (1960) with some modification. COs evolution was carried out as described by Sanadi, al (1952). -et -*'

53

BIOCHEMICAL

Vol. 17, No. 1, 1964

which

migrated

pH 8.6)

toward

at 4OC.

the

highly

in Spinco coefficients

purified

Diffusion

studies

using

and 1.0

be approximately

now under

ml per

1952)

acid

autoclaving

15 lb for

3 hours

contained

respectively

The absorption absorption sodium

dithionite.

Streptococcus in 0.1 N sodium

under

nitrogen

weights

were

of 0.6

X

calculated

to

a partial

specific studies

are

weights

and shapes.

Fig.2. pattern per ml, r.p.m.;

Ultracentrifuge schlieren obtained with KGDC, 10 mg after 25 min. at 43,700 temperature, 8.5OC. manometrically

hydroxide

in an open

test-tube

et al.,

1956).

shoulder

et al., at

PDC and KGDC

of (+)-lipoic,acid

a broad

1 and 2 :

(Dso,w)

(Gunsalus,

spectrum

54

H Electrophoresis

was determined

400 to 500 sq~ was significantly The absorption

model

lCC!l cells

moles

of PDC showed

in Figures

faecalis

(Wagner,

5 and 2 X lo-'

spectrum between

of the complexes

with

shown

and light-scattering

molecular

the

The patterns,

(KGDC) .assuming

hydrodynamic

the exact

content

acid-deficient

after

X lo6

Ultracentrifuge schlieren obtained with PDC, 6.6 mg after 24 min. at 34,450 temperature, 6.7OC.

The lipoic lipoic

and 3.3

S.

(ptO.06,

upon with

coefficients

The molecular

Further

g.

in a Spinco diffusion

X 10"cm2sec-I. (PDC)

S and 36.5 are

buffer

homogenous

Ultracentrifuge

complexes,

optics

in Verona1

essentially

of 67.5

gave corrected

way to confirm

Fig.1. pattern per ml, r.p.m.;

with

schlieren

10 X 10'

of 0.73

also

of both

RESEARCH COMMUNICATIONS

boundary

E analytical

(S"so,w)

apparatus

lo-'cm2sec"

as a single

model

preparations

and Diffusion

volume

anode

The PDC and KGDC were

ultracentrifugation sedimentation

AND BIOPHYSICAL

from

decreased

of KGDC showed

per

mg protein.

400 to 460 ~qr; the after

addition

maxima at 350,

of 415

Vol. 17, No. 1, 1964

and 455 w, of sodium

BIOCHEMICAL

and a shoulder dithionite

The flavin

D-amino

complexes

were

et al.,

of Kajiro

(1957)

thiamine*

1.5 (PDC)

and 2.5

Thiamirm+P

and the activity

of thiaminemin

a little

response

assay;

its

in the

dismutation

in the dismutation determined

PDC showed

assay.

Neither

fluorometrically of the

of 1.24

and 1.40,

nucleic

acid.

Lipid,

extracted

petroleum

ether,

amounted

into

preparations.

purified

suggesting

PDC contains

and 15 moles

of bound

bound

acid,

lipoic Electron

progress graphs

its

C showed,

structural

hot

by thiamine-PB.

X 10m6 M.

dependence

on added

a detectable

KGDC showed

approximately

staining

various

with

aspects

55

at 280 and 260 q 1.55

and 0.7%

(2:l)

and then weight

of protein-bound 6 moles

of the

lipoic

acid

of protein-

of thiaminePP. PDC and KGDC are procedures. buffered of regular

respectively.

complexes

organization.

Prep-

of the dry

KGDC contains

stained

et al ' (1947). - -..*

methanol-chloroform

of the purified

CoA and DPN DPN as

of approximately

0.8$

All

bound

ratios

50 moles

of both

of

The dissociation

mg protein.

approximately

and positive

studies

PDC was free

per

absorbancy

to approximately

400 s in diameter,

microscope

procedure

was restored

was 4.2

of Levitas,

of FAD and 6 moles

so far,

purified

purification.

showed

presence into

studies

negative

600 1 and

electron

8 moles

the

by the modified

moles

absolute

showed

of PDC and KGDC negatively

cytochrome about

FAD.

microscope

by the

X lo-'

complex

complexes

per mg protein

the c+ketoglutarate.carboxylase

by the method

arations

of recrystallized The FAD contents

moles

assay

assay

was 1.5

of examined

1939).

Highly

PDC during

thiami=PPin

addition

activati4n

was determined

from

after

as FAD by paper

(KtKDc) X lo-'

(1941).

thiamine-PPcontent

preparations

et al.,

the dismutation

to added

identified

and by full

et al.,

was dissociated

constant

1957)

(Negelein,

spectrum

PDC.

tentatively

system

and Green,

thiamixm+PP

for

--et al.,

1957).

RESEARCH COMMUNICATIONS

and difference

to that has been

apo-oxidase

(Beinert,

Thus

similar

(Huennekens,

acid

of the

at 480 w

component

chromatography

AND BIOPHYSICAL

should

now in

Electron

micro-

phosphotungstate globular

structure

The details shed further

and

of light

on

Vol. 17, No. 1, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Reference

Beinert, II., and Page, E., J. Biol. Chem., 225, 479 1957) Goldman, D. S., Biochim, Biophys+ Acta, 32, 60 (1959 Green, D. E., Herbert, D., and Subrahmanyan, V., J. Biol. Chem., 138, 327 1941 Gunsalus, I. C., Dolin, 116.I., and Struglia, L., J. Biol. Chem., 194, 649 1952 I 1 The mechamismof enzyme Gunsalus, I. C., in W. D. McElroy and B. Glass (Editors), action, Johns Hopkins Press, Baltimore, 1954, p. 545. Huennekens, F. M., and Felton, S. P., 'in S. P. Colowick, and N. 0. Kaplan (Editors), Methods in enzymology, Vol. III, Academic Press, Inc., New York, 1957, Pg 950. Jagannathan, V., and Schweet, R. S., J. Biol. Chem., 196, 551 (1952 Kaufman, S., Gilvarg, C., Cori, O., and Ochoa, S., J. Biol. Chem., 03, a 869 (1953) Kaziro, V., J. Biochem., 44, 627 (1957) Koike, M., Reed, L. J., and Carroll, W. R., J. Biol. Chem., 235, 1924 (1960a) Koike, Id., and Reed, L. J., J. Biol. Chem., 235, 1931 (196Ob) Koike, M., Shah, P. C., and Reed, L. J., J. Biol. Chem., 235, 1939 (196Oc Koike, Y., Reed, L. J., and Carroll, W. R., J. Biol. Chem., 238, 30 (1963 1 Korkes, S., de1 Campillo, A., Gunsalus, I. C., and Ochoa, S., J. Bid. Chem., 193, 721 (1951) Korkes, S., de1 Campillo, A., and Ochoa, S., J. Biol. Chem., 195, 541 (1952) Levitas, N., Robinson, J., Rosen, F., Huff, J. W., and Perlsweig, W. A-, J- Bide Chem., 167, 169 (1947) Massey, V., Biochim. Biophys. Acta, 36, 447 1960) Negelein, E., and Bromel, H., Biochem. Z., 3,6 225 (1939) Price, V. E., and Greenfield, R. E., J. Biol. Chem., 209, 363 (1954) J. w., and Bock, R. M., J. Biol. Chem., 197, 851 (1952) Sanadi, D. R., Littlefield, Schweet, R. s., Katchman, B., Bock, R. M., and Jagannathan, V., J- BidChem., 196, 563 (1952) Wagner, A. F., Walton, E., Boxer, G. E., Pruss, K P., Holly, F. W-9 and Folk-s, K* J. Am Chem. Sot., 76, 5079 (1956)

56