Purification and properties of beef heart muscle “cytoplasmic” malic dehydrogenase

Vol.&

No.3

BIOCHEMICAL

,PURIFICATION

AND

AND

PROPERTIES

Sasha

Englard,

September

We have of beef Englard,

muscle

1960).

used

for

reported which

and

1959).

BUcher,

dehydrogenase

and

1960).

**

which

from

in agreement

BUcher,

kinetically

powders

by

acetone-dried

reported

Wolfe

and

of ox heart Neilands

differences powders

from the and from

(19%)

in molecular of ox heart

National

the National

Predoctoml Fellow on Interdisciplinary Health, United States Public Health

similar

Kun

mitochdndria, from

whole

properties mitochondria

Institutes of Health, Science Foundation

Gmnt Service. 323

12M-6418,

report-

malic beef

by Straub

for

thus

isolated

more

closely

pig

heart

acetone

the

and

resembled powders.

dehydrogenase “mitochondrial”

United States (G-9748). National

(Siegel obtained,

and

and

heart

(1942)

the enzyme

of malic

are

&r&ch

of whole

dehydrogenase

(1957)

inhibition

“mitochondrial”

described

and

procedures

Schimassek,

like

malic

for

observations

powders

originally

(Siegel

dmstic

DelbrfJck,

of acetone-dried

procedure

in minces

capacity

relatively with

1959;

behaved

extmcts

of the

of the marked

by gmnts (RG-4428)

and

dehydrogenases by oxaloacetate

when

and

acetone-dried

Supported Service

Zebe

are

only

by Davies

from

*

findings

minces

those

purified

prepared

fresh

of the

from

from

MALIC

Breiger

by its strong

properties

greatly

isolated

H.

malic

recognized

The molecular

differed

In view

from

The enzyme

the essentials

enzyme

“CYTOPLASMIC”

to inhibition

enzyme

(Delbrllck,

was purified

Englard,

Hilda

of two

In sensitivity

These

by following

the

differ

soiubilization. tissues

and

the occurrence

is extracted

ed for other

MUSCLE

1960

Department of Biochemistry Einstein College of Medicine Yeshiva University New York 61, New York

A “mltochondrial”

by oxaloacetate,

Siegel**

Sept.

COMMUNICATIONS

9, 1960

recently

heart

RESEARCH

OF BEEF HEART DEHYDROGENASE* Lewis

Albert

Received

EIOPHYSICAL

Institutes

Public

Health

of Mental

Vol.3,No.3

BIOCHEMICAL

enzyme

obtained

heart,

AND

from

acetone-dried

it was of interest

“cytoplasmlc”

enzyme. of the

therefore,

reports

the

mince

beef

from

hearts

stirred

layers

of cheese

cloth

with

solid

the

pH wlls

The

40 to 82 percent

ammonium

with

brought

between

(EDTA)

pH 7.4

solution

fate

fraction

ws

and

subsequently

dialyzed

column traces

equilibrated

by this

A similar

unretarded

cellulose

has recently

procedure behavior been

raised

0.035!

wlth

water.

O.OOSE enzyme

since of rat reported

this

enzyme

liver

was maintained

saturation

with sulfate

was passed

at this

was not

retained mallc The

was added

EDTA

sul-

pH 6.4

through

a DEAE-

EDTA

pH 6.4. were

DEAE-cellulose.

dehydrogenase

“cytoplasmic”

ammonium

of purification

by the

by

ammonium

+ 0.0005M stage

was

was removed

solution phosphate

to 35 per-

solution

+ 0.0035&

present

1960). 324

the

hydroxide.

supematant

phosphate

“mitochandrial”

(Thome,

clear

The 45 to 72 percent

potassium still

fmctionations

brought

and ammonlum

This

several

+ O.OOlM -

precipitate

to 45 percent

potassium

with

the

pH 7.6

ammonium

dialysis

and

The

saturation

sulfate

phosphate

prior

bath

grinder.

82 percent

of dilute

the bulky

saturation.

of “mitochondrial”

eliminated

coaling,

to 72 percent

fold

addition

water

by centrifugation

seven

“cytoplasmic”

through

ammonium

w,ithaut

removed

against

diluted

communication,

triethanolamine

-

40 and

centrifugatlon,

in a boiling

supernatant

solution

heart

proposed

present

was decanted

potassium

and

After

the

was

the

beef

beef

a mechanical

+ O.OlM

subsequent

In 0.05fi

sulfate.

through

sucrose

by the

was dissolved

by immersion

precipitate

for

of purified

between

and 7.4

and

supematant

last

and

After

to the

The

this

7.2

passed

suspension

During

10 minutes.

The

of whole

of the

The

properties

(r.25M The

for

sulfate.

cellulose

of cold

62 to 64O

centrifugation

and

fractionated

ammonium

to 600

properties

consideration.

filtrate

between

tetraacetate

saturation

rapidly

the

residue

minces

is a prerequisite

kinetic

diced

15 minutes.

sulfate.

maintained

ethylenedlamine cent

and

a study

some

defatted,

for

washed

Sept. 1968

heart.

in 5 volumes

mechanically

of thoroughly

under

and

COMMUNICATIONS

physico-chemical

such

enzymes

beef

were

was suspended

and

various

preparation

dehydragenase

Fresh

the

In addition,

studies

RESEARCH

powders

to investigate

structural

malic

BIOPHYSICAL

malic

on DEAEdehydro-

Vol.3,No.3

genase mte

was eluted

with

were

pooled

enzyme

barbital component

purified

hour.

Fractions

of an increased

and

the

enzyme

isolated

ever,

unlike

potassium affected

the

anode

presence (0.5M -

showed

enzyme at high

“cytoplasmic” x 10m2M -

sulfate.

strength

electrophoretic + 0.001

specific

beef

heart

muscle

increased

activity

M - EDTA.

activity

purified

when

“mitochondrial”

(Table

I).

How-

in 0.05M -

“cytoplasmic”

enzyme

is un-

x 10m4M -

Englard,

in O.lM

tris

with

1960).

an anamolous

appears

malic

(Siegel

to be sensitive

(hydroxymethyl)

the enzyme

acceleration Englard, to levels aminomethane

as 1.9

is in marked

dehydrogenase,

evi-

purified

In addition,

and

as high

This

enzyme.

of “mitochondrial” of 2.7

and

of oxaloacetate

“cytoplasmic”

of L-malate

dehydrogenase assayed

ionic

as a single

the

or

ammonium

in 0.038

than

concentrations

on the

exhibits

concentrations

malic when

phosphate),

(Siegel

which

of 5 fold

with

phosphate

the

at a flow

phosphate.

inhibition

heart

activity

1955)

showed

Sept. 1960

pH 6.4

had a higher

of whole

which

concentration

beef

potassium

glycylglycine),

effect

characteristic

of whole

“mitochondrial” reduction

or without

potassium

moved

(Ochoa,

powders

of added

at an oxaloacetate

powders

glycylglycine

no inhibitory

the

obtained

dehydrogenase

enzyme

specific

electrophoresis

in 0.05E

malic

acetone-dried

(with

thus

EDTA

by precipitation

block

enzyme

at pH 7.1

in 0.025M -

phosphate*

with

by starch

The

+ 0.002M -

concentmted

“mitochondrial”

At pH 6.7

contmst

purified

from

in the

x 10m3M -

enzyme

“cytoplasmic”

at pH 7.5

*

per

the

assayed

1.6

phosphate

at pH 8.5.

toward

The

dent

potassium

was further

buffer

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0.02M -

of 10 to 12 ml.

greater The

BIOCHEMICAL

unlike of the 1960)

mte

from the beef

acetone heart

of DPN+

the beef

of L-malate

heart exceeding

at pH 8.4.

The malic dehydrogenase obtained from whole pig heart acetone powders behaves in o similar manner and this effect has also been obtained with sulfate, arsenate, maleate, EDTA, etc. and appears to be due to the increased ionic strength of the assay medium. The enzyme obtained directly from acetone-dried powders of beef heart mitochondria also shows increased activity when assayed in glycylglycine in the presence of potassium phosphate. It thus appears that the stimulatory effect of added salts is characteristic for the enzymes which behave kinetically like “mitochondrial” malic dehydragenase.

325

Vol.3, No.3

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Sept.

1960

Table 1 Comparison

of Purified Beef Purified “Mitochondrial” Acetone-Dried

Heart

“Cytoplasmic” Malic Dehydragenase Malic Dehydrogenase Isolated from Powders of Whole Beef Heart Turnover

Conditions

of Assays

“Cytoplasmic” enzyme

with

Number* “Mltochondrial” enzyme**

DPNH OXIDATION WITH OXALOACETATE: a) in 0.025M glycylglycine pH 7,5(l) b) in 0.025% glycylglycine + 0.05M potas.siuGphosphate pH 7.5(T) c) in 0.05M triethanolamine pH 7.6(2) d) in O.Srpotassium phosphate pH 6. %3) -

817

585

817 701 487

1493 1088 738

DPNH OXIDATION WITH MESOXAIATE: a) in 0.5M potassium phosphate pH 6. 7(4)

13

63

94

527

87

115

DPN+ REDUCTION WITH L-MALATE: a) in 0.09M glycine-NaOH pH 9.9(5) b) in 0.1 firis (hydroxymethyl) aminorYi&ane pH 8.4(6) DPN+ REDUCTION WITH a) in 0. lIvJglycine-NaOH

MESO-TARTRATE: pH 9.9(7)

0.32

2.33

DPN+ REDUCTION WITH a) in 0.1 M glycine-NaOH -

L-TARTRATE: pH 9.9(8)

0.44

4.27

*

**

Expressed as pmoles of either DPNH oxidized protein, as calculated from the initial reaction photometer. cf. Siegel and Englard (1960).

the

or DPN+ reduced rates recorded

per minute with a Cary

per mg of Spectro-

In addition, each of the above reaction mixtures contained: (1) 0.76 pmoles of oxaloacetate and 0.26 mg of DPNH in a total volume of 3.02 ml. (2) 0.38 pmoles of oxalaacetate and 0.39 mg of DPNH in a total volume of 3.02 ml. (3) 6.4 pmoles of EDTA, 0.76 pmoles of oxalaacetate and 0.31 mg of DPNH for the “cytoplasmic” enzyme assay and 0.26 mg of DPNH for the “mitochondrial” enzyme asSay, in a total volume of 3.02 ml. (4) 6.4 pmoles of EDTA, 25.0 pmoles of mesoxalate and 0.26 mg of DPNH in a total volume of 3.01 ml. (5) 300 pmoles of sodium-L-malate and 7.36 pmoles of DPN+ in a total volume of 3.02 or 3.05 ml. The concentmtion of L-malate used was somewhat inhibitory for the “cytoplasmic” enzyme. Under the same conditions in the presence of only 50 pmoles of sodium-L-malate the “cytoplasmic” enzyme yielded a turnover number of 118. (6) 250 pmoles of sodium-L-malate and 8.61 pmoles of DPN+ for the “cytoplasmic” enzyme assay and 11.77 pmoles of DPN+ for the “mitochondrial” enzyme assay, in a total volume of 16.0 or 16.1 ml. (7) (8)

500 pmoles of sodium-meso-tartmte 3.02 or 3.1 ml. 500 pmoles of sodium-L-tartmte or3.1 ml.

and and

7.36

8.37

326

pmoles

pmoles

of DPN+

of DPN+

in a total

in a total

volume

volume

of

of 3.02

Vol.3,

No.3

BIOCHEMICAL

Table plarmic” the

I also and

oxidation

several

AND

shows

other

kinetic

“mitochondrial” and

of DPNH

as assayed

RESEARCH

differences

enzymes

reduction

substrates

BIOPHYSICAL

between

as evidenced and

in various

COMMUNICATIONS

by the

DPN+,

buffers

the

purified different

respectively, at different

Sept. 1960

beef

heart

relative

in the

“cyto-

rotas

presence

for

of

pH values.

References Davies,

D.D.,

and

Kun,

Delbrtlck,

A.,

Zebe,

DelbrUck,

A.,

Schimassek,

Ochoa,

E.,

Biochem.

and

BLlcher,

H.,

J., T.,

Bartsch,

g,

307

L., and (1960).

Stmub,

F.B.,

Z.

Physiol.

Thome,

C. J. R.,

Biochem.

R.G.,

Englard,

and

Neilands,

S.,

Biochem.

Chem.,

K.,

and

J.B.,

331, -

BUcher,

T.,

Biophys.

-275,63

273

Biol.

z, Chem.,

327

175

(1959).

Biochem.

Z.,

331, -

297

Methods

in Enzymology,

1, 735,

Research

Communications,

vol.

(1942). Acta,

J.

Z.,

N.O.,

and

et Biophys.

(1957).

Biochem.

S., in Colowick, S.P., and Kaplan, Academic Press, Inc., New York (1955).

Siegel,

Wolfe,

E.,

(1960). -221,61

(1956).

(1959).

3, %,