The role of malic enzyme in bovine adrenal cortex mitochondria

Vol. 31, No. 1, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE ROLEOF MALIC ENZYMEIN BOVINEADRENALCORTEXMITOCHONDRIA* Evan R. Simpson+, Wendy Cammerz,and Ronald W. Estabrook Department of Biophysics and Physical Biochemistry Johnson Research Foundation University of Pennsylvania, Philadelphia 19104

Received March 7, 1968 The oxidative first

decarboxylation

of malate to pyruvate and carbon dioxide was

observed by Moulder, Vennesland, and Evans (1945) and by Ochoa, Mehler,

and Kornberg (1947). reverse direction

The possibility to effect

that the reaction might operate in the

the fixation

Ochoa, Mehler, and Kornberg (1.948).

of carbon dioxide was suggested by

The enzyme catalyzing

enzyme (L-malate : NADPoxidoreductase (decarboxylating) fied frcm pigeon liver

and characterized

was found to occur in the cytosol fraction intracellular

location,

this reaction, 1.1.1.40),

by Hsu and Lardy (1967). of pigeon liver.

malic

was puriThe enzyme

Because of this

Young, Shrago, and Lardy (1964) suggested that in liver

and adipose tissue malic enzyme activity

was important as a source of NADPHfor

lipogenesis. Steroid mixed-function

oxidases also require NADPH,but in the adrenal

cortex three of these enzyme reactions are mitochondrial, hydroxylase terol

(Sweat, 1951; Brownie and Grant, 1954), 18-hydroxylase

side-chain cleavage system (Psychoyos, Tallan,

Halkerston,

namely the llg-

Eichhorn, and Hechter, 1961).

and choles-

and Greengard, 1966;

The source of NADPHfor these sys-

tems is not known, but evidence has been presented to suggest that the mixedfunction

oxidase electron transport

linked to the main respiratory

chain of adrenal cortex mitochondria is

chain by an energy-dependent transhydrogenase,

* Supported in part by U.S.P.H.S. Research Grants GMI2202 and 2G277. + Permanent address: Department of Biochemistry, Medical School, University Edinburgh, Great Britain. $ Present address: Department of Pharmacology, Albert Einstein College of Medicine, The Bronx, New York

113

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BIOCHEMICAL

and that reducing equivalents

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

generated during succinate oxidation

coupled to this by reversed electron of the respiratory 1966; Koritz,

flow along the NADH-dehydrogenaseportion

chain (Harding and Nelson, 1966; P&on, McCarthy, and Guerra,

1966; and Hall,

1967).

In contrast,

the generation of NADPHfor l&3-hydroxylation enzyme activity

can be

Grant (1956) suggested that

might be associated with malic

in the mitochondria of the adrenal cortex.

The purpose of this communication is to show that bovine adrenal cortex contains two distinct

malic enzymes, one in the cytoaol and one in the mito-

chondria, and to present evidence that the mitochondrialmalic is the source of NADPHfor llg-hydroxylation. linked reactions

enzyme activity

The hypothesis that energy

occurring during auccinate oxidation

play a significant

in the generation of NADPHis not supported by the results

role

presented in this

paper. Methods Bovine adrenal cortex mitochondria were prepared as previously (Cammerand Eatabrook, 1967).

Malic enzyme activity

NADPHformation using an Bppendorf fluorimeter ment as described by Estabrook and Maitra

described

was assayed by meansof

with compensating voltage attach-

(1962).

Alternatively,

the increase

in absorption at 340 rnp was observed using an Aminco-Chance dual wavelength split

beam spectrophotameter.

oxygen electrode

Oxygen uptake was determined using a Clark

(Clark, Wolf, Granger, and Taylor,

was by the method of Estabrook and Maitra

1963).

Pyruvate estimation

(lg&).

Results and Discussion -Differential

centrifugation

of a hanogenate of bwine adrenal cortex

revealed that malic enzyme activity

was located in the mitochondria as well as

in the cytosol.

in the mitochondria was about one-fifth

The total

activity

that in the cytoaol, the cytoaol respectively).

whereas the specific activity was about one-half that in -1 -1 -1 mg prot-1 and 40 rnp moles (25 rnp moles min min mg prot The mitochondrial

activity,

howwer, was only manifest when the

mitochondria were subjected to ultraaonication,

114

which aolubilized

the enzyme,

Vol. 3 1, No. 1, 1968

thus indicating

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that the enzyme was located within

not merely absorbed on the outer surface, i.e.

the mitochondrion and was

the mitochondrial

activity

was

not due to contamination fran the cytosol enzyme. The mitochondrial have been purified

and cytoplasmic malic enzymes of bovine adrenal cortex

by ammoniumsulphate fractionation

Sephadex G-100 and DEAE-cellulose. purified

seventy fold.

In this way, the mitochondrial

That the bimodal distribution

in the tissue was due to two distinct their

and by chrcmatography on

of malic enzyme activity

species of enzyme protein was shown by

chromatographic behaviour on DEAE-cellulose (Fig.

behaved similarly elution

to the pigeon liver

of the mitochondrial

Kinetic mitochondrial

enzyme was

1).

The cytosol enzyme

enzyme of Hsu and Lady

enzyme required a buffer

studies indicated a further

difference

enzymes. Measurements of the ratio

(1967), but

of higher ionic strength. between the cytoplasmic and

of the maximuminitial

veloci-

ties for the forward and reverse reactions:

malate + TPN+indicated marked differences

forward

pyruvate

+ CO2 + TPNH+ H+

for the two enzymes. The ratio

PJmax1reverSe for the mitochondrial

Wmax)fomara/

enzyme was 105, whereas the ratio

for the

cytoplasmic enzyme was 2.5. An interesting

property

of the mitochondrial

tion observed in the presence of the respiratory dicumarol.

The inhibition

enzyme was the strong inhibiuncouplers dinitrophenol

by dicumarol was apparently

to malate with a Ki of 15 $I.

(Ki for dicumarol = 51 $4).

presence of a malic enzyme in the mitochondrial

fraction

uncoupling agents raised the question of the validity that NADPHwas generated during succinate oxidation electron transport, critical

competitive with respect

The supernatant enzyme was also inhibited

these reagents, but to a much smaller extent

inhibited

The

by

via energy-linked

reverse

considered most

of this pathway, is the inhibitory

115

by

of the interpretation

since one of the experimental results,

for the evaluation

and

effect

of

Vol. 31, No. 1, 1968

BIOCHEMICAL

8

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1’6 24

32 40

Fraction

Number

48

54

Figure 1. Chrcmatography of a mixed sample of mitochondrial and cytosol malic enzymes on DEAE-cellulose. O.lml samples fran each fraction were assayed in a final volume of 3 ml in the presence of 8 mML-malate, 300 fl TPN, and 5 mM Mgc12. s-

cytosolmalic

M- mitochondrial

enzyme. malic enzyme.

uncouplers on the succinate-supported and McCarthy,

ll&hydroxylation

of DOC(Guerra, P&on,

1966).

Experiments were then carried out to measure pyruvate formation during malate-

or succinate-supported

mitochondria. cxidase

and rotenone to inhibit

in Figure 2, the llg-hydroxylation of pyruvate

rate of pyruvate

situation

reaction,

NADHdehydrogenase.

As

pyruvate can be seen

of DOCin the presence of succinate produced

formation as a result

formation was sufficient

the hydroxylation A similar

of DOCby adrenal cortex

Arsenite was added to the reaction mediumto inhibit

activity,

a stimulation

l&3-hydrcncylation

of malic enzyme activity.

to provide all the IiADPH required for

as measured by the stimulation

was found if rotenone was omitted.

obtained when malate was used as substrate.

The

of oxygen uptake.

Canparable results were

Thus bovine adrenal cortex contains,

in addition to the normal cytoplasmic enzyme, a second malic enzyme located in

116

Vol. 31, No. 1, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Mitochondrio

/JLx7?&

Minutes

Figure 2. Oxygen uptake and pyruvate formation during succinate-supported IL@hydroxylation of DCC. Oxygen uptake was measured in the chamber of a Clark oxygen electrode. In a parallel experiment sampleswere taken for pyruvate Concentrations in the final mixture were: estimation as described in the text. 8m~ succinate; 135 $4 DOC; 2 mMarsenite; and 3 pg/ml of rotenone. Oxygen uptake is shown as a continuous line, pyruvate formation as solid circles. The num!frs on the figure are rates of oxygen uptake or pyruvate formation in $4 min .

the mitochondria, mixed-function

the function

reactions.

uncoupler-sensitive

of which is to provide NADPHfor mitochondrial

Previous conclusions regarding the role of an

energy-dependent succinate-linked

reduction of NAD+,

coupled with an energy-dependent transhydrogenase for formation of NADPH,need not be considered as an obligatory

requirement for the mixed-function

oxidase

reaction.

REFERENCFaS Brownie, A. C., and Grant, J. K., Biochem. J. x, 255 (1954). Cammer,W., and Estabrook, R. W., Arch. Biochem. Biophys. z, 721 (1967). Clark, L. C. Jr., Wolf, R., Granger, D., and Taylor, Z., J. Appl. Physiol. _6, 189 (1953). Estabrook, R. W., and Maitra, P. K., Anal. Biochem. 2, 369 (1962). Grant, J. K., Biochem. J. 64, 559 (1956). Guerra, F., P&on, F. C., and McCarthy, 3. L., Biochim. Biophys. Acta llJ, 433 (1966). Halkerston, I. D. IL, Eicbhorn, J., and Hechter, O., J. Biol. C&m. 236, 374

(1961).

Hall, P. F., Biochem. Biopbys. Res. Ccmmun.6, 320 (1967). Harding, B. W., and Nelson, D. H., J. Biol. Chem.2&, 2212 (1966).

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Hsu, R. Y., and Lardy, H. A., J. Biol. Chem.242, 520 (1967). Koritz, S. B., Biochem. Biophys. Res. Ccmmuu.3, 485 (1966). Moulder, J. W., Vennesland, B., and Evans, E. A. Jr., J. Biol.

Chem.160, 305

Ochoa(1z45)* ., Mehler, A. H., and Kornberg, A., J. Biol. Chem. Ochoa' S Mehlrr, A. H., and Kornberg, A., (l&j: P&on: FI'C., McCarthy, J. L., and Guerra, I?., Biochim. Biophys. Acta 3, (1966).

Psychoybs, S., Tallan, H. H., and Greengard, P., J. Biol. Chem.2& 2949 0966). Sweat, M. L., J. Am. Chem. Sot. z, 4056 (1~1). Young, J. W., Shrago, E., and Lardy, H. A., Biochemistry 2, 1687 (1964).

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