The separation and properties of two forms of carnitine palmitoyltransferase from ox liver mitochondria

Vol. 42, No. 5, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE SEPARATIONANDPROPERTIES OF TWOFORMSOF CARNITINE PALMITOYITRANSFERASE FROMOX LIVER MITOCHONDRIA D.W. West, J.F.A. University

Chase and P.K. Tubbs of Cambridge

Department of Biochemistry, Tennis Court Road, Cambridge, England. ReceivedJanuary 22, 1971 SUUlfIl~~ Ox liver mitochondria contain two types of carnitine palmitoyltransferase, which have been separated by ion exchange chromatography and isoelectric focusing. One is readily obtained in soluble form, and is inactivated by 2-bromopalmitoyl derivatives of CoA or carnitine. The other is tightly bound to membranematerial, from which it may be released by butanol, and uses the bromopalmitoyl compoundsas substrates. Other differences in substrate specificity are reported. It is proposed that the former enzyme is the "outerl' transferase of intact mitochondria, which is accessible to added acyl-CoA, while the latter represents the %nner" transferase available only to CoA in the mitochondrial matrix. Undamagedmitochondria will fatty

oxidise the CoenzymeA derivatives

acids only in the presence of carnitine;

mitochondria require carnitine

furthermore,

for the oxidation

acids when these are added in low concentrations albumin.

Fritz

to CoA but not to carnitine

oxidation,

of long-chain fatty in the presence of serum

acids in an outer compartment of the mitochondria,

was separated from the enzymes of P-oxidation

barrier,

isolated

and Yue (1963) proposed a schemein which acyl-CoA, formed

from long-chain fatty

of carnitine

of

or its esters.

palmitoyltransferase

would permit the transfer carnitine

by a barrier

The existence of two pools

(EC 2.3.1.23),

one on each side of the

of acyl groups to the site of S-

acting as a fatty

acyl carrier:

Palmitoyl-CoA + carnitine j palmitoylcarnitine Outer transferase CoASH+ palmitoylcarnitine

impermeable

j palmitoyl-CoA Inner transferase

912

+ CoASH

+ carnitine

Vol. 42, No. 5, 1971

BIOCHEMICAL

AND BIOPHYSICAL

Further work from a number of laboratories

RESEARCH COMMUNICATIONS

has provided support

for a schemeof this general type (for a discussion, see Greville Tubbs, 1968).

Tubbs and Chase (1967) reported that soluble carnitine

palmitoyltransferase

from ox liver

CoA in the presence of carnitine Chase and Tubbs (l%g)), otidation

and

by 2-bromoacyl-

(for the probable mechanismof this see

but not of palmitoylcarnitine,

These results,

that someof the carnitine is inaccessible

inhibited

and that these acyl-CoA analogues abolish the

of palmitoyl-CoA,

mitochondria.

was powerfully

by rat liver

and those of Garland and Yates (19671 show

palmitoyltransferase

to added acyl-CoA,

of intact

mitochondria

in accord with the proposal of Fritz

end Yue. Wenow wish to report the separation end partial two carnitine solubilised

palmitoyltransferases and is inhibited

from ox liver.

to membranematerial,

and Yue;

is not inhibited

represent the inner transferase. palmitoyltransferases their

intramitochondrial

differ

of

One, which is readily

by 2-bromopalmitoyl-CoA,

,ponds to the outer enzyme of Fritz

purification

probably corres-

the other is tightly

bound

by bromoacyl-CoA and is likely

to

It appears that the two carnitine

in their

enzymic properties

as well as in

location. EXPF,RIMEWTAL

Carnitine p"

by following

palmitoyltransferase

the increase in extinction

taining 38 PM palmitoyl-CoA, (2-nitrobenzoic

activity

acid),

was routinely

at 412 nm of a system con-

1.25 mML-carnitine,

100 mMtris-HCl

assayed at

125 ~045,5*-dithio-bis

(pH 8.0) and enzyme. 1 unit of

enzyme catalysed the release of 1 pmole of CoASHper minute in this system. Separation of carnitine Frozen ox liver with 3 vol.

palmitoyltransferase

isoenzymes

(58 g.) was minced and then homogenisedfor 5 min.

of water at 0' using a Polytron overhead blender (Kinematica

GmbH,Lucerne, Switzerland).

After

the supernatant was equilibrated

centrifugation

at 20,000 g for 25 min.,

with 5 mMpotassium malonate, pH 5.7, by 913

Vol. 42, No. 5, 1971

BIOCHEMICAL

gel filtration.

The extract

of carboxymethylcellulose

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(190 ml> was applied to a column (4 x 20 cm.)

equilibrated

with the samemalonate buffer.

column was washed with 300 ml of 5 mMmalonate and further with a gradient of !5+0 collected.

mMmalonate, pH 5.7;

Three peaks of carnitine

protein

50 ml fractions

pslmitoyltransferase

A, B and C in Fig. 1) were found and the fractions

The

eluted

were

activity

‘(labelled

corresponding to each

peek combined.

TUBE No.

Figure 1.

Separation of ox liver carboxymethylcellulose;

carnitine palmitoyltransferases for details see text.

on

Samples of enzyme were preincubated with 2-bromopslmitoyl-CoA in the presence of Lcernitine

before the addition

the transferase reaction

(for conditions see Fig. 2).

B was almost completely inhibited, remained active.

of palmitoyl-CoA

to initiate

The enzyme in peak

but the material from peek A and C

Peek A was a cloudy suspension from which all

palmitoyltransferase

activity

carnitine

could be sedimented by centrifugation

100,000 g for 30 min. at pH 5.7 or 7.1.

at

Peaks B and C contained only

soluble protein. A similar

pattern

of carnitine

obtained when a 0.5% Triton

palmitoyltransferase

X-100 extract

914

of frozen ox liver

activity

was

mitochondria

Vol. 42, No. 5, 1971

BIOCHEMICAL

AND BIOPHYSICAL

I

RESEARCH COMMUNICATIONS

I

/

2 Ill,”

Pm CoA

Cn &Pm CoA

/

Pm CoA

“INNER-ENZYME

Figure 2.

“OUTER“EN~YME

Effect of bromopalmitoyl-CoA on carnitine palmitoyltransferases. The routine assay was used; when present, the concentration of i&2-bromopalmitoyl-CoA was 3 pM. (a) purified %nner" enzyme (see text) incubated with palmitoyl-CoA for 2 min. before starting the transferase reaction with carnitine. (b) %rnerll enzyme incubated with bromopalmitoyl-CoA plus cernitine for 2 min.; reaction started with palmitoyl-CoA. (c> As (a), but using purified "outer" enzyme. (d) As (b), but using "outertl enzyme.

was subjected to electrofocusing

for 3 days at 15' in an LKB-8101 electro-

focusing column using Ampholine pH 3-10 in a 20-60% glycerol.

Enzyme activity

(29%)

points of pH 4.8

and pH 7.5

to inhibition

(66% of the activity

re-

Under the conditions described in

(5%).

Fig. 2, the enzyme preparations with iso-electric 7.5.were insensitive

gradient of

was focused into three regions, corresponding

to material with iso-electric covered), pH 5.7

(v/v)

points at pH 4.8 and

by 2-bromopalmitoyl-CoA,

whereas the

pH 5.7 material was inactivated. Purification

of two forms of carnitine

a> Membranebound enzyme.

paltzitoyltransferase

The material sedimented on centrifugation

of peak A (Fig. 1) at 100,OCC g for 30 min. was suspended in 20 mM triethanolamine-HCl, and recentrifuged.

pH 7.5,

homogenisedwith 0.3 vol.

A clear aqueous solution activity

of cold n-butanol

containing nearly all

the carnitine

palmitoyltransferase

equilibration

with 5 mMmalonate containing 20$ glycerol, 915

of

was obtained, which, after passed un-

BIOCHEMICAL

Vol. 42, No. 5, 1971

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

retarded through a carboxymethylcellulose was

column. Further purification

achieved by chromatography on DE&Z-cellulose at pH 7.5, followed by

adsorption onto and elution

from calcium phosphate gel.

transferase with a specific

activity

in this way;

of 0.4 units&.

it was not inhibited

Carnitine palmitoyl-

protein was obtained

by 2-bromopalmitoyl-CoA (Fig. 2).

b) Soluble enzyme. The supernatant obtained from a homogenateof frozen liver

by centrifuging

at 20,OCOg for 20 min. was treated with 5 mMlead

acetate and the resultant of carnitine

precipitate

palmitoyltransferase,

discarded.

This gave a soluble form

which was purified

chromatography on DEAE- and carbozqmethyl-cellulose, Further purification

and concentration

gels yielded enzyme with a specific protein,

and which was totally

CoA (Fig. 2); that it Nl

this,

by

successive

and cellulose

phosphate.

using calcium phosphate and alumina

activity

inactivated

in excess of 10 units/mg. by treatment with bromopalmitoyl-

and the enzyme's chromatographic behaviour,

indicated

corresponded to peak B of Fig. 1. details

of the purification

pslmitoyltransferase

will

of the two forms of carnitine

be published elsewhere. DISCUSSION

The above results

demonstrate that extracts

of whole liver

mitochondria contain at least two forms of carnitine One of these (peak A of Fig. 1) is tightly This enzyme, even when solubilised

or of

palmitoyltransferase.

bound to membranematerial.

by butanol treatment,

is not inhibited

by incubation with bromopalmitoyl-CoA in the presence of carnitine.

The

other major enzyme form (peak B, Fig. 1) is in a soluble form in crude extracts

and is inactivated

activity

(peak C, Fig. 1;

inhibited

by bromopslmitoyl-CoA. also observed after

by bromopalmitoyl-Cob;

A minor peak of

electrofocusing)

was not

this may perhaps represent material of

peak A type which has become separated from acidic membranecomponents. It

seemslikely

that the enzyme inhibited

corresponds to the "outer't pool of transferase 916

by bromopalmitoyl-CoA in intact

mitochondria,

Vol. 42, No. 5, 1971

i.e.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the enzyme accessible to added acyl-CoA.

Thus both Tubbs and Chase

(1967) and Garland and Yates (1967) found carnitine-dependent acyl-CoA by rat liver palmitoylcarnitine

oxidation,

palmitoyltransferase It

mitochondria to be inhibited

pool to be active,

of

by bromoacyl-CoA, while

which requires only the "inner"

is known that nearly all

of liver

oxidation

carnitine

was unimpaired.

of the carnitine

palmitoyltransferase

mitochondria is associated with the inner-membrane fraction

(Haddock, Yates and Garland, 19701, and that the "inner"

transferase pool

represents the major part of this enzyme in such mitochondria (Garland and Yates, 1967). acylcarnitine inhibiting inhibit

analogue 2-bromomyristoyl-thiocarnitine, the mitochondrial

the "inner"

chondria. carnitine

Furthermore, Tubbs end Chase (1970) found that the

oxidation

pool of carnitine

although powerfully

of palmitoylcarnitine, palmitoyltransferase

did not in intact

mito-

Similar results have been obtained with 2-bromopalmitoyl-L(Eiochem. J. in preparation)

and we have found that,

whereas the

TABLE 1 Acylcarnitine

Specificity

of Carnitine

Palmitoyltransferase

Wutertf Enzyme LCarnitine

Ester

*'Innern Enzyme

K,(M)I

Km(W)

LX t

Acetyl Propionyl mtyry1 Hexanoyl Octanoyl Dodecanoyl Palmitoyl DL-24romopalmitoyl

10 ;z 101

320

2: 100*

:i

Isoenzymes

0 0 0

200

Inhibited

19

19 93 152 100* 22

z 426 60 73

*V max with palmitoylcarnitine taken as 100 (arbitrary units). Rates were measured at 30° in a 2.0 ml system containing 100 mMtris-HCl, pH 8.0, 120 PM CoASH, varied amounts of acylcarnitine and enough of the appropriate enzyme (purified as in the text) to give an extinction change of 0.005-0.050 per minute at 232 nm in a 10 mmlight path.

917

Vol. 42, No. 5, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

peak B (Fig. 1) enzyme is inhibited

by bromopalmitoylcarnitine

presence of CoASH, the peak A enzyme is not. support the view that the non-inhibited "inner"

mitochondrial

Substrate specificity.

transferase

All these considerations

enzyme of peak A represents the

pool.

Table 1 shows the results

of the activity

of the two carnitine

esters of fatty

acids of different

"inner"

of an investigation

palmitoyltransferases chain-length.

enzyme use6 as substrate6 only carnitine

enzyme, however, is active

with fatty

using carnitine

The partially derivatives

with 6 or more carbon atoms; bromopalmitoylcarnitine The "outer"

in the

purified of fatty

acids

is also a substrate.

acids containing 2-12

carbon atoms and, as mentioned above, is inactivated

by bromopalmitoyl-

carnitine. We thank the Medical Research Council for an expenses grant and the Broodbank Fund for a grant to D.W.W. REFERENCES Chase, J.F.A. and Tubbs, P.K. (1969). Biochem. J. 111, 225. Fritz, I.B. and Yue, K.T.N. (1963). J. Lipid Res. -(279. Garland,, P.B. and Yates, D.W. (1967). Mitochondrial Structure and Compartmentation. teds. Quagliarello, E., Papa, S., Slater, E.C. and Tager, J.M. 1, p.385. Bari: Adriatica Edit-rice. Greville, G.D. and Tubbs, P.K. (1968). Essays in Biochemistry (eds. Campbell, P.N. and Greville, G.D.) 4, 155. Academic Press. 565. Baddock, B.A., Yates, D.W. and Garland, P.B. (1970). Biochem. J. 2, Tubbs, P.K. and Chase, J.F.A. (1967). Abstr. 4th. Meeting Fed. Eur. Biochem. sots., Oslo, p.135. Tubbs, P.K. and Chase, J.F.A. (1970). Biochem. J. 116, 34P.

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