ARCHIVES OFBIOCHEMISTRYAND BIOPHYSICS

Vol. 201, No. 2, May, pp. 674-677, 1980

Binding of Creatine Kinase to Heart and Liver Mitochondria in Vitro’

NORMAN HALL AND MARLENE DELUCA

Departments of Medicine and Chemistry, University of California, San Diego, La Jolla, California 92037

Received February 19, 1980

Phosphate extraction of heart mitochondria results in the release of creatine kinase. Under appropriate conditions phosphate-extracted mitochondria are able to rebind the creatine kinase, either from crude extracts or as the purified enzyme. Heart mitochondria are able to bind up to sevenfold more creatine kinase than they originally contained. The association is specific since the cytoplasmic isozyme from heart (MM) does not bind, and does not interfere with the binding of the mitochondrial isozyme even when MM is present in large excess. It is interesting that although liver mitochondria do not contain the mitochondrial isozyme of creatine kinase they are able to bind approximately the same amount of the enzyme as the heart mitochondria.

The existence of a distinct isozyme of creatine kinase (CK,* EC 2.7.3.2) in mitochondria from mammalian heart has been known for some time (1, 2). Evidence suggests that it is located on the outer face of the inner mitochondrial membrane (3, 4). It has been demonstrated that the role of this enzyme is to catalyze the phosphorylation of creatine by ATP produced in the mitochondria so that the end product of oxidative phos- phorylation is phosphocreatine (4, 5). It is therefore an important enzyme in regulating the export of high- energy phosphate compounds from heart mitochondria. The enzyme has been purified and shown to exist in two electrophoretically separable forms. These have been designated m-l, a slowly migrating cathodal form, and m-2, a more rapidly migrating cathodal form (6). While these forms have not been well characterized, it has been suggested that they are a mixture of dimers and polymers which are in equilibrium. This equilibrium is affected by protein concentration and by the presence of a reducing environment (7).

Recently Vial et al. (8) have reported that CK is released from heart mitochondria when they are ex- posed to 20 mM phosphate buffer at pH 7.4. The enzyme reassociates with mitoplasts or partially purified inner membranes if either the phosphate concentra-

1 This research was supported by National Institutes of Health Grant HL17682 awarded by the National Heart, Lung, and Blood Institute, U. S. Public Health, Department of Health, Education and Welfare.

2 Abrreviations used: CK, creatine kinase; CPK, creatine phosphokinase; buffer A, 20 mM potassium phosphate, pH 7.2, 1 mM p-mercaptoethanol; buffer B, 2 mM potassium phosphate, pH 7.2, 1 mM pmercapto- ethanol.

tion or the pH is lowered. Whether this release and rebinding is physiologically important is not clear.

In this paper we report that phosphate-extracted mitochondria from both heart and liver can bind heart mitochondrial CK, both from crude extracts and as the purified enzyme (7). We further report that the binding of CK is specific, since only the mitochondrial isozyme, and not the cytoplasmic MM isozyme, is bound. The binding of the mitochondrial CK is not affected by the presence of excess amounts of the MM isozyme.

MATERIALS AND METHODS

Beef heart and liver mitochondria were isolated by differential centrifugation and washed three times in 50 mM Tris-HCl, pH 7.4, 250 mM sucrose, 1 mM EDTA, and 1 mM ,&mercaptoethanol. Protein was assayed by the method of Bradford (9). CK was assayed by the coupled enzyme method (lo), using Calbiochem CPK- single vial reagent. Mitochondrial CK was purified as previously described (‘7), and the CK-MM was partially purified by DEAE Sephadex chromatography (11).

Mitochondrial extract was prepared by suspending heart mitochondria at 5 mg protein/ml in 20 mM

potassium phosphate, pH 7.2,l mM /3-mercaptoethanol (buffer A), and incubating at 10°C for 20 min. After 10 min centrifugation at SOOOg, the supernatant frac- tion was dialyzed against 2 mM potassium phosphate, pH 7.2, 1 mM &mercaptoethanol (buffer B). Purified mitochondrial CK and CK-MM were dialyzed against buffer B, and the extracted mitochondria were re- suspended and brought to their original volume in buffer B. Extracted liver mitochondria were prepared in the same way.

Binding experiments were carried out using 0.050 ml

0003-9861/80/060674-04$02.00/O Copyright Q 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

674

BINDING OF CREATINE KINASE TO MITOCHONDRIA IN VITRO 675

G;ir-,:-- k-y-y ; 0 4 0 12 0 4 6 12 16 20

Units CK added per mg mitochondrbal protein

FIG. 1. Binding of mitochondrial creatine kinase to mitochondria in vitro. (A) Heart mito-

chondria + mitochondrial extract. (B) Heart mitochondria + purified mitochondrial CK. (C) Liver mitochondria + mitochondrial extract. (D) Liver mitochondria + purified mitochondrial CK.

extracted mitochondria (0.25 mg protein) in a total volume of 1.25 ml buffer B. After 20 min incubation

at 10°C the samples were centrifuged for 5 min at SOOOg and the supernatant fractions were removed. The mitochondria were then resuspended in buffer A.

In the experiments using MM, the mitochondria were washed with 1 ml buffer B before resuspension in

0.5 ml buffer A. Enzyme activities in the supernatant fractions as well as the resuspended mitochondria were

assayed, and the total recovery did not deviate sig- nificantly from 100%.

Separation and quantitation of creatine kinase iso-

zymes on cellulose acetate strip electrophoresis was carried out as previously described (12).

RESULTS AND DISCUSSION

Heart and liver mitochondria were extracted in 20 mM phosphate and subsequently resuspended in

2 mM phosphate. These extracted mitochondria were then incubated with increasing amounts of either crude or purified mitochondrial CK. The amount of CK which

bound to the mitochondria was determined by assaying

the amount of activity which remained with the mito- chondrial pellet. Typical results are shown in Fig. 1. In all cases, the CK activity found to sediment with

the mitochondria rose to a maximum level and then fell slightly with increasing concentrations of mito- chondrial extract or purified enzyme. Both liver and heart mitochondria bound more of the purified enzyme

than crude. The amount of CK released or bound was quite variable for different mitochondrial preparations.

However, the maximal CK activity which would re- associate with heart mitochondria was found to be

about sevenfold the amount extracted from these same mitochondria.

Vial et al. (8) have concluded that the association of mitochondrial CK with the mitochondrial membrane is ionic in nature. Our findings indicate that while the binding sites are not unique to the heart mito-

chondria, they are present in limited numbers on both heart and liver mitochondrial membranes. The binding appears to show Michaelis-Menten saturation kinetics,

with no evidence of the cooperativity reported for the binding of yeast cytochrome b, to rat liver mito-

plasts (13). The reason for the drop in binding at higher con-

centrations of enzyme is not known, but might be

related to the ability of the mitochondrial CK isozyme to form a high molecular weight polymer at high con-

centrations (6, 7). Mitochondrial malate dehydrogenase has been reported to associate with phospholipid

vesicles and with mitoplasts as a monomer, but not when in the polymer form (14, 15). Dissociation into

subunits has also been reported to enhance binding of rat liver mitochondrial ATPase (16).

Cellulose acetate strip electrophoresis was per- formed on a sample of heart mitochondria which had re- bound the maximum amount of CK from the mito-

N

i

1

I I I I I 0 1 2 3

Distance from origin (cm)

FIG. 2. Cellulose acetate strip electrophoresis of CK

bound and resolubilized from beef heart mitochondria. Electrophoresis was carried out for 2l~ h at 300 V, fol- lowed by staining with a coupled enzyme assay mix-

ture, and fluorescent scanning to quantitate the NADPH produced (12).

676 HALLANDDELUCA

20

OltZC’ -a 0 50 100 950 200 250 300

Untts CK-MM added per mg mitochondrml protean

FIG. 3. The binding of mitochondrial (open circles) and MM (closed circles) isozymes of CK to heart mito- chondria. Each incubation mixture contained 4.6 units of purified mitochondrial CK and increasing amounts of MM as indicated. The increase in the mitochon- drially associated activity of each isozyme over that for controls in which no isozymes were added is expressed as the percentage of the activity of that isozyme added to the incubation mixture.

chondrial extract, and was then resolubilized in 20 mM phosphate (Fig. 2). Four peaks were found, with 73% of the activity contained in the two mito- chondrial forms (52% m-2, 21% m-l). CK-MM com- prised 12%, and unsolubilized activity remaining at the origin comprised 15% of the activity. In our experience, even well-washed mitochondria have sig- nificant amounts of CK-MM which is released in phos- phate much more slowly than is the mitochondrial isozyme. Whether this MM is associated with mitochon- dria, or with contaminating myofibrils, is not known.

As a control for the specificity of binding, experi- ments were performed to test whether MM would also bind under similar conditions, or if it would interfere with the binding of the mitochondrial iso- zyme. Extracted heart mitochondria were incubated in mixtures containing a concentration of purified mito- chondrial CK sufficient to bind the maximum activity, plus partially purified MM isozyme in increasing amounts. Electrophoresis of the bound and resolubilized CK revealed four peaks as described above. The rela- tive proportions of the MM and the mitochondrial isozyme activities bound were calculated. The results in Fig. 3 show little or no binding of MM. It is also apparent that the presence of excess MM does not interfere with the binding of mitochondrial CK. Each incubation mixture contained 4.6 units of purified mitochondrial CK per milligram mitochondrial protein. With 64-fold as much MM activity added, more than 70% of the mitochondrial isozyme remained bound. Less than 0.5% of the added MM was found bound to the mitochondria. Similar results were obtained when the dialyzed extract, rather than purified mitochondrial isozyme, was used.

The ability of mitochondria to bind the mitochondrial

CK isozyme, but not the cytoplasmic MM isozyme, is similar to the case of aspartate aminotransferase. This enzyme is found to cross the inner mitoehondrial membrane in response to certain effector molecules (17,18). Addition of purified enzyme from mitochondria causes an increase in intramitochondrial enzyme activity, but no increase is seen when purified cyto- plasmic isozyme is used (19). This contrasts with the case of tyrosine aminotransferase, where the cyto- plasmie isozyme is seen to be taken up by mito- chondria in vivo under certain conditions. This is ap- parently due to an alteration in the cytoplasmic isozyme, rather than in the mitochondria (20).

The role of the release and reassociation of the mito- chondrial isozyme in the physiological function of mitochondria in vivo is unclear (8). The ability to release and rebind the isozyme in vitro may, however, provide a tool for the further experimental investiga- tions of the function of the enzyme in mitochondria and the nature of its association.

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