Cardiovascular Research, 1977, 11, 250-259

Subcellular compartmentation of creatine kinase isoenzymes in guinea pig heart' E . A. O G U N R O , T. J. PETERS, A N D D . J . H E A R S E 2 From the Cardiovascular Research Unit and Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, and the Myocardial Metabolism Research Laboratories, The Rayne Institute, St Thomas' Hospital, London SUMMARY In an attempt to determine whether a subcellular compartmentation of creatine kinase exists and if so, whether there is a differential distribution of the 3 isoenzymes of CK in the cell, studies were carried out with the guinea pig heart which had been subfractionated by either isopycnic density gradient centrifugation or differential pelleting. Isoenzyme analysis of C K in the isolated subcellular fractions by electrophoresis on agarose gels, revealed that the MM isoenzyme occurred in the cytosol, myofibrils, and the microsomes while the MB isoenzyme (which is cardiospecific) was only found in the cytosol. Trace amounts of the BB isoenzyme were detected in the cytosol. Considerable CK activity was associated with the mitochondria, this did not represent the MM, the MB, or the BB isoenzymes but was a distinct and additional mitochondrial-specific form of CK. pH optima and kinetic studies were carried out t o characterise and distinguish the mitochondria] isoenzyme from other CK isoenzyme activity. The evidence for a differential compartmentation of MM, MB, BB, and mitochondrial CK is discussed in relation to their possible cellular roles.

Current interest in the mechanisms of cellular damage and enzyme release, together with speculation concerning the possible cellular roles of the various creatine kinase (ATP: creatine phosphotransferase, EC isoenzymes has led us to investigate whether a subcellular compartmentation of creatine kinase (CK) exists and whether there may be a differential function-related distribution of the various isoenzymes within any compartment. Creatine kinase catalyses the reversible transfer of a phosphate group from ATP-Mg2- to creatine: ATP-Mga- fcreatine + ADP-Mg- fcreatine phosphatea- H+ The reaction proceeding in the direction of creatine phosphate synthesis has been designated the forward reaction whilst that proceeding in the direction of ATP synthesis has been designated the reverse react ion. At least 2 specific roles have been assigned to CK i n mammalian heart cells. Firstly cytoplasmic CK

is responsible for mediating cellular energy stores where high energy phosphate groups are transferred from creatine phosphate to ADP, producing ATP for cellular energy requirements (Jacobus and Lehninger, 1973a). Secondly, it has been suggested (Gudbjarnason et al., 1970; Vial and Gautheron, 1973; Jacobus and Lehninger, 1973a; Saks et al.. 1974) that mitochondrial CK is involved in the transfer of intramitochondrial high energy phosphate to the extra-mitochondria1sites of energy utilisation. Cytosolic CK has been shown to exist as 3 distinct isoenzymes (Dawson et al., 1968) designated the MM, the MB, and the BB forms (Dawson et a/., 1965) and these exhibit differing tissue distribution patterns (Dawson and Fine, 1967; Smith, 1972). A mitochondrial CK has been described and although it had previously been thought (Dawson. 1970) that this represents cytoplasmic CK absorbed to the mitochondria, electrophoretic studies (Sobel et al., 1972; Farrell et al., 1972; Saks et al., 1974; Somer et a/., 1974) suggest that this form may be distinct 'This work was carried out with the aid of grants from the from any of the cytoplasmic isoenzymes. Attention British Heart Foundation, the Wellcome Trust and the has been focused on CK by the observation that Vandervell Foundation. cellular damage results in its leakage from the cell 'Address for correspondence and reprints: Dr D. J. Hearse, into the circulating blood and its measurement is Myocardial Metabolisni Research Laboratories, The Rayne routinely used in the diagnosis and assessment of a Institute, St Thomas' Hospital, London SEI 7EH. 250


Subcellular compartmentation of CK isoenzymes

variety of disease states. The reported cardiospecificity (Van Der Veen and Willebrands, 1966; Smith, 1972; Roberts et a/., 1975) of the MB isoenzyme makes it potentially very valuable for the specific detection and assessment of myocardial cell damage (Roberts et al., 1975; Ogunro et al., 1976). In an attempt to learn more about the nature and progression of cellular damage, it would be valuable to know whether there is any differential loss of the various isoenzymes from damaged cells and whether in fact they may exhibit any intracellular compartmentation, possibly with different isoenzymes being associated with different metabolic roles. The aim of the studies reported in this paper were threefold: firstly, to compare the activity of CK present in each of the subcellular fractions isolated from mammalian heart cells; secondly, to identify the isoenzymes associated with each subcellular fraction ; and thirdly to characterise any differences in the pH optima and kinetics of the cytosolic and mitochondria1 isoenzymes.

Materials and methods


Hearts were rapidly excised from guinea pigs which had been anaesthetised with a mixture of halothane and air; 1 g of left ventricle was sectioned and homogenised in 10 cmy of ice cold 250 mmol.litre-1 sucrose containing I mmol*litre-* disodium EDTA. pH 7.4 and 20 mmol.litre-l ethanol (SVE medium) with a loose fitting type A Dounce homogeniser. was centrifuged at 800g for The homogenate (HI) 10min and the post nuclear supernatant was decanted and stored on ice. The pellet (PI) was rehomogenised in 5cm3 of SVE medium and the resulting homogenate (H,) was again centrifuged at 800g for 10 rnin. The supernatants were cornbined, heparin was added to give a final concentration of 50 I U - C ~(in- ~order to improve resolution of the organelles; Bloomfield and Peters, 1974) and the pellet (Pl)which contained the myofibrils, unbroken cells and connective tissue was retained for preparation of the myofibrillar fraction. The pooled supernatant fraction was further subfractionated by 1 of 2 methods: ( I ) lsopycnic density gradient centrifugation or (2) differential pelleting.


Male guinea pigs (weighing 400-600 g) of the Dunkin-Hartley strain were obtained from Bantin and Kingman Ltd, Grimston. All chemicals were of the highest analytical grade commercially available and were obtained from either Sigma Ltd (London), British Drug Houses Ltd (Poole) or Koch-Light Laboratories Ltd (Colnbrook). Biochemicals and enzymes were obtained from The Boehringer Corporation (London) Ltd, radioactively labelled biochemicals were obtained from The Radiochemical Centre Ltd (Amersham) and agarose (A-37 indubiose) was obtained from Microbiolabs Ltd (London). UV spectrophotometry was carried out with a Cecil 272 system spectrophotometer, fluorometry was carried out with a Farrand Mark I spectrofluorometer and a Beckman scintillation counter (type LS250) was used for the determination of radioactivity. Homogenisation procedures employed the Dounce (Kontes Glass Co, Vineland, New Jersey) or the Polytron (Kinematica GmbH, Lucerne, Switzerland) PT 10 OD homogenisers, refractometry was carried out with an Abbe refractometer (Bausch and Lomb Incorporated, Rochester, New York) and electrophoresis was carried out using a Shandon U77 electrophoresis chamber with a refrigerated platten (Shandon Southern Instruments Ltd, Camberley). A Vokam dc power supply (Shandon Southern Instruments Ltd) was used to deliver a constant voltage of 110 V at a current of 25-35 mA per gel.

( I ) Isopycrric density gradient centrifugatioti Two types of gradient were employed: (a) Steep gradient: 5.0 cm3 of the postnuclear supernatant fraction was layered onto a 24 cmy sucrose gradient (containing 50 IU of heparin per cm3)extending linearly with respect to volume, from a density of 1.05-1.28 and resting on a sucrose cushion of density 1.32 in a Beaufay automatic zonal rotor (Beaufay, 1966). The rotor was run at 35000rpm for 35min at 4°C. Sixteen fractions were collected and weighed. After careful mixing, the density of the sucrose in each tube was determined indirectly by refractometry. (b) Shallow gradient: 5.0cm3 of the postnuclear supernatant fraction was layered onto a 24cm3 sucrose gradient (containing 50 IU of heparin per cm3) extending linearly with respect to volume from a density of 1.05-1.20 and resting on a sucrose cushion of density 1.32 in a Beaufay automatic zonal rotor. The subsequent experimental procedure was identical to that described in (a). A number of marker enzymes (characteristic of the principal organelles) were asssyed throughout the sucrose density gradient in order to determine the distribution of the organelles themselves. Cytochrome oxidase was assayed as a marker for mitochondria, 5’ nucleotidase for plasma membrane, neutral a-glucosidase for endoplasmic reticulum and a - hydroxybutyrate dehydrogenase for cytosol (Bloomfield and Peters, 1974). In addition protein, CK (by the reverse reaction), and CK isoenzymes

252 were assayed. CK isoenzyme distribution was compared with that of the marker enzymes for the individual organelles. As will be discussed later in relation to the myofibrillar marker enzyme, a completely specific enzymic marker is not always available for each subcellular organelle. While a number of good microsomal marker enzymes (eg, glucosed-phosphatase and rotenone insensitive cytochrome c reductase) are available for liver and other tissues, these enzymes are not present in significant amounts in the heart. Therefore neutral a-glucosidase, an enzyme which exhibits significant amounts of activity in both cardiac microsomes and cytosol (Bloomfield and Peters, 1974) was used as the microsomal marker enzyme. Subsequent interpretation of the results was therefore made in the light of this fact. (2) Differential pelleting The postnuclear supernatant was subfractionated into mitochondria1 and microsomal fractions by centrifugation (4°C) at 25 OOOg for 10 min and 105 OOOg for 60min respectively. The mitochondrial and microsomal pellets were each washed twice with the SVE medium to minimise the cross contamination of these fractions with cytosol proteins. The myofibrillar fraction was prepared by a modification of 2 methods (Ottaway, 1976; Potter, 1974). The pellet (P,) was rehomogenised for 1 min (Polytron PT 10 OD, maximum speed setting) in 20cm3 of the SVE medium. The homogenate was filtered through 4 layers of fine mesh to remove connective tissue and then centrifuged at 800g for 10min. This procedure was repeated a further 6 times (omitting filtration) until the activity of CK in the supernatant was negligible ( < 10 This ensured that any CK activity in the final myofibrillar pellet was not caused by contamination by cytosolic CK. The myofibrillar pellet was resuspended in the SVE medium (5.0cm3) and the suspension was centrifuged at lOOg for 5 min to remove any remaining intact cells and connective tissue. The supernatant comprised the myofibrillar fraction. Marker enzymes, total CK, CK isoenzymes, and protein were assayed in the homogenate and in the 4 subcellular fractions isolated, together with azide resistant calcium-activated adenosine triphosphatase which was used as the myofibrillar marker enzyme. ASSAY P R O C E D U R E S

(1) Creatine kinase (a) Forward reaction: The enzyme was assayed according to the Boehringer method (Catalogue No:15992).The reaction mixture (pH 9.0, 3.0cm3) contained the following final concentrations of substrates: glycine buffer

E. A . Ogunro, T. J. Peters, and D. J . Hearse

100 mmol.litre-l, creatine 28.30 mmol.litre-', sodium chloride 80 mmol.litre-l, magnesium chloride 3.30 mmol.litre-l, adenosine triphosphate 0.83 mmol * litre-l, phosphoenol pyruvate 0.72 mmol.litre-l, reduced nicotinamide adenine dinucleotide 0.24 mmol.litre-l, lactate dehydrogenase (EC 7.5 I U * C ~ and - ~ pyruvate kinase (EC 2.6 I U * C ~ -The ~ . reaction was initiated by the addition of 0.1 cm3 enzyme and the decrease in absorbance at 340 nm was measured at 25°C. (b) Reverse reaction: This was performed by a modification (Hearse et al., 1973) of the method of Oliver (1955). Thereaction mixture(pH=7.0, 1.2cm3) contained the following final concentrations of substrate: triethanolamine buffer 100 mmol.litre-', glucose sodium EDTA 1.O rnmol.litre-l, 20 rnmol*litre-l, magnesium acetate 10 mmol.litre-', adenosine diphosphate 1.O mmoI.litre-l, adenosine monophosphate 10 rnmol*litre-', nicotinamide adenine dinucleotide phosphate 0.6 mmol.litre-l, creatine phosphate 35 mmol*litre-', glutathione 9.0 mmol.litre-', hexokinase (EC 7 and glucose-6-phosphate dehydrogenase (EC 3.5 IUe~rn-~. The reaction was initiated by the addition of 0.1 cm3 enzyme sample and the increase in absorbance at 340 nm was measured at 25°C. (2) Creatine kinase isoenzymes These were separated by electrophoresis on a 1.2% w/v agarose gel in 25 mmol4itre-l sodium barbitone buffer pH 8.6. Following electrophoresis, the gel was overlayed with a substrate gel and incubated at 37°C for I h. After incubation, the isoenzymes were visualised under UV light (350 nm), fluorescent NADPH (produced as a result of isoenzyme activity) was eluted from the gel and the isoenzymes were quantitated fluorometrically (Ogunro et al., 1976). Before isoenzyme analysis, each subcellular fraction was treated with 0.5 % w/v sodium deoxycholate to solubilise any protein to which the isoenzymes may have been bound. (3) Azide resistant calcium-activated adenosine rriphosphatase (EC was assayed as described by Avruch and Wallach (1971) by using [ Y - ~ ~TrisP] adenosine triphosphate. In order to inhibit the considerable amounts of calcium-activated adenosine triphosphatase which is found in the mitochondria (Chandler et al., 1967) and therefore increase the selectivity of this enzyme as a myofibrillar marker, the assay was performed in the presence of sodium azide. The incubation medium contained : imidazole buffer 10 mmol.litre-', calcium chloride 10 mmol litre-', sodium azide Tris-adenosine triphosphate 10 mmol * litre-l, 2 mmol.litre-l and tracer [y-"P] Tris-adenosine


Subcellular compartmentation of CK isoenzymes triphosphate. The enzyme activity was corrected for basal adenosine triphosphatase activity. (4) Cytochrome oxidase ( E C I .9.3.1) was assayed by a modification (Peters et al., 1972) of the method of Cooperstein and Lazarow (1951). (5) a-Hydroxybutyrate dehydrogenase ( E C I . I . 1.61) was assayed by the standard method (Rosalki and Wilkinson, 1960). (6) 5’ Nucleotidase (EC was assayed by a modification of the method of Avruch and Wallach (1971)by using [WH]adenosine 5‘ monophosphate as substrate (Kane and Peters, 1975). (7)Neutral a-glucosidase (EC was assayed fluorometrically by a modification (Peters et al., 1972) of the method of Mead et al. (1955). ( 8 ) Protein was determined by 2 methods: the procedure of Lowry et al. (1951) was applied to the protein determinations on the fractions prepared by differential centrifugation. In the sucrose density gradient studies a modification of the fluorometric technique of Hiraoka and Glick (1963) was used (Peters et al., 1972). This technique has been reported to be more sensitive than the method of Lowry et al. (1951)and to be unaffected by the high sucrose concentrations (up to 60% w/v) encountered in the sucrose density gradient studies. Crystalline bovine serum albumin was used as a standard for both methods of protein estimation.

Results (1 ) Isopycnic density gradient centrifugation

(a) Steep gradient: Fig. 1 shows typical distribution profiles for the marker enzymes of the principal subcellular organelles isolated by centrifugation in a steep sucrose density gradient. Cytochrome oxidase was concentrated over a narrow density span with a modal density of 1.17,5’ nucleotidase and neutral a-glucosidase showed peaks with modal densities of 1.12 and 1.14 respectively, while a-hydroxybutyrate dehydrogenase remained in the sample layer. The distribution of CK was bimodal, with most of the enzyme activity following the distribution of ahydroxybutyrate dehydrogenase. The remaining CK activity showed a peak with a modal density of 1.17 which suggested localisation of a small amount of this enzyme in the mitochondria. (b) Shallow gradient: the results in Fig. 1 show some CK activity in the density range 1.10 to 1.25.This activity may represent either a spread from the cytosolic fractions or may represent the Occurrence of CK associated with the plasma membrane (since the plasma membrane marker enzyme was detected in this region). To determine whether the CK activity resulted from an overlap of cytosolic CK into the plasma membrane fraction or whether it


Fig. 1 lsopycnic centrifugation in steep sucrose gradient (see text for details) of post nuclear supernatant isolated from guinea pig lefr ventricle. Graphs show jiequency-distribution histogramsfor various marker enzymes and for protein throughout the gradient. Frequency is defined as fraction of recovered activity present in subcellular fraction divided by density span covered. Enzyme activities in top fractions (up to sucrose density 1.10) relate mainly to activity in the cytosol of cell and have been pooled and plotted over an arbitrary interval of 1.05 to 1.10. Enzyme recoveries were: 5’ nucleotidase. 87%; cytochrome oxidase, 81 %; CK, 89 %; a-hydroxybutyrate dehydrogenase, 97% ;neutral a-glucosidase.90 %; protein. 79 %.

represented true plasma membrane activity, it was necessary to achieve complete separation of these 2 fractions. This was accomplished by centrifugation of the postnuclear supernatant in shallow sucrose gradients. The results of this study (Fig. 2) showed that the distribution of CK was distinct from that of 5’ nucleotidase, the plasma membrane marker enzyme. Isoenzyme analysis revealed (Fig. 2) that the MM, MB, and trace amounts of the BB isoenzyme were cytosolic but that a separate isoenzyme which migrated towards the cathode (Fig. 3) was characteristic of mitochondria1 C K activity. Of the total CK activity present in the cytosol fraction, 84% was represented by the MM isoenzyme, 15% by the MB isoenzyme, and approximately 1 % by the BB isoenzyme.


1 1.00 - 1

E. A . Ogunro, T. J. Peters, and D . J . Hearse




Fig. 3 Photograph of separation of creatine kinase isoenzymes in ( a ) mitochondrial. and ( b ) cytosol fractions isolated from guinea pig myocardial homogenate by isopycnic centrifugation.

of CK in the 4 fractions isolated. Cytochrome oxidase, a-hydroxybutyrate dehydrogenase, and neutral a-glucosidase showed highest specific activity 11-f CYTOCHROME lA,, in the mitochondria, cytosol, and microsomes respectively. Azide resistant calcium-activated adenosine triphosphatase (the myofibrillar marker enzyme) showed highest specific activity in the 0 microsomes with an appreciable amount in the 0 20 LO 60 80 100 0 20 10 60 00 100 /'. VOLUME /'. VOLUME myofibrillar fraction. The higher specific activity of this enzyme which was observed in the microsomes Fig. 2 Isopycnic centrifugation in shallow sucrose was not unexpected, since the microsomal fraction gradient (see text for details) of post nuclear supernatant essentially represents sarcoplasmic reticulum (tofraction isolated from guinea pig left ventricle. Graphs show sucrose density. relative activities of CK isoenzymes, gether with some mitochondria, plasma membrane selected marker enzymes and protein, plotted as functions fragments and golgi apparatus), and has been of the percentage of total volume of gradient. Enzyme reported (Macknnan, 1970) to contain substantial recoveries were: total CK, 71 %; CK M M ,74%; C K MB, amounts of calcium-activated adenosine triphospha67%: a-hydroxybutyrate dehydrogenase, 84 %; 5' tase. This together with the low protein content of nucleotidase, 82 %: protein, 81 %: mitochondria1 C K . the microsomal fraction accounts for the high I01 %; cytochrome oxidase. 69%. specific activity of this enzyme in the microsomes. CK exhibited highest specific activity in the cytosol (12 400 protein) with smaller amounts in (2) Diferential pelleting the microsomes (2800, mitochondria The subcellular distribution of the enzymes in this (1 800 and myofibrils ( 1 100 The analytical results expressed in the form of part of the study are expressed in 2 forms. Firstly, in terms of the absolute specific activity of each percentage distribution show greatest distribution enzyme in the 4 fractions (irrespective of the per- of cytochrome oxidase, a-hydroxybutyrate dehydrocentage of the cell which is occupied by that fraction), genase, and azide resistant calcium-activated adenoand secondly, as a recovery of the total enzyme sine triphosphatase in the mitochondria, cytosol, activity in any single fraction, expressed as a per- and myofibrils respectively. Neutral a-glucosidase centage of the total activity recovered in all the sub- (which is both microsomal and cytosolic) exhibited cellular fractions (irrespective of variations in the a greater distribution in the cytosol fraction. The relative efficiency of preparation of each fraction or apparent discrepancy between highest specific possible activation or inactivation of any enzyme activity and greatest percentage distribution of this enzyme in the microsomes and cytosol (as was within that fraction). Table I shows the specific activity and percentage observed with azide resistant calcium-activated distribution of each marker enzyme and also that adenosine triphosphatase in microsomes and myo-

Subcellular compartmentation of C K isoenzymes


Table 1 Fractionation of guinea pig left ventricle by differentialpelleting Enzyme Cytochrome oxidase

Homogenare (SA) 551 *I55 ( %)

a-hydroxybutyrate dehydrogenase (SA) Azide resistant calcium-activated adenosine triphosphatase Neutral a-glucosidase

1072 i 126

( %)

(SA) 24.5 f 10.3 ( %)

(SA) 0.17 It0.02 ( %)

Creatine kinase Protein (mgg-' . - - tissue)

(SA) 6000f920 ( %)

(SA) ( %)

109 f 12.5





246 122.5 (8) 10.0 i 2 . 0

2362 1 169

370 148.4

0.10 fO.07



40.0 i3.0

83.0 125.0




57.0 *7.7 (60) 0.03 *0.01 (4) 1100f130 (4) 10.9 f 1.3 (18)

12.8 *9.1 (14) 0.07 fO.01

412 123.3

2950 350 (99) 0. I7 10.09



(1 1)

I800 i90.0 (6) 15.5 f 1.3 (26)

2.30 10.03 (18) 2800 f80.0 (1)

1.2 *0.13 (2)



0.20 10.02 (67) 12 400 + 1800 (89) 32.0*3.1 (54)

(SA)=specific activity (' protein), (%)=percentage distribution. Specific activities quoted are the mean f S D for n = 4 experiments. Units of enzyme activity represent nmol of substrate hydrolysed/min (mlU) except for cytochrorne oxidase, where the units correspond to those o f Cooperstein and Lazarow (1951). Percentage distribution is defined as the enzyme activity recovered in any individual fraction expressed as a percentage of the total activity recovered in all 4 fractions.

fibrils) results from the large difference in protein content between these 2 fractions. The distribution of CK showed 4, 6, 1, and 89% of the total recovered activity in the myofibrils, mitochondria, microsomes, and cytosol respectively. The percentage of CK in the myofibrillar fraction is probably lower than the 4% quoted, since the preparative procedure involved preparation of the myofibrillar fraction from all the cells, while mitochondria, microsomes, and cytosol were isolated only from the cells which were disrupted by the initial homogenisation procedure (see Methods). It should be emphasised that the percentages quoted above represent only an approximation to the situation which exists in the cell. Clearly it is not possible to definitively deduce the absolute percentage of the total cellular activity of any enzyme in any single fraction. However, the 2 forms of data presentation (ie specific activity and percentage distribution) give the best indication of the most probable location of major enzyme activity. Isoenzyme analysis (see Fig. 4) showed that MM CK together with a small amount of the mitochondrial isoenzyme was associated with the myofibrils and the microsomes. The presence of the rnitochondrial isoenzyme in these fractions probably resulted from minor contamination. Isoenzyme profiles obtained for the cytosolic and mitochondrial fractions were essentially similar to those obtained by the isopycnic studies, with the exception that the mitochondrial fraction showed slightly more MM isoenzyme activity.

( 3 ) Isoenzyme characterisation studies Having confirmed that an isoenzyme distinct from MM, MB, and BB CK was present in the mito-

chondria, we compared some of the properties of the cytosolic and mitochondrial isoenzymes to investigate any possible differences in their catalytic properties. In these studies, we employed the cytosol and mitochondrial fractions previously isolated by differential centrifugation. Comparison of p H optima of cytosolic and mitochondrial CK. In order to characterise any differ-

ences in the pH optima of cytosolic and mitochondrial CK, pH optimum curves were compared in both directions of the CK reaction using optimised enzyme assays. The results (see Fig. 5 ) revealed that the pH optimum curves for cytosolic and mitochondrial CK were similar, showing pH optima of approximately 9.0 and 6.8 when assayed by the forward and reverse react ions respectively .

Fig. 4 Photograph of separation of creatine kinase isoenzymes in (a) myofibrillar. (b) microsomal, ( c ) mitochondrial. and ( d ) cytosol fractions isolated from guinea pig myocardial homogenate by differential pelleting.

E. A . Ogunro, T. J. Peters, and D. J. Hearse








ASSAY pH (b)


* 20-


in the absence of activators. This avoided any potential differential activation of cytosolic or mitochondrial CK when assayed by either reaction. The assay of mitochondrial CK was potentially subject to large errors which originated from the fact that both adenosine triphosphatase and NADH oxidase were capable of interfering with the CK assay (Saks et al., 1975). These potential artefacts were therefore eliminated by the liberation of mitochondrial CK with 50 mmol.litre-' sodium phosphate buffer pH 7.2. Subsequent assay of the liberated mitochondrial CK (by the forward reaction) in the absence of creatine substrate, revealed that there was no NADH oxidation and therefore verified the absence of adenosine triphosphatase or NADH oxidase interference.







Fig. 5 pH-activity curves for cytosolic and mitochondrial CK assayed by forward and reverse reactions. (a) Mitochondria1 CK ( 0 )forward reaction ( 0 )reverse reaction (b) Cytosolic CK ( 0 )forward reaction ( 0 ) reverse reaction

Comparison of the activity ratio: activity measured by the forward reactionlactivity measured by the reverse reaction. for cytosolic and mitochondrial CK. In order to verify whether there was any difference in catalytic capacity between cytosolic and mitochondrial CK (in either direction), enzyme activity in these fractions was measured by the forward and reverse reactions using optimkd enzyme assays. Calculation of the above ratio for cytosolic and mitochondrial CK yielded values of 0.1 1 fO.O1 and 0.35 f0.02 respectively (mean fSD, n=5 experiments, P ~0.01). As glutathione was incapable of activating CK (when measured by the forward reaction), both the forward and reverse reaction assays were performed

The effect of 50 mmol-litre-l sodium phosphate bufer on the activity of mitochondrial CK. Since mitochondrial CK was liberated with phosphate buffer, it was necessary to ensure that there was no differential change in mitochondrial CK activity (caused by the phosphate buffer) in either direction of assay. The activity of a fixed amount of mitochondrial CK in phosphate buffer (test) was therefore compared with the activity of an equivalent amount of CK in distilled water (control). Using the forward reaction, CK exhibited an activity of 126.8f6.9 (meanISD, n=4) in distilled water and an activity of 110.5f2.3 in sodium phosphate buffer. The corresponding values for the reverse reaction were 253.6* 13.0 (distilled water) and 219.0f9.1 (sodium phosphate buffer). These results show that the phosphate buffer had a slight inhibitory effect on the activity of mitochondrial CK which was approximately 14% in either direction of assay. The similar extent of inhibition observed for both reactions therefore had no overall effect on the value of the activity ratio determined for mitochondrial CK.

Discussion The results presented in this paper provide evidence for the compartmentation of the CK isoenzymes in guinea pig heart cells. Of the total activity of CK in the cell, approximately 89% was found in the cytosol, 6% in the mitochondria, 4% in the myofibrils, and 1% in the microsomes. The MM isoenzyme accounted for 84% of the total CK activity in the cytosol and was also found to be associated with the myofibrils and the microsomes. The MB and BB isoenzymes were detected only in the cytosol, where they represented 15 % and approximately 1 % of the total CK activity respectively. A distinct and additional isoenzyme was found to be characteristic

Subcellular compartmentation of CK isoenzymes

of the mitochondria. Comparison of the pHactivity curves for cytosolic and mitochondrial CK resulted in similar pH optima for CK in both compartments. However, there was a significant difference in the value of the activity ratio determined for CK in the cytosolic and mitochondrial compartments. Since a ratio of less than 1.0 is indicative of a greater catalytic capacity in the reverse reaction, it is evident that both cytosolic and mitochondrial CK catalyse the reverse reaction more efficiently. However, since the activity ratio of 0.35 (obtained for mitochondrial CK) is greater than the corresponding value of 0.11 obtained for cytosolic CK, these results suggest that the mitochondrial isoenzyme exhibits a greater capacity than the cytosolic isoenzymes in catalysing the forward reaction. Our results show that an additional fourth isoenzyme of CK is present in guinea pig heart in the mitochondria. Previous investigators have demonstrated the presence of this isoenzyme in mitochondria isolated from rat (Jacobs et al., 1964; Scholte, 1973; Saks, 1974; Somer et al., 19741, rabbit (Sobel et al., 1972), pigeon (Jacobs et al., 19641, and beef heart (Farrell et al., 1972). The presence of this isoenzyme in human heart has not been reported. Despite the differences in animal species employed, the data presented in this paper are in agreement with the findings of Sobel et al. (1972) and Somer et al. (1974) that only a small ( < 10%) amount of the total CK in the cell is associated with the mitochondria. At present the function of mitochondrial C K is not clear. It has been suggested (Jacobs et al., 1964; Gudbjarnason et al., 1970; Vial and Gautheron, 1973; Jacobus and Lehninger, 1973a; Saks et al., 1974) that this isoenzyme mediates the transfer of high energy phosphate from the mitochondrial compartment to the cytosol compartment, by catalysing the conversion of ATP (produced intramitochondrially) into freely diffusible creatine phosphate which readily passes into the cytosol where it is transphosphorylated back into ATP (by cytosolic CK) to meet cellular energy requirements. This suggested function has been postulated on the basis of results from kinetic studies (Jacobus and Lehninger, 1973a; Saks et al., 1974), studies with intact animals (Gudbjarnason et al., 1970) and observations on the creatine content of heart mitochondria (Vial and Gautheron, 1973; Altschuld et al., 1975). The hypothesis that CK in the mitochondrial compartment mediates this energy transfer process requires that mitochondrial CK catalyses the kinetically less favourable (Saks et al., 1975) forward reaction, ie the reaction in the direction of creatine phosphate synthesis. Conversely CK in the cytosol compartment is required by this hypothesis to catalyse the reverse reaction, since the enzyme in

257 this compartment is mainly involved in the regeneration of ATP for use in the myofibrillar contractionrelaxation cycle and in other extramitochondrial energy requiring processes. The data presented in this paper show that CK in the mitochondrial compartment functions more efficiently by the forward reaction than CK in the cytosol compartment and therefore supports the suggested function of cytosolic and mitochondrial CK. In view of the proposed function of mitochondrial CK, it would appear that the proportion of the total cellular activity which is represented by this isoenzyme is relatively low ( < 10%). However, if mitochondrial CK is localised within the intermembranous space of the mitochondrion (as it has been suggested by Klingenberg and Pfaff, 1966). it is conceivable that the concentration of the enzyme within this space is comparable with that of CK in the cytosol (since the latter clearly occupies a much larger volume). Further evidence for the compartmentation of the CK isoenzymes is provided by the localisation of MM C K in the myofibrils and the microsomes. The association of CK with myofibrillar fractions has previously been reported (Ottaway, 1967; Jacobus and Lehninger, 3973b; Scholte, 1973) and recent reports (Eppenberger et al., 1975) have demonstrated interactions of the CK isoenzymes with specific areas of the myofibril. The results presented in this paper, in contrast to some previous reports (Jacobus and Lehninger, 1973b; Scholte, 1973) suggest association of only a small proportion ( < 5 % ) of the total cellular CK activity with this fraction. It is possible, however, that the previously reported percentage total CK values of 33% (Scholte, 1973) and 50% (Jacobus and Lehninger, 1973b) have resulted from the use of myofibrillar preparations which were contaminated with unbroken .cells- and therefore contained substantial amounts of cytosolic CK. In the present study, the myofibrillar fraction was repeatedly homogenised and washed in order to ensure that any CK activity remaining in the final pellet represented a true myofibrillar localisation and did not result from contamination by cytosolic CK. The physiological significance of a myofibrillar localisation of CK would probably be in the regeneration of ATP from creatine phosphate. However, since the proportion of the total cellular activity of CK associated with this fraction was low, it is unlikely that the enzyme in this location plays an important role in the regeneration of ATP for muscular contraction. Other than the work of Kleine (1965), little evidence is available on the association of CK with the microsoma1 fraction. Although microsomes exhibit little CK activity (approximately 1 % of the total cellular


content), it has been suggested (Baskin and Deamer, 1970) that CK in this compartment could function to provide a local source of ATP for calcium transport when the overall ATP concentration of the cell is relatively low as a result of myofibrillar contract ion. In conclusion, apart from the reported (Eppenberger et al., 1967; Witteveen et al., 1974; Wong and Smith, 1976) kinetic differences between the MM, MB, and the BB isoenzymes, there is little evidence of any difference in functional role between the CK isoenzymes in the cytosol compartment (with particular reference to the cardiospecific MB form) or between MM CK in the myofibrils, microsomes and the cytosol. Clearly further studies are necessary in order to fully elucidate the possible functional implications of a differential CK isoenzyme distribution within the cardiac cell. The help and advice of Professor J. P. Shillingford, Dr E. A. Welman, and Miss A. Long are gratefully acknowledged. References Altschuld, R. A., Merola, A. J., and Brierley, G. P. (1975). The permeability of heart mitochondria to creatine. Journal of Molecular and Cellular Cardiology. I, 451-462. Avruch, J., and Wallach, D. F. H. (1971). Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells. Biochimica et Biophysics Acta, 233, 334-347, Baskin, R. J., and Deamer, D. W. (1970). A membrane bound creatine phosphokinase in fragmented sarcoplasmic reticulum. The Journal of Biological Chemistry, 245, 1345-1347.

Beaufay, H.(1966). La centrifugation en gradient de densitt. Thlse d’agrkgation de I’ensignement supkrieur. Universitt catholique de Louvain, Louvain, Belgium. Centerick, Louvain, Belgium, Bloomfield, F. J., and Peters, T. J. (1974). Analytical subcellular fractionation of guinea pig myocardium with special reference to the localization of adenosine triphosphatases. Biochemical Society Transactions. 2, 10981101.

Chandler, B. M., Sonnenblick, E. H., Spann, J. F., and Pool, P. E. (1967). Association of depressed myofibrillar adenosine triphosphatase and reduced contractility in experimental heart failure. Circulation Research, 21, 717-725. Cooperstein, S. J., and Lazarow, A. (1951). A microspectrophotometric method for the determination of cytochrome oxidase. Journal of Biological Chemistry, 189, 665-670.

Dawson, D. M. (1970). Creatine kinase from brain. Kinetic aspects. Journal of Neurochemistry. 11, 65-74. Dawson, D. M., Eppenberger, H. M., and Eppenberger, M. E. (1968). Multiple molecular forms of creatine kinases. Annals of the New York Academy of Sciences. 151, 616626.

Dawson, D. M., Eppenberger, H. M., and Kaplan, N. 0. (1965). Creatine kinase: Evidence for a dimeric structure. Biochemical and Biophysical Research Communications, 21, 346-353.

Dawson, D. M., and Fine, 1. H. (1967). Creatine kinase in

E. A. Ogunro, T. J . Peters, and D . J . Hearse human tissues. Archives of Neurology, 16, 175-180. Eppenberger, H. M., Dawson, D. M., and Kaplan, N. 0. (1967). The comparative enzymology of the creatine kinases. I. Isolation and characterisation from chicken and rabbit tissues. The Journal of Biological Chemistry. 242, 204-209.

Eppenberger, H. M., Wallimann, T., Kuhn, H. J., and Turner, D. C. (1975). Isoenzymes. Academic Press: New York. Farrell, E. C., Baba, N., Brierley, G. P., and Griimer, H. D. (1972). On the creatine phosphokinase of heart musclc mitochondria. Laboratory Investigation, 21, 209-2 13. Gudbjarnason, S., Mathes, P., and Ravens, K. G. (1970). Functional compartmentation of ATP and creatine phosphate in heart muscle. Journal of Molecular and Cellular Cardiology. 1, 325-339. Hearse, D. J., Humphrey, S. M., and Chain, E. 8. (1973). Abrupt reoxygenation of the anoxic potassium-arrested perfused rat heart: a study of myocardial enzyme release. Journal of Molecular and Cellular Cardiology. 5. 395407. Hiraoka, T., and Glick, D. (1963). Studies in histochemistry. LXXI. Measurement of protein in millimicrogram amounts by quenching of fluorescence dye. Analytical Biochemistry, 5, 497-504. Jacobs, H., Heldt, H. W.. and Klingenberg, M. (1964). High activity of creatine kinase in mitochondria from muscle and brain and evidence for a separate mitochondrial isoenzyme of creatine kinase. Biochemical and Biophysical Research Communications, 16, 5 1 6 5 2 I. Jacobus. W. E., and Lehninger, A. L. (1973a). Creatine kinase of rat heart mitochondria. Coupling of creatine phosphorylation to electron transport. The Journal of Biological Chemistry, 248, 4 8 0 3 4 8 10. Jacobus, W. E., and Lehninger, A. L. (1973b). Mechanism of phosphocreatine production coupled to cardiac mitochondrial electron transport. The American Journal of Cardiology, 31, 139 (Abstract). Kane, S. P., and Peters, T. J. (1975). Analytical subcellular fractionation of human granulocytes with reference to the localization of vitamin BIB-binding proteins. Clinicaf Science and Molecular Medicine, 49, 171-1 82. K1eine.T. 0.(1965). Localization of creatine kinase in microsomes and mitochondria of human heart and skeletal muscle and cerebral cortex. Nature, 207, 1393-1 394. Klingenberg, M., and Pfaff, E. (1966). Regularion of metabolic processes in mitochondria. Elsevier: New York. Lowry, 0 . H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with folin phenol reagen 1. Journal of Biological Chemistry, 193, 265-275. MacLennan, D. H. (1970). Purification and properties of an adenosine triphosphatase from sarcoplasmic reticulum. Journal of Biological Chemistry. 245, 4 5 0 8 4 5 18. Mead, J. A. R., Smith, J. N., and Williams, R. T. (1955). Studies on detoxication. LXVII. The biosynthesis of the glucuronides of umbelliferone and 4-methyl-umbelliferone and their use in fluorometric determination of P-glucuronidase. Biochemical Journal. 61, 569-574. Ogunro, E. A., Hearse, D. J., and Shillingford, J. P. (1977). Creatine kinase isoenzymes: their separation and quantitation. Cardiovascular Research. 1I, 94-101. Oliver, 1. T. (1955). A spectrophotometric method for the determination of creatine phosphokinase and myokinase. Biochemical Journal, 61, I 16-1 22. Ottaway, J. H. (1967). Evidence for the binding of cytoplasmic creatine kinase to structural elements in heart muscle. Nature, 215, 521-522. Peters, T. J., Miiller, M., and De Duve, C. (1972). Lyosomes of the arterial wall. 1. Isolation and subcellular fractionation of cells from normal rabbit aorta. The Journal of Experimental Medicine. 136, 1 1 17-1 139.


Subcellular compartmentation of CK isoenzymes Potter, J. D. (1974). The content of troponin, tropomyosin, actin and myosin in rabbit skeletal muscle myofibrils. Archives of Biochemistry and Biophysics. 162, 436-441. Roberts, R., Henry, P. D., and Sobel, B. E. (1975). Am improved basis for enzymatic estimation of infarct size. Circulation. 52, 743-754. Rosalki, S. B., and Wilkinson, J. H. (1960). Reduction of a-ketobutyrate by human serum. Nature. 188, 1 110-1 1 1 1. Saks, V. A., Cbernousova, G. B., Gukovsky, D. E., Smirnov, V. N., and Cbazov, E. I. (1975). Studies of energy transport in heart cells. Mitochondria1 isoenzyme of creatine phosphokinase: kinetic properties and regulatory action of Mg*+ ions. European Journal of Biochemistry. 57, 273290.

Saks, V. A., Chernousova, G. B., Voronkov, Iu. I., Smirnov, V. N., and Chazov, E. 1. (1974). Study of energy transport mechanism in myocardial cells. Circulution Research, 34 a n d 3 5 (suppl. 111). 111-138 and 111-149. Scholte, H. R. (1973). On the triple localization of creatine kinase in heart and skeletal muscle cells of the rat: evidence for the existence of myofibrillar and mitochondrial isoenzymes. Biochimica et Biophysica Arm. 305, 413 4 2 7 . Smith, A. F. (1972). Separation of tissue and serum creatine

kinase isoenzymes on polyacrylamide gel slabs. Clinica Chimica Acta, 39, 351-359. Sobel, B. E., Shell, W. E., and Klein. M. S. (1972). An isoenzyme of creatine phosphokinase associated with rabbit heart mitochondria. Journal of Molecular and Cellular Cardiology. 4, 367-380. Somer, H., Uotila, A., Konttinen, A., and Saris, N. E. (1974). Creatine kinase activity and its isoenzyme pattern in heart mitochondria. Clinica Chimica Acta. 53, 369-372. Van Der Veen, K. J., and Willibrands, A. F. (1966). Isoenzymes of creatine phosphokinase in tissue extracts and in normal and pathological sera. Clinica Chimica Actu, 13, 312-316. Vial, C., and Gautheron, D. C. (1973). Some properties of pig heart mitochondrial creatine kinase. Recent Advances in Studies in Cardiac Structure and Metabolism. 3, 81-89. Witteveen, S. A. G. J., Sobel, B. E., and De Luca, M. (1974). Kinetic properties of the isoenzymes of human creatine phosphokinase. Proceedings of the National Academy of Sciences ( U S A ) , 71, 1384-1387. Wong, P.C. P., and Smith, A. F. (1976). Biochemical differences between the MB and MM isoenrymes of creatine kinase. Clinica Chimica Acta. 68, 147-158.

Announcement The first International Congress on Cardiac Rehabilitation will be held in Hamburg from 12-14 September 1977. The general principle of the Congress is to critically display the present concepts of cardiac rehabilitation and to collect information on the on-going activities. For further information contact the Secretary: Prof Dr med Kurt Konig, Herz-Kreislauf-Klinik Waldkirch, Kandelstrasse 41, Postfach 270, D-7808 Waldkirch, West Germany.

Subcellular compartmentation of creatine kinase isoenzymes in guinea pig heart.

Cardiovascular Research, 1977, 11, 250-259 Subcellular compartmentation of creatine kinase isoenzymes in guinea pig heart' E . A. O G U N R O , T. J...
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