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Mitochondrial isoenzyme of creatine phosphokinase in frog heart
Comp. Biochem. PhysioL Vol. 8911,No. 2, pp. 251-255, 1988 Printed in Great Britain
0305-0491/88 $3.00+ 0.00 © 1988PergamonJournals Ltd
MITOCHONDRIAL ISOENZYME OF CREATINE PHOSPHOKINASE IN FROG HEART ABDELKHALEQ LEGSSYERand MAnrINE ARRIO-DuPONT* Laboratoire de Physiologic Cellulaire Cardiaque. INSERM U-241 Universit6 de Paris-Sud, batiment 443, 91405 Orsay Cedex, France (Tel: 1-6941-7774) (Received 30 March 1987)
Abstract--1. Frog heart has a partially glycolytic metabolism; nevertheless a mitochondrial form of creatine phosphokinase (CPK) has been identified. 2. The CPK isoenzymepatterns (electrophoresis at pH 9.2 on celluloseacetate strips) in heart are similar to those of other vertebrates, with some variations. 3. The BB and MB isoenzymes are found in large quantities. 4. The electrophoretic mobilities of the mitochondrial isoform and that of the MB-form are the same, but the isoelectric point of the mitochondrial form (pHi 7) is not the same as that of the MB isoenzyme (pHi 5.6). 5. The activity of the CPK-form bound to mitochondria is strongly coupled to the ADP/ATP translocase.
INTRODUCTION Creatine phosphokinase (CPK, EC 2.7.3.2) is an enzyme involved in cellular distribution of energy, mainly occurring in organs such as heart, muscle or brain. It catalyses the transphorylation reaction between creatine phosphate and ADP: CrP + ADP + H+~-ATP + Cr It has been extensively studied in terms of its subcellular distribution in vertebrates (Eppenberger et al., 1967; Jacobs et al., 1964; Scholte, 1973). Different isoforms have been observed. The soluble isoenzymes are homo or heterodimers, namely MM, MB and BB according to the tissues where they are predominantly found: MM-CPK in skeletal muscle and BB-CPK in brain. Another isoform, the mitochondrial one (m-CPK), has also been observed: it is bound to the external face of the internal membrane of mitochondria (Jacobus and Lehninger, 1973; Scholte et aL, 1973) and it is implicated in the synthesis of creatine phosphate. In the case of amphibians, the soluble isoforms MM, MB and BB have been identified (Eppenberger et al., 1967) and the distribution of the isoforms in eggs and selected stages of development has been studied (Klemann and Pfohl, 1982). However the mitochondrial form has never been identified. The existence of such a form may be questionable. In these animals, the cardiac metabolism is more glycolytic than in terrestrial vertebrates and it is possible that mitochondrial metabolism is not as important in frog heart as in adult mammal heart. In animals such as the rat or the
sheep, the development of the mitochondrial creatine kinase is linked to the appearance of mitochondria and oxidative metabolism (Hall and Deluca, 1975; Norwood et al., 1983; Van Brussel et al., 1983). Amphibians (frog) have a metabolism similar to intermediate stages of mammal development and it is possible that there is no mitochondrial CPK in these animals. In the present work, the different isoforms of CPK have been identified in heart and compared to skeletal muscle and brain extracts. For a better identification of the mitochondrial isoform, mitochondria have been isolated and checked for the presence of CPK activity. The repartition of the CPK activity in the main cellular compartments: soluble cytosol, mitochondria and myofilament has been determined in isolated myocytes from ventricles.
MATERIALSAND METHODS a. Tissue extracts Mature frogs (Rana esculenta) were killed by decapitation and the following tissues removed: cardiac muscle, skeletal muscle (gastrocenemius) and brain. For cardiac muscle, the heart was previously washed with standard Ringer solution to eliminate blood cells. The tissues were cut into small pieces in 20 mM Na phosphate pH 7.4, containing 0.I mM dithiothreitol, at 2°C. The suspension was homogenized with a Potter-Elvehjem (600 rpm, 1 min at 2°C). The homogenates were centrifuged at 8000g for 10 rain at 4°C and the supernatant fluids were retained for analysis by either electrophoresis or eiectrofocusing. b. Cell isolations. Fast fractionation Ventricular cells were isolated as previously described (Arrio-Dupont and De Nay, 1985) by perfusion with collagenase and trypsin. The main cellular compartments were separated by fast digltonin (Zuurendonk et al., 1979) or Triton (Arrio-Dupont and De Nay, 1986) fractionation, in
*To whom correspondence should be addressed. Abbreviations used---CPK, creatine phosphokinase; MK, myokinase; MPS, morpholino propane sulfonic acid; CrP, creatine phosphate; Cr, creatine. 251
252
ABDELKI-IALEQLEGSSYERand MART1NEARRIO-DUPONT
a medium containing 20 mM MPS, 3 mM EDTA and either 230 mM mannitol (low I medium) or 115 mM KC1 (high I medium), for which the pH was adjusted to 6.75 at 22°C. After 10 sec at 2°C in the presence of the lysis medium, the cell suspension was centrifuged through silicon oil. The enzymatic activities were determined in the supernate and in the pellet after treatment by Triton in low I medium. The total intracellular activities were obtained in the pellet by omitting digitonin or Triton in the lysis medium. As previously described (Arrio-Dupont and De Nay, 1986), the top and bottom fractions obtained after centrifugation contained respectively broken cells plus cytosol and mitochondria plus myofilament after digltonin treatment, and broken cells plus mitochondria plus cytosol and myofilaments after Triton lysis. According to the ionic strength of the lysis medium, CPK is either detached or not detached from mitochondria and myofdaments.
c. Mitochondria Mitochondria were isolated by a method analogous to that described by Scarpa and Grazzioti (1975). Small pieces of ventricular tissue, from two hearts, were cut in 20 mM MPS buffer pH7.4, containing 55mM sucrose, 16mM mannitol, 3 mM EDTA and 0.1 mM dithiothreitol, at 2°C and homogenized with a Potter-Elvejhem homogenizer (600 rpm for 1 min at 2°C). The homogenate was then centrifuged for 10 min at 1000 g. After centrifugation of the supernatant for 20 min at 12,000g, the mitochondria were obtained in the pellet. After two further steps of washing and centrifugation, the mitochondria were suspended in 200/~1 of the same buffer and kept in an ice-bath. The final concentration corresponded to about 10 mg/ml protein. The obtained mitochondria were checked for their respiratory control and their ADP/O ratio, by using a Clark oxygen electrode (YSI) at 20°C. The buffer used to study oxygen consumption was 16mM Tris-HCl, containing 105raM KCI and 2 mM KH2PO 4 pH 7.4, with 3.5 mM pyruvate and 0.35 mM malate as substrates. The respiration was stimulated by 0.2 mM ADP. d. Electrophoresis, electrofocusing Electrophoresis was performed on cellulose acetate strips. The electrophoretic buffer was 50 mM Tris--veronal pH 9.2; three gels (Cellogel, 14 x 5.7 era) were run simultaneously by applying 200 V and 10mA for 3 hr. A Sebia electrophoresis cuvette (S 60) was used. Electrofocusing was performed on PGA plates LKB, pH either 3-9.5 or 5.5-8.5 at 6°C, under constant 20 W power, for 4 hr. A 2117 multiphor from LKB was used. For both electrophoresis and electrofocusing, a power supply 2197 from LKB was used. e. Enzymatic activities, protein determination Creatine kinase activity was determined at 22°C, by a coupled enzymatic assay and the formation of NAD(P)H was studied. The reaction mixture contained 4.2raM MgSO4, 1 mM ADP, 10 mM creatine phosphate, 0.6 mM NADP, 20 mM glucose, 1 U/ml hexokinase, 1 U/ml glucose6-phosphate dehydrogenase in 50 mM Tris--acetate buffer, pH 7.4. In spectrophotometer, the NADPH formation was determined by its absorbance at 340 nm; on the gels, it was detected by the formazan reaction and observed by incubating the strips with an overlay of the same support medium containing the substrates, the enzymes, phenazine methosuifate and nitroblue tetrazolium. The contamination by myokinase (MK, EC 2.7.4.3) activity (2 A D P ~ A T P + AMP) was determined under the same conditions by omitting creatine phosphate in the reaction mixture. In some cases, the myokinase activity was inhibited by an excess of AMP (13 mM). The MK activity was determined with a coupled enzymatic assay similar to that used for CK activities, with no ereatine phosphate in the medium. Proteins were determined after 0.1 M NaOH dena-
Table 1. Compartmentation of creatine kinase activity in isolated ventrieular cells Percentage of activity* Lysis medium Top fraction Bottomfractiont None 10-15 85-90 Digitonin high I 65-70 30-35 Digitonin low I 85 15 Triton high I 75-80 20-25 Triton low I 100 0 *The total CK activity (100%) was determined in a medium of low ionic strength to detach the enzyme bound to myofilamentsand in the presence of either Triton X-100 or Lubrol WX. fin the bottom fractions, the activity was determined after treatment of the pellets with Triton, in a medium of low ionic strength. In all the fractions, the myokinase activity observed by omitting creatine phosphate in the medium has been deducted. turation, by the Lowry et al. method (1951) using bovine serum albumin as a standard.
f. Materials Enzymes were from Boehringer. Pyruvate, L-malate, phenazine methosulfate, nitroblue tetrazolium, NADP, ATP, ADP and AMP were from Sigma. RESULTS
A. Compartmentation of creatine kinase activity By using the lysis medium of either high or low ionic strength, on isolated ventricular cells, it was possible to estimate the repartition of the creatine kinase activity in the main cellular compartments. The activities observed in the top and bottom fractions after lysis with 0.5 mg/ml digitonin for 10 see at 2°C or with 2% Triton X-100 for 15 see at the same temperature are indicated in Table 1. In the medium of low ionic strength containing Triton, 100% of the creatine kinase activity is observed in the supernatant, because in this medium all the membranes are destroyed and creatine kinase is detached from the myofilaments ( M o r i m o t o and Harrington, 1972; Turner et al., 1973). The total C P K activity determined either in 2% Triton of low ionic strength or in Lubrol WX, for nine cellular isolations, was 0 . 6 3 _ 0.08 (SD) I U / m g cellular protein. In Triton medium of high ionic strength, 80-85% of the total C P K activity is observed in the supernatant and about 15% in the bottom. F r o m this result, it can be deduced that 15-20% of the creatine kinase activity is bound to the myofilaments. The use of digitonin in a low ionic strength medium gives access to creatine kinase activity bound to the mitochondria: it represents about 15% of the total activity. The remaining activity, 65-70%, is the free cytosolic creatine kinase. B. Total heart extracts. Comparison with extracts o f skeletal muscle and brain The total C P K activity of whole heart extracts in 2 0 m M N a phosphate buffer was 1.2 _+ 0.2 I U / m g protein and the total myokinase activity represents 2 - 5 % of this activity. These activities correspond to several isoforms, as shown by electrophoresis on cellulose acetate strips at p H 9.2 (Fig. 1). When the creatine kinase activity on the gel is revealed in the presence of 13 m M A M P , three bands migrating toward the anode are observed. The myokinase activity revealed in medium without A M P and creatine
Mitochondrial creatine kinase of frog heart
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phosphate, gave a band of lower mobility than the creatine kinase bands. For comparison, an extract of rat heart was submitted to the same electrophoresis (not shown). Under these conditions, the rat BB-form migrates out of the gel into the anode buffer. The rat M M has the same mobility as the frog myokinase and the mobility of the MB isoform of rat is higher than that of the three forms of frog creatine kinase. The rat mitochondrial form migrates towards the cathode and none of the frog creatine kinase forms migrate in the same way as the rat mitochondrial form. The identification of the frog isoenzymes of creatine kinase was performed by comparison with extracts of skeletal muscle and brain (Fig. 2) where the main isoforms are respectively M M and BB. This comparison indicates that in frog heart, the three forms MM, MB and BB are observed. It seemed that no mitochondrial form was observable under these conditions, although the digitonin fractionation had indicated the presence of 15% of the total creatine kinase activity in the mitochondrial fraction.
C. Identification of the mitochondrialform of frog creatine kinase Mitochondria were isolated from two hearts. The respiratory control and the ADP/O ratio of these mitochondria were determined. These are shown in Fig. 3 (A). The maximum respiration rate was 133 ng atoms O/min per mg protein in the presence of A D P 0.2 mM. The existence of a creatine kinase activity is demonstrated by the experiment shown in Fig. 3 (B). The maximum respiration rate is possible to obtain in
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Fig. l. Electrophoresis on cellulose acetate at pH 9.2 of whole heart extracts in 20 mM Na phosphate buffer. A: creatine kinase activity revealed in the presence of 13 mM AMP to inhibit myokinase. B: myokinase activity determined in the absence of creatine phosphate.
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Fig. 3. Oxygen consumption of frog heart mitochondria. A: mitochondria were suspended in a medium containing 108raM KC1, 2raM KH2PO 4 and 16mM Tris acetate buffer pH 7.4. B: 20 mM creatine and 2 mM Mg acetate were added to the medium. M fmitochondria, 1.ling protein/ml. S = 3.5 mM pyruvate and 0.35 mM malate. a medium containing no ADP, but 20 mM creatine and 0.2 mM ATP-Mg. The isolated mitochondria were submitted to an electrophoresis at pH 9.2 and compared with total heart extracts (Fig. 2). The mitochondrial creatine kinase was observed at the same place as the MB isoform. To obtain a separation between MB isoform and mitochondrial creatine kinase, total heart extracts ~.
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Fig. 2. Creatine kinase and myokinase activity observed on cellulose acetate strips after electrophoresis of various tissue extracts and isolated heart mitochondria.
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Fig. 4. Electrofocusing on acrylamide gels of whole frog heart extracts and mitochondrial fraction. A: whole heart extract in MOPS, mannitol, EDTA, KC1 buffer plus 1% Triton. The creatine kinase activity is revealed in the presence of 13 mM AMP and the myokinase activity by omitting creatine phosphate in the mixture. B and C comparative electrofocusing of: B=whole heart extracts, C = mitochondrial fractions.
254
ABDELKHALEQLEGSSYERand MARTINEARRIO-DuPONT
and mitochondrial fractions were submitted to isoelectric focusing (Fig. 4). On the eleetrofocusing plate shown in Fig. 4A, the activity was revealed with and without creatine phosphate and in the presence of AMP, so that either myokinase or creatine kinase activities or both were observed. On the plate shown in Fig. 4B and C, whole heart extracts and mitochondrial fraction were compared. In these experiments, creatine kinase, but not myokinase, is very sensitive to acidic denaturation by the anode electrode solution. The contribution of creatine kinase is underestimated. Three forms of myokinase are observed, one is encountered in the supernatant after a 8000 g centrifugation with a pHi of 7.1. The others, with p h i 6.8 and 6.3, are observed in the pellet and are very likely to be mitochondrial. In the purified mitochondria, we have observed that the myokinase activity is of the same order of magnitude as the creatine kinase activity. The MM, MB and BB isoforms of creatine kinase have pHi between 5.5 and 5.8 and this was confirmed by comparison with brain and skeletal muscle extracts (not shown). The mitochondrial creatine kinase, observed in the pellet obtained after the 8000 g centrifugation, has a pHi of 7.0, i.e. much higher than the isoelectric point of the MB form.
Frog heart has a metabolism which is more glycolytic than that of mammalian heart. The quantity of mitochondria is lower in the frog than in mammals (Scarpa and Grazzioti, 1973; Hoerter et al., 1981). Nevertheless, a mitochondrial creatine kinase is evident in frog heart. During development of mammals, the mitochondrial creatine kinase activity appears several days after birth (Hall and Deluca, 1975; Norwood et al., 1983; Van Brussel, 1983), so it seems difficult to compare adult frog tissues to neonatal mammal tissues. In adult frog heart, the creatine kinase in mitochondria is very likely to play the same role as in mammals (Jacobus, 1985), i.e. by rephosphorylation of creatine at the expense of newly synthetized mitochondrial ATP. This can be shown by the maximum respiration rate observed in the presence of ATP and creatine.
DISCUSSION
REFERENCES
Using starch gel electrophoresis, Eppenberger et al. (1967) found a single band of CPK activity as M M - C P K in crude isosmotic extracts of skeletal muscle from Rana pipiens. In cardiac extracts, the same authors found three bands. The most electronegative isoenzyme, BB-CPK, stained with the greatest intensity. Klemann and Pfohl (1982) have studied the CPK isoenzyme patterns of low osmolarity extracts of various tissues of Rana pipiens, by electrophoresis on acrylamide gels at pH 6.7. Surprisingly, these authors did not observe a mitochondrial form of CPK neither in brain nor in heart extracts, though in high vertebrate species, these tissues contain' mitochondrial C P K (Jacobs et al., 1964; Scholte, 1973). Isozyme patterns of CPK from pipid frogs (Wolff and Kobel, 1985) are more intricate than patterns observed for Rana pipiens, but a particulate form ("CPK I") was shown. Our electrophoretic patterns of CPK obtained for Rana esculenta seemed to indicate the absence of a mitochondrial form and the presence of the three isoenzyrnes MM, MB and BB-CPK in whole heart extracts. Nevertheless, in isolated cells permeabilized with digitonin as well as in purified mitochondria, creatine kinase activity was observed, for which the mobility was similar to that of the MB form. The mitochondrial creatine kinase has an isoelectric point very different from that of the MB form. MM, MB and BB isoenzymes of frog CPK have p h i between 5.8 and 5.5, but the mitochondrial form has a pHi of 7.0. It is possible that, because the migration of the mitochondrial CPK of Rana pipiens is similar to the migration of one of the soluble forms, it cannot be seen in the electrophorograms. This may explain why " M M - C P K " was observed in the nervous tissue of Rana pipiens by Klemann and Pfohl (1982): a plausible possibility is the presence of mitochondrial CPK in nervous tissue.
Arrio-Dupont M. and De Nay D. (1985) High yield preparation of calcium-tolerant myocytes from frog ventricles. Some properties of the isolated cells. Biol. Cell. 54, 163-170. Arrio-Dupont M. and De Nay D. (1986) Compartmentation of high energy phosphates in resting and beating heart cells. Biochim. biophys. Acta 851, 249-256. Eppenberger H. M., Dawson D. M. and Kaplan N. O. (1967) The comparative enzymology of creatine kinases-I. Isolation and characterization from chicken and rabbit tissues. J. biol. Chem. 242, 204-209. Eppenberger M. E., Eppenberger H. M. and Kaplan N. O. (1967) Evolution of creatine kinase. Nature 214, 239-241. Hall N. and Deluca M. (1975) Developmental changes in creatine phosphokinase isoenzymes in neonatal mouse hearts. Biochem. Biophys. Res. Commun. 66, 988-994. Hoerter J., Mazet F. and Vassort G. (I 98 I) Perinatal growth of the rabbit cardiac cell', possible implication for the mechanism of relaxation. J. molec. Cell. Cardiol. 13, 725-740. Jacobs H., Heldt H. W. and Klingenberg E. M. (1964) High activity of creatine kinase in mitochondria from muscle and brain and evidence for a separate mitochondrial isoenzyme of creatine kinase. Biochem. Biophys. Res. Commun. 16, 516--521. Jacobus W. E. and Lehninger A. L. (1973) Creatine kinase of the rat heart mitochondria. Coupling of phosphorylation to electron transport. J. biol. Chem. 248, 4804-4810. Jacobus W. E. (1985) Respiratory control and the integration of heart high-energy phosphate metabolism by mitochondrial ci'eatine kinase. A. Rev. Physiol. 47, 707-725. Klemann S. W. and Pfhol R. J. (1982) Creatine phosphokinase in Rana pipiens: Expression in embryos, early larvae and adult tissues. Comp. Biochem. Physiol. 73B, 907-914. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Morimoto K. and Harrington W. F. (1972) Isolation and physical properties of an M-line protein from skeletal muscle. J. biol. Chem. 247, 3052-3061.
Acknowledgements--This work has been supported by the Centre National de la Recherche Scientifique (Action th~matique Biorner#tique). We would like to thank Doctor G. Vassort, director of the laboratory, for his constant interest for this work, Professor D. C. Gautheron and Doctor C. Vial for fruitful discussions and suggestions and S. Rakotondrainibe for secretarial assistance.
Mitochondrial creatine kinase of frog heart Norwood W. I., Ingwall J. S., Norwood C, R. and Fossel E. T. (1983) Developmental changes of creatine kinase metabolism in rat brain. Am. J. Physiol. 244, C205-C210. Scarpa A. and Grazziotti P. (1973) Mechanisms for intracellular Ca regulation in heart--I. Stopped flow measurements of Ca + + uptake by cardiac mitochondria. J. Gen. Physiol. 62, 756-772. 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 myofibriUar and mitochondrial isoenzymes. Biochim. biophys. Acta 305, 413-427. Scholte H. R., Weijers P. J. and Wit-Peeters E. M. (1973) The localization of mitochondrial creatine kinase and its use for the determination of the sidedness of the submitochondrial particles. Biochim. biophys. Acta 291, 764-773. Turner D. C., Wallimann T. and Eppenberger H. M. (1973) A protein that binds specifically to the M-line of skeletal
255
muscle is identified as the muscle form of creatine kinase. Proc. natn. Acad. Sci. U.S.A. 70, 702-705. Van Brussel E., Yang J. J. and Seraydarian M. W. (1983) Isozymes of crcatine kinase in mammalian cell cultures. J. Cell. Physiol. 116, 221-226. Watts D. C. (1973) Creatine kinase (adenosine 5'-triphosphate-creatine phosphotransferase). In The Enzymes (Edited by Boyer P. D.) Vol. 8, pp. 383-455. Academic Press, New York. Wolff J. and Kobel H. R. (1985) Creatine kinas¢ isozymes in pipid frogs: their genetic bases, gene expressional differences and evolutionary implications. J. exp. Zool. 234, 471-480. Zuurendonk P. F., Tischler M. E., Akerboom T. P. M., Van Der Mccr R., Williamson J. R. and Tater J. M. (1979) Rapid separation of particulate and soluble fractions from isolated cell preparations (digitonin and cell cavitaticYn procedures). Methods Enzymol. 56, 207-223.
0305-0491/88 $3.00+ 0.00 © 1988PergamonJournals Ltd
MITOCHONDRIAL ISOENZYME OF CREATINE PHOSPHOKINASE IN FROG HEART ABDELKHALEQ LEGSSYERand MAnrINE ARRIO-DuPONT* Laboratoire de Physiologic Cellulaire Cardiaque. INSERM U-241 Universit6 de Paris-Sud, batiment 443, 91405 Orsay Cedex, France (Tel: 1-6941-7774) (Received 30 March 1987)
Abstract--1. Frog heart has a partially glycolytic metabolism; nevertheless a mitochondrial form of creatine phosphokinase (CPK) has been identified. 2. The CPK isoenzymepatterns (electrophoresis at pH 9.2 on celluloseacetate strips) in heart are similar to those of other vertebrates, with some variations. 3. The BB and MB isoenzymes are found in large quantities. 4. The electrophoretic mobilities of the mitochondrial isoform and that of the MB-form are the same, but the isoelectric point of the mitochondrial form (pHi 7) is not the same as that of the MB isoenzyme (pHi 5.6). 5. The activity of the CPK-form bound to mitochondria is strongly coupled to the ADP/ATP translocase.
INTRODUCTION Creatine phosphokinase (CPK, EC 2.7.3.2) is an enzyme involved in cellular distribution of energy, mainly occurring in organs such as heart, muscle or brain. It catalyses the transphorylation reaction between creatine phosphate and ADP: CrP + ADP + H+~-ATP + Cr It has been extensively studied in terms of its subcellular distribution in vertebrates (Eppenberger et al., 1967; Jacobs et al., 1964; Scholte, 1973). Different isoforms have been observed. The soluble isoenzymes are homo or heterodimers, namely MM, MB and BB according to the tissues where they are predominantly found: MM-CPK in skeletal muscle and BB-CPK in brain. Another isoform, the mitochondrial one (m-CPK), has also been observed: it is bound to the external face of the internal membrane of mitochondria (Jacobus and Lehninger, 1973; Scholte et aL, 1973) and it is implicated in the synthesis of creatine phosphate. In the case of amphibians, the soluble isoforms MM, MB and BB have been identified (Eppenberger et al., 1967) and the distribution of the isoforms in eggs and selected stages of development has been studied (Klemann and Pfohl, 1982). However the mitochondrial form has never been identified. The existence of such a form may be questionable. In these animals, the cardiac metabolism is more glycolytic than in terrestrial vertebrates and it is possible that mitochondrial metabolism is not as important in frog heart as in adult mammal heart. In animals such as the rat or the
sheep, the development of the mitochondrial creatine kinase is linked to the appearance of mitochondria and oxidative metabolism (Hall and Deluca, 1975; Norwood et al., 1983; Van Brussel et al., 1983). Amphibians (frog) have a metabolism similar to intermediate stages of mammal development and it is possible that there is no mitochondrial CPK in these animals. In the present work, the different isoforms of CPK have been identified in heart and compared to skeletal muscle and brain extracts. For a better identification of the mitochondrial isoform, mitochondria have been isolated and checked for the presence of CPK activity. The repartition of the CPK activity in the main cellular compartments: soluble cytosol, mitochondria and myofilament has been determined in isolated myocytes from ventricles.
MATERIALSAND METHODS a. Tissue extracts Mature frogs (Rana esculenta) were killed by decapitation and the following tissues removed: cardiac muscle, skeletal muscle (gastrocenemius) and brain. For cardiac muscle, the heart was previously washed with standard Ringer solution to eliminate blood cells. The tissues were cut into small pieces in 20 mM Na phosphate pH 7.4, containing 0.I mM dithiothreitol, at 2°C. The suspension was homogenized with a Potter-Elvehjem (600 rpm, 1 min at 2°C). The homogenates were centrifuged at 8000g for 10 rain at 4°C and the supernatant fluids were retained for analysis by either electrophoresis or eiectrofocusing. b. Cell isolations. Fast fractionation Ventricular cells were isolated as previously described (Arrio-Dupont and De Nay, 1985) by perfusion with collagenase and trypsin. The main cellular compartments were separated by fast digltonin (Zuurendonk et al., 1979) or Triton (Arrio-Dupont and De Nay, 1986) fractionation, in
*To whom correspondence should be addressed. Abbreviations used---CPK, creatine phosphokinase; MK, myokinase; MPS, morpholino propane sulfonic acid; CrP, creatine phosphate; Cr, creatine. 251
252
ABDELKI-IALEQLEGSSYERand MART1NEARRIO-DUPONT
a medium containing 20 mM MPS, 3 mM EDTA and either 230 mM mannitol (low I medium) or 115 mM KC1 (high I medium), for which the pH was adjusted to 6.75 at 22°C. After 10 sec at 2°C in the presence of the lysis medium, the cell suspension was centrifuged through silicon oil. The enzymatic activities were determined in the supernate and in the pellet after treatment by Triton in low I medium. The total intracellular activities were obtained in the pellet by omitting digitonin or Triton in the lysis medium. As previously described (Arrio-Dupont and De Nay, 1986), the top and bottom fractions obtained after centrifugation contained respectively broken cells plus cytosol and mitochondria plus myofilament after digltonin treatment, and broken cells plus mitochondria plus cytosol and myofilaments after Triton lysis. According to the ionic strength of the lysis medium, CPK is either detached or not detached from mitochondria and myofdaments.
c. Mitochondria Mitochondria were isolated by a method analogous to that described by Scarpa and Grazzioti (1975). Small pieces of ventricular tissue, from two hearts, were cut in 20 mM MPS buffer pH7.4, containing 55mM sucrose, 16mM mannitol, 3 mM EDTA and 0.1 mM dithiothreitol, at 2°C and homogenized with a Potter-Elvejhem homogenizer (600 rpm for 1 min at 2°C). The homogenate was then centrifuged for 10 min at 1000 g. After centrifugation of the supernatant for 20 min at 12,000g, the mitochondria were obtained in the pellet. After two further steps of washing and centrifugation, the mitochondria were suspended in 200/~1 of the same buffer and kept in an ice-bath. The final concentration corresponded to about 10 mg/ml protein. The obtained mitochondria were checked for their respiratory control and their ADP/O ratio, by using a Clark oxygen electrode (YSI) at 20°C. The buffer used to study oxygen consumption was 16mM Tris-HCl, containing 105raM KCI and 2 mM KH2PO 4 pH 7.4, with 3.5 mM pyruvate and 0.35 mM malate as substrates. The respiration was stimulated by 0.2 mM ADP. d. Electrophoresis, electrofocusing Electrophoresis was performed on cellulose acetate strips. The electrophoretic buffer was 50 mM Tris--veronal pH 9.2; three gels (Cellogel, 14 x 5.7 era) were run simultaneously by applying 200 V and 10mA for 3 hr. A Sebia electrophoresis cuvette (S 60) was used. Electrofocusing was performed on PGA plates LKB, pH either 3-9.5 or 5.5-8.5 at 6°C, under constant 20 W power, for 4 hr. A 2117 multiphor from LKB was used. For both electrophoresis and electrofocusing, a power supply 2197 from LKB was used. e. Enzymatic activities, protein determination Creatine kinase activity was determined at 22°C, by a coupled enzymatic assay and the formation of NAD(P)H was studied. The reaction mixture contained 4.2raM MgSO4, 1 mM ADP, 10 mM creatine phosphate, 0.6 mM NADP, 20 mM glucose, 1 U/ml hexokinase, 1 U/ml glucose6-phosphate dehydrogenase in 50 mM Tris--acetate buffer, pH 7.4. In spectrophotometer, the NADPH formation was determined by its absorbance at 340 nm; on the gels, it was detected by the formazan reaction and observed by incubating the strips with an overlay of the same support medium containing the substrates, the enzymes, phenazine methosuifate and nitroblue tetrazolium. The contamination by myokinase (MK, EC 2.7.4.3) activity (2 A D P ~ A T P + AMP) was determined under the same conditions by omitting creatine phosphate in the reaction mixture. In some cases, the myokinase activity was inhibited by an excess of AMP (13 mM). The MK activity was determined with a coupled enzymatic assay similar to that used for CK activities, with no ereatine phosphate in the medium. Proteins were determined after 0.1 M NaOH dena-
Table 1. Compartmentation of creatine kinase activity in isolated ventrieular cells Percentage of activity* Lysis medium Top fraction Bottomfractiont None 10-15 85-90 Digitonin high I 65-70 30-35 Digitonin low I 85 15 Triton high I 75-80 20-25 Triton low I 100 0 *The total CK activity (100%) was determined in a medium of low ionic strength to detach the enzyme bound to myofilamentsand in the presence of either Triton X-100 or Lubrol WX. fin the bottom fractions, the activity was determined after treatment of the pellets with Triton, in a medium of low ionic strength. In all the fractions, the myokinase activity observed by omitting creatine phosphate in the medium has been deducted. turation, by the Lowry et al. method (1951) using bovine serum albumin as a standard.
f. Materials Enzymes were from Boehringer. Pyruvate, L-malate, phenazine methosulfate, nitroblue tetrazolium, NADP, ATP, ADP and AMP were from Sigma. RESULTS
A. Compartmentation of creatine kinase activity By using the lysis medium of either high or low ionic strength, on isolated ventricular cells, it was possible to estimate the repartition of the creatine kinase activity in the main cellular compartments. The activities observed in the top and bottom fractions after lysis with 0.5 mg/ml digitonin for 10 see at 2°C or with 2% Triton X-100 for 15 see at the same temperature are indicated in Table 1. In the medium of low ionic strength containing Triton, 100% of the creatine kinase activity is observed in the supernatant, because in this medium all the membranes are destroyed and creatine kinase is detached from the myofilaments ( M o r i m o t o and Harrington, 1972; Turner et al., 1973). The total C P K activity determined either in 2% Triton of low ionic strength or in Lubrol WX, for nine cellular isolations, was 0 . 6 3 _ 0.08 (SD) I U / m g cellular protein. In Triton medium of high ionic strength, 80-85% of the total C P K activity is observed in the supernatant and about 15% in the bottom. F r o m this result, it can be deduced that 15-20% of the creatine kinase activity is bound to the myofilaments. The use of digitonin in a low ionic strength medium gives access to creatine kinase activity bound to the mitochondria: it represents about 15% of the total activity. The remaining activity, 65-70%, is the free cytosolic creatine kinase. B. Total heart extracts. Comparison with extracts o f skeletal muscle and brain The total C P K activity of whole heart extracts in 2 0 m M N a phosphate buffer was 1.2 _+ 0.2 I U / m g protein and the total myokinase activity represents 2 - 5 % of this activity. These activities correspond to several isoforms, as shown by electrophoresis on cellulose acetate strips at p H 9.2 (Fig. 1). When the creatine kinase activity on the gel is revealed in the presence of 13 m M A M P , three bands migrating toward the anode are observed. The myokinase activity revealed in medium without A M P and creatine
Mitochondrial creatine kinase of frog heart
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phosphate, gave a band of lower mobility than the creatine kinase bands. For comparison, an extract of rat heart was submitted to the same electrophoresis (not shown). Under these conditions, the rat BB-form migrates out of the gel into the anode buffer. The rat M M has the same mobility as the frog myokinase and the mobility of the MB isoform of rat is higher than that of the three forms of frog creatine kinase. The rat mitochondrial form migrates towards the cathode and none of the frog creatine kinase forms migrate in the same way as the rat mitochondrial form. The identification of the frog isoenzymes of creatine kinase was performed by comparison with extracts of skeletal muscle and brain (Fig. 2) where the main isoforms are respectively M M and BB. This comparison indicates that in frog heart, the three forms MM, MB and BB are observed. It seemed that no mitochondrial form was observable under these conditions, although the digitonin fractionation had indicated the presence of 15% of the total creatine kinase activity in the mitochondrial fraction.
C. Identification of the mitochondrialform of frog creatine kinase Mitochondria were isolated from two hearts. The respiratory control and the ADP/O ratio of these mitochondria were determined. These are shown in Fig. 3 (A). The maximum respiration rate was 133 ng atoms O/min per mg protein in the presence of A D P 0.2 mM. The existence of a creatine kinase activity is demonstrated by the experiment shown in Fig. 3 (B). The maximum respiration rate is possible to obtain in
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Fig. l. Electrophoresis on cellulose acetate at pH 9.2 of whole heart extracts in 20 mM Na phosphate buffer. A: creatine kinase activity revealed in the presence of 13 mM AMP to inhibit myokinase. B: myokinase activity determined in the absence of creatine phosphate.
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Fig. 3. Oxygen consumption of frog heart mitochondria. A: mitochondria were suspended in a medium containing 108raM KC1, 2raM KH2PO 4 and 16mM Tris acetate buffer pH 7.4. B: 20 mM creatine and 2 mM Mg acetate were added to the medium. M fmitochondria, 1.ling protein/ml. S = 3.5 mM pyruvate and 0.35 mM malate. a medium containing no ADP, but 20 mM creatine and 0.2 mM ATP-Mg. The isolated mitochondria were submitted to an electrophoresis at pH 9.2 and compared with total heart extracts (Fig. 2). The mitochondrial creatine kinase was observed at the same place as the MB isoform. To obtain a separation between MB isoform and mitochondrial creatine kinase, total heart extracts ~.
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Fig. 2. Creatine kinase and myokinase activity observed on cellulose acetate strips after electrophoresis of various tissue extracts and isolated heart mitochondria.
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Fig. 4. Electrofocusing on acrylamide gels of whole frog heart extracts and mitochondrial fraction. A: whole heart extract in MOPS, mannitol, EDTA, KC1 buffer plus 1% Triton. The creatine kinase activity is revealed in the presence of 13 mM AMP and the myokinase activity by omitting creatine phosphate in the mixture. B and C comparative electrofocusing of: B=whole heart extracts, C = mitochondrial fractions.
254
ABDELKHALEQLEGSSYERand MARTINEARRIO-DuPONT
and mitochondrial fractions were submitted to isoelectric focusing (Fig. 4). On the eleetrofocusing plate shown in Fig. 4A, the activity was revealed with and without creatine phosphate and in the presence of AMP, so that either myokinase or creatine kinase activities or both were observed. On the plate shown in Fig. 4B and C, whole heart extracts and mitochondrial fraction were compared. In these experiments, creatine kinase, but not myokinase, is very sensitive to acidic denaturation by the anode electrode solution. The contribution of creatine kinase is underestimated. Three forms of myokinase are observed, one is encountered in the supernatant after a 8000 g centrifugation with a pHi of 7.1. The others, with p h i 6.8 and 6.3, are observed in the pellet and are very likely to be mitochondrial. In the purified mitochondria, we have observed that the myokinase activity is of the same order of magnitude as the creatine kinase activity. The MM, MB and BB isoforms of creatine kinase have pHi between 5.5 and 5.8 and this was confirmed by comparison with brain and skeletal muscle extracts (not shown). The mitochondrial creatine kinase, observed in the pellet obtained after the 8000 g centrifugation, has a pHi of 7.0, i.e. much higher than the isoelectric point of the MB form.
Frog heart has a metabolism which is more glycolytic than that of mammalian heart. The quantity of mitochondria is lower in the frog than in mammals (Scarpa and Grazzioti, 1973; Hoerter et al., 1981). Nevertheless, a mitochondrial creatine kinase is evident in frog heart. During development of mammals, the mitochondrial creatine kinase activity appears several days after birth (Hall and Deluca, 1975; Norwood et al., 1983; Van Brussel, 1983), so it seems difficult to compare adult frog tissues to neonatal mammal tissues. In adult frog heart, the creatine kinase in mitochondria is very likely to play the same role as in mammals (Jacobus, 1985), i.e. by rephosphorylation of creatine at the expense of newly synthetized mitochondrial ATP. This can be shown by the maximum respiration rate observed in the presence of ATP and creatine.
DISCUSSION
REFERENCES
Using starch gel electrophoresis, Eppenberger et al. (1967) found a single band of CPK activity as M M - C P K in crude isosmotic extracts of skeletal muscle from Rana pipiens. In cardiac extracts, the same authors found three bands. The most electronegative isoenzyme, BB-CPK, stained with the greatest intensity. Klemann and Pfohl (1982) have studied the CPK isoenzyme patterns of low osmolarity extracts of various tissues of Rana pipiens, by electrophoresis on acrylamide gels at pH 6.7. Surprisingly, these authors did not observe a mitochondrial form of CPK neither in brain nor in heart extracts, though in high vertebrate species, these tissues contain' mitochondrial C P K (Jacobs et al., 1964; Scholte, 1973). Isozyme patterns of CPK from pipid frogs (Wolff and Kobel, 1985) are more intricate than patterns observed for Rana pipiens, but a particulate form ("CPK I") was shown. Our electrophoretic patterns of CPK obtained for Rana esculenta seemed to indicate the absence of a mitochondrial form and the presence of the three isoenzyrnes MM, MB and BB-CPK in whole heart extracts. Nevertheless, in isolated cells permeabilized with digitonin as well as in purified mitochondria, creatine kinase activity was observed, for which the mobility was similar to that of the MB form. The mitochondrial creatine kinase has an isoelectric point very different from that of the MB form. MM, MB and BB isoenzymes of frog CPK have p h i between 5.8 and 5.5, but the mitochondrial form has a pHi of 7.0. It is possible that, because the migration of the mitochondrial CPK of Rana pipiens is similar to the migration of one of the soluble forms, it cannot be seen in the electrophorograms. This may explain why " M M - C P K " was observed in the nervous tissue of Rana pipiens by Klemann and Pfohl (1982): a plausible possibility is the presence of mitochondrial CPK in nervous tissue.
Arrio-Dupont M. and De Nay D. (1985) High yield preparation of calcium-tolerant myocytes from frog ventricles. Some properties of the isolated cells. Biol. Cell. 54, 163-170. Arrio-Dupont M. and De Nay D. (1986) Compartmentation of high energy phosphates in resting and beating heart cells. Biochim. biophys. Acta 851, 249-256. Eppenberger H. M., Dawson D. M. and Kaplan N. O. (1967) The comparative enzymology of creatine kinases-I. Isolation and characterization from chicken and rabbit tissues. J. biol. Chem. 242, 204-209. Eppenberger M. E., Eppenberger H. M. and Kaplan N. O. (1967) Evolution of creatine kinase. Nature 214, 239-241. Hall N. and Deluca M. (1975) Developmental changes in creatine phosphokinase isoenzymes in neonatal mouse hearts. Biochem. Biophys. Res. Commun. 66, 988-994. Hoerter J., Mazet F. and Vassort G. (I 98 I) Perinatal growth of the rabbit cardiac cell', possible implication for the mechanism of relaxation. J. molec. Cell. Cardiol. 13, 725-740. Jacobs H., Heldt H. W. and Klingenberg E. M. (1964) High activity of creatine kinase in mitochondria from muscle and brain and evidence for a separate mitochondrial isoenzyme of creatine kinase. Biochem. Biophys. Res. Commun. 16, 516--521. Jacobus W. E. and Lehninger A. L. (1973) Creatine kinase of the rat heart mitochondria. Coupling of phosphorylation to electron transport. J. biol. Chem. 248, 4804-4810. Jacobus W. E. (1985) Respiratory control and the integration of heart high-energy phosphate metabolism by mitochondrial ci'eatine kinase. A. Rev. Physiol. 47, 707-725. Klemann S. W. and Pfhol R. J. (1982) Creatine phosphokinase in Rana pipiens: Expression in embryos, early larvae and adult tissues. Comp. Biochem. Physiol. 73B, 907-914. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Morimoto K. and Harrington W. F. (1972) Isolation and physical properties of an M-line protein from skeletal muscle. J. biol. Chem. 247, 3052-3061.
Acknowledgements--This work has been supported by the Centre National de la Recherche Scientifique (Action th~matique Biorner#tique). We would like to thank Doctor G. Vassort, director of the laboratory, for his constant interest for this work, Professor D. C. Gautheron and Doctor C. Vial for fruitful discussions and suggestions and S. Rakotondrainibe for secretarial assistance.
Mitochondrial creatine kinase of frog heart Norwood W. I., Ingwall J. S., Norwood C, R. and Fossel E. T. (1983) Developmental changes of creatine kinase metabolism in rat brain. Am. J. Physiol. 244, C205-C210. Scarpa A. and Grazziotti P. (1973) Mechanisms for intracellular Ca regulation in heart--I. Stopped flow measurements of Ca + + uptake by cardiac mitochondria. J. Gen. Physiol. 62, 756-772. 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 myofibriUar and mitochondrial isoenzymes. Biochim. biophys. Acta 305, 413-427. Scholte H. R., Weijers P. J. and Wit-Peeters E. M. (1973) The localization of mitochondrial creatine kinase and its use for the determination of the sidedness of the submitochondrial particles. Biochim. biophys. Acta 291, 764-773. Turner D. C., Wallimann T. and Eppenberger H. M. (1973) A protein that binds specifically to the M-line of skeletal
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muscle is identified as the muscle form of creatine kinase. Proc. natn. Acad. Sci. U.S.A. 70, 702-705. Van Brussel E., Yang J. J. and Seraydarian M. W. (1983) Isozymes of crcatine kinase in mammalian cell cultures. J. Cell. Physiol. 116, 221-226. Watts D. C. (1973) Creatine kinase (adenosine 5'-triphosphate-creatine phosphotransferase). In The Enzymes (Edited by Boyer P. D.) Vol. 8, pp. 383-455. Academic Press, New York. Wolff J. and Kobel H. R. (1985) Creatine kinas¢ isozymes in pipid frogs: their genetic bases, gene expressional differences and evolutionary implications. J. exp. Zool. 234, 471-480. Zuurendonk P. F., Tischler M. E., Akerboom T. P. M., Van Der Mccr R., Williamson J. R. and Tater J. M. (1979) Rapid separation of particulate and soluble fractions from isolated cell preparations (digitonin and cell cavitaticYn procedures). Methods Enzymol. 56, 207-223.