Ultrastructural Distribution of the M Form of Creatine Phosphokinase in Human Muscle by lmmunogold Labeling JOHN R. DANKERT, GEORGE P. PAPADI, AND ROBERT P. SHIELDS Department of Comparative and Experimental Pathology, Health Science Center, College of Veterinary Medicine University of Florida, Gainesville, Florida 3261 0


Creatine, Creatine phosphokinase, Ultrastructure

ABSTRACT Creatine phosphokinase regenerates ATP from ADP using creatine phosphate. Isoenzymes of creatine phosphokinase are bound t o certain cellular structures or are compartmentalized in areas of the cell, and this has been used as a basis for defining the role of these isoenzymes in energy metabolism. The M isoenzyme of creatine phosphokinase has been morphologically associated with the M-line of striated muscle in many species. In this present study the ultrastructural distribution and the relative concentration of the M form of creatine phosphokinase in human muscle tissue was determined using immunogold and electron microscopy. The M-line of the sarcomere, comprising only 3-4% of the sarcomere area, was found to contain over 20%of the total M isoenzyme signal of the entire sarcomere. This technique represents a quantitative, ultrastructural method to study the subcellular distribution of this isoenzyme. These data suggest that localized concentrations of M-CPK may be important for normal energy metabolism, and may also serve as a foundation for a better understanding of the relationship between abnormal creatine metabolism and the pathogenesis of neuromuscular disease. INTRODUCTION The enzyme creatine phosphokinase (CPK) is an integral part of the phosphagen system of mammals. It has been proposed that creatine phosphate serves as a shuttle metabolite between an area of ATP synthesis and an area of ATP utilization or need (Bessman and Carpender, 1985; Wallimann and Eppenberger, 1990). Energy stored as creatine phosphate is unique in that it is the only storage form of phosphate-bond energy in mammals (Ennor and Morrison, 1958). Three isoenzymes of CPK have been isolated from skeletal muscle: the M or muscle form, the B or brain form, and the Mi or mitochondrial form (Pennington, 1988; Schlegel et al., 1988). The M and B isoenzymes are postulated to form homodimers; however a heterodimer MB complex has also been isolated (Pennington, 1988). For the Mi form, dimers and more complex oligomers (octamers) have been reported (Schlegel et al., 1988).Adult skeletal muscle contains primarily the M and the Mi-forms, with the M form primarily in MM dimers (MM-CPK) (Turner et al., 1973). Studies of the subcellular distribution of CPK indicate that part of the total muscle enzyme activity can be found in a soluble form within the sarcoplasm and part in a form bound to intracellular structures (Baskin and Deamer, 1970; Lane et al., 1986; Saks et al., 1977; Turner et al., 1973). A “compartmentalized’ arrangement has been proposed for the mitochondrial form of CPK (Wallimann and Eppenberger, 1990). Although subcellular localizations of CPK have suggested specific functions for its isoenzymes, it is not clear if subcellular binding is essential for normal, physiological enzyme function. For example, finding the Mi-form of CPK between the inner and outer membranes of mitochondria supports a



role for the Mi-form of the enzyme participating in a high energy phosphate shuttle between mitochondria and the cytosol (Biermans et al., 1989).Finding the M form bound or associated with the M-line of myofibers has suggested a role for this form in the contraction process (Wallimann et al., 1977). An increase in plasma levels of the CPK enzyme is considered a sensitive biochemical index of muscle disease (Pennington, 1988). The metabolic defect(s) responsible for the increased plasma levels of CPK has not been clearly defined. A non-specific leak in the plasma membrane might explain this increase nevertheless; recent work does not support the concept that there is a global breakdown in membrane permeability (Bank et al., 1971; Fitch et al., 1968; Fitch and Sinton, 1964; Pennington, 1988; Silverman et al., 1976). Abnormally high plasma levels of CPK have been used with some success to detect asymptomatic carriers of Duchenne’s muscular dystrophy, and such increases may occur long before clinical signs of muscle disease are evident (Heyck et al., 1966; Pearce et al., 1964).In patients with Duchenne’s muscular dystrophy, the isoenzyme pattern of CPK within muscles changes and increased amounts of the MB and B forms of CPK have been reported (Jacobs and Kuby, 1980; Somer et al., 1976). In addition, in cells cultured from patients with Duchenne’s dystrophy it was reported that the binding of the M form of CPK to the M-line was impaired (Askanas et al., 1987). Received April 1. 1991; accepted in revised form June 5,1991. Address reprint requests to John R. Dankert, Ph.D., Department of Comparative and Experimental Pathology, Box 5-145, University of Florida, Gainesville, FL 32610.



Figs. 1 and 2. (Legendson facing page.)



Fig. 3. Electron micrograph of distribution of gold-labeled secondary antibody on muscle with primary antibody to human M-creatine phosphokinase. Scale bar represents 0.5 pm.

Explanations of the role of M-CPK in muscle energy metabolism have been based, at least initially, on the intracellular location of this enzyme. Therefore, in this study a method was sought to quantitatively determine the distribution of the M form of CPK in a normal human muscle sarcomere. Any abnormal distribution of this isoenzyme within defined areas of an individual sarcomere that might occur during neuromuscular disease could then be compared to the normal distribution. This could help to clarify the relationship between “abnormal creatine metabolism” and neuromuscular disease as well as increase our understanding of the role of M-CPK in energy metabolism.


Fig. 1. Electron micrograph of human skeletal muscle stained with gold-labeled (10 nm gold particles) secondary antibody using pre-immune normal rabbit serum as the source of primary antibody. Thin, labeled lines represent sample longitudinal components of defined areas for particle counting. Letter labels: M, M-line; A, A-band; I, I-band; and Z, Z-disc. Each sarcomere contains one M-line area, two areas of A-bands, and two areas of I-bands. Scale bar represents 0.5 Pm.

Fig. 2. Electron micrograph of distribution of gold-labeled (10nm gold particles) secondary antibody on muscle pre-incubated with primary antibody to myosin. Scale bar represents 0.5 pm.

MATERIALS AND METHODS Chemicals and Reagents LR White acrylic resin, nickel grids (200 mesh), uranyl acetate, and the fixing agents (glutaraldehyde and paraformaldehyde) were obtained from the Ernest Fullam Co., Latham, NY. Polyclonal anti-human M-CPK antibodies were obtained from Chemicon International, Inc., El Segundo, CA.; anti-myosin antibody from Sigma Chemical Co., St. Louis, MO.; and Janssen gold-labeled secondary antibodies from Amersham Inc., Arlington Heights, IL. All other chemicals and buffers were obtained from Fisher Scientific, Pittsburgh, PA. Preparation of Muscle Normal human muscle samples were obtained from tissue submitted for histopathology, but were negative for neuromuscular disease. Fresh muscle samples were placed in cacodylate buffer (pH 7.4) which contained 2% paraformaldehyde and 0.5% glutaraldehyde, trimmed to approximately 2 x 2 x 10 mm, and were allowed to fix in this solution for 1.5 h with gentle shaking. After rinsing in cacodylate buffer (3 x 1, the tissues were stored in the same buffer a t 5°C. Selected strips of muscle were dehydrated in 50% ethanol (15 m i d , 70%ethanol (15 min), and then infiltrated for 12 to 16 h at 5°C in a 1:1 mixture of 70% ethanol and LR White embedding media with mild rocking agitation.



The tissue was transferred to 100% LR White for 5 h and embedded in fresh LR White media in a size 00 Beem capsule for 24 h a t 50°C. Tissues were oriented for transverse or longitudinal sections. Sectioned tissue (LKB Ultratome 111) was placed on nickel grids for staining. 4 Immunostaining The antibody against human M-CPK was checked for specificity by immunoblotting of tissue homogenized in a 10% solution of sodium dodecyl sulfate and 2 centrifuged (9,OOOg, 10 min) to remove insoluble debris, which revealed one major band from human muscle with a relative mobility of about 40,000 MW (data not shown). Thin sections on nickel grids were incubated a t 23°C (20 min) in a solution of 5% normal goat 0 serum and 0.25%ovalbumin in a buffer consisting of 10 M-Line I-Band A-Band mM Tris, 0.1% bovine serum albumin, 0.5 M NaC1, 0.05% Tween 20, and 0.2 mM NaN, at pH 8.2. This was Muscle Zone followed by incubation for 2 h in the same buffer containing 1%normal goat serum and 0.25% ovalbumin Fig. 4. Calculated particle densities of gold-labeled secondary ansupplemented with 20% of a primary antibody (either tibody particle counts found in the M-line, A-bands, or I-bands using rabbit anti-human M-CPK, rabbit anti-human myosin, primary antibody to either anti-M-CPK (dark bars) or anti-myosin bars). Error bars represent SEM. Background counts obtained or pre-immune normal rabbit serum). Sections were (light using pre-immune rabbit serum as the source of primary antibody then washed 3 times (for 5 min each wash) in buffer would result in a decrease of the value of each zone of less than 0.2 and incubated for 20 min with a 5% solution of 10 nm units of particle density. gold-labeled goat anti-rabbit IgG secondary antibody. This was followed by washing 3 times in buffer, and 3 times in distilled water. Sections were then stained with uranyl acetate and lead citrate and were imaged body produced micrographs with many more gold parusing a Zeiss EM-10 transmission electron microscope. ticles (Fig. 2) and with the majority of the label located in the A-bands. This indicated that the techniques used Distribution of Gold Particles to prepare the sections for labeling had not destroyed Electron microscopic photographs of longitudinal the ability of the antibody to recognize the correspondsections of muscle were used to measure the tissue area ing antigenic site. Sections of normal human skeletal muscle were inin each of the following zones: the entire sarcomere, the A-band, the I-band, and the M-line. Gold particles were cubated with anti-M-CPK antibodies followed by the counted and the number of particles per unit area in secondary gold-labeled antibody. Micrographs of these each zone was calculated. This total was then divided sections illustrate that many gold particles could be by the total number of particles per the entire sarco- seen in the sarcomeres (Fig. 3). It was noted, however, mere to produce the particle density value. Micro- that the distribution of the particles was not random graphs of tissue sections from muscle incubated with since the particles appeared more numerous at the cenpre-immune serum (normal rabbit serum) were tral M-line. The number of gold particles counted in the counted to determine the background level of binding. M-line was found to be 23.8% (21.0 SEM) of the total Total sarcomere was taken as the area from outer edge number in the sarcomere. The measured area of the of a Z-disc to the inner edge of the opposite Z-disc; I- M-line relative to the area of the sarcomere was only band as the two areas from the Z-disc to the start of the 4.8% (*0.2 SEMI. This indicated that 23.8% of the gold darker A-band; A-band as the two areas from end of particles occupied only 4.8% of the sarcomere area and I-band to the dark edges of the M-line; and M-line as thus, supported the idea that the concentration of the the dark central area between the two A-bands. M-CPK antigen was elevated at the M-line. An understanding of differences in intracellular MRESULTS CPK concentrations could be of importance in elucidatA micrograph of a section of normal human skeletal ing a possible role of M-CPK dimers or polymers in in muscle fixed and incubated as in “Materials and Meth- vivo enzyme activity (Schlegel et al., 1988). In order to ods” in the presence of non-specific primary antibody quantitate these differences in concentration and also source (normal rabbit serum) is presented in Figure 1. to account for the differences in the level (i.e., total The morphologic features of normal skeletal muscle number) of gold-staining particles per sarcomere, parcan be readily identified (Fitzsimons and Sewry, 1985). ticle density was determined. The gold particle density The low number of gold particles (mean = 1.2 per sar- for M-CPK and for myosin in the M-line, I-bands, and comere) demonstrates the low reactivity of the pre-im- A-bands is shown in Figure 4. It is evident that the mune and the gold-labeled secondary antibody for this density of particles designating the M-CPK antigen is tissue. Sections incubated with anti-myosin antibodies greater in the M-line relative to that of the other zones. prior to reaction with the gold-labeled secondary anti- For the M-CPK label, the mean density for the M-line





















Particle Density Fig. 5. Frequency distribution of M-line particle density values of anti-M-CPKin human muscle. Densities were grouped as defined on the abscissa into units of 0.5, with 8 + grouping all values above 8. The ordinate depicts the frequency of values in each group. Total number of values = 150. Mean = 4.94. SD = 1.5.

was 4.94, with values of 1.1 and 0.65 for the I and A-bands, respectively. Comparing the mean values obtained for the M-CPK, it can be seen that the M-line particle density value is over 5 times greater than that of the I-bands, and over 7 times greater than that of the A-bands. The number of gold particles found in the mitochondria or Z-disc areas of the micrographs using anti-M-CPK antibody did not exceed that seen using pre-immune serum (background control). Values measured for three different individuals were included in Figure 4; however, no set of values for any single individual was significantly different (mean M-line particle density values for M-CPK of 4.75,4.82,and 5.02 for the three individuals, all within the standard error of the total mean). In contrast to M-CPK, the particle density values for myosin (Fig. 4) display a higher concentration of myosin in the A-bands and M-line of the sarcomere than in the I-bands. The particle density values suggest that there are elevated levels of M-CPK antigen in the M-line of a sarcomere; nevertheless, a comparison of individual Mline values from one sarcomere to another could vary from a value of 0 to a value of over 8. It was important therefore, to determine whether the mean M-line value presented in Figure 4 was the result of a normal distribution or was a result of a population in which certain values were more frequent than others. Figure 5 presents the frequency distribution of the individual particle densities measured, with values grouped into sections of 0.5 units. Using the standard deviation value of 1.5 obtained for the 150 measurements presented, 68% of the values fall within 1 SD about the mean. DISCUSSION The chemical fixation and tissue processing methods used in this present study were selected to preserve the


cytological structure as well as the immunological signal. More specifically, the use of osmium tetroxide and long exposures to high concentrations of other chemical fixatives which might block immunocytochemical reactions (Cullen and Landon, 1988; Fitzsimons and Sewry, 1985; Jorgensen and McGuffee, 1987; Neville, 1973) were avoided. Tissues were embedded in LR White since the hydrophilic nature of this medium reportedly aids tissue penetration (Causton, 1984). Immuno-gold was selected because it would permit higher magnification than fluorescence and could be more easily differentiated from cellular structures than the reaction product of immunoperoxidase (Romano and Romano, 1984). The direct coupling of creatine phosphate with the expenditure of phosphate-bond energy for muscle contraction was demonstrated by Infante and Davies (1965), and has more recently been integrated with other cellular energy systems and described as a “creatine phosphate shuttle” (Bessman and Carpender, 1985) or as a “phosphoryl-circuit” model (Wallimann and Eppenberger, 1990). These systems are based to some extent upon the subcellular distribution of CPK isoenzymes. Fluorescence microscopy (Turner et al., 1973) and immunoenzymatic techniques (Biermans et al., 1989) have been used to determine the subcellular location of CPK isoenzymes. Biochemical examination of the intracellular distribution of M-CPK in chicken muscle indicated that approximately 3 -5% of the total CPK activity of the muscle was bound to the myofibrils and this amount of enzyme was sufficient to produce the ATP needed for muscle contraction (Wallimann et al., 1977). Fluorescence microscopy demonstrated that M-CPK could be visualized along the M-lines of the chicken myofibrils (Wallimann and Eppenberger, 1990). The M-line of rabbit skeletal muscle has also been found to contain a protein that has the capacity to bind to M-CPK (Mani and Kay, 1978). The intracellular distribution of the M-CPK has not been studied as extensively in muscle from humans as in other species. M-CPK has been visualized, however, along the M-line of human muscle using fluorescence microscopy (Feit et al., 1983). In this present work, fixed human muscle and immuno-gold staining techniques were used to provide electron microscopic evidence of the uniquely high concentration of M-CPK at the M-line. Multicomponent complexes of CPK isoenzymes have been implicated as a possible regulatory factor of its activity (Schlegel et al., 1988). Elevated concentrations of M-CPK could be of great importance in maintaining dimer or larger polymer complexes of this isoenzyme. The estimation of particle density permits comparisons to be made between individual sarcomeres and, since each sarcomere serves as a control for the total signal, this provides an estimate of the concentration in one area of the sarcomere relative to that within the whole sarcomere. It should be pointed out, however, that any conclusions drawn would be based on the assumptions that 1) the antigenic characteristics of M-CPK have not been destroyed by tissue processing and that 2) M-CPK antigenicity was homogeneous throughout the sarcomere. Immuno-enzymatic stain-



and contracting cultured muscle fibers of Duchenne muscular dystrophy patients. Life Sci., 41:927-933. Bank, W.J., Rowland, L.P., and Ipsen, J. (1971) Amino acids in plasma and urine in diseases of skeletal muscle. Arch. Neurol., 24:176-186. Baskin, J.R., and Deamer, D.W. (1970) A membrane-bound creatine phosphokinase in fragmented sarcoplasmatic reticulum. J . Biol. Chem., 245:1345-1347. Bessman, S.P., and Carpender, C.L. (1985) The creatine-creatine phosphate energy shuttle. Annu. Rev. Biochem., 54:831-862. Biermans, W., Bernaert, I., De Bie, M., Nijs, B., and Jacob, W. (1989) Ultrastructural localization of creatine kinase activity in the contact sites between inner and outer mitochondria1 membranes of rat myocardium. Biochim. Biophys. Acta, 974:74-80. Causton, B.E. (1984) The choice of resins for electron immunochemistry. In: Immunolabeling for Electron Microscopy. J.M. Polak and LM. Varnell, eds. Elsevier, New York, pp. 29-36. Cullen, M.F., and Landon, D.N. (1988) The ultrastructure of the motor unit. In: Disorders of Voluntary Muscle, 5th edition. J. Walton, ed. Churchill Livingstone, New York, pp. 27-73. Ennor, A.H., and Morrison, J.F. (1958) Biochemistry of phosphagens and related guanidines. Physiol. Rev., 38631-674. Feit, H., Fuseler, J., and Cook, J.D. (1983) Myofibrillar creatine kinase in Duchenne and avian muscular dystrophy. Biochem. Med., 29:355-359. Fitch, C.D., Lucy, D.D., Bornhofen, J.H., and Dalrymple, G.V. (1968) Creatine metabolism in skeletal muscle. 11. Creatine kinetics in man. Neurology, 1832-42. Fitch, C.D., and Sinton, D.W. (1964)A study of creatine metabolism in diseases causing muscle wasting. J. Clin. Invest., 43:444-452. Fitzaimons, R., and Sewry, C.A. (1985) Immunocytochemistry. In: Muscle Biopsy: A Practical Approach. V. Dubwitz, ed. Bailliere and Tindall, Philadelphia, pp. 184-207. Heyck, H., Laudahn, G., and Carsten, P.M. (1966) Enzymaktivitatsbestimmungen bei Dystrophia musculorum progressiva. IV. Mitteilung. Die Serumenzymkinetik im praklinischen Stadium des Typus Duchenne wahrend der ersten Lebensjahre. Klin. Wochenschr., 44:695-700. Infante, A.A., and Davies, R.E. (1965) The effect of 2,4-dinitrofluorobenzene on the activity of striated muscle. J . Biol. Chem., 240: 3996-4001. Jacobs, H., and Kuby, S. (1980) Studies on muscular dystrophy. J. Biol. Chem., 255:8477-8482. Jorgensen, A.O., and McGuffee, L.J. (1987) Immunoelectron microscopic localization of sarcoplasmic reticulum proteins in cryofixed, freeze-dried, and low temperature-embedded tissue. J. Histochem. Cytochem., 35:723-732. Lane, R.J.M., Watmough, N.J., Campaneria, S., and Pennington, R.J.T. (1986) Creatine kinase activity in human skeletal-muscle Trans., 14:126-127. membranes. Biochem. SOC. Mani, R.S., and Kay, C.M. (1978) Isolation and characterization of the 165,000 dalton protein component of the M-line of rabbit skeletal muscle and its interaction with creatine kinase. Biochim. Biophys. Acta, 533:248-256. Neville, H.E. (1973) Ultrastructural changes in muscle disease. In: Muscle Biopsy: A Modern Approach. V. Dubowitz and M.H. Brooke, eds. W.B. Saunders Co., Philadelphia, pp. 383-444. Pearce, J.M.S., Pennington, R.J.T., and Walton, J.N. (1964) Serum enzyme studies in muscle disease: Part 111. Serum creatine kinase activity in relatives of patients with the Duchenne type muscular dystrophy. J. Neurol. Neurosurg. Psychiatry, 27:181-185. Pennington, R.J.T. (1988)Biochemical aspects of muscle disease with particular reference to the muscular dystrophies. In: Disorders of Voluntary Muscle, 5th edition. J. Walton, ed. Churchill Livingstone, New York, pp. 455-486. Romano, E., and Romano, M. (1984) Historical aspects. In: ImmunoACKNOWLEDGMENTS labelling for Electron Microscopy. J . Polak and I. Varndell, eds. Elsevier, New York, pp. 3-15. We wish to thank Dr. W. Ballinger, University of Florida, for helpful consultations. This work was sup- Saks, V., Lipina, N., Sharov, V., Smirnof, V., Chazov, E., and Grosse, T.R. (1977) The localization of the MM isoenzyme of creatine phosported by the International Amyotrophic Lateral Sclephokinase on the surface membrane of myocardial cells and its rosis Foundation, the University of Florida Division of functional coupling to ouabain-inhibited (Na,K)-ATPase.Biochem. Sponsored Research, and the College of Veterinary Biophys. Acta, 465550-558. Schlegel, J., Zurbriggen, B., Wegmann, G., Wyss, M., Eppenberger, Medicine, University of Florida. H., and Wallimann, T. (1988) Native mitochondrial creatine kinase forms octameric structures. J. Biol. Chem., 263:16942-16953. REFERENCES Silverman, L.M., Mendell, J.R., Sahenk, Z., and Fontana, M.B. (1976) Askanas, V., Martinuzzi, A,, Engel, W., Kobayashi, T., Stern, L., and Significance of creatine phosphokinase isoenzymes in Duchenne Hsu, J . (1987) Accumulation of CK-MM is impaired in innervated dystrophy. Neurology, 16:561-564.

ing techniques that deposit electron-dense material are dependent on enzymatic turnover number, diffusion artifacts, and the ability to differentiate the electrondense product of the enzyme reaction from other electron-dense cellular structures. The immuno-gold technique used in this study provided information on relative particle density data without the above factors associated with immuno-enzymatic methods. If M-line associated M-CPK represents a fraction that is bound to another protein it could be expected that the presentation of some antigenic sites on the molecule are masked. In addition, if M-CPK were present at high local concentrations it could hinder access of antibody molecules to the bound CPK. Taken together, it could be argued that the data presented here represents a minimal estimate of relative concentration of M-CPK within the M-line of skeletal muscle. The M-CPK particle density reported for the I-band could be due to the M-CPK bound to or “trapped” within the sarcotubular system which is located in the area of the I-band (Baskin and Deamer, 1970; Lane et al., 1986). Approximately 20% of the total cellular M-CPK found by the immuno-gold technique described here was associated with the M-line, which is much higher than that accounted for using biochemical techniques (Lane et al., 1986; Wallimann et al., 1977). This could be explained if M-CPK that was associated with the M-line was lost during extraction procedures used to biochemically isolate enzyme activity. Another explanation is that enzyme activity may have been destroyed during extraction (Lane et al., 1986; Wallimann et al., 1977) or, that the immuno-gold labeling used here has detected an enzymatically inactive form of M-CPK. To explain either case it would be important to understand the normal distribution of M-CPK antigen. The distribution of the isoenzyme forms of CPK in different disease states of muscle is unknown. Determination of this distribution would be of value in any future work on the function of these isoenzymes in vivo and could provide a basis for diagnosis andlor better understanding of the pathogenesis of neuromuscular diseases. The ability to examine single cells is of vital importance in this latter regard since, as is the case for muscular dystrophy, assays of whole tissue extracts (enzymatic or immunologic) might not detect a small number of individual muscle fibers that may be altered in the early stages of the disease. In addition, any future study of the functional characteristics of any “abnormal” isoenzymes will be critically dependent upon ascertaining the location of the enzyme in the cell.

ULTRASTRUCTURAL LOCALIZATION OF HUMAN M-CPK Somer, H.,Dubowitz, V., and Donner, M. (1976)Creatine kinase isoenzymes in neuromuscular diseases. J. Neurol. Sci., 29:129-136. Turner, D., Wallimann, T., and Eppenberger, H.M.A. (1973)A protein that binds specifically to M-line of skeletal muscle is identified as the muscle form of creatine kinase. Proc. Nat. Acad. Sci. U.S.A., 79:702-705. Wallimann, T.,and Eppenberger, H.M.A. (1990)The subcellular com-


partmentalization of creatine kinase isozymes as a precondition for a proposed phosphoryl-creatine circuit. In: Isozymes: Structure, Function, and Use in Biology and Medicine. Z. Ogita and C.L. Markert, eds. Wiley-Liss, Inc., New York,pp. 877-889. Wallimann, T., Turner, D.C., and Eppenberger, H.M.(1977)Localization of creatine kinase isoenzymes in myofibrils. J. Cell Biol., 75: 297-317.

Ultrastructural distribution of the M form of creatine phosphokinase in human muscle by immunogold labeling.

Creatine phosphokinase regenerates ATP from ADP using creatine phosphate. Isoenzymes of creatine phosphokinase are bound to certain cellular structure...
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