Biochemical Genetics, VoL 15, Nos. 9/10, 1977

Creatine Phosphokinase Activity in Dysgenic (mdg/mdg) Mouse Muscle Francine B. Essien 1 and Cheryl L. Biber 1,2

Received 18 Nov. 1975--Final25 Mar. 1977 The specific activity of creatine phosphokinase (CPK) was measured in the muscle of mdg/mdg and control embryos of 14-18 days' gestation. CPK specific activity values were similar in mutant and normal embryos at tke earliest stages examined (14-15 days). However, after 15½ days, tke specific activity of tke enzyme in tke mdg/mdg embryos was approximately 50% lower tkan in the controls. The dysgenic and normal muscle extracts exkibited comparable stability after storage at - 85 C. CPK activity levels in tke muscle of adult keterozygotes (+/mdg) and wild-type ( + / + ) controls were found to be statistically identical. Tke findings suggest tkat tke mdg mutation does not kave a primary or direct effect on CPK activity. KEY WORDS: creatine phosphokinase (CPK); dysgenic (mdg/mdg);muscle. INTRODUCTION The recessive lethal mdg (muscular dysgenesis) mutation in the mouse causes abnormal skeletal muscle differentiation and maintenance in affected homozygotes, which exhibit no spontaneous or induced movements (Pal, 1965a,b). The experiments of Bowden-Essien (1972) further showed that even though dysgenic (mdg/mdg) cells undergo morphological differentiation in vitro they still fail to contract spontaneously or in response to mechanical or electrical stimulation. Since lack of contractility is characteristic of homozygous mdg This research was supported by grants from the Charles and Johanna Busch Memorial Fund and Research Council of Rutgers University and by a grant from the Muscular Dystrophy Association of New York. Department of Biological Sciences, Douglass Campus, Rutgers University, New Brunswick, N.J. 2 Present address: New Jersey School of Dentistry, Jersey City, N.J. 963 This journal is copyrighted by Plenum. Each article is avMlable for $7.50 from Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. I001 I,


Essien and Biber

cells in situ and in culture, it was considered of interest to analyze creatine phosphokinase in the dysgenic cell in view of its importance as an enzyme involved in muscle contraction. Creatine phosphokinase (CPK), or ATP: creatine phosphotransferase (E.C., mediates energy production in muscle cells by catalyzing the transfer of phosphate in the form of phosphocreatine. The reverse reaction yields free ATP: ATP + creatine ~ ADP + phosphocreatine Several studies have demonstrated that CPK specific activity increases significantly during skeletal muscle differentiation in the chick, rat, and guinea pig (Reporter et al., 1963; Eppenberger et al., 1964; Ziter, 1974; Prochazka and Wachsmuth, 1972). In addition, analysis of cultured avian and mammalian muscle cells reveals a similar rise in CPK activity in fusionblocked myoblasts (Tarakis and Schubert, 1974; Turner et al., 1976) or in multinucleated myotubes (Reporter et al., 1963; Coleman and Coleman, 1968; Shainberg et al., 1971 ; Morris and Cole, 1972; Turner et al., 1974). In the present report, CPK activity levels in the limb muscle of control and dysgenic (mdg/mdg) embryos of various gestational ages are compared. Data on CPK specific activity in heterozygous (+/mdg) and wild-type ( + / + ) adults are also presented. MATERIALS AND METHODS

Dysgenic (mdg/mdg) and control embryos were obtained from timed matings of heterozygous adults (+/mdg x +/mdg). Females were examined at 9:00 A.M. and 5:00 P.M. for the presence of a vaginal plug; detection of the plug was considered as day zero of pregnancy. At appropriate stages, the females were sacrificed by cervical dislocation, and the uterus was briefly rinsed and then dissected in Hanks' balanced salt solution. The ages of the embryos were verified by morphological criteria, and phenotypes were recorded. Each embryo was then processed individually. After removal of the skin and paws from the four limbs, the muscle was dissected from the bones in a depression slide containing 0.5 ml of buffer consisting of 0.1 M KC1 and 0.014 M 2mercaptoethanol. The muscle tissue was then transferred to a homogenizer, and the slide was rinsed with 0.5 ml of buffer, which was added to the homogenizer tube to yield a final volume of 1.0 ml. After homogenization, the sample was placed in a centrifuge tube and spun at 48,000g for 20 rain at 4 C. (This latter procedure reduces the nonspecific oxidase activity which competes with pyruvate for N A D H in the CPK assay.) Supernatants were transferred to chilled vials and kept on ice during the assay procedure. CPK activity was measured by the modified method reported by Coleman and Coleman (1968). Enzyme activity was monitored indirectly by

C P K Activity in Dysgenic Mouse Muscle


coupling of the phosphorylation of creatine with oxidation of an equimolar amount of NADH by pyruvate: Creatine + ATP ~ creatine phosphate + ADP ADP + phosphoenolpyruvate ~- ATP + pyruvate Pyruvate + NADH + H + ~ lactate + NAD ÷ A 1.0-ml reference cuvette and experimental cuvette were prepared for each extract. The assay mixture contained 0.2 ml of 3.75/~M phosphenolpyruvate, 2.05 #M ATP made up in 0.1 M glycine buffer (pH 9.0), 0.02 ml of 20/~M Mg acetate, 0.2 ml of 1.45 M glycine, 0.05 ml pyruvate kinase (60 units/ml), 0.05 ml lactate dehydrogenase (100 units/ml); 0.09 ml of 0.1 M glycine buffer (pH 9.0), 0.1 ml of 1.5/~M NADH, and 0.1 ml muscle extract. After a 3-min stabilization period, the reaction in the experimental cuvette was initiated by the addition of 0.28 ml of 0.107 mg creatine in glycine buffer (pH 9.0). An equivalent volume of buffer was added to the reference cuvette. Optical density was monitored at 340 nm at 1-min intervals to measure disappearance of NADH. The protein concentration of each sample was determined by the method of Lowry et al. (1951). The linear portion of OD v. time curves was used for calculation of enzyme activity. CPK specific activity was determined as

A OD (experimental)-A OD (reference) /rag protein At x volume The CPK activity in muscle of adult heterozygotes and normal homozygotes was also measured. Females of various ages were killed by cervical dislocation and the hind limbs were swabbed with 70% ethanol and placed in a petri dish containing cold dissection buffer (0.1 M KC1 with 0.014 M 2mercaptoethanol). Skin and paws were removed, and the gastrocnemius muscle was teased from the bone and placed in 1.5 ml of cold buffer. A homogenate was prepared with a Vertis "45" homogenizer (A. Thomas, Philadelphia) and centrifuged; CPK activity was assayed as described above. RESULTS

CPK Specific Activity in Embryonic Muscle CPK activity was detected in normal and dysgenic (rndg/mdg) skeletal muscle at 14 days' gestation, the earliest stage at which mutant and control embryos could be individually processed to yield significant amounts of material. The levels of activity observed were statistically identical for both phenotypes (Fig. 1). Significant differences between CPK values in control and mutant


Essien and Biber 1.6

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~ 0.4 n vQ. 0.2 0 I












Fig. 1. Specific activity of creatine phosphokinase in skeletal muscle of control ( + / + and +/mdg) and dysgenic (mdg/ mdg) embryos. Units/mg protein are plotted against gestational age. Closed circles (e) and solid lines ( ) indicate control embryos; open circles (o) and dashed lines ( - - - ) indicate mutants. Each point represents the mean for several litters, and 1 SD is indicated.




mdg) mouse muscle.

Biochemical Genetics, VoL 15, Nos. 9/10, 1977 Creatine Phosphokinase Activity in Dysgenic (mdg/mdg) Mouse Muscle Francine B. Essien 1 and Cheryl L. B...
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