Enzyme alterations in muscle cells from mice with hereditary dystrophy

Experimental

Cell

ENZYME

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35, 219-229

ALTERATIONS IN MUSCLE CELLS FROM MICE WITH HEREDITARY DYSTROPHY1 J. P. TASSONI,

Seton

Hall

219

(1964)

College

L. MANTEL2

of Medicine and Dentistry, Memorial Laboratory, Bar Received

and Jersey Harbor,

P. J. HARMAN City, N. J. and Roscoe Maine, U.S.A.

B. Jackson

June 21, 19633

morphologic changes occurring in the muscle cell have been carefully described in human muscular dystrophy [l ] but an understanding of the progression of cellular and connective tissue alterations from quasi normal to frankly deranged states is hampered by the difficulties inherent in obtaining graded stages from the human patient. The discovery of hereditary muscular dystrophy in an inbred strain of laboratory mice [25] has made possible a study of the origin and progression of cellular changes in close sequence within a comparatively short period of time. Concomitant with the histopathologic alterations which have proven to bear a remarkable similarity to those found in human dystrophy [4, 12, 14, 30, 381 many chemical alterations occur. Previous investigations of mice with hereditary muscular dystrophy have demonstrated several altered chemical systems and pathways in skeletal muscle homogenates. These studies include succinic dehydrogenase [36] cytochrome oxidase [37], phosphorylase [al], phosphoglucomutase [15], and amino acids [18, 341. Enzyme alterations have also been reported in human hereditary muscular dystrophy [ll, 331. Variations in dystrophic metabolic systems have been demonstrated histochemically in both experimental animals [lo, 121 and in humans [4, 5, 6j. The present communication deals with progressive chemical changes of actomyosin adenosine triphosphatase, 5-nucleotidase and phosphorylase in muscle cells with an aim toward correlation of chemical and morphological changes during the progress of the disease. Such correlations, by expanding our knowledge of activities at the cellular level during a series of pathologic events, also contribute to the understanding of the normal cell. It has been often _____THE

1 This study was supported by separate grants from the Muscular Dystrophy Association of America to Seton Hall College of Medicine and Dentistry and the Rosoce B. Jackson Memorial Laboratory; by PHS grants NB-01222 and NB-01634 to Seton Hall College of Medicine and Dentistry; and grant CRT-5013 from the National Cancer Institute to the Roscoe B. Jackson Memorial Laboratory. 2 Present address: Albert Einstein College of Medicine, New York, N. Y., U.S.A. 3 Revised version received February 3, 1964. 15 -

641804

Experimental

Cell

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35

J. P. Tassoni,

L. Mantel

and P. J. Harman

observed in biology that the existence of normal activities are first revealed by alterations occurring during disease. Thus a unit of heredity involving an enzyme system may be mlsuspected until a mutation with loss of enzymic activity occurs with deleterious consequences. In such cases the study of the abnormal precedes an appreciation of the normal.

MATERIALS

AND

METHODS

Actomyosin rind A TP.-Twenty pairs of dystrophic mice and unaffected littermate controls (strain 129/Re+dy, R. B. Jackson Memorial Laboratory) ranging in age from 7 weeks to 23 weeks mere processed simultaneously by pairs as follows. EXTRACTION AND PURIFICATION OF ACTOJIYOSIN. The extraction and purification of the actomyosin protein was accomplished by subjecting minced muscle samples to strong salt solutions, then precipitating the protein by reducing the ionic strength of the solution. Since the amount of skeletal muscle that can be conveniently obtained from an individual mouse is on the order of 1 g, standard techniques [26] which were developed for much larger quantities of tissue had to be adjusted. All steps of the preparation of the actomyosin protein were carried out at 4°C and all reagents were used at this temperature. The animals were sacrificed by cervical dislocation, the hindlimbs were massaged to eliminate excess blood, then amputated and frozen at ~20°C for 10 min. Skeletal muscle from both hindlimbs of an individual animal was removed, minced with surgical scissors, and added to 10 ml of extraction solution containing: 0.6 fif KCI, 0.04 M NaHCO,, and 0.01 fif Na,CO,. Extraction was carried out for a 24-hr period with continual magnetic stirring. The extracts were centrifuged at 2000 x 9 and 4°C for 5 min, the supernate strained through several layers of gauze, and the protein precipitated from the extract by the addition of 15 vol of water. The protein suspension then was centrifuged at 2000 x g and 4°C for 15 min; the supernate decanted, and the precipitated protein redissolved with 0.5 h1 KCl. Precipitation and purification of the protein were repeated and the final precipitate diluted with water to permit transfer by pipette. ASSAY OF ACTOMYOSIN ATPASE ACTIVITY. Actomyosin ATPase activity was assayed by a calorimetric technique modified from Perry [29]. This method involved the determination of the inorganic phosphate released by the ATPase-catalyzed reaction: ATP -I H,O+ADP I Pi. One ml aliquots of each protein solution was incubated in the following medium preheated at 25°C for 5 min. 1 ml 1.0 M glycine buffer adjusted to pH 9.4 with NaOH; 1 ml 0.1 &I CaCl,; 1 ml 1.0 M KCl; 1 ml 0.005 IM adenosine triphosphate (ATP). For determination of the non-enzymatic phosphate level a vessel including water instead of ATP was simultaneously incubated. Incubation was carried out in a Dubnoff metabolic shaking incubator for 10 min at 25°C. The reaction was terminated by the addition of 5 ml cold 10 per cent trichloroacetic acid (TCA) to each vessel, bringing the final concentration of TCA to 5 per cent. Aliquots of each incubated solution were assayed for inorganic phosphate by the Erperimentul

Cell Rescnrch

35

Enzymes

in mouse dystrophy

221

method of Allen [3]. The phosphate released as a result of the enzymic hydrolysis of ATP was calculated by subtracting the value of the non-ATP containing media from that which contained ATP. Determination of the protein content of each protein solution was accomplished by the biuret method as described by Layne [19]. The data are expressed as milligrams phosphate per milligram protein. Statistical analysis of the data were carried out by paired comparisons using the appropriate “t” test [24]. &NucZeotidase.-Strain 129 mice of the Roscoe B. Jackson Memorial Laboratory Bar Harbor, Maine, in which the dystrophic gene is segregating [25] were used. Thirty pairs of animals were tested; a dystrophic mouse and a littermate control constituted each pair. The animals were evenly divided between two age groups: 2-4 weeks and 5-9 weeks. All mice were on the same diet of Purina Laboratory Chow, and water ad lib. Paired dystrophic and control animals were processed simultaneously. Approximately 150 mg (wet weight) of the hindlimb muscle was excised after sacrifice blcervical dislocation and massage of the limbs to eliminate excess blood. The samples were coarsely minced with surgical scissors and homogenized in 5 ml of cold distilled water with a chilled mortar and pestle. Commercially prepared adenosine monophosphate (AMP) was used as the substrate to assay the level of 5-nucleotidase activity; distilled water was substituted for AMP in one aliquot from each animal to determine the free phosphorus level of the incubation solutions. The incubation solutions were made up according to a modification of the method described hy Heppel and Hilmoe [16]; 1 ml muscle homogenate, 1 ml 1.0 M glycine buffer at pH 8.5, 1 ml 0.1 M MgCl,, and 1 ml 0.0005 A1 AMP or 1 ml distilled water. Controls using mill concentrations of NiZf, a specific inhibitor of 5-nucleotidase activity [2], in each of the above incubation solutions were incubated at the same time. Samples were incubated in a Dubnoff metabolic shaking incubator at a constant temperature of 37.5”C for 15 min, 30 min, 1 hr, 2 hr, and 4 hr. At the end of each incubation period, 4 ml of 10 per cent trichloroacetic acid mere added to each sample. The samples were then centrifuged and aliquots reserved for estimation of phosphorus by the amidol technique as described by Allen [3]. The amount of phosphorus released by the reaction: AMP YmH,O+-Adenosine ! I’, is given in the data as mgjml 121) of aliquot. Increased phosphorus is a direct indication of the rate of hydrolysis of the substrate. The phosphorus content of the aliquots was determined for all supernates. The quantity of P present in the samples containing no substrate (the free phosphorus of the samples) and that determined for samples containing the inhibitor were subtracted from these which contained substrate but no inhibitor of adenosine monophosphatase activity. Phosphor&se.-Mice from the R. B. Jackson Memorial Laboratory’s (129/R+ Dydyx ~57 B1/6-“ydy ) I<‘, hybrid colony were used. Dystrophic mice (dydy) and unaffected littermate controls (Dy-) were assayed for muscle phosphorylasc by the method previously described by Stetten [35] and Leonard [ZO, 211 with modifications personally communicated. Age of the animals ranged from 11 to 17 days. Paired dystrophic and littermate controls were processed simultaneously by pairs as follows. The animals were sacrificed by decapitation and the hindlimbs skinned, removed and frozen with dry ice. Approximately 200 mg wet weight of hindlimb Experimental

Cell

Rcsctrrch

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J. P. Tassoni, L. Mantel and P. J. Harman

222

skeletal muscle were excised, minced and then homogenized in 10 ml of cold 0.1 M NaF. The homogenates were centrifuged at 4°C and 2000 xg for 30 min. The supernates were decanted and aliquots taken for active and total phosphorylase assay and determination of protein content. The reaction vessel for determination of active phosphorylase (phosphorylase a) consisted of the following: 0.5 ml 2 per cent glycogen, 0.2 ml distilled water, and 1.0 ml of the muscle homogenate supernate. These were preheated for 7 min in a Dubnoff metabolic shaking incubator at 37.5”C after which the enzyme reaction was initiated by the addition of 0.5 ml 0.06 M glucose-l-phosphate (G-l-P) adjusted to pH 6.1 with succinic acid. After a IO min incubation period at 37.5% the reaction was terminated by the addition of 2 ml cold IO per cent trichloroacetic acid. Total phosphorylase was simultaneously determined by the same procedure except that 0.2 ml of 0.0014 M adenosine-5-monophosphate(5’-AMP) was substituted for the water. A vessel for determination of non-enzymatic phosphate level was prepared similar to that for total phosphorylase except that the G-l-P was not added until after the incubation period and the addition of the trichloroacetic acid. All vesselswere then brought to a total volume of 10 ml with distilled water and centrifuged for 20 min at 2000 xg. Aliquots of the supernates were taken and determination of inorganic phosphate content was made by a method described by Allen [3]. Rates of reaction were based on the amount of inorganic phosphate liberated from G-l-P during synthesis of glycogen. Protein determinations were carried out on aliquots of the muscle homogenate supernate by the biuret method described by Layne [19]. The data are expressedas mg of phosphate per mg of protein. Statistical analysis of the data was accomplished by the appropriate f-test for paired comparisons [24]. RESULTS Table I shows the results of the twenty paired analyses for actomyosin ATPase activity levels. In sixteen of the twenty pairs of animals (dystrophic with littermate control) the dystrophic preparation exhibited greater ATPase activity (33 to 1372 per cent) while the controls exhibited slightly increased activity (8 to 37 per cent) in only four comparisons. The mean value of mg phosphate liberated from ATP per mg protein was 0.0399 for the dystrophic animals and 0.0176 for the controls: the mean difference was 0.0223 with a standard error of 0.0077. By the appropriate f test analysis for paired comparisons, the increase in ATPase activity of dystrophic actomyosin preparations as compared to littermate controls was shown to be statistically significant (P < 0.01). The data for 5-nucleotidase, contained in Fig. 1, were analyzed by application of the f-test for mean differences of small samples [22]. Significance was implied for P-c 0.025. 5-Nucleotidase activity levels (Fig. 1) were significantly greater in the dystrophic animals when compared with their littermate controls at each Experimental

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Enzymes

223

in mouse dystrophy

hour of incubation and for both age groups. The 5-g-week-old groups of animals showed lower enzyme activity levels when compared with the 2-4week-old groups on both dystrophies and controls. These differences are significant at the 4 hr incubation period for the dystrophies and the 1 and 4 hr incubation periods for the controls. The remaining differences did not meet the criteria for significance since P > 0.20. The phosphorylase data for nine paired comparisons (dystrophic animal with unaffected littermate control) are presented in Table II. In all nine comparisons, the dystrophic animal exhibited decreased activity levels of both active phosphorylase and total phosphorylase when compared with unaffected littermate controls. Mean phosphorylase a activity expressed as mg phosphate liberated in 10 min at 37.5”C from G-l-P per mg protein was 0.081 for the controls and 0.050 for the dystrophies; the mean difference TABLE Pair No.

I. Actomyosin ATPase activity Dystrophic? activity

Age days

levels.

Controla activity

Dystrophic minus control

76 change

1

49

0.0546

0.0385

0.0161

42

2

51

0.0497

0.0232

0.0265

114

3

55

0.0536

0.0777

- 0.0241

-31

4

58

0.0190

0.0301

- 0.0111

-37

5

63

0.0298

0.0182

0.0116

6

65

0.0530

0.0036

0.0494

7

66

0.0512

0.0166

0.0346

208

8

71

0.1264

0.0277

0.0987

356

9

71

0.0160

0.0174

- 0.0014

-8

10

78

0.0065

0.0049

0.0016

33

11

93

0.1269

0.0151

0.1118

740

97

12

64 1372

0.0147

0.0182

- 0.0035

13

129

0.0130

0.0062

0.0068

14

131

0.0071

0.0047

0.0024

51

15

139

0.0220

0.0103

0.0117

114

16

143

0.0186

0.0075

0.0111

148

17

146

0.0213

0.0142

0.0071

50

18

157

0.0295

0.0067

0.0228

340

19

162

0.0662

0.0080

0.0582

728

20

163

0.0127

0.0036

0.0091

253

0.0399

0.0176

0.0223

Mean a Expressed protein.

as mg

phosphate

(Pi)

liberated

in

10 min

at 25’C

from

ATP

Experimental

- 19 110

per

mg

actomyosin

Cdl

Research

35

224

J. P. Tassoni,

L. Mantel

and P. J. Harman

was 0.031 with a standard error of 0.0050. Likewise, mean phosphorylase t activity values were 0.104 for the controls and 0.069 for the dystrophies; the mean difference was 0.035 with a standard error of 0.0028. The decreases in phosphorylase a and phosphorylase t activity in skeletal muscle of dys0.014 1

0012 0.010 $0.006 2 ~0.006h d,

Fig. l.-5-Nucleotidase activity levels for skeletal muscle of mice with hereditary muscular dystrophy. Pre-weaning (2-4 weeks) and post-weaning (5-8 wee&) periods’ are bAuded. 6,‘2-4 weeks; l , 5-9 weeks; DY, control; dy, dystrophic.

= 0.004. 0.002-

00 INCU@ATION (HOURS)

trophic mice as compared to unaffected littermate controls was statistically significant (PC 0.001). The ratio of phosphorylase a to phosphorylase t activity was decreased in the dystrophic in seven of the comparisons and slightly increased in two. The mean ratio (a/t x 100) for the controls was 77.0 and 68.7 for the dystrophies; the mean difference was 8.3 with a standard error of 2.94. This decrease of the a/f ratio in the dystrophic animals as compared with littermate controls was on the borderline of statistical significance (P =0.05).

DISCUSSION

In metabolic diseases it is often difficult to establish reference points or baselines of enzyme activity because of variations in morphology and rate of pathologic progresses usually found in such disorders. Various enzymes have shown marked alterations in mouse muscular dystrophy [14] but these have been based variously on wet weight [21, 36, 391, dry weight [23], total protein [37], and non-collagenous nitrogen protein [15, 371. Each has dealt with muscle as a composite tissue rather than with the muscle cell itself. In our assays of actomyosin ATPase we have attempted to deal with an EsperimPntnl

Cell Research

35

Enzymes entity located only described in mice this seemed only This approach

Phosphorylase NO.

1

2 3 4 5 6 7 8 9

II. Phosphorylase aa

activity

Phosphorylase

ta

a/f x 100 c----7 Cont.

Dyst.

0.124 0.045 0.040 0.043 0.060 0.066

23 29 23 15 48 55 38 50 30

79.4 73.2 89.1 80.1 81.6 86.4 47.8 72.7 83.0

63.3 79.7 85.7 79.8 66.7 70.0 48.8 53.3 71.2

20 -9 4 1 18 19 -2 27 14

0.069

34

77.0

68.7

11

w Cont.

Dyst.

% Dec.

Cont.

Dyst.

11 11 15 15 15 15 17 17 17

0.081 0.071 0.114 0.117 0.071 0.076 0.033 0.088 0.078

0.050 0.055 0.084 0.099 0.030 0.028 0.021 0.032 0.047

38 23 26 15 58 63 36 64 40

0.102 0.097 0.128 0.146 0.087 0.088 0.069 0.121 0.094

0.079

0.081

0.050

38

0.104

’ Expressed

levels.

5

Age days

Mean

225

in the muscle cell. Since the hereditary muscular dystrophy is indicated as primary in the muscle cells themselves [SS] logical. cannot attempt to solve all the problems inherent in such TABLE

Pair

in mouse dystrophy

as mg phosphate

liberated

% Dec.

0.069 0.098

in 10 min at 37.5”C

from

G-1-P

% Dec.

per mg protein.

a study but can eliminate some, such as comparisons based on wet weight which often distort the facts primarily by ignoring the definite change in protein content which accompanies the progression of the disease [37]. It should be noted here, however, that the data for 5-nucleotidase activity are presented on a wet weight basis. Actual protein determinations were carried out in this laboratory and others [37] and all indicate an actual decrease in non-collagenous protein in dystrophic muscle tissue. Therefore by using the wet weight basis the differences are presented in their minimal form. On the basis of non-collagenous protein the differences between dystrophic and control would have been even greater than the 100 per cent reported. Although the investigation of actomyosin ATPase measured enzyme activity against a definite baseline (myotibrillar protein), it still was not possible to establish a general reference point of “normal” activity levels nor one for the diseased tissue. However, it was established that actomyosin ATPase activity of dystrophic mouse skeletal muscle was significantly greater than that of littermate controls on an animal to animal basis. Investigations [lo, 131 by histochemical techniques of ATPase activity in dystrophic mouse Experimental

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J. P. Tassoni,

L. Mantel

and P. J. Harman

skeletal muscle suggest alterations, but the results are conflicting and as yet inconclusive. The cause(s) of altered ATPase activity in dystrophic animals is (are) still unknown, but myofibrillar protein associated with ATPase activity from dystrophic mice exhibits polydispersity with physical characteristics (molecular weight, UV absorption spectrum, and Z-average radius of gyration) different from unaffected control animals [27, 281. Associated with this, abnormalities including primary loss of contractile elements in the muscle cell and random distribution of the sites of origin of degeneration have been observed with the electron microscope [30]. The picture [30, 381 of the seemingly random distribution of the afflic,tion to various muscle cells in any particular preparation demonstrates that it is difficult to ascertain the progression of the disease to a definite degree. Therefore rate and extent of the progression of the disease in any given sample can be variable. The seemingly extreme variations in amount of activity noted in a few cases in both the diseased and controls has no definite answer at this point though there are strong implications, viz.; the alteration [27, 28, 401 and possible progressive alteration of myofibrillar protein as the disease progresses and the variability of the sample with any progressive disease. The problem of the controls in this instance is very complex for if we assume that the dydy genetic state acts either primarily or, what is more probable, secondarily in affecting chemical structure and activity [7] then it is possible that a heterozygote (Dydy) could have an intermediate enzyme activity. It is impossible at this time to determine which littermates are homozygous (DyDy) or heterozygous (Dydy). The confusion which exists in the literature concerning phosphorylase activity would best be portrayed in an extensive review article. However, a brief notation of the major problems is necessary here. Much of the difficulty surrounds the estimation of activity and the form of activity, i.e., a or t, plus the specific activators and cofactors involved. Phosphorylase a is indicated as the predominant form by Cori [S] while phosphorylase t is indicated as the predominant form by Krebs and Fischer [17]. Both used rabbit skeletal muscle. The wide discrepancies observed for a/t [al, 321 in dystrophic mice cannot be totally resolved by attributing them to technique differences, nor is there merit in ascribing them to age differences for these latter [al, 321 were done on animals in fairly advanced stages of the disease. The present study was done with younger animals in which the disease was in its early stages and probably beginning the more rapid phase of progression in contrast to the latter [al, 321 well-advanced stages where many Experimental

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Enzymes

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more problems are introduced. Added to this is the factor of wet weight comparisons [20, 21, 31, 321 versus results based on protein content. The importance of relating enzyme activity to protein is well established and is very clearly demonstrated as being necessary [9] in diseases of this type where protein wasting, fatty infiltration and coagulation necrosis are established processes involved in the particular pathology. We must add to this one further source or basis for discrepancy. Much of the determination of phosphorylase is based upon the utilization of AMP as the activator for total phosphorylase activity. Recent work [37] indicates the possibility that another nucleotide, cyclic 3’,5’-adenosine monophosphate should be considered the primary activator. Despite the technical and theoretical problems, however, a definite pattern has emerged which shows very clearly that a significant decrease occurs in the phosphorylase activity of the dystrophic animals when compared to littermate controls [21, 321. The present work corroborates this finding and further demonstrates slight differences in a/f ratios. These results correlate, favorably, with findings in the human disease [ll 1. Concerning 5-nucleotidase activity, the present experiment was designed particularly to test whether enzymic differences found in the later stages (i.e., 5-9 weeks) of the disease were established at the earlier ages (2-4 weeks), since an earlier study [36] demonstrated that elevated enzymic activity in well established dystrophy may not be correlated with heightened activity in the incipient stages of the disease. The data here presented show AMPase activity to be higher in the dystrophic as compared to littermates, at all ages studied. Elevated AMPase activity in dystrophic animals is thus more parallel to the etiology of the disease than those enzymic alterations which are not observed until the later ages. This finding re-affirms the importance of the clinical progression of the disease to the interpretation of any differences found between dystrophic and control muscle. Heckett and Rourne [4] observed reactions for AMPase in the connective tissue of both dystrophic and normal patients. Since relative and absolute increases in the connective tissue of dystrophic mice have been reported [38], it would be expected that if AMPase were localized in the connective tissue of mice, there would be an increase in activity levels with progression of the disease. The present data, however, show a decrease in activity in the older group of animals. This observation correlates with histochemic data on dystrophic mouse muscle which reports strong reactions for AMPase not in the connective tissue but in the nerve fibers and blood vessels [lo, 121. This disparity in histochemic staining between human and mouse dystrophic Experimental

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J. P. Tassoni,

228

L. Mantel

and P. J. Harman

connective tissue does not necessarily imply a difference in the pathology of the disease in the two species. The histologic [38] and biochemic [ll, 361 similarities between mouse and human muscular dystrophy strongly indicate that the two diseases are analogous in many important respects.

SUMMARY

1. Actomyosin was extracted from mice with hereditary muscular dystrophy. It was further purified by slight modifications of existing procedures, then assayed for ATPase activity. In these assays animals of various ages and stages of development of dystrophy were utilized. The preponderance of evidence suggests an altered enzyme activity. This coupled with physicochemical and biochemical results of others demonstrates a possible chemical lesion in contractile proteins involving altered structure and function. 2. 5-Nucleotidase activity levels, using adenosine monophosphatase (AMP) as substrate, were studied in skeletal muscle homogenates of hereditarily dystrophic mice. Enzymic activity levels decreased with age in both dystrophic and littermate controls. At all ages observed (2-9 weeks) dystrophic animals demonstrated significantly elevated AMPase activity levels when compared with littermate controls. 3. Skeletal muscle from dystrophic mice and littermate controls was assayed for phosphorylase activity. Both phosphorylase Q and t are significantly lowered in the dystrophic mouse but the a/t ratios may not be significantly altered. The writers wish to express their appreciation to Dr. Elizabeth Roscoe B. Jackson Memorial Laboratory for her courtesies.

Russell of the

REFERENCES

R.

1. ADAMS,

D., DENNY-BROWN, D. and PEARSON, C. M., Diseases B. Hoever Inc., New York, 1954. AHMED, A. and REIS, J. L., Biochem. J. 69, 386 (1958). ALLEN, R. J. L., Biochem. J. 34, 858 (1940). BECKETT, E. B. and BOURNE, G. H., J. Neuropath. Exptl Neural.

of Muscle,

pp.

253-254.

Paul

2. 3. 4. 17, 199 (1958). 5. __ Science 126, 357 (1957). 6. BOURNE, G. H. and GOLARZ, M. N., Nature 183, 1741 (1959). 7. COLEMAN, D. L. and ASHWORTH, M. E., Am. J. Physiot. 197, 839 (1959). 8. CORI, G. T., J. Biol. Chem. 158, 333 (1945). 9. CORI, G. T., ILLINGWORTH, B. and KELLER, P. J., Methods in Enzymology, Vol. Academic Press Inc., New York, 1955. 10. CURTIS, R. L. and HOLLINSHEAD, M. B., Anat. Rec. 136, 179 (1960). 11. DREYFUS, J. C., SCHAPIRA, G. and SCHAPIRA, F., J. Ctin. Innest. 33, 794 (1954). 12. GOLARZ, M. N., Anat. Rec. 136, 198 (1960).

Experimental

Cell Research

35

1, p. 200.

Enzymes 13. 14. 15. 16. 17. 18.

19. 20. 21. 22. 23. 24.

25.

in mouse dystrophy

229

M. N. and BOURNE, G. H., Acta Anat. 43, 193 (1960). P. J., TASSONI, J. P., CURTIS, R. L. and HOLLINSHEAD, M. B., Muscular Dystrophy in Man and Animals, pp. 408-456. S. Karger, New York, 1963. HAZZARD, W. R. and LEONARD, S. L., Proc. Sot. Explf Dial. Med. 102, 720 (1959). HEPPEL, L. A. and HILMOE, R. J., Methods in Enzymology. Vol. 2, p. 546. Academic Press Inc., New York, 1955. KREBS, E. G. and FISCHER, E. H., J. Biol. Chem. 216,113 (1955). KRUH,.J., DRBYFUS, J., &APIRA, G. and GEY, G. 0.; J. Chin. inuesf. 39, 1180 (1960). LAYNE, E., Methods in Enzymology. Vol. 3, p. 450. Academic Press Inc., New York, 1955. LEONARD, S. L., Endocrin. 60, 619 (1957). __ Proc. Sot. Exptl Biof. Med. 96. 720 (1957). LINDQUIST, E. F., Statistical Analysis in Ednc&ional Research. Houghton Mifflin Co., New York, 1940. MCCAMAN, M. W., Science 132, 621 (1960). MCNEMAR, Q., Psychological Statistics, pp. 104-114. John Wiley and Sons, New York, 1955. MICHELSON, A. M., RUSSELL, E. S. and HARMAN, P. J., Proc. Naff. Acad. Sci. U.S. 41, 1079 GOLARZ, HARMAN,

(1955). 26. 27. 28. 29. 30.

MOXIMAERTS, W. F. H. M., Methods in Med. Res. 7, 1 (1955). OPPENHEII\IER, H., BARANY, K., TERRY, H. and FORSYTH, E., Fed. OPPENHEIMER, H., TERRY, H. R., FORSYTH, E. and MILHORAT, A. PERRY, S. V., Methods in Enzymol. 2, 582 (1955). Ross, M. H., PAPPAS, G. D. and HARMAN, P. J., Lab. Inuesf. 9, 388 RULON, R. R., SCHOTTELIUS, D. D. and SCHOTTELIUS, B. A., Am. J.

31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

ibid.

SCHAPIRA, 313, SIMON, E. STETTEN, TASSONI, WEINSTOCK, WEST, W. ZIERLER, ZYIMARIS;

Proe. T.,

20,299 (1961). ibid. 18, 116 (1959).

(1960). Physiof.

200,1236

(1961).

202, 821 (1962). G., DREYFUS, J. C., SCHAPIRA, F. and KRUH, J., Proc. Third Med. Conf. MDAA, 1954. J., GROSS, C. S. and LESSELL, I. M., Arch. Biochem. Biophys. 96, 41 (19G2). D., JR. and STETTEN, M. R., Physiol. Rev. 40, 505 (1960). J. P. and HARMAN, P. J., J. Neuropath. Exptf Neurof. 20, 158 (1961). I. M., EPSTEIN, S. and MILHORAT. A. T., Proc. Soe. Expll Biol. Med. 99.272 (1958). , \ , T. and.MuRpHY; E. D., Anal. Ret: 137, i79 (1960). . K. L., Bull. Johns Hopkins Hosp. 102, 17 (1958). M. C.; EPSTEIN, N., S.&FER, A.,-ARON~ON, ‘S. M: and VOLK, 8. W., Am. J. Physiol.

196, 1093 (1959).

Experimental

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