A study of platelet protein phosphorylation in duchenne muscular dystrophy

Journal of the Neurological Sciences, 1984, 64:21-32

21

Elsevier

A STUDY OF PLATELET PROTEIN PHOSPHORYLATION IN D U C H E N N E M U S C U L A R DYSTROPHY Further Evidence against the Generalised Membrane Defect Theory

G.A. NICHOLSON,J.G. McLEOD and J.W. SUGARS Department of Medicine, The Universityof Sydney, Clinical Sciences Building, Concord Hospital, Concord 2139. N. S. 14/. (Australia)

SUMMARY As a test of the generalised defect theory for Duchenne muscular dystrophy (DMD), basal and calcium-dependent platelet protein phosphorylation was examined in order to determine if the increased concentration of calcium in D M D skeletal muscle is reflected in D M D platelets. Protein phosphorylation was quantitated by gradient slab gel electrophoresis and autoradiography. The number of phosphoproteins in each phosphoprotein peak was determined by comparison with two-dimensional gel electrophoresis. Many phosphoprotein peaks were present in unstimulated platelet preparations both in whole platelet homogenates and in intact platelets. Two of these phosphoprotein peaks were calciumdependent, one was a single phosphoprotein, the other consisted of 4 phosphoproteins. No disease-related differences were observed in either basal or calcium-stimulated phosphoproteins. These results do not support previous reports of platelet abnormalities in DMD, and provide further evidence that the biochemical defect in Duchenne muscular dystrophy is neither generalised nor a membrane defect. The biochemical defect in DMD should be regarded as a skeletal muscle abnormality until proved otherwise.

Key words: Duchenne muscular dystrophy - M e m b r a n e defect theory - Platelet protein phosphorylation.

This work was supported by grants from the National Health and Medical Research Council of Australia. 0022-510X/84/$03.00 © 1984 Elsevier Science Publishers B.V.

22 INTRODUCTION

Research into the fundamental defect of Duchenne muscular dystrophy (DMD) has now turned a complete circle. The pioneering clinical studies of Duchenne and others suggested that the disease prirnarily involved skeletal muscle (Meryon 1852; Duchenne 1868; more recently Walton 1964). Early biochemical studies also pointed to a muscle defect (Schapira et al. 1953). The best documented muscle abnormalities include muscle enzyme "leakage" and increased calcium concentrations in muscle (Bodensteiner and Engel 1978). Over the last 10 years concurrently with the change of emphasis of biochemistry from metabolic disorders to disorders of membrane architecture, a number of investigators proposed that in DMD the muscle defect may not be limited to muscle and may be expressed as a generalised disorder of cell membrane (Kunze et al. 1973; Matheson and Howland 1974; Rodan et al. 1974; Roses et al. 1975). The generalised membrane defect theory can be regarded as a special case of a generalised defect theory for DMD. The generalised membrane defect theory may have been prompted by studies of myotonic dystrophy where clinical and other evidence suggested a membrane disorder affecting many cell membranes. However, the membrane theory for D M D was not new and had been proposed earlier to explain muscle aldolase leakage to serum (Schapira et al. 1953). In support of the generalised defect theory for DMD there is some clinical evidence of involvement of tissues other than muscle e.g. the association of DMD with mild mental retardation and there have been some reports of gastrointestinal motility disorders in Duchenne muscular dystrophy (for review see Nowak et al. 1982). Although there is no true animal model for Duchenne muscular dystrophy, studies of various animal dystrophies have paralelled studies of the human disorder. Alterations in membrane structure and function have been observed in dystrophic mice (De Kretser and Livett 1977; Strickland et al. 1979). In one of the few studies of platelets in the muscular dystrophies, Mizobe and Livett (1982), examined membrane-bound acetylcholine esterase in two forms of muscular dystrophy in mice. No abnormalities of this membrane enzyme were found and these authors came to the conclusion that their results did not substantiate a membrane defect theory. We have chosen to study platelets as they have an extensive membrane and contractile system which in some respects resembles muscle. Platelets can therefore be used to test the validity of both the generalised defect and the generalised membrane defect theories for DMD. Two platelet proteins (approximately 20 and 40 kdaltons) are phosphorylated when platelets are exposed to collagen, thrombin or the calcium ionophore A23187 (Lyons et al. 1975; Haslam et al. 1979). Adelstein and Conti (1975) identified the 20 kdalton protein to be a light chain of myosin. These phosphorylation reactions are closely associated with the platelet release reaction and are directly or indirectly mediated by increases in the concentration of calcium in the platelet cytosol (Haslam and Lynham 1978). If there is a generalised abnormality in DMD, with raised intracellular calcium levels, the abnormality may be reflected as altered calcium-dependent protein phosphorylation. Preliminary results of this study have been presented to the Australian Biochemical Society (Nicholson and Sugars 1981).

23 METHODS

Platelet isolation Paired blood samples (15 ml heparinised venous blood) were obtained from age-matched DMD and control subjects and were coded for subsequent analysis. One or two pairs of samples were analysed in a single experiment. Results were accumulated over a number of weeks. Platelet-rich plasma was prepared by centrifuging plasma for 15 min at 250 x g, following which the platelets were pelleted at 1750 × g for 15 min. The platelet pellet was resuspended and washed twice with 0.15 M NaC1, 0.01 M Tris-HC1, 5 mM EDTA, pH 7.4 the final pellet was resuspended in 500 #1 of washing buffer and the protein concentration was determined by the Lowry method.

Phosphorylation reactions Phosphorylation of platelet homogenates Platelet homogenates were prepared by resuspending the platelet pellet in 1.5 ml of hypotonic lysis buffer (5 mM of sodium acetate, 0.5 mM EGTA, pH 7.4) and sonication with 3 5-s bursts. This procedure resulted in disruption of all platelets as determined by phase contrast microscopy. Protein phosphorylation was carried out using endogenous protein kinases by a modification of the method of Steiner (1976) with incubation in 15 mM sodium acetate, 0.3 mM EGTA buffer, pH 6.5, 10 mM magnesium chloride using 150 #g of platelet protein and 1 #M [32p]ATP, 5-20 × 103 cpm in a total volume of 200 #1. The phosphorylation reaction was initiated by addition of platelet homogenate and was stopped with 20 #1, 10~o SDS, 6 mM 2-mercaptoethanol, 5 mM EDTA buffer. The time course of the reaction was linear up to 1 min for all phosphoroprotein peaks and there was a linear relationship of 32p incorporation over a protein range of 5-250 #g of platelet protein. A 1-min end-point and 100 #g protein were used in all experiments for comparison of normal and DMD platelets.

Phosphorylation of whole platelets Platelets were preincubated in buffer (0.15 M NaC1, 0.01 M Tris-HC1, 5 mM EDTA, pH 7.4) containing [32p]orthophosphate, specific activity 0.5 mCi/ml for 60 min at 37 ° C. This time period was found necessary to approach isotopic equilibrium. After 60 min, aliquots (160 #g platelet protein) were removed and incubated in A23187 (1 #M) for 15 s at 37 °C. Control platelets were incubated with incubation buffer in place of A23187. The reaction was stopped by both the addition of stop solution (10 #I 10~o SDS, 6 mM 2-mercaptoethanol, 5 mM EDTA, per 100 #g total protein) and heating for 3 min at 100 °C. Samples were stored until analysed at - 2 0 °C.

Gradient slab gel electrophoresis A 7.5~o to 12.5 or 15~o linear gradient gel with a 4~/o stacking gel was prepared as described by Laemmli (1970). Platelet samples (100 #g) were run for 4-6 h until the dye front reached the bottom of the gel. The gels were stained with Coomassie blue G250 and destained in ethanol/acetic acid. After soaking the gels in 10~o acetic acid, 2~o

24 glycerol for 30 min the gels were dried on a Bio-Rad dryer. Autoradiographs were prepared using Kodak OM-1 film in a Kodak X-Omatic cassette with a Kodak Lanex regular intensifying screen at - 7 0 ° C. Gels were stained for protein with Coomassie blue and autoradiographs were quantitated by scanning with an LKB laser densitometer. Apparent molecular weights ofplatelet proteins were determined from Pharmacia low-molecular weight standards, using a regression plot of log molecular weight prepared from the molecular weight standards. Molecular weights are cited as apparent molecular weight expressed in kdaltons.

Two-dimensional gel electrophoresis Two-dimensional gels were prepared following the method described by Ames and Nikaido (1976). Protein samples were solubilized in 9.5 M urea, 4~o N P40, 5~o 2-mercaptoethanol and 2~o LKB pH 3-10 ampholines. The ratio of NP40 to SDS was maintained at 8 : 1. Two hundred #g of platelet protein were loaded on to each disc gel. Isoelectric focussing was carded out as prescribed by O'Farrell (1977) using 0.01 M ethylenediamine as top buffer (cathode solution) and 0.1 M iminodiacetic acid as bottom buffer (anode solution) in a Bio-Rad model 155 gel electrophoresis cell. Samples were applied to the top of the gel without any pre-running procedure as described by Ames, covered with sample overlay solution (6 M urea, 2~o pH 3-10 ampholytes, 4~o NP40). The gels were run at 400 V overnight with cooling at I0 °C following which the voltage was raised to 800 V for 1 h to give a total of 7000 to 8000 V h. Gels were then removed and equilibrated in 5 ml of equilibration buffer (1.5 ~/o Tris-HCl pH 6.8, 5 ~ 2-mercaptoethanol, 10~ glycerol, 2.3~o SDS) for 30 min as described by Anderson and Anderson (1978). Gels were then frozen at - 70 °C until the second dimension was run. A linear gradient gel (7.5-15~o) was prepared as described above. After unfreezing, the isoelectric focussing gel was set into the top of the gradient gel in 0.7~/o agarose and allowed to set using a Bio-Rad vertical slab gel apparatus. Electrophoresis was carried out at 50 V at 15 °C until the dye front reached the bottom of the gel with a Tris/glycine/SDS, pH 8.3, buffer system as described by Anderson and Anderson (1978). Staining and destaining, drying and quantitation were carried out as described above for the gradient slab gels.

Analysis of results and statistics Results for protein phosphorylation were determined from quantitation of the area under each peak of the scanned autoradiographs and were expressed in arbitary units. Responses for 6-7 paired DMD and normal platelets were compared using both a paired t-test and the Wilcoxon sum of ranks test in order to overcome possible differences due to variation in assay conditions from day to day.

25 RESULTS

There were no differences in the protein profiles of platelets from DMD and normal subjects. Approximately 25 proteins were resolved on gradient gels, and 50 on two-dimensional gels. Two sets of protein phosphorylation experiments were carried out. Protein phosphorylation was examined in platelet homogenates using endogenous protein kinase and [32p]ATP as substrate, and also in whole platelets preincubated in [ 32p ] ATP and stimulated with A23187. In homogenised platelets, calcium-dependent protein phosphorylation could not be demonstrated as no difference was obtained using added calcium (0.1-1.0 mM), low calcium buffer (0.1 mM EGTA), trifluperazine (1/~m), or added calcium plus calmoTABLE 1 C O M P A R I S O N OF P H O S P H O P R O T E I N S IN D M D AND NORMAL H O M O G E N I Z E D PLATELET PREPARATIONS Comparison of the 6 principal phosphoprotein peaks in 4 pairs of normal and D M D homogenized platelets, separated on 5-12.5 % polyacrylamide gradient gels. Phosphorylation was quantitated from autoradiographic density as described in the Methods section. Means have not been calculated as results for each experiment (paired normal and D M D platelets) have been compared using a paired t-test. Peak molecular weight (kdaltons)

Normal

DMD

t

P

110-125

0.189 0.964 0.262 0.458

0.664 0.894 0.695 0.515

1.651

>0.1

50-88

1.570 3.333 5.149 5.229

0.441 3.478 0.290 1.916

2.052

> 0.1

40-46

3.246 7.237 3.769 11.752

9.614 10.630 2.492 8.017

0.522

> 0.6

34-40

0.772 4.703 2.384 11.742

4.186 4.274 2.050 8.017

0.185

> 0.8

25-34

1.44 1 4.560 2.377 5.051

4.856 5.952 2.437 2.162

0.384

> 0.7

17.5-19.5

0.412 0.750 0.914 4.217

0.446 2.311 0.951 3.951

0.827

> 0.4

26 dulin (1/~g/ml). No disease-related differences were found in two sets of experiments with homogenised platelets using both linear 11.5 ~o polyacrylamide slab gels (6 paired DMD and normal preparations) and 5-12.5 ~o polyacrylamide gradient slab gels, either in terms of individual proteins, or in protein phosphorylation of the 6 principal phosphoprotein peaks (Table 1). Inspection of two-dimensional gels and autoradiographs of the same platelet preparations revealed no differences in the protein-staining pattern or in the pattern of phosphorylation in autoradiographs. Approximately 60 different phosphoproteins were resolved. Whole platelets incubated in [32p]orthophosphate showed no disease-related differences either in basal phosphorylation of 15 phosphoprotein peaks (Table 2) or in the two A23187-stimulated protein phosphorytation peaks (Table3, Fig. 1). Twodimensional gels showed that the higher molecular weight calcium-dependent phosphorylation peak (40 kdaltons) consisted of 4 phosphoproteins and the 17 kdalton peak was a single phosphoprotein (Fig. 2). The approximate number of phosphoproteins (as determined by two-dimensional gel electrophoresis) in each phosphoprotein peak quanTABLE 2 C O M P A R I S O N O F P H O S P H O P R O T E I N S IN D M D A N D N O R M A L W H O L E PLATELETS Comparison of basal [32P]phosphoproteins in 7 pairs of normal and D M D whole platelets incubated with [a2p]orthophosphate as described in the Methods section. Fitteen peaks were resolved. Results are expressed as the area under each peak in arbitary units. There were no differences between D M D and normal platelets in any of the peaks. M e a n s are not calculated, as results for each experiment were analysed in pairs (normal and D M D ) using a paired t-test. Dashes indicate where a peak was incompletely resolved on a particular 7-15 % polyacrylamide gel. Molecular weight (kdaltons)

Molecular weight (kdaltons)

Normal

t

P

0.209 0.106 0.179 0.193 0.386 0.309

0.868

> 0.4

0.384 0.221 -0.325 0.245 0.345 0.313

0.103 --0.314 0.100 0.772 0.335

0.020

>0.9

-0.345 0.726 -0.723 -0.815

-0.188 0.502 -0.882 -1.279

0.381

>0.7

Normal

DMD

t

P

17.4

0.428 1.650 0.217 0.324 0.122 0.058

0.328 0.546 0.110 0.955 0.335 0.226

0.213

> 0.8

21.3

0.267 0.283 0.229 0.169 0.245 0.137

25.0

0.226 0.303 0.400 0.605 0.318 0.243 0.243

0.079 0.238 0.344 0.145 0.483 0.878 0.329

0.177

>0.8

30.0

37.4

0.908 1.139 1.288 0.775 0.324 0.723

1.264 0.511 1.117 0.306 0.534 1.842 --

0.266

>0.8

40.3

DMD

27 TABLE 2 (continued) 47.2

-0.663 0.509 0.498 0.405 0.367 0.403

-0.181 0.672 0.348 0.571 0.931 0.825

1.I59

> 0.2

50.5

0.593 0.265 1.404 0.806 1.071 0.861 0.835

1.277 1.039 1.425 0.307 1.152 2.154 1.368

1.850

>0.1

56.2

0.896 0.997 1.838 1.655 1.433 1.220 1.298

1.348 0.495 1.756 0.599 1.810 2.174 1.872

0.391

>0.7

66.0

4.099 6.072 6.116 7.570 5.466 4.850 4.904

6.326 2.749 6.921 3.578 8.229 7.716 6.669

0.406

>0.6

-0.629 2.491 1.285 2.663 2.412 2.271

0.263

> 0.8

82.2

-6.828 8.765 6.934 4.811 5.586 6.399

-4.809 6.219 5.727 7.997 7.977 6.384

0.036

>0.9

-0.444 0.916 1.104 2.285 3.705 2.121

0.596

>0.5

-1.113 2.407 4.117 2.764 ! .948 2.126

-0.899 4.632 1.857 10.048 5.244 4.525

1.599

>0.1

1.319 0.567 2.541 1.694 1.845 0.756 1.054 3.427 2.224 0.848

1.019 1.886 2.507 0.953

0.611

>0.5

1.301 1.079

3.256 2.051

74.7

-

-

2.471 2.124 1.843 1.612 1.465 1.510 103

170

-

-

118

titated in Tables 1 and 2 are given in Table 4. Visual inspection of the two-dimensional gels showed n o differences in single phosphoproteins in D M D a n d n o r m a l platelets. DISCUSSION The m e m b r a n e defect hypothesis for D M D has been reviewed by Lucy (1980). I n general most reports of n o n - m u s c l e m e m b r a n e defects were single studies showing m i n o r changes. N o reproducible disorder of cell m e m b r a n e s has so far been described which shows a large (i.e. greater than 50~o) variation from normals. Most reported abnormalities have been just outside the 2 s t a n d a r d deviations or the 95 ~ n o r m a l range chosen for each study. A possible explanation for the m a n y reports of m i n o r m e m b r a n e abnormalities is selection for publication, of positive rather than negative findings. This

TABLE3 COMPARISON OF A23187-STIMULATED PHOSPHOPROTEINS IN DMD AND NORMAL WHOLE PLATELETS Comparison of the two A23187-stimulated phosphoprotein peaks in 7 pairs of DMD and normal platelets, The response to A23187 (1 #M, 15 s incubation) are expressed as a ratio relative to an unstimulated control. Phosphoproteins were quantitated as described in the Methods section (see Fig. 1). Means have not been calculated as results for each experiment (normal and DMD pairs) have been compared by a paired t-test. Peak molecular weight (kdaltons)

Normal

DMD

t

P

17.4

4.798 2.057 10.402 7.996 6.764 6.846 21.154

19.361 6.488 1.698 15.674 9.414 9.288 9.598

0.479

>0.6

40.3

2.528 5.749 3.688 4.005 3.852 5.391 3.978

5.667 9.290 3.747 9.192 4.442 3.813 5.506

2.031

>0.05

TABLE 4 COMPARISON OF PHOSPHOPROTEIN PEAKS IN ONE DIMENSION (GRADIENT GELS) WITH PHOSPHOPROTEINS RESOLVED BY TWO*DIMENSIONAL ELECTROPHORESIS The phosphoprotein peaks quantitiated in Tables 1, 2 and 3 in 7.5-12.5~ polyacrylamide gradient gels are compared with the number of phosphoproteins resolved by two-dimensional eleetrophoresis in each molecular weight range. Results are expressed as the maximum number ofphosphoproteins resolved in each molecular weight range as determined from 14 two-dimensional eleetrophoresis runs of 7 normal platelet preparations. Gradient gel phosphoprotein peak (kdaltons)

Total

Number of phosphoproteins resolved by two-dimensional electrophoresis

17 21 25 30 37 40 47 50 56 66 75 82 103 118 170

1 1 3 1 4 4 4 7 4 4 7 7 4 4 3

15

58

29

a

S | 17

40

66 82

170

Fig. 1. Densitometry (a and b) of platelet autoradiograph (c and d). Whole platelets were incubated with [?-32P]ATP as described in the Methods section and exposed to the calcium ionophore A23187 (b and d). Control platelets are shown in a and c. The figures shown below the autoradiogram indicate molecular weights, expressed in kdaltons, of some of the autoradiograph bands. Stimulation of two phosphoproteins of 17 and 40 kdaltons is clearly seen. Figures in Tables 1, 2 and 3 were derived from the integrated peak areas of similar densitographic recordings.

would result in publication of the one out of twenty studies (95 ~o normal range) which by chance shows a positive result. Subsequent studies o f the same "defect" are therefore likely to produce a negative result. The results reported in this study give indirect evidence that the calcium abnormality described in muscle is not shared by platelets. However, it has not been determined whether the raised total calcium levels in D M D muscle represent an elevated intracellular resting calcium concentration or increased concentrations o f calcium in intracellular stores such as the sarcoplasmic reticulum. If the calcium defect was generalised in D M D , platelets could also have raised levels of calcium in intracellular calcium stores, e.g. the platelet-dense bodies, as has been suggested by the work o f

30 pH

7

6743 ~ KD

20" a

14 -

4

946743KD 30-

2014-

Fig. 2. Autoradiograms of5-12% polyacrylamide two-dimensional elvctrophoresis gels. In b platelets were exposed to A23187 as described the in Methods section; a shows control unstimutatvd platcle~,s. The principal A23187-stimuhted phosphoprotein is sven as a single spot at approximately 17 kdldtoas. The 40 kdalton calcium-stimulated phosphoprotein appears as 4 incompletely resolved spots.

31 Yarom et al. (1980). In the only other study relating to platelet-dense bodies in DMD, Adornato et al. (1979) reported decreased numbers of dense bodies in DMD. The calcium ionophore A23187 is thought to act by releasing calcium from both extracellular and intracellular stores. It would be expected that, if calcium concentrations in dense bodies were elevated, stimulation with A23187 should produce increased calciumdependent phosphorylation. It is therefore concluded from these experiments that platelets do not share the muscle calcium abnormality. A similar conclusion was made by Szibor et al. (1981) who examined metabolic responses of red cells to A23187 and to the potassium ionophore, valinomycine, and concluded that there was no evidence for a generalised calcium defect affecting DMD red cells. An additional reason for the choice of platelets for these studies is that platelets contain a calcium-activated contractile system which in some respects resembles that in muscle. Although the pathway of calcium activation of contraction in platelets and smooth muscle cells is through myosin light-chain kinase a similar pathway has recently been described in skeletal muscle (Barany and Barany 1977; Stull et al. 1980). If the disturbance in contractile function in DMD muscle involved this pathway, it should be shared with platelets. Our results show no differences in basal or calcium-stimulated phosphorylation of the 17 kdalton peak which is probably the light chain of myosin. Our results therefore provide evidence that the myosin light-chain kinase pathway is not disturbed in DMD. Platelets were also chosen for study because membrane proteins represent a relatively large proportion of total platelet protein. If a disorder of membrane protein phosphorylation was present in DMD platelets similar to that described in DMD red cells by Roses et al. (1976), it should be detected by the technique used. The results of these experiments therefore do not support the generalised hypothesis for DMD. REFERENCES Adelstein, R. S. and M. A. Conti (1975) Phosphorylation ofplatelet myosin increases actin-activated myosin ATPase activity, Nature (Lond.), 256: 597-598. Adornato, B.T., J.C. Corash, B. Shaper, H. Stark, D. Murphy and W.K. Engel (1979) Abnormality of platelet dense bodies in Duehenne dystrophy, Neurology, 29: 567. Ames, G.F.-L. and K. Nikaido (1976) Two-dimensional gel electrophoresis of membrane proteins, Biochemistry, 15: 616-623. Anderson, N.G. and N.L. Anderson (1978) Analytical techniques for cell fractions, Anal. Biochem., 85: 331-354. B arany, K. and M. Barany (1977) Phosphorylation of the 18,000 dalton light chain of myosin during a single tetanus of frog muscle, J. Biol. Chem., 252: 4752-4754. Bodensteiner, J.B. and A.G. Engel (1978) Intracellular calcium accumulation in Duchenne dystrophy and other myopathies - - A study of 567,000 muscle fibres in 114 biopsies, Neurology, 28: 439-446. Bradley, W. G. (1980) Cell membrane abnormalities and muscular dystrophy (Editorial), Muscle and Nerve, 3: 1-2. De Kretser, T.A. and B.G. Livett (1977) Skeletal-muscle sarcolemma from normal and dystrophic mice, Biochem. J., 168: 229-237. Duchenne, G.B. (1868) Recherches sur la paralysie musculaire pseudo-hypertrophique ou paralysie myoscl~rosique, Arch. G~n. M~d., 2: 5, 179, 305, 421,552. Haslam, R. J. and J.A. Lynham (1978) Relationship between phosphorylation of blood platelet proteins and secretion of platelet granule constituents, Part 2 (Effects of different inhibitors), Thromb. Res., 12: 619-628.

32 Haslam, R. J., J. A. Lynham and J. E. B. Fox (1979) Effects of collagen, ionophore A23187 and prostaglandin E on the phosphorylation of specific proteins in blood platelets, Biochem. J., 178: 397-406. Kunze, D., E. Reichmann, G. Egger, H. Levschner and H. Eckhardt (1973) Erythrozytenlipide bei progressiver Muskeldystrophie, Clin. Chim. Acta, 43: 333-341. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4~ Nature (Lond.), 227: 680-685. Lucy, J.A. (1980) Is there a membrane defect in muscle and other cells? Brit. Med. Bull., 36: 187-192. Lyons, R. M., N. Stanford and P.W. Majerus (1975) Thrombin-induced protein phosphorylation in human platelets, J. Clin. Invest., 56: 924-936. Matheson, D. W. and J. L. Howland (1974) Erythrocyte deformation in human muscular dystrophy, Science, 184: 165-166. Meryon, E. (1852) On granular and fatty degeneration of the voluntary muscles, Med.-chir. Trans. (Lond.), 35: 73. Mizobe, F. and B.G. Livett (1982) Evidence against a generalised membrane defect in dystrophic mice platelets, Muscle and Nerve, 5: 387-395. Nowak, T.V., V. Ionasescu and S. Anuras (1982) Gastrointestinal manifestations of the muscular dystrophies, Gastroenterology, 82: 800-810. O'Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins, J. Biol. Chem., 250: 4007-4021. Rodan, S. B., R.L. Hintz, R.I. Sha'afi and G.A. Rodan (1974) The activity of membrane bound enzymes in muscular dystrophic chicks, Nature (Lond.), 252: 589-59t. Roses, A.D., M. Herbstreith and S.H. Appel (1975) Membrane protein kinase alteration in Duchenne muscular dystrophy, Nature (Lond.), 254: 350-351. Roses, A. D., M. Herbstreith, B. Metcalf and S.H. Appel (1976) Increased phosphorylated components of erythrocyte membrane spectrin band II with reference to Duchenne dystrophy, J. Neurol. Sci., 30: 167-178. Rowland, L.P. (1980) Biochemistry of muscle membranes in Duchenne muscular dystrophy, Muscle and Nerve, 3: 3-20. Schapira, G., J.C. Dreyfus and F. Schapira (1953) L'616vation du taux de l'aldolase s6rique - - Test biochimique de myopathies, Sem. H6p. Paris, 29: 1917-1921. Steiner, M. (1976) Effect of thrombin on phosphorylation of platelet membrane proteins, Thrombos. Haemos., 35: 635-642. Strickland, K.P., A.J. Hudson and J.H. Thakar (1979) Biochemical studies in dystrophic mouse muscle, Ann. N. Y. Acad. Sci., 317: 187-205. Stull, J.T., D. R. Manning, C.W. High and P. K. Blumenthal (1980) Phosphorylation of contractile proteins in heart and skeletal muscle, Fed. Proc., 39: 1552-1556. Szibor, R., U. Till, W. Losche and V. Steinbicker (1981) Red cell response to A23187 and valinomycine in Duchenne muscular dystrophy, Acta Biol. Med. Germ., 40:1187-1190. Walton, J.N. (1964) Muscular dystrophy - - Some recent advances in knowledge, Brit. Med. J., 1: 1344-1348. Yarom, R., J. Blatt, R. Gorodetsky and G.C. Robin (1980) Microanalysis and X-ray fluorescence spectrometry of platelets in diseases with elevated muscle calcium, Europ. J. Clin. Invest., 10: t43-147.