002iI-711X/92$5.00+ O.O!l Fcrgamon Press pIc
br. J. Biocirem.Vol. 24, No. 4, pp. 579-584, 1992 Printed in Great Britain
MYOFIBRILLAR AND MEMBRANE-BOUND ENZYMES IN SKELETAL MUSCLE FROM MYODYSTROPHIC MICE Y.
S. REDDY,’
N. B.
REDDY,*
B.
A. MOBLEYI and R. C. BEESLEY’
‘Department of Physiology and Biophysics, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190, U.S.A. [ZW. (405)271-2226; Fnx (405)271-31811 and ‘Department of Neurology, Medical College of Pennsylvania, Philadelphia, Pa, U.S.A. (Received
7 July 1991)
Altstrac--1. Experiments were carried out to examine the biochemical changes, such as contractile protein biochemistry and membrane bound enzyme alterations associated with skeletal muscles of myd/myd. 2. Our studies demonstrate that there was a progressive decline in myofibrillar ATPase activity, and this decrease is greatest in 30 weeks old animals of myd/myd as compared to controls. 3. The proteolytic activity of myofibrils isolated from myd~myd was si~ifi~ntly higher than controls. 4. There was no significant difference in Ca 2+ ATPase activity of myosin and actin-activated myosin ATPase activity of myd/myd and their controls. 5. Mg* + ATPase and Na + + K + -ATPase of myodystrophic SL showed significant increase compared to controls. 6. Isoproterenol stimulated adenytate cyclase activity was significantly lower in the SL of dystrophic mice compared to controls. 7. GTP + isoproterenol stimulate adenylate cyclase was significantly higher in control SL and SR when compared to SL and SR isolated from myd/myd. 8. Guanylate cyclase activity was greater in myodystrophic mice both in the absence and presence of Triton X- 100. cGMP and CAMP phosphodiesterase activities were greater in dystrophic mice as compared to controls. 9. These observations suggest that there are significant changes in myofibrillar ATPase, myofibrillar protease and membrane bound enzymes of myd/myd compared to control,
INTRODUCTION Lane et ai. (1976) identifi~ mouse myopathy resulting from genetic mutation of chromosome (Goldberg ef al., 1975). The histopathological studies of myodystrophic mice (myd/myd) is similar to that of dysttophia muscularis (129/dy/dy), a disease resulting from genetic mutation of chromosome 4 (Neymark et al., 1980). In case of myd/myd all skeletal muscles were affected whereas in dy/dy only the hindlimbs are affected. Mobley et al. (1985) showed previously a decrease in myofib~ll~ ATPase activity but identical force generating capacity of myodystrophic skeletal muscle compared to control muscle. Neymark et al. ( 1980) found that sarcoplasmic reticular membranes isolated from mydlmyd exhibited an increase of basal ATPase activity, no change in Ca* +ATPase activities and a decrease in phosphoenzyme concentrations. Nutting et al. (1980) described elevated levels of Cal + in the diaphragm of myd/myd. We carried out the experiments on contractile protein biochemistry and membrane bound enzyme changes in myd/myd and their controls. Our main goal was to discover the possible defects in contractile protein ATPase activities and determine if membrane bound enzyme alterations may be responsible for the myodystrophy in all skeletal muscles of myd/myd. Part of this work was published previously in abstract form (Robinson et al., 1981).
METHODS AND MATERIALS ~yo~rii
preparation
Myodystrophic (myd/myd) mice and their sibling controls were purchased from Jackson Laboratories (Bay Harbor, Maine). Mice were sacrificed by cervical dislocation and the muscles of the hind legs were excised as quickly as possible and washed in cold 0.9% NaCl. Skeletal muscie mvo~b~ls were prepared according to the procedure of Solaio et af. (1971). Briefly, the procedure involves homogenizing the tissue in 0.3 M sucrose containing 10 mM imidazole @H 7.0) for 3-S set intervals using a Brinkman polytron (settinE 7). The homogenate was ~n~ifu~ and the resulting pelle? was washed 3 times with standard buffer &OmM KCl. 30 mM imidazole, 2 mM MgCl,, pH 7.0). The pellet was then washed once with standard buffer containing 2mM ethylene glycol 6is@-amino-ethyl ether)-N-N-tetra-acetic acid (EGTA), then twice with standard. buffer containing 1% Triton X-100. Finallv. the &let was washed 3 times with standard buffer. Aliwashe’s consisted of suspension/ homogenization of the pellet with a glass Dual1 homogenizer followed by centrifugation. The final pellet was suspended in a reaction medium consisting of 50 mM Tris-HCl, 5 mM NaN,, 2.5 mM MgCl,, pH 7.6. The myofibrillar protein concentrations were determined as described by Lowry er al. (1951). Comaerile
protein ATPase acriviry
Myofibrillar ATPase activity was measured in 1 ml of reaction medium containing 5OmM Tris-HCl (pH 7.6)
5 mM NaN,, 2.5 mM MgCl,, 2.5 mM ATP, 10mM 579
Y. S.
580
REDDY et al.
Ca-EGTA buffer and 2OOpcn myofibrillar protein. A &,,, = 1 x IO’ was used to &lculate the [Caj+] in the Ca-EGTA buffers (Martel. 1971). The reaction was started with the addition of ATP and stopped after 5 min at 3O’C with 1.0 ml of cold 10% TCA (tr~~hloroacetic acid). The denatured protein was removed by centrifugation and the amount of inorganic phosphate was measured in 1.0 ml of the supernatant using the method of Fiske and Subba Row (1925). Myosin was isolated by the procedure of Shiverick et al. (1975). Actin was purified from rabbit skeletal muscle acetone powder by the method of Spudich and Watt (1971). The protein content was determined by the procedure of Lowry et al. (1951) using bovine serum albumin as standard. Myosin Cal+ -ATPase activity was determined using 150mM Tris-maleate buffer (pH 6.5), 5 or IOmM CaCl,, 5 mM ATP, and the incubation was performed at 30°C for 5 min. The reaction was terminated by addition of 10% TCA and the inorganic phosphate present in the supernatant was determined (Hoh and Salafsky, 1972). Actinactivated myosin ATPase activity was determined using lOO/cg of myosin, 100~1 of reaction buffer containing 200 mM Tris-HCl, 50 mM MgCl,, 100 ~1 of 5 mM DDT, 50 ~1 of 50 mM ATP and different concentrations of actin in 1 ml of total reaction medium. The reaction was initiated by the addition of 50~1 of 50mM Na, ATP and stopped after 5 mitt of incubation at 37°C by the addition of I ml of 10% cold TCA. Inorganic phosphate (Pi) present in the supernatant was determined by the method of Fiske and Subba Row (1925). Caseinolylic activity of myqfibrils
The caseinolytic activity of myofibrils was measured using the method of Kar and Pearson (1967). The 1ml incubation mixtures containing 50 mM Tris-HCI (PH 8.0), 2 mg caseinyellow (CalBiochem) and l-5 mg myofibrillar protein were incubated at 37°C for 18 hr. The incubation was stopped by the addition of l.Oml of 10% HCIO,. The samples were centrifuged and 2.5 ml of 10% KOH was added to the supernatants. The samples were centrifuged again and the absorbance of the resulting supernatants was measured at 428 nm. A change in absorbance of 0,001 per mg protein after 18 hr at 37°C was defined as one unit of caseinolytic activity.
10
Membrane preparation
Sarcoplasmic reticulum (SR) and sarcolemma were isolated from normal and dystrophic mice as described previously (Reddy ef al., 1978a). SL membranes, without further puri~cation on sucrose density gradients were used in the present studies. Membrane-bound
enzyme assays
The membrane-bound ATPase activity was determined using [y-“P]ATPase as substrate and by measuring the liberated ‘*Pi by Cerenkov counting as previously described (Lane et al., 1976). The standard reaction medium in a final volume of 0.5 ml contained 30 mM imid~ol~glycylglycine, DH 7.5. 3 mM Tris-ATP (0.2 uCi IV-“*PlATP). 0.5 mM EGTA, 20-50 pg of membrane protein and indicated concentrations of different ions in individual experiments. Each reaction was allowed to proceed for 15 min at 37°C. In all ATPase assays the reaction was linear with time and protein, and not more than 15% of the added ATP was hydrolyzed under our assay conditions. Mg2’-ATPase activity was essentially similar in the presence or absence of ouabain. Na + + K + -ATPase activity was obtained by subtracting Mg* + -ATPase from that observed in the presence of 5 mM Mg*+, 100mM Na+, and 20 mM K + , The Ca2+ -ATPase was obtained by subtracting Mg2+-ATPase from the activity obtained in the presence of 5 m Mgr + and 0.1 mM Ca’+. EGTA was omitted from the tubes that contained Ca?f. Adenylate and guanylate cyclase activities were assayed as previously described (Reddy er al., 1978b). The reaction medium (0.1 ml) contained 50 mM Tri-HCl, pH 7.5,0.5 mM [y-32P]ATP (adenylate cyclase) or 0.1 mM [y-‘2P]GTP (guanylate cyclase), 5 mM MgCI,, 1OmM KCI, 1OmM theophylline, IO mM creatinine phosphate, 1 unit of creatinine phosphokinase, and SO-100 pg membrane protein. The reaction was ahowed to proceed for 10 min at 37°C and it was stopped by adding 0.1 ml of 2% SDS, 1 mM unlabeled ATP and 1 mM [“Hjcyclic AMP (- 10,000 cpm) in the case of adenylate cyclase, or with 0.1 ml of 5 M HCl and boiling in the case of guanylate cyclase. After cooling, 0.1 ml of 1 mM [‘H]cyclic GMP (C 10,OOOcpm)was added to the guanylate cyclase assay tubes. The newly formed [32P]cyclic AMP (adenylate cyclase) or [32P]cyclic GMP (guanylate cyclase) was separated by sequential elution on Dowex
AGE (weeks) (weeks) Fig. 1. Myofibrillar ATPase (A) and caseinolytic (B) activities of myofibrils from skeletal muscle of control and myodystrophic mice of different ages. One unit of caseinolytic activity is equal to a change in absorbance of 0.001 per mg protein after incubation for 18 hr at 37°C. Values are the mean + SD for four or five preparations. *P < 0.01 AGE
Contractile protein ATPases
581
and alumina columns as described by Salomon ef al. (1974) with minor modifications (Reddy et al., 1978b). Recovery for [‘HIcyclic AMP ranged between 70-85% and that for 13Hlcvchc GMP between 58-75%. Assay of cyclic AMP- and cyclic GMP-phosphodiesterase were performed essentially according to the two-step procedure of Thompson et al. (1974) using SO-1OOpg of membrane protein. The reaction was performed at 30°C for 20 min.
J
=
Control
I
2 Myodystrophlc
RESULTS
The myofibrillar ATPase activity (pCa 6 or greater) of both control and myodystrophic (myd/myd) mouse skeletal muscle increased to age 17 weeks compared to 10 week old animals [Fig. l(A)]. Control myofibrillar ATPase activity of 30 week old animals remained the same as that of 17 week old animals. The myofibrillar ATPase activity of 10 week old myd/ myd mice was significantly less than ATPase activity for their sibling controls (P < 0.01). The difference between the myofibrillar ATPase activity of control and myd/myd mice increased with age of the mice. Compared to their controls, 10 week old myd/myd mice have 10% less myofibrillar ATPase activity; 17 week old myd/myd mice have 14% less activity; and 30 week old myd/myd mice have 23% less activity. Results from studies on the caseinolytic activity of skeletal myofibrils from control and myd/myd mice are shown in Fig. l(B). While both control and myd/ myd myofibrils hydrolyze casein-yellow under the alkaline assay conditions, the myd/myd myofibrils had significantly higher caseinolytic activity than the controls (P < O.Ol).The hydrolysis of casein of these myofibrils was completely inhibited by the addition of 1 mM PMSF (phenylmethyl sulfonyl fluoride) to the incubation system. There was no statistical significant differences between the Ca2+ activated myosin ATPase activity of control and myd/myd mice. Similarly, there was no significant difference in actin-activated myosin ATPase activities of control and myd/myd mice (data now shown).
Mg2’
Na’+K-
ca2-
hlg”
CC?-
Fig. 2. ATPase activities of sarcolemma (SL) and sarcoplasmic reticulum (SR) from skeletal muscle of control and myodystrophic mice as measured in the presence of different cations. Values are the mean + SD for three preparations each assayed in duplicate. *P i 0.01.
B
F
I
GTP
B
F
I
GTP
Fig. 3. Adenylate cyclase activities of sarcolemma (SL) and sarcoplasmic reticulum (SR) from skeletal muscle of control and myodystrophic mice. Basal activity (B) and activity in the presence of 10 mM NaF (F), 10 PM isoproterenol (I) or 10pM isoproterenol plus 1OlM GTP (GTP) were determined. Values are the mean f SD for three preparations each assayed in triplicate. *P < 0.01.
Figure 2 shows ATPase activities in SL and SR of control and myodystrophic mice Mg*+-ATPase activity of myodystrophic SL showed marked increase over the control SL (P < 0.01). Na+ + K+ -ATPase also was significantly increased in myodystrophic SL compared to controls (P < 0.01). It should be noted that we did not detect any Na + + K+ -ATPase activity in SR, indicating that our SR fraction is not contaminated with SL membranes. While Ca2+-ATPase activity of both SR and SL of control mice were greater than those for myd/myd mice the differences were not statistically significant. Likewise there is no statistical significant difference in Mg*+-ATPase activities of SR isolate from control and myd/myd mice. Basal and NaF-stimulated adenylate cyclase activities were slightly less in the SL of dystrophic mice compared to control SL (Fig. 3). By contrast, those activities were slightly greater in SR of dystrophic mice. However, these differences were not statistically significant. Isoproterenol and isoproterenol + GTP stimulated adenylate cyclase activities of myd/myd SL were significantly less as compared to control animals (P < 0.01). GTP had no effect on the basal enzyme activity in the absence of isoproterenol (data not shown). There was no significant difference in isoproterenol stimulated adenylate cyclase activity of SR isolated from control animal compared to SR isolated from myd/myd. However, GTP + isoproterenol stimulated adenylate cyclase activity of control SR was significantly greater than that in SR of myd/myd (P < 0.01). We determined guanylate cyclase activity in SL, SR and cytosol fractions of control and myodystrophic mice (Fig. 4). There was no statistically significant difference in basal guanylate cyclase activities of SL between control and myd/myd. However, basal guanylate cyclase activity of SR and cytosol fractions from myd/myd were significantly greater than those for controls (P < 0.02). Guanylate cyclase activity was also significantly greater in all three fractions from myodystrophic mice in the presence of Triton X-100 (P < 0.01).
582
Y. S. REDDYet al, 0
=
CollCrol
I
= Myodystrophlc
Fig. 4. Guanylate cyclase activities of sarcolemma (SL), sarcoplasmic reticulum (SR) an cytosol (Cytosol) from skeletal muscle of control and myodystrophic mice. Basal activity (B) and activity in the presence of OS% Triton X-100 (T) was determined, Values are the mean f SD for three preparations each assayed in duplicate. *P -c 0.02, **p < 0.01.
Cyclic GMP dependent phosphodiesterase activities (PDE) were significantly higher in SL, SR and homogenate from myd/myd mice as compared to control (Fig. 5). Similarly cyclic AMP dependent PDE was higher in SL and homogenate of myd/myd mice compared of control mice. There was no statistically significant difference in cyclic AMP dependent PDE between control and myd/myd SR. DISCUSSION
Several studies have shown that the dystrophic mouse skeletal muscle exhibit many abnormalities. Neymark ef al. (1980) reported increased levels of basal ATPase activities, no change in Ca2+-ATPase and decreased levels of phosphoenzyme activity in myd/myd compared to control animals. We have shown previously that there was a significant difference in force generating capacity between control and myd/myd muscle, and a decrease in myofibrillar
Fig. 5. Cyclic nucleotide phosphodiesterase activities of sarcolemma (SL), sarcoplasmic reticulum (SR) and homogenate (Horn) of skeletal muscle from control and myodystrophic mice. Phosphodiesterase activity was determined using CAMP or cGMP as substrate. Values are the mean *SD for three preparations each assayed in duplicate. *P < 0.02, **p < O*OI*
ATPase activity which could be due to a greater percentage of type 1 fibers (slow fibers) associated with the myd/myd as compared to controls (Mobley et al., 1985). In the present investigation we have found an age dependent decrease in myofibrillar ATPase activity and the greatest percentage of decrease in myofibrillar ATPase activity was seen in 30 week old myd/myd as compared to controls. Furukawa and Peter (1971) showed that the skeletal muscle actomyosin isolated from patients affected with Duchenne muscular dystrophy exhibited tower actomyosin ATPase activity compared to controls. John (1976) reported a decrease in myofibrillar ATPase activity of mouse skeletal muscle affected by muscular dystrophy. Thus the decreased myofibrillar ATPase activity of myd/myd shown here supports the work of Furukawa and Peter (1971) and John (1976). Previously, we have explained that the decrease in myofibrillar ATPase activity may be due to an increase in density of slow (type 1) fibers in mydjmyd (Mobley et al., 1985). An alternative explanation for the decrease in ATPase activity of myd/myd may be due to an increase in myofibrillar protease activity. Indeed, we have shown in Fig. l(3) a significant increase in proteolytic activity associated with the myofibrils isolated from the mydjmyd mice. The characteristic clinical feature of muscular dystrophy is a progressive muscle atrophy and weakness, both processes presumably due to the continuous loss of both soluble and myofibrillar proteins. The decrease in muscle proteins is largely due to proteolytic action of enzyme@). One such enzyme extensively studied was the Ca2+ activated neutral proteases (CANP). Sodium dodecyl sulfate gel electrophoresis of chicken myofibrils treated with purified CANP exhibited loss of myofibrillar proteins such as myosin heavy chain, troponin-I and troponin-T. This pattern of loss of muscle proteins is similar to those observed in muscle of patients having Duchenne muscular dystrophy (Sugita et al., 1980). In the dystrophic chicken skeletal muscle Stracher et al. (1979) found that a decrease in LC3 component of myosin was due to a protease action on the LCJ component of myosin. Myofibrillar alkaline protease activity has been reported by several authors (Nutting et al., 1980; Pennington, 1972; Penniston and Green, 1968). These proteases degrade myofibrillar proteins (Sugita et al., 1980; Warnes et al., 1981), resulting in loss of Mg2+ATPase activity and Ca*+ sensitivity of myofibrils (Kuo and Bhan, 1980; Murakami and Uchida, 1979). Thus, a decrease in myofibrillar ATPase activity observed in myd/myd may be due to loss of myofibrillar protein components, as a result of action of protease on the myofibrillar proteins. The increase in protease activity reported here [Fig. l(B)] may be responsible for the decrease in myofibrillar ATPase in myd/myd mice. Mg2 + -ATPase activity of myodystrophic SL showed marked increase (76% over the normal SL). In red blood cells, correlation of endocytosis and vacuole formation with levels of Mg2 + -ATPase activity has been made (Nutting et al., 1980; Penniston and Green, 1968). It is possible that in the dystrophic state, increased Mg*+ -ATPase may be involved in enhanced permeability (endo- or pinocytosis) of
583
Contractile protein ATPases muscle SL to nutrients. Na+ + K+-ATPase also showed about a 50% increase in myodystrophic SL compared to controls. It is known that intracellular concentrations of Na + and K + of skeletal muscle are altered in pathologic states (Hoh and Salafsky, 1972; Horvath et al., 1955). Since Na+ + K+-ATPase is involved in maintaining normal levels of cellular Na+ and K+ (Albers, 1976), and enhanced Na+ + K+ATPase activity may reflect an adaptive response of the muscle to correct any imbalance in Na + and K + levels that occurs in the myodystrophic condition. It should be noted that we did not detect any Na+ + K+-ATPase activity in SR, indicating that our SR fraction is not contaminated with SL membranes. Ca2 +-ATPase activity showed no significant change in dystrophic muscle. SR of both normal and dystrophic muscle had greater levels of Ca2 + -ATPase activity than SL reflecting higher calcium uptake activity in SR compared to SL. GTP is known to enhance adenylate cyclase response to different hormones, including catecholamines in several tissues (Rodbell et al., 1975). GTP by itself had no effect on the basal enzyme activity in the absence of isoproterenol. However, the stimulation of adenylate cyclase by GTP plus isoproterenol was greater in SL, and SR from controls than in SL and SR from myd/myd. Since isoproterenol activates adenylate cyclase through an initial interaction with beta adrenergic receptor, the receptor+nzyme interaction may be altered in the myodystrophic muscle. Guanylate cyclase activity is known to be present both in membrane and cytosol fractions of different tissues (Goldberg et al., 1975) including skeletal muscle (Blosser and Appel, 1978; Reddy and Engel, 1978). Accordingly, we determined this enzymatic activity in SL, SR and cytosol fractions of control annd myodystrophic mice. Guanylate cyclase activity was significantly greater in all the three fractions of myodystrophic mice. The difference was even more striking in the presence of Triton X-100, which stimulates guanylate cyclase (Sugita et al., 1980). Since cyclases involved in the synthesis of CAMP and cGMP were different in the skeletal muscle of myodystrophic mice compared to controls, it was of interest to see whether the activity of phosphodiesterases, which are involved in the breakdown of those cyclic nucleotides, were also different in the dystrophic muscle. SL and ;SR from dystrophic mice showed significantly greater cyclic GMP-phosphodiesterase activity than controls while cyclic AMP-phosphodiesterase activity was significantly greater in SL of dystropic mice than controls. Phosphodiesterases are known to be present at a higher level in the cytoplasm than in the membranes of various tissues (Appleman er al., 1973). Therefore, we determined the phosphodiesterase activity in whole-muscle homogenates to obtain a measure of the cytoplasmic enzyme activity, and found that both the phosphodiesterases were markedly greater in dystrophic mice compared to normal mice. Lower levels of adenylate cyclase activity and greater levels of cyclic AMP-phosphodiesterase activity means that the cyclic AMP content will be lower in myodystrophic mice skeletal muscle than in controls. Because cyclic AMP plays an important role in several cellular processes, a change in its content in
muscle may lead to other metabolic changes in that tissue. Higher levels of guanylate cyclase as well as cyclic GMP-phosphodiesterase indicate a greater turnover of cyclic GMP in the myodystrophic mice compared to controls. The functions of cyclic GMP in skeletal muscle are not known at present. Acknowledgement-We
thank Mrs Lula Rhoton for her expert secretarial assistance. REFERENCES
Albers R. W. (1976) The Enzymes of Biological Membranes Vol. 17, pp. 283-301. Plenum Press, New York. Appleman M. M., Thompson W. J. and Russel T. R. (1973) Cyclic nucleotide phosphodiesterases. Ado. Cyclic Nucleotide Res. 3, 65-98.
Baker N. H., Bland H. W. and Hart P. (1958) Concentrations of K and Na in skeletal muscle of mice with a hereditary myopathy. Am. J. Physiol. 193, 530-533. Blosser J. C. and Appel S. H. (1978) Properties and distribution of mammalian skeletal muscle guanylate cyclase. J. biol. Chem. 253, 3088-3093. Edmunds T. and Pennington R. J. T. (1981) Mast cell origin of “Myofibrillar Protease” of rat skeletal and heart muscle. Biochem. biophys. Acta 661, 28-31. Fiske C. H. and Subba Row Y. (1925) The calorimetric determination of phosphorus. J. biol. Chem. 66,375-380. Furukawa T. and Peter J. B. (1971) Superprecipitation and adenosinetriphosphatase activity of myosin B in Duchenne muscular d&trophy. Neurology 2i, 920-924. Goldberg N. D.. O’Dea R. F. and Haddox M. K. (1975) ’ Cyclic-GMP. cyclic Nucleotide Res. 3, 155-223. \ Hoh J. F. Y. and Salafsky B. (1972) Intracellular electrolytes in dystrophic mouse muscle. Expl Neural. 37, 639-642. Horvath B., Berg L., Cummings D. J. and Shy G. M. (1955) Muscular dystrophy, cation concentrations in residual muscle. J. appl. Physiol. 8, 22-30. John H. A. (1976) Myofibrillar proteins of developing and dystrophic skeletal muscle. FEBS Left. 64, 116-121. Kar N. C. and Pearson C. M. (1967) A calcium-activated neutral protease in normal and dystrophic human muscle. Clin. chim. Acta 73 293-297.
Kuo T. and Bhan K. (1980) Studies of a myosin-cleaving protease from dystrophic hamster heart. Biochem. biophys. Res. Commun. 92, 570-576.
Lane P. W., Beamer T. C. and Myers D. D. (1976) Myodystrophy, a new myopathy on chromosome 8 of the mouse. J. Hered. 67, 135-138. Lowry 0. 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. Martell A. E. (1971) Stability Constants of Metallim Complexes, Part 11, Vol. 17, p. 651. Mayer M., Amin R., Milholland R. J. and Rosen F. (1976) Possible significance of myofibrillar protease in muscle catabolism. Exp. molec. Pathol. 25, 9-19. Mobley B. A., Reddy Y. S., Feeback D. L., Bodensteiner J. B., Bokhari M., Robinson B. S. and Clark R. (1985) Control of myofibrillar ATPase activity and force in myodystrophic mice. Muscle Nerve 8, 93-98. Murakami F. and Uchida K. (1979) Degradation of rat cardiac myofibrils and myofibriilar proteins by a myosin cleaving protease. J. Biochem. 86, 553-562. Neymark M. A., Kopacz S. J. and Lee C. P. (1980) Characterization of ATPase in sarcoplasmic reticulum from two strains of dystrophic mice. Muscle Nerue 3, 316-325. Nutting D. F., MacPike A. D. and Meier H. (1980) The calcium content of various tissues from myodystrophic and dystrophic mice. J. Hered. 71, 15-18.
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Penniston J. R. (1972) Endocytosis by erythrocyte ghosts; dependence upon ATP hydrolysis. Archs Biochem. Biophys. 153, 410-412. Penniston J. R. and Green D. E. (1968) The confirmational basis of energy transformation in membrane systems. Archs Biochem. Biophys. 128, 339-350. Reddy N. B. and Engel W. K. (1978) Increased guanylate cyclase (Gc) activity in denervated rat skeletal muscle. Cyclic Nucleotide Res. 9, 743. Reddy N. B., Oliver K. L., Festoff B. W. and Engel W. K. (1978a) Adenylate cyclase system of human skeletal muscle subcellular distribution and general properties. Biochem. biophys. Acta 540, 371-388. Reddy N. B., Oliver K. L., Festoff B. W. and Engel W. K. (1978b) Adenylate cyclase system of human skeletal muscle. Characterization of catecholamine stimulation and nucleotide regulation. Biochem. biophys. Acfa 540, 389-401. Robinson R. D., Reddy Y. S. and Mobley B. A. (1981) Myofibrillar ATPase and myosin ATPase activity in myodystrophic mice. Fedn. Proc. 40, 2218. Rodbell M., Lin M. C., Salamon Y., Londos C., Harwood J. P., Martin B., Rendel M. and Berman M. (1975) The role of adenine and guanine nucleotides in the activity and response of adenylate cyclase systems to hormones: evidence for multisite transition state. Adv. Cyclic Nucleotide Res. 5, 3-29. Salomon Y., Londos C. and Rodbell M. (1974) A highly
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sensitive adenylate cyclase assay. Analyt. Biochem. Ss, 541-548. Shiverick K. T., Thomas L. L. and Alpert N. R. (1975) Purification of cardiac myosin. Application to hypertrophied myocardium. Biochem. biophys. Acta 245, 124-133. Solar0 R. J., Pang D. C. and Briggs F. N. (1971) The purification of cardiac myofibrils with triton X-100. Biochem. biophys. Acta 245, 259-262. Spudich J. A. and Watt S. (1971) The regulation of rabbit skeletal muscle contraction. J. biof. Chem. 246, 4866-487 1. Stracher A., McGowen E. B., Siemenkowski L., Molak V. and Shafig S. A. (1979) Relationship between myosin structure and muscle degeneration. Ann. N. Y. Acad. Sci. 317, 349-35s. Sugita H., Ishiura S., Suzuki K. and Imahori K. (1980) Car+-activated neutral protease and its inhibitors: in vitro effect on intact myofibrils. Muscle Nerve 3, 335-339. Thompson W. J., Brooker G. and Appleman M. M. (1974) Assay of cyclic nucleotide phosphodiesterase with radioactive substrates. Meth. Enzym. 38, 205-212. Warnes D., Tomas F. M. and Ballard J. F. (1981) Increased rates of myofibrillar protein breakdown in muscle-wasting diseases. Muscle Nerve. 4, 62-66. Yasogawa N., Sanada Y. and Katunuma N. (1978) Susceptibilities of various myofibrillar proteins to muscle serine protease. J. Biochem. 83, 1355-1360.
br. J. Biocirem.Vol. 24, No. 4, pp. 579-584, 1992 Printed in Great Britain
MYOFIBRILLAR AND MEMBRANE-BOUND ENZYMES IN SKELETAL MUSCLE FROM MYODYSTROPHIC MICE Y.
S. REDDY,’
N. B.
REDDY,*
B.
A. MOBLEYI and R. C. BEESLEY’
‘Department of Physiology and Biophysics, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190, U.S.A. [ZW. (405)271-2226; Fnx (405)271-31811 and ‘Department of Neurology, Medical College of Pennsylvania, Philadelphia, Pa, U.S.A. (Received
7 July 1991)
Altstrac--1. Experiments were carried out to examine the biochemical changes, such as contractile protein biochemistry and membrane bound enzyme alterations associated with skeletal muscles of myd/myd. 2. Our studies demonstrate that there was a progressive decline in myofibrillar ATPase activity, and this decrease is greatest in 30 weeks old animals of myd/myd as compared to controls. 3. The proteolytic activity of myofibrils isolated from myd~myd was si~ifi~ntly higher than controls. 4. There was no significant difference in Ca 2+ ATPase activity of myosin and actin-activated myosin ATPase activity of myd/myd and their controls. 5. Mg* + ATPase and Na + + K + -ATPase of myodystrophic SL showed significant increase compared to controls. 6. Isoproterenol stimulated adenytate cyclase activity was significantly lower in the SL of dystrophic mice compared to controls. 7. GTP + isoproterenol stimulate adenylate cyclase was significantly higher in control SL and SR when compared to SL and SR isolated from myd/myd. 8. Guanylate cyclase activity was greater in myodystrophic mice both in the absence and presence of Triton X- 100. cGMP and CAMP phosphodiesterase activities were greater in dystrophic mice as compared to controls. 9. These observations suggest that there are significant changes in myofibrillar ATPase, myofibrillar protease and membrane bound enzymes of myd/myd compared to control,
INTRODUCTION Lane et ai. (1976) identifi~ mouse myopathy resulting from genetic mutation of chromosome (Goldberg ef al., 1975). The histopathological studies of myodystrophic mice (myd/myd) is similar to that of dysttophia muscularis (129/dy/dy), a disease resulting from genetic mutation of chromosome 4 (Neymark et al., 1980). In case of myd/myd all skeletal muscles were affected whereas in dy/dy only the hindlimbs are affected. Mobley et al. (1985) showed previously a decrease in myofib~ll~ ATPase activity but identical force generating capacity of myodystrophic skeletal muscle compared to control muscle. Neymark et al. ( 1980) found that sarcoplasmic reticular membranes isolated from mydlmyd exhibited an increase of basal ATPase activity, no change in Ca* +ATPase activities and a decrease in phosphoenzyme concentrations. Nutting et al. (1980) described elevated levels of Cal + in the diaphragm of myd/myd. We carried out the experiments on contractile protein biochemistry and membrane bound enzyme changes in myd/myd and their controls. Our main goal was to discover the possible defects in contractile protein ATPase activities and determine if membrane bound enzyme alterations may be responsible for the myodystrophy in all skeletal muscles of myd/myd. Part of this work was published previously in abstract form (Robinson et al., 1981).
METHODS AND MATERIALS ~yo~rii
preparation
Myodystrophic (myd/myd) mice and their sibling controls were purchased from Jackson Laboratories (Bay Harbor, Maine). Mice were sacrificed by cervical dislocation and the muscles of the hind legs were excised as quickly as possible and washed in cold 0.9% NaCl. Skeletal muscie mvo~b~ls were prepared according to the procedure of Solaio et af. (1971). Briefly, the procedure involves homogenizing the tissue in 0.3 M sucrose containing 10 mM imidazole @H 7.0) for 3-S set intervals using a Brinkman polytron (settinE 7). The homogenate was ~n~ifu~ and the resulting pelle? was washed 3 times with standard buffer &OmM KCl. 30 mM imidazole, 2 mM MgCl,, pH 7.0). The pellet was then washed once with standard buffer containing 2mM ethylene glycol 6is@-amino-ethyl ether)-N-N-tetra-acetic acid (EGTA), then twice with standard. buffer containing 1% Triton X-100. Finallv. the &let was washed 3 times with standard buffer. Aliwashe’s consisted of suspension/ homogenization of the pellet with a glass Dual1 homogenizer followed by centrifugation. The final pellet was suspended in a reaction medium consisting of 50 mM Tris-HCl, 5 mM NaN,, 2.5 mM MgCl,, pH 7.6. The myofibrillar protein concentrations were determined as described by Lowry er al. (1951). Comaerile
protein ATPase acriviry
Myofibrillar ATPase activity was measured in 1 ml of reaction medium containing 5OmM Tris-HCl (pH 7.6)
5 mM NaN,, 2.5 mM MgCl,, 2.5 mM ATP, 10mM 579
Y. S.
580
REDDY et al.
Ca-EGTA buffer and 2OOpcn myofibrillar protein. A &,,, = 1 x IO’ was used to &lculate the [Caj+] in the Ca-EGTA buffers (Martel. 1971). The reaction was started with the addition of ATP and stopped after 5 min at 3O’C with 1.0 ml of cold 10% TCA (tr~~hloroacetic acid). The denatured protein was removed by centrifugation and the amount of inorganic phosphate was measured in 1.0 ml of the supernatant using the method of Fiske and Subba Row (1925). Myosin was isolated by the procedure of Shiverick et al. (1975). Actin was purified from rabbit skeletal muscle acetone powder by the method of Spudich and Watt (1971). The protein content was determined by the procedure of Lowry et al. (1951) using bovine serum albumin as standard. Myosin Cal+ -ATPase activity was determined using 150mM Tris-maleate buffer (pH 6.5), 5 or IOmM CaCl,, 5 mM ATP, and the incubation was performed at 30°C for 5 min. The reaction was terminated by addition of 10% TCA and the inorganic phosphate present in the supernatant was determined (Hoh and Salafsky, 1972). Actinactivated myosin ATPase activity was determined using lOO/cg of myosin, 100~1 of reaction buffer containing 200 mM Tris-HCl, 50 mM MgCl,, 100 ~1 of 5 mM DDT, 50 ~1 of 50 mM ATP and different concentrations of actin in 1 ml of total reaction medium. The reaction was initiated by the addition of 50~1 of 50mM Na, ATP and stopped after 5 mitt of incubation at 37°C by the addition of I ml of 10% cold TCA. Inorganic phosphate (Pi) present in the supernatant was determined by the method of Fiske and Subba Row (1925). Caseinolylic activity of myqfibrils
The caseinolytic activity of myofibrils was measured using the method of Kar and Pearson (1967). The 1ml incubation mixtures containing 50 mM Tris-HCI (PH 8.0), 2 mg caseinyellow (CalBiochem) and l-5 mg myofibrillar protein were incubated at 37°C for 18 hr. The incubation was stopped by the addition of l.Oml of 10% HCIO,. The samples were centrifuged and 2.5 ml of 10% KOH was added to the supernatants. The samples were centrifuged again and the absorbance of the resulting supernatants was measured at 428 nm. A change in absorbance of 0,001 per mg protein after 18 hr at 37°C was defined as one unit of caseinolytic activity.
10
Membrane preparation
Sarcoplasmic reticulum (SR) and sarcolemma were isolated from normal and dystrophic mice as described previously (Reddy ef al., 1978a). SL membranes, without further puri~cation on sucrose density gradients were used in the present studies. Membrane-bound
enzyme assays
The membrane-bound ATPase activity was determined using [y-“P]ATPase as substrate and by measuring the liberated ‘*Pi by Cerenkov counting as previously described (Lane et al., 1976). The standard reaction medium in a final volume of 0.5 ml contained 30 mM imid~ol~glycylglycine, DH 7.5. 3 mM Tris-ATP (0.2 uCi IV-“*PlATP). 0.5 mM EGTA, 20-50 pg of membrane protein and indicated concentrations of different ions in individual experiments. Each reaction was allowed to proceed for 15 min at 37°C. In all ATPase assays the reaction was linear with time and protein, and not more than 15% of the added ATP was hydrolyzed under our assay conditions. Mg2’-ATPase activity was essentially similar in the presence or absence of ouabain. Na + + K + -ATPase activity was obtained by subtracting Mg* + -ATPase from that observed in the presence of 5 mM Mg*+, 100mM Na+, and 20 mM K + , The Ca2+ -ATPase was obtained by subtracting Mg2+-ATPase from the activity obtained in the presence of 5 m Mgr + and 0.1 mM Ca’+. EGTA was omitted from the tubes that contained Ca?f. Adenylate and guanylate cyclase activities were assayed as previously described (Reddy er al., 1978b). The reaction medium (0.1 ml) contained 50 mM Tri-HCl, pH 7.5,0.5 mM [y-32P]ATP (adenylate cyclase) or 0.1 mM [y-‘2P]GTP (guanylate cyclase), 5 mM MgCI,, 1OmM KCI, 1OmM theophylline, IO mM creatinine phosphate, 1 unit of creatinine phosphokinase, and SO-100 pg membrane protein. The reaction was ahowed to proceed for 10 min at 37°C and it was stopped by adding 0.1 ml of 2% SDS, 1 mM unlabeled ATP and 1 mM [“Hjcyclic AMP (- 10,000 cpm) in the case of adenylate cyclase, or with 0.1 ml of 5 M HCl and boiling in the case of guanylate cyclase. After cooling, 0.1 ml of 1 mM [‘H]cyclic GMP (C 10,OOOcpm)was added to the guanylate cyclase assay tubes. The newly formed [32P]cyclic AMP (adenylate cyclase) or [32P]cyclic GMP (guanylate cyclase) was separated by sequential elution on Dowex
AGE (weeks) (weeks) Fig. 1. Myofibrillar ATPase (A) and caseinolytic (B) activities of myofibrils from skeletal muscle of control and myodystrophic mice of different ages. One unit of caseinolytic activity is equal to a change in absorbance of 0.001 per mg protein after incubation for 18 hr at 37°C. Values are the mean + SD for four or five preparations. *P < 0.01 AGE
Contractile protein ATPases
581
and alumina columns as described by Salomon ef al. (1974) with minor modifications (Reddy et al., 1978b). Recovery for [‘HIcyclic AMP ranged between 70-85% and that for 13Hlcvchc GMP between 58-75%. Assay of cyclic AMP- and cyclic GMP-phosphodiesterase were performed essentially according to the two-step procedure of Thompson et al. (1974) using SO-1OOpg of membrane protein. The reaction was performed at 30°C for 20 min.
J
=
Control
I
2 Myodystrophlc
RESULTS
The myofibrillar ATPase activity (pCa 6 or greater) of both control and myodystrophic (myd/myd) mouse skeletal muscle increased to age 17 weeks compared to 10 week old animals [Fig. l(A)]. Control myofibrillar ATPase activity of 30 week old animals remained the same as that of 17 week old animals. The myofibrillar ATPase activity of 10 week old myd/ myd mice was significantly less than ATPase activity for their sibling controls (P < 0.01). The difference between the myofibrillar ATPase activity of control and myd/myd mice increased with age of the mice. Compared to their controls, 10 week old myd/myd mice have 10% less myofibrillar ATPase activity; 17 week old myd/myd mice have 14% less activity; and 30 week old myd/myd mice have 23% less activity. Results from studies on the caseinolytic activity of skeletal myofibrils from control and myd/myd mice are shown in Fig. l(B). While both control and myd/ myd myofibrils hydrolyze casein-yellow under the alkaline assay conditions, the myd/myd myofibrils had significantly higher caseinolytic activity than the controls (P < O.Ol).The hydrolysis of casein of these myofibrils was completely inhibited by the addition of 1 mM PMSF (phenylmethyl sulfonyl fluoride) to the incubation system. There was no statistical significant differences between the Ca2+ activated myosin ATPase activity of control and myd/myd mice. Similarly, there was no significant difference in actin-activated myosin ATPase activities of control and myd/myd mice (data now shown).
Mg2’
Na’+K-
ca2-
hlg”
CC?-
Fig. 2. ATPase activities of sarcolemma (SL) and sarcoplasmic reticulum (SR) from skeletal muscle of control and myodystrophic mice as measured in the presence of different cations. Values are the mean + SD for three preparations each assayed in duplicate. *P i 0.01.
B
F
I
GTP
B
F
I
GTP
Fig. 3. Adenylate cyclase activities of sarcolemma (SL) and sarcoplasmic reticulum (SR) from skeletal muscle of control and myodystrophic mice. Basal activity (B) and activity in the presence of 10 mM NaF (F), 10 PM isoproterenol (I) or 10pM isoproterenol plus 1OlM GTP (GTP) were determined. Values are the mean f SD for three preparations each assayed in triplicate. *P < 0.01.
Figure 2 shows ATPase activities in SL and SR of control and myodystrophic mice Mg*+-ATPase activity of myodystrophic SL showed marked increase over the control SL (P < 0.01). Na+ + K+ -ATPase also was significantly increased in myodystrophic SL compared to controls (P < 0.01). It should be noted that we did not detect any Na + + K+ -ATPase activity in SR, indicating that our SR fraction is not contaminated with SL membranes. While Ca2+-ATPase activity of both SR and SL of control mice were greater than those for myd/myd mice the differences were not statistically significant. Likewise there is no statistical significant difference in Mg*+-ATPase activities of SR isolate from control and myd/myd mice. Basal and NaF-stimulated adenylate cyclase activities were slightly less in the SL of dystrophic mice compared to control SL (Fig. 3). By contrast, those activities were slightly greater in SR of dystrophic mice. However, these differences were not statistically significant. Isoproterenol and isoproterenol + GTP stimulated adenylate cyclase activities of myd/myd SL were significantly less as compared to control animals (P < 0.01). GTP had no effect on the basal enzyme activity in the absence of isoproterenol (data not shown). There was no significant difference in isoproterenol stimulated adenylate cyclase activity of SR isolated from control animal compared to SR isolated from myd/myd. However, GTP + isoproterenol stimulated adenylate cyclase activity of control SR was significantly greater than that in SR of myd/myd (P < 0.01). We determined guanylate cyclase activity in SL, SR and cytosol fractions of control and myodystrophic mice (Fig. 4). There was no statistically significant difference in basal guanylate cyclase activities of SL between control and myd/myd. However, basal guanylate cyclase activity of SR and cytosol fractions from myd/myd were significantly greater than those for controls (P < 0.02). Guanylate cyclase activity was also significantly greater in all three fractions from myodystrophic mice in the presence of Triton X-100 (P < 0.01).
582
Y. S. REDDYet al, 0
=
CollCrol
I
= Myodystrophlc
Fig. 4. Guanylate cyclase activities of sarcolemma (SL), sarcoplasmic reticulum (SR) an cytosol (Cytosol) from skeletal muscle of control and myodystrophic mice. Basal activity (B) and activity in the presence of OS% Triton X-100 (T) was determined, Values are the mean f SD for three preparations each assayed in duplicate. *P -c 0.02, **p < 0.01.
Cyclic GMP dependent phosphodiesterase activities (PDE) were significantly higher in SL, SR and homogenate from myd/myd mice as compared to control (Fig. 5). Similarly cyclic AMP dependent PDE was higher in SL and homogenate of myd/myd mice compared of control mice. There was no statistically significant difference in cyclic AMP dependent PDE between control and myd/myd SR. DISCUSSION
Several studies have shown that the dystrophic mouse skeletal muscle exhibit many abnormalities. Neymark ef al. (1980) reported increased levels of basal ATPase activities, no change in Ca2+-ATPase and decreased levels of phosphoenzyme activity in myd/myd compared to control animals. We have shown previously that there was a significant difference in force generating capacity between control and myd/myd muscle, and a decrease in myofibrillar
Fig. 5. Cyclic nucleotide phosphodiesterase activities of sarcolemma (SL), sarcoplasmic reticulum (SR) and homogenate (Horn) of skeletal muscle from control and myodystrophic mice. Phosphodiesterase activity was determined using CAMP or cGMP as substrate. Values are the mean *SD for three preparations each assayed in duplicate. *P < 0.02, **p < O*OI*
ATPase activity which could be due to a greater percentage of type 1 fibers (slow fibers) associated with the myd/myd as compared to controls (Mobley et al., 1985). In the present investigation we have found an age dependent decrease in myofibrillar ATPase activity and the greatest percentage of decrease in myofibrillar ATPase activity was seen in 30 week old myd/myd as compared to controls. Furukawa and Peter (1971) showed that the skeletal muscle actomyosin isolated from patients affected with Duchenne muscular dystrophy exhibited tower actomyosin ATPase activity compared to controls. John (1976) reported a decrease in myofibrillar ATPase activity of mouse skeletal muscle affected by muscular dystrophy. Thus the decreased myofibrillar ATPase activity of myd/myd shown here supports the work of Furukawa and Peter (1971) and John (1976). Previously, we have explained that the decrease in myofibrillar ATPase activity may be due to an increase in density of slow (type 1) fibers in mydjmyd (Mobley et al., 1985). An alternative explanation for the decrease in ATPase activity of myd/myd may be due to an increase in myofibrillar protease activity. Indeed, we have shown in Fig. l(3) a significant increase in proteolytic activity associated with the myofibrils isolated from the mydjmyd mice. The characteristic clinical feature of muscular dystrophy is a progressive muscle atrophy and weakness, both processes presumably due to the continuous loss of both soluble and myofibrillar proteins. The decrease in muscle proteins is largely due to proteolytic action of enzyme@). One such enzyme extensively studied was the Ca2+ activated neutral proteases (CANP). Sodium dodecyl sulfate gel electrophoresis of chicken myofibrils treated with purified CANP exhibited loss of myofibrillar proteins such as myosin heavy chain, troponin-I and troponin-T. This pattern of loss of muscle proteins is similar to those observed in muscle of patients having Duchenne muscular dystrophy (Sugita et al., 1980). In the dystrophic chicken skeletal muscle Stracher et al. (1979) found that a decrease in LC3 component of myosin was due to a protease action on the LCJ component of myosin. Myofibrillar alkaline protease activity has been reported by several authors (Nutting et al., 1980; Pennington, 1972; Penniston and Green, 1968). These proteases degrade myofibrillar proteins (Sugita et al., 1980; Warnes et al., 1981), resulting in loss of Mg2+ATPase activity and Ca*+ sensitivity of myofibrils (Kuo and Bhan, 1980; Murakami and Uchida, 1979). Thus, a decrease in myofibrillar ATPase activity observed in myd/myd may be due to loss of myofibrillar protein components, as a result of action of protease on the myofibrillar proteins. The increase in protease activity reported here [Fig. l(B)] may be responsible for the decrease in myofibrillar ATPase in myd/myd mice. Mg2 + -ATPase activity of myodystrophic SL showed marked increase (76% over the normal SL). In red blood cells, correlation of endocytosis and vacuole formation with levels of Mg2 + -ATPase activity has been made (Nutting et al., 1980; Penniston and Green, 1968). It is possible that in the dystrophic state, increased Mg*+ -ATPase may be involved in enhanced permeability (endo- or pinocytosis) of
583
Contractile protein ATPases muscle SL to nutrients. Na+ + K+-ATPase also showed about a 50% increase in myodystrophic SL compared to controls. It is known that intracellular concentrations of Na + and K + of skeletal muscle are altered in pathologic states (Hoh and Salafsky, 1972; Horvath et al., 1955). Since Na+ + K+-ATPase is involved in maintaining normal levels of cellular Na+ and K+ (Albers, 1976), and enhanced Na+ + K+ATPase activity may reflect an adaptive response of the muscle to correct any imbalance in Na + and K + levels that occurs in the myodystrophic condition. It should be noted that we did not detect any Na+ + K+-ATPase activity in SR, indicating that our SR fraction is not contaminated with SL membranes. Ca2 +-ATPase activity showed no significant change in dystrophic muscle. SR of both normal and dystrophic muscle had greater levels of Ca2 + -ATPase activity than SL reflecting higher calcium uptake activity in SR compared to SL. GTP is known to enhance adenylate cyclase response to different hormones, including catecholamines in several tissues (Rodbell et al., 1975). GTP by itself had no effect on the basal enzyme activity in the absence of isoproterenol. However, the stimulation of adenylate cyclase by GTP plus isoproterenol was greater in SL, and SR from controls than in SL and SR from myd/myd. Since isoproterenol activates adenylate cyclase through an initial interaction with beta adrenergic receptor, the receptor+nzyme interaction may be altered in the myodystrophic muscle. Guanylate cyclase activity is known to be present both in membrane and cytosol fractions of different tissues (Goldberg et al., 1975) including skeletal muscle (Blosser and Appel, 1978; Reddy and Engel, 1978). Accordingly, we determined this enzymatic activity in SL, SR and cytosol fractions of control annd myodystrophic mice. Guanylate cyclase activity was significantly greater in all the three fractions of myodystrophic mice. The difference was even more striking in the presence of Triton X-100, which stimulates guanylate cyclase (Sugita et al., 1980). Since cyclases involved in the synthesis of CAMP and cGMP were different in the skeletal muscle of myodystrophic mice compared to controls, it was of interest to see whether the activity of phosphodiesterases, which are involved in the breakdown of those cyclic nucleotides, were also different in the dystrophic muscle. SL and ;SR from dystrophic mice showed significantly greater cyclic GMP-phosphodiesterase activity than controls while cyclic AMP-phosphodiesterase activity was significantly greater in SL of dystropic mice than controls. Phosphodiesterases are known to be present at a higher level in the cytoplasm than in the membranes of various tissues (Appleman er al., 1973). Therefore, we determined the phosphodiesterase activity in whole-muscle homogenates to obtain a measure of the cytoplasmic enzyme activity, and found that both the phosphodiesterases were markedly greater in dystrophic mice compared to normal mice. Lower levels of adenylate cyclase activity and greater levels of cyclic AMP-phosphodiesterase activity means that the cyclic AMP content will be lower in myodystrophic mice skeletal muscle than in controls. Because cyclic AMP plays an important role in several cellular processes, a change in its content in
muscle may lead to other metabolic changes in that tissue. Higher levels of guanylate cyclase as well as cyclic GMP-phosphodiesterase indicate a greater turnover of cyclic GMP in the myodystrophic mice compared to controls. The functions of cyclic GMP in skeletal muscle are not known at present. Acknowledgement-We
thank Mrs Lula Rhoton for her expert secretarial assistance. REFERENCES
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Baker N. H., Bland H. W. and Hart P. (1958) Concentrations of K and Na in skeletal muscle of mice with a hereditary myopathy. Am. J. Physiol. 193, 530-533. Blosser J. C. and Appel S. H. (1978) Properties and distribution of mammalian skeletal muscle guanylate cyclase. J. biol. Chem. 253, 3088-3093. Edmunds T. and Pennington R. J. T. (1981) Mast cell origin of “Myofibrillar Protease” of rat skeletal and heart muscle. Biochem. biophys. Acta 661, 28-31. Fiske C. H. and Subba Row Y. (1925) The calorimetric determination of phosphorus. J. biol. Chem. 66,375-380. Furukawa T. and Peter J. B. (1971) Superprecipitation and adenosinetriphosphatase activity of myosin B in Duchenne muscular d&trophy. Neurology 2i, 920-924. Goldberg N. D.. O’Dea R. F. and Haddox M. K. (1975) ’ Cyclic-GMP. cyclic Nucleotide Res. 3, 155-223. \ Hoh J. F. Y. and Salafsky B. (1972) Intracellular electrolytes in dystrophic mouse muscle. Expl Neural. 37, 639-642. Horvath B., Berg L., Cummings D. J. and Shy G. M. (1955) Muscular dystrophy, cation concentrations in residual muscle. J. appl. Physiol. 8, 22-30. John H. A. (1976) Myofibrillar proteins of developing and dystrophic skeletal muscle. FEBS Left. 64, 116-121. Kar N. C. and Pearson C. M. (1967) A calcium-activated neutral protease in normal and dystrophic human muscle. Clin. chim. Acta 73 293-297.
Kuo T. and Bhan K. (1980) Studies of a myosin-cleaving protease from dystrophic hamster heart. Biochem. biophys. Res. Commun. 92, 570-576.
Lane P. W., Beamer T. C. and Myers D. D. (1976) Myodystrophy, a new myopathy on chromosome 8 of the mouse. J. Hered. 67, 135-138. Lowry 0. 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. Martell A. E. (1971) Stability Constants of Metallim Complexes, Part 11, Vol. 17, p. 651. Mayer M., Amin R., Milholland R. J. and Rosen F. (1976) Possible significance of myofibrillar protease in muscle catabolism. Exp. molec. Pathol. 25, 9-19. Mobley B. A., Reddy Y. S., Feeback D. L., Bodensteiner J. B., Bokhari M., Robinson B. S. and Clark R. (1985) Control of myofibrillar ATPase activity and force in myodystrophic mice. Muscle Nerve 8, 93-98. Murakami F. and Uchida K. (1979) Degradation of rat cardiac myofibrils and myofibriilar proteins by a myosin cleaving protease. J. Biochem. 86, 553-562. Neymark M. A., Kopacz S. J. and Lee C. P. (1980) Characterization of ATPase in sarcoplasmic reticulum from two strains of dystrophic mice. Muscle Nerue 3, 316-325. Nutting D. F., MacPike A. D. and Meier H. (1980) The calcium content of various tissues from myodystrophic and dystrophic mice. J. Hered. 71, 15-18.
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Penniston J. R. (1972) Endocytosis by erythrocyte ghosts; dependence upon ATP hydrolysis. Archs Biochem. Biophys. 153, 410-412. Penniston J. R. and Green D. E. (1968) The confirmational basis of energy transformation in membrane systems. Archs Biochem. Biophys. 128, 339-350. Reddy N. B. and Engel W. K. (1978) Increased guanylate cyclase (Gc) activity in denervated rat skeletal muscle. Cyclic Nucleotide Res. 9, 743. Reddy N. B., Oliver K. L., Festoff B. W. and Engel W. K. (1978a) Adenylate cyclase system of human skeletal muscle subcellular distribution and general properties. Biochem. biophys. Acta 540, 371-388. Reddy N. B., Oliver K. L., Festoff B. W. and Engel W. K. (1978b) Adenylate cyclase system of human skeletal muscle. Characterization of catecholamine stimulation and nucleotide regulation. Biochem. biophys. Acfa 540, 389-401. Robinson R. D., Reddy Y. S. and Mobley B. A. (1981) Myofibrillar ATPase and myosin ATPase activity in myodystrophic mice. Fedn. Proc. 40, 2218. Rodbell M., Lin M. C., Salamon Y., Londos C., Harwood J. P., Martin B., Rendel M. and Berman M. (1975) The role of adenine and guanine nucleotides in the activity and response of adenylate cyclase systems to hormones: evidence for multisite transition state. Adv. Cyclic Nucleotide Res. 5, 3-29. Salomon Y., Londos C. and Rodbell M. (1974) A highly
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sensitive adenylate cyclase assay. Analyt. Biochem. Ss, 541-548. Shiverick K. T., Thomas L. L. and Alpert N. R. (1975) Purification of cardiac myosin. Application to hypertrophied myocardium. Biochem. biophys. Acta 245, 124-133. Solar0 R. J., Pang D. C. and Briggs F. N. (1971) The purification of cardiac myofibrils with triton X-100. Biochem. biophys. Acta 245, 259-262. Spudich J. A. and Watt S. (1971) The regulation of rabbit skeletal muscle contraction. J. biof. Chem. 246, 4866-487 1. Stracher A., McGowen E. B., Siemenkowski L., Molak V. and Shafig S. A. (1979) Relationship between myosin structure and muscle degeneration. Ann. N. Y. Acad. Sci. 317, 349-35s. Sugita H., Ishiura S., Suzuki K. and Imahori K. (1980) Car+-activated neutral protease and its inhibitors: in vitro effect on intact myofibrils. Muscle Nerve 3, 335-339. Thompson W. J., Brooker G. and Appleman M. M. (1974) Assay of cyclic nucleotide phosphodiesterase with radioactive substrates. Meth. Enzym. 38, 205-212. Warnes D., Tomas F. M. and Ballard J. F. (1981) Increased rates of myofibrillar protein breakdown in muscle-wasting diseases. Muscle Nerve. 4, 62-66. Yasogawa N., Sanada Y. and Katunuma N. (1978) Susceptibilities of various myofibrillar proteins to muscle serine protease. J. Biochem. 83, 1355-1360.
