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Studies on myofibrillar protein synthesis in chicken muscular dystrophy
Life Sciences Vol . 20, pp . 1091-1096, 1977 . Printed in the U .S .A .
Pergamou Press
STUDIES ON MYOFIBRILLAR PROTEIN SYNTHESIS IN CHICKEN MUSCULAR DYSTROPHY Jeremiah J . Morrissey* and S . S . Rerwar# Roche Institute of Molecular Biology Nutley, N . J . 07110 (Received in final form February 15, 1977) Myofibrillar proteins synthesised in vitro by normal and dystrophic chicken muscle polysomes werepurified and analysed by SDS gel electrophoresis . No substantial difference in the synthesis of myofibrillar proteins could be detected . These observations suggest that the loss of muscle mss that is observed in muscular dystrophy is not related to a translational defect in the dystrophic polysones . Muscular Dystrophies are a group of hereditary diseases characterised by the progressive wasting of skeletal muscle (1) . This process results in a large decrease in the concentratioA of myofibrillar proteins and, in certain con ditions, a replacement of muscle mass by connective tissue or fat (2) . Although several hypotheses have been proposed to explain the redaction of muscle mss (3,4), it appears that the cellular mechanisms that control the synthesis or degradation of myofibrillar proteins are deranged . Thus, it is not clear whether the reduction in the amount of myofibrillar proteins in the affected muscle is related to a decrease in their synthesis and/or increase in their breakdown . Human dystrophic muscle is not readily available, therefore two models have been used for studies on muscular dystrophy : the Jackson Laboratory muse (5) and the dystrophic chicken (6) . Genetic analysis has been conducted (7,S) suggesting that studies with these models are relevant to some of the human dystrophies . Previous investigations (9,11) have compared total protein synthesis in dystrophic chicken muscle with that of normal chickens and found that protein synthesis was higher in the dystrophic muscle . However, the synthesis of myofibrillar proteins was not determined . Therefore, these studies did not rule out that the reduced content of nyofibrillar proteins observed in dystrophy was due to a specific defect in their synthesis . The present report has examined the synthesis in vitro , of myofibrillar proteins by polysomes prepared from normal and dystrophic chick muscle . METHODS [ 3H] or [14C] labeled leucine and proline were obtained from New England Nuclear Corporation, Boston, Massachusetts . Rabbit liver tRNA was a product. of General Biochemicals, Chagrin Falls, Ohio . Fertilised eggs of dystrophic white leghorn chickens were obtained from Dr . L . Pierro, Department of Animal Present Address : * National Heart, Lung and Blood lust . NIH, Bethesda, Md .20014 # Metabolic Disease Therapy Research Section, Lederle Laboratories, A Division of American Cyanamid Company, Pearl River, New York 10965 . All correspondence to this address . 1091
1092
Myofibrillar Protein Synthesis
Vol . 20, No . 6, 1977
Genetics, University of Connecticut, Storrs, Connecticut . These eggs were incubated at 37 0 for hatching in a standard egg incubator. Normal chick embryos or chickens were obtained from a local supplier (Spring Lake Farms, Wyckoff, New Jersey) . Polyribosomes from 15 day embryonic breast muscle, dissected free of bone, were prepared as described previously (12) while polysomee from the breast muscle of hatched chickens was prepared as above except that the homogenization of the tissue was performed with a polytron homogenizer (Bria1®ann Instruments) . A crude preparation of initiation factors was prepared from rab bit reticulocyte ribosomee as described by Miller and Schweet (13) . A preincubated S-30 fraction was prepared from Ehrlich Ascitea cells by the method of Aviv _et al . (14) . Carrier myofibrillar proteins were prepared from normal chicken breast muscle as described by Wilman-Coffelt et al . (15) . The initial precipitate of myofibrils was dissolved in a minimal ;ohe of 2 M KC1 to which an equal volume of glycerol was added. Assays for total protein synthesis in an Ascites S-30 were as follows : polysomal pellets were rinsed and resuspended in homogenizing buffer devoid of heparin and sucrose (9) . Assay for total protein synthesis was conducted in a total volume of 0.075 ml and contained: 20 nM Tris-C1, pH 7 .5 ; 120 mM RC1 ; 5 MM MgC12 ; 7 mM DTT ; 1 mM ATP; 0.2 nM GTP ; 0 .6 mM CTP; 6 mM PEP; 0 .4 u of rabbit liver tRNA ; 0.24 mM of each of the 19 amino acids; 15 n moles of [§H]-leucine (380 cpm/p mole) or 15 n moles of [14C]-leucine (410 cpm/p mole) ; 50 jig of protein in the crude reticulocyte initiation factors and a saturating amount of the preincubated ascites S-30 preparation . Polysomes from normals were incubated with [3H]-leucine and polysomes from dystrophic incubated with [14C] leucine. The individual reaction mixtures were incubated for 10 minutes at 370 and the amount of leucine incorporated into hot trichloroacetic acid precipitable protein was determined . Incorporation of labeled leucine into hot trichloroacetic acid precipitable protein by both polysome preparations was linear for at least 15 minutes . Assays for myofibrillar protein synthesis was carried out in a 5 fold scaled up mixture. After a 30 min. incubation at 37 0 at which time the incorporation of leucine into total protein was essentially complete, an aliquot was removed for the determination of total leucine incorporation into hot trichloroacetic acid precipitable radioactivity. RC1 was added to both mixtures to 0 .5 M to solubilize the myofibrillar proteins synthesized in vitro . The normal and dystrophic polysomal incubation mixtures from the same developmental stage were combined and 2 mg of carrier myofibrillar proteins were added . The mixture was centrifuged at 150,000 x g for 30 minutes to re move ribosomes and polysomes. The supernatant was adjusted to pH 11 .0 with FOH, mixed for 10 minutes and readjusted to pH 7 .5 . This step facilitated dissociation and reassociation of myosin light chains (16) . The myofibrillar proteins were purified from other proteins by two cycles of coprecipitation (16) . The myosin and actin of dystrophic chicken breast muscle has been shown to be identical to normal myosin and actin (17,20) . Therefore, the synthesized myofibrillar proteins from normal and dystrophic muscle should copurify with the added carrier protein to the same extent . The myofibrillar protein precipitate was dissolved in 22 SDS-0 .35 M mercaptoethanol and were separated .into the various myofibrillar proteins by SDS acrylamide gel electrophoresis as described by Weber and Osborne (21) . The gels were sectioned into 2 mm slices with the aid of a gel crusher (Gilman Instruments) . After addition of 0 .2 ml of O.1S SDS and 10 ml of Instabrays (Yorktown Research) the slices were -lyzed for [14C] and [ 3H] . RESULTS AND DISCUSSION The phenotypic expression common to muscular dystrophy in humans, chickens and mice is the reduced content of myofibrillar proteins . Earlier investigation with chicken muscle minces suggested that the dystrophic muscle was more active than normal in the ability to incorporate labeled amino acids into pro-
Vol . 20, No . 6, 1977
Myofibrillar Protein.Synthesis
109 3
tains (9,10) . Using a purified system, Batelle and Florini (11) reported that polysoms prepared frbm breast muscle of newly hatched dystrophic chicksas were less active in protein synthesis than those from normal muscle, while polysomes from older dystrophic chickens were more active than the corresponding controls when using the control supernatant enzymes . However, the activity of the polysomal preparations in the synthesis of myofibrillar proteins was not determined in these previous studies . The present investigation has specifically examined the activity of normal and dystrophic chicken polysomes in myofibrillar protein synthesis using an ascites S-30 . This system should give a fairly nondiscriminatory picture of the types and amounts of proteins preprogrammed onto the ribosome in vivo . Muscle supernatant factors that may differentially regulate the syntheiis of individual nyofibrillar proteins in vivo, would not be assayed . 7,p general dystrophic polyribosomes were found to be slightly more active than normal polyribosoms (Table 1) in the rates of labeled leucine incorporated into total protein and there was a decrease in activity with age . TABLE 1 Specific Activity of Normal and Dystrophic Polyribosomss in the Incorporation of Labeled Leucine into Total Protein p moles Leucine Incorporated per A260 Polyribosoms/10 sin .
NORMAL
DYSTROPHIC
15 Day Embryo Breast Muscle
35 .5 t 2 .0a
51 .0 t 3 .0
8 Day Hatched Breast Muscle
42 .5 t 3 .0
49 .0 t 3 .0
52 Day Hatched Breast Muscla
17 .5 t 1 .0
25 .0 t 2 .0
a
Represents the average of 3 determinations on separate polyribosome preparations t standard error of the peen . Incubations were for 10 min . during which period the incorporation of leucine was linear .
This is consistent with the results of other investigators using muscle minces (9,10) . The purified myofibrillar protein was separated by SDS-acrylamide electrophoresis to identify the heavy and light chains of myosin, actin, troponin and tropomyosin . The results are presented in Figure 1 for the 15 day embryo, 8 and 52 day hatched breast muscle . At all developmental sta"s exmined, there was no substantial difference in nyofibrillar protein synthesis between the normal and dystrophic polyribosomss . The slight increase in total protein synthesis with the dystrophic polyribosoms resulted in a uniform increase in the synthesis of all the myofibrillar proteins and a differential effect on one of the protein components was not observed . In general, there was a decrease in myosin and actin synthesis with age and an apparent maintenance of troponin, troposyosin and myosin light chains synthesis . The concentrations of polyribosoms did not vary significantly between the normal and dystrophic breast muscle through the course of the study (Morrissey and Hsrwar, unpublished observations) . This is consistent with the observations of Oppsnheimr and Markiswuica (22) on the total polyribosomes concentrations in normal and dystrophic chickens up to 2 maths of age . Thus, a low polyribosom concentration would not seam to be a likely cause for the decreased
1094
Vol. 20, No . 6, 1977
Hyofibrillar Protein Synthesis
myofibrillar content in dystrophic chicken breast muscle . Therefore, the decrease in the amount of myofibrillar proteins in the dystrophic muscle cannot be attributed to an inherent translational defect in the diseased muscle, at least through the 52 days of this study . Watts and Reid (23) and F.itchin and Wate (24) have found no real difference in total protein synthesis between the normal and dystrophic souse in vitro and in vivo . Some differences in the turnover of individual water soluFIG. 1
z
z
ó 400
a o a z o
i é'
z
ó
mi
11~ 1 11 400
z
-
1111
si 2
z
400
ó
ié
111 d 11
W
u
N N, 200 d V 100
SLICE NUMBER
Figure 1. SDS-gel electrophoresis of the purified myofibrillar proteins syn thesized in vitro using an ascites S-30 fraction . E 15-15 day embryonic breast muscle ; H 8-8 day hatched breast muscle ; H 52-52 day hatched breast muscle . Proteins synthesised by normal muscle ("-4 " -0) and dystrophic muscle (0--0--0) . LC1, LC2 and LC3 are myosin light chains ; TNT is a troponin subunit . ble muscle proteins were observed in the dystrophic souse . Batelle and Florini have made a similar observation concerning the synthesis of soluble muscle proteins in the dystrophic chicken (11) . Our study with polysomes has demonstrated little apparent change in the synthesis of relatively insoluble myofibrillar proteins in the chicken prior to and after the physical onset of dystrophy . The decrease in myofibrillar proteins seen in dystrophic chicken muscle say probably be the result of the much greater than normal proteolytic
Vol . 20, No . 6, 1977
Myofibrillar Protein Synthesis
109 5
activity observed in dystrophic muscle (25,26) . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 .
F .H . TYLER, in "The Metabolic Basis of Inherited Diseases ", (p .939) edited by J .B . Stanbury, J .B . Wyngaarden and D .S . Fredrickson, McGrawHill, (1973) . J .C . DREYFUS, G . SCHAPIRA and F . SCHAPIRS, J . Clin . Invest . 33, 794 (1954) . V . DUBOWITZ, J . Neurol . Neurosburg . Psychiat . 30, 99 1967) . V .A . McKUSICK, J . Chronic, Die . 3, 360 (1956) . A .M . MICHELSON, B .S . RUSSEL and P .T . HERMAN, Proc . Nat . Aced . Sci . 41, 1074 (1955) . V .S . ASMUNDSON and L .M . JULIAN, J . Heredity 47, 248 (1956) T .W . West and W .G . HOAG, Ann . N .4 . Aca d-Sci . _138, 4 (1966) . V .A . ASHUNDSON, F .H . KRATZER and L .M . JULIAN, Ann . N . Y . Aced . Sci . 138, 49 (1966) . .F D . MORGAN and H . HERRMANN, Proc . Soc . Ezptl . Biol . Mad . 120, 68 (1965) . I .M . WEINSTOCK, T .S . JOH, H .A .FREEDMAN and M .E . CUTLER, Biochemical Mad . 2, 345 (1969) . B.A . BATTELLE and J .R . FLORINI, Biochem . 12, 635 (1973) . T .M . BURNS, C .L . SPEARS and S .S . KERWAR, Arch . Biochem . Biophys . 159, R . MILLER and R .L . SCHWEET, Arch . Biochem . Biophys . 125, 632 (1968). H . AVIV, I . BODE and P . LEDER, Proc . Nat . Aced . Sci . 68, 2303 (1971) . J . WIRMAN-COFFELT, R . ZELIS, C . FENNER and D .T . MASON, Biochem. Biophys . Ras . Commun . 51, 1097 (1927) . S . SARKAR aad P .H. COOKE, Biochem. Biophys . Res . -Commun . 41, 918 (1970) . W .E . BRENNOCK and W .O . READ, Res . Commun . Chem. Pathol . Pharmacol . 3, 417 (1972) . J . S . MOREY, K . TARCZY-HARNACH and E .G . RICHARDS, Arch . Biochem . Biophys . 119, 491 (1967) . K .S . MOREY, & . TARCZY-HORNACH and W .D . BROWN, Arch . Biochem . Biophys . 124, 521 (1968) . H . OPPENHEIMER, S . HASSELRI and A .T . MILHORAT, in "Muscle Diseases ", (p .266) edited by J .N . Walton, N . Canal, G . Scarlato and M . Gleave . Excerpts Medica, Amsterdam (1970) . K . WEVER and M . OSBORNE, J . Biol . Chem . 244, 4406 (1969) . H . OPPENHEIMER and L . MARKIEWICZ, Biochemical Mad . 7, 479 (1973) . D .C . WATTS and J .D . REID, Biochem. J . 115, 37T--(196--9) . S .E . KITCHIN and D .C . WATTS, Biochem. J .136, 1017 (1973) . R .J . PENNINGTON, in "Muscle Dieesees", (p.252) edited by J .N . Walton, N . Canal, G . Scarlato an ave . Excerpts Medica, Amsterdam (1970) . A .A . IODICE, S . PERKER and I .M. WEINSTOCK, in "Múec1e Diseases", (p .313) edited by J .N . Walton, N . Canal, G . Scarlato M . G Bave . Excerpts, Medica, Amsterdam (1970) .
Pergamou Press
STUDIES ON MYOFIBRILLAR PROTEIN SYNTHESIS IN CHICKEN MUSCULAR DYSTROPHY Jeremiah J . Morrissey* and S . S . Rerwar# Roche Institute of Molecular Biology Nutley, N . J . 07110 (Received in final form February 15, 1977) Myofibrillar proteins synthesised in vitro by normal and dystrophic chicken muscle polysomes werepurified and analysed by SDS gel electrophoresis . No substantial difference in the synthesis of myofibrillar proteins could be detected . These observations suggest that the loss of muscle mss that is observed in muscular dystrophy is not related to a translational defect in the dystrophic polysones . Muscular Dystrophies are a group of hereditary diseases characterised by the progressive wasting of skeletal muscle (1) . This process results in a large decrease in the concentratioA of myofibrillar proteins and, in certain con ditions, a replacement of muscle mass by connective tissue or fat (2) . Although several hypotheses have been proposed to explain the redaction of muscle mss (3,4), it appears that the cellular mechanisms that control the synthesis or degradation of myofibrillar proteins are deranged . Thus, it is not clear whether the reduction in the amount of myofibrillar proteins in the affected muscle is related to a decrease in their synthesis and/or increase in their breakdown . Human dystrophic muscle is not readily available, therefore two models have been used for studies on muscular dystrophy : the Jackson Laboratory muse (5) and the dystrophic chicken (6) . Genetic analysis has been conducted (7,S) suggesting that studies with these models are relevant to some of the human dystrophies . Previous investigations (9,11) have compared total protein synthesis in dystrophic chicken muscle with that of normal chickens and found that protein synthesis was higher in the dystrophic muscle . However, the synthesis of myofibrillar proteins was not determined . Therefore, these studies did not rule out that the reduced content of nyofibrillar proteins observed in dystrophy was due to a specific defect in their synthesis . The present report has examined the synthesis in vitro , of myofibrillar proteins by polysomes prepared from normal and dystrophic chick muscle . METHODS [ 3H] or [14C] labeled leucine and proline were obtained from New England Nuclear Corporation, Boston, Massachusetts . Rabbit liver tRNA was a product. of General Biochemicals, Chagrin Falls, Ohio . Fertilised eggs of dystrophic white leghorn chickens were obtained from Dr . L . Pierro, Department of Animal Present Address : * National Heart, Lung and Blood lust . NIH, Bethesda, Md .20014 # Metabolic Disease Therapy Research Section, Lederle Laboratories, A Division of American Cyanamid Company, Pearl River, New York 10965 . All correspondence to this address . 1091
1092
Myofibrillar Protein Synthesis
Vol . 20, No . 6, 1977
Genetics, University of Connecticut, Storrs, Connecticut . These eggs were incubated at 37 0 for hatching in a standard egg incubator. Normal chick embryos or chickens were obtained from a local supplier (Spring Lake Farms, Wyckoff, New Jersey) . Polyribosomes from 15 day embryonic breast muscle, dissected free of bone, were prepared as described previously (12) while polysomee from the breast muscle of hatched chickens was prepared as above except that the homogenization of the tissue was performed with a polytron homogenizer (Bria1®ann Instruments) . A crude preparation of initiation factors was prepared from rab bit reticulocyte ribosomee as described by Miller and Schweet (13) . A preincubated S-30 fraction was prepared from Ehrlich Ascitea cells by the method of Aviv _et al . (14) . Carrier myofibrillar proteins were prepared from normal chicken breast muscle as described by Wilman-Coffelt et al . (15) . The initial precipitate of myofibrils was dissolved in a minimal ;ohe of 2 M KC1 to which an equal volume of glycerol was added. Assays for total protein synthesis in an Ascites S-30 were as follows : polysomal pellets were rinsed and resuspended in homogenizing buffer devoid of heparin and sucrose (9) . Assay for total protein synthesis was conducted in a total volume of 0.075 ml and contained: 20 nM Tris-C1, pH 7 .5 ; 120 mM RC1 ; 5 MM MgC12 ; 7 mM DTT ; 1 mM ATP; 0.2 nM GTP ; 0 .6 mM CTP; 6 mM PEP; 0 .4 u of rabbit liver tRNA ; 0.24 mM of each of the 19 amino acids; 15 n moles of [§H]-leucine (380 cpm/p mole) or 15 n moles of [14C]-leucine (410 cpm/p mole) ; 50 jig of protein in the crude reticulocyte initiation factors and a saturating amount of the preincubated ascites S-30 preparation . Polysomes from normals were incubated with [3H]-leucine and polysomes from dystrophic incubated with [14C] leucine. The individual reaction mixtures were incubated for 10 minutes at 370 and the amount of leucine incorporated into hot trichloroacetic acid precipitable protein was determined . Incorporation of labeled leucine into hot trichloroacetic acid precipitable protein by both polysome preparations was linear for at least 15 minutes . Assays for myofibrillar protein synthesis was carried out in a 5 fold scaled up mixture. After a 30 min. incubation at 37 0 at which time the incorporation of leucine into total protein was essentially complete, an aliquot was removed for the determination of total leucine incorporation into hot trichloroacetic acid precipitable radioactivity. RC1 was added to both mixtures to 0 .5 M to solubilize the myofibrillar proteins synthesized in vitro . The normal and dystrophic polysomal incubation mixtures from the same developmental stage were combined and 2 mg of carrier myofibrillar proteins were added . The mixture was centrifuged at 150,000 x g for 30 minutes to re move ribosomes and polysomes. The supernatant was adjusted to pH 11 .0 with FOH, mixed for 10 minutes and readjusted to pH 7 .5 . This step facilitated dissociation and reassociation of myosin light chains (16) . The myofibrillar proteins were purified from other proteins by two cycles of coprecipitation (16) . The myosin and actin of dystrophic chicken breast muscle has been shown to be identical to normal myosin and actin (17,20) . Therefore, the synthesized myofibrillar proteins from normal and dystrophic muscle should copurify with the added carrier protein to the same extent . The myofibrillar protein precipitate was dissolved in 22 SDS-0 .35 M mercaptoethanol and were separated .into the various myofibrillar proteins by SDS acrylamide gel electrophoresis as described by Weber and Osborne (21) . The gels were sectioned into 2 mm slices with the aid of a gel crusher (Gilman Instruments) . After addition of 0 .2 ml of O.1S SDS and 10 ml of Instabrays (Yorktown Research) the slices were -lyzed for [14C] and [ 3H] . RESULTS AND DISCUSSION The phenotypic expression common to muscular dystrophy in humans, chickens and mice is the reduced content of myofibrillar proteins . Earlier investigation with chicken muscle minces suggested that the dystrophic muscle was more active than normal in the ability to incorporate labeled amino acids into pro-
Vol . 20, No . 6, 1977
Myofibrillar Protein.Synthesis
109 3
tains (9,10) . Using a purified system, Batelle and Florini (11) reported that polysoms prepared frbm breast muscle of newly hatched dystrophic chicksas were less active in protein synthesis than those from normal muscle, while polysomes from older dystrophic chickens were more active than the corresponding controls when using the control supernatant enzymes . However, the activity of the polysomal preparations in the synthesis of myofibrillar proteins was not determined in these previous studies . The present investigation has specifically examined the activity of normal and dystrophic chicken polysomes in myofibrillar protein synthesis using an ascites S-30 . This system should give a fairly nondiscriminatory picture of the types and amounts of proteins preprogrammed onto the ribosome in vivo . Muscle supernatant factors that may differentially regulate the syntheiis of individual nyofibrillar proteins in vivo, would not be assayed . 7,p general dystrophic polyribosomes were found to be slightly more active than normal polyribosoms (Table 1) in the rates of labeled leucine incorporated into total protein and there was a decrease in activity with age . TABLE 1 Specific Activity of Normal and Dystrophic Polyribosomss in the Incorporation of Labeled Leucine into Total Protein p moles Leucine Incorporated per A260 Polyribosoms/10 sin .
NORMAL
DYSTROPHIC
15 Day Embryo Breast Muscle
35 .5 t 2 .0a
51 .0 t 3 .0
8 Day Hatched Breast Muscle
42 .5 t 3 .0
49 .0 t 3 .0
52 Day Hatched Breast Muscla
17 .5 t 1 .0
25 .0 t 2 .0
a
Represents the average of 3 determinations on separate polyribosome preparations t standard error of the peen . Incubations were for 10 min . during which period the incorporation of leucine was linear .
This is consistent with the results of other investigators using muscle minces (9,10) . The purified myofibrillar protein was separated by SDS-acrylamide electrophoresis to identify the heavy and light chains of myosin, actin, troponin and tropomyosin . The results are presented in Figure 1 for the 15 day embryo, 8 and 52 day hatched breast muscle . At all developmental sta"s exmined, there was no substantial difference in nyofibrillar protein synthesis between the normal and dystrophic polyribosomss . The slight increase in total protein synthesis with the dystrophic polyribosoms resulted in a uniform increase in the synthesis of all the myofibrillar proteins and a differential effect on one of the protein components was not observed . In general, there was a decrease in myosin and actin synthesis with age and an apparent maintenance of troponin, troposyosin and myosin light chains synthesis . The concentrations of polyribosoms did not vary significantly between the normal and dystrophic breast muscle through the course of the study (Morrissey and Hsrwar, unpublished observations) . This is consistent with the observations of Oppsnheimr and Markiswuica (22) on the total polyribosomes concentrations in normal and dystrophic chickens up to 2 maths of age . Thus, a low polyribosom concentration would not seam to be a likely cause for the decreased
1094
Vol. 20, No . 6, 1977
Hyofibrillar Protein Synthesis
myofibrillar content in dystrophic chicken breast muscle . Therefore, the decrease in the amount of myofibrillar proteins in the dystrophic muscle cannot be attributed to an inherent translational defect in the diseased muscle, at least through the 52 days of this study . Watts and Reid (23) and F.itchin and Wate (24) have found no real difference in total protein synthesis between the normal and dystrophic souse in vitro and in vivo . Some differences in the turnover of individual water soluFIG. 1
z
z
ó 400
a o a z o
i é'
z
ó
mi
11~ 1 11 400
z
-
1111
si 2
z
400
ó
ié
111 d 11
W
u
N N, 200 d V 100
SLICE NUMBER
Figure 1. SDS-gel electrophoresis of the purified myofibrillar proteins syn thesized in vitro using an ascites S-30 fraction . E 15-15 day embryonic breast muscle ; H 8-8 day hatched breast muscle ; H 52-52 day hatched breast muscle . Proteins synthesised by normal muscle ("-4 " -0) and dystrophic muscle (0--0--0) . LC1, LC2 and LC3 are myosin light chains ; TNT is a troponin subunit . ble muscle proteins were observed in the dystrophic souse . Batelle and Florini have made a similar observation concerning the synthesis of soluble muscle proteins in the dystrophic chicken (11) . Our study with polysomes has demonstrated little apparent change in the synthesis of relatively insoluble myofibrillar proteins in the chicken prior to and after the physical onset of dystrophy . The decrease in myofibrillar proteins seen in dystrophic chicken muscle say probably be the result of the much greater than normal proteolytic
Vol . 20, No . 6, 1977
Myofibrillar Protein Synthesis
109 5
activity observed in dystrophic muscle (25,26) . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 .
F .H . TYLER, in "The Metabolic Basis of Inherited Diseases ", (p .939) edited by J .B . Stanbury, J .B . Wyngaarden and D .S . Fredrickson, McGrawHill, (1973) . J .C . DREYFUS, G . SCHAPIRA and F . SCHAPIRS, J . Clin . Invest . 33, 794 (1954) . V . DUBOWITZ, J . Neurol . Neurosburg . Psychiat . 30, 99 1967) . V .A . McKUSICK, J . Chronic, Die . 3, 360 (1956) . A .M . MICHELSON, B .S . RUSSEL and P .T . HERMAN, Proc . Nat . Aced . Sci . 41, 1074 (1955) . V .S . ASMUNDSON and L .M . JULIAN, J . Heredity 47, 248 (1956) T .W . West and W .G . HOAG, Ann . N .4 . Aca d-Sci . _138, 4 (1966) . V .A . ASHUNDSON, F .H . KRATZER and L .M . JULIAN, Ann . N . Y . Aced . Sci . 138, 49 (1966) . .F D . MORGAN and H . HERRMANN, Proc . Soc . Ezptl . Biol . Mad . 120, 68 (1965) . I .M . WEINSTOCK, T .S . JOH, H .A .FREEDMAN and M .E . CUTLER, Biochemical Mad . 2, 345 (1969) . B.A . BATTELLE and J .R . FLORINI, Biochem . 12, 635 (1973) . T .M . BURNS, C .L . SPEARS and S .S . KERWAR, Arch . Biochem . Biophys . 159, R . MILLER and R .L . SCHWEET, Arch . Biochem . Biophys . 125, 632 (1968). H . AVIV, I . BODE and P . LEDER, Proc . Nat . Aced . Sci . 68, 2303 (1971) . J . WIRMAN-COFFELT, R . ZELIS, C . FENNER and D .T . MASON, Biochem. Biophys . Ras . Commun . 51, 1097 (1927) . S . SARKAR aad P .H. COOKE, Biochem. Biophys . Res . -Commun . 41, 918 (1970) . W .E . BRENNOCK and W .O . READ, Res . Commun . Chem. Pathol . Pharmacol . 3, 417 (1972) . J . S . MOREY, K . TARCZY-HARNACH and E .G . RICHARDS, Arch . Biochem . Biophys . 119, 491 (1967) . K .S . MOREY, & . TARCZY-HORNACH and W .D . BROWN, Arch . Biochem . Biophys . 124, 521 (1968) . H . OPPENHEIMER, S . HASSELRI and A .T . MILHORAT, in "Muscle Diseases ", (p .266) edited by J .N . Walton, N . Canal, G . Scarlato and M . Gleave . Excerpts Medica, Amsterdam (1970) . K . WEVER and M . OSBORNE, J . Biol . Chem . 244, 4406 (1969) . H . OPPENHEIMER and L . MARKIEWICZ, Biochemical Mad . 7, 479 (1973) . D .C . WATTS and J .D . REID, Biochem. J . 115, 37T--(196--9) . S .E . KITCHIN and D .C . WATTS, Biochem. J .136, 1017 (1973) . R .J . PENNINGTON, in "Muscle Dieesees", (p.252) edited by J .N . Walton, N . Canal, G . Scarlato an ave . Excerpts Medica, Amsterdam (1970) . A .A . IODICE, S . PERKER and I .M. WEINSTOCK, in "Múec1e Diseases", (p .313) edited by J .N . Walton, N . Canal, G . Scarlato M . G Bave . Excerpts, Medica, Amsterdam (1970) .