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Changes in light chains of myosin during animal development
CHANGES
Institute
IN LIGHT CHAINS OF MYOSIN ANIMAL DEVELOPMENT
of Physiology.
Czechoslovak Academy of Sciences, BudEjovickB 1083. Czechoslovakia (Rrceid
22 Srprrmher
DURING
14220 Prague
4.
1978)
Abstract-l. CaZt-ATPase activity of myosin. prepared from the longissimus dorsi muscle of adult rat or rabbit is higher than that of embryonic ones. 2. The relative amount of LC, of myosin decreases and that of LC, increases during development. 3. It is suggested that at early developmental stages only the homodimer of myosin containing LC, and LCI-is present.
INTRODUCTION It is now clearly
established
that
fast and slow
Weeds ef u/., 1975) three myosin isoenzymes were predicted : two homodimers and a heterodimer. It was shown that subfragment I, heavy meromyosin and myosin can be resolved into two fractions. one rich in alkali 1 and the other rich in alkali 2 light chains (Holt & Lowey, 1977; Trayer et (II., 1977; Yagi et al., 1977; Weeds & Taylor, 1975) and this fractionation has provided direct evidence for the existence of two isoenzymic populations in adult vertebrate skeletal myosin. As the amount of LC, increases during development, it can be speculated whether the ratio of the two myosin isoenzymes does not alter during development. It this is the case, then the amount of LC, should decrease at the same time. In this report we present evidence of the decrease of LC, of myosin from logissimus dorsi of the rat and rabbit during development.
skeletal
muscles differ considerably, as far as changes in myosin during development are concerned. Mammalian muscles, which are in adult animals slow, contain both fast and slow types of myosin before and after birth; during subsequent development the fast type of myosin is eliminated (Margreth, 1975; PelloniMueller et ul., 1976a,b; Syrovjr, 1977; Syrovjl & Gutmann. 1977; Rubinstein & Kelly, 1978). When the isolated from mammalian properties of myosin. muscles which are fast in adult animals, were studied during development, no such heterogeneity of myosin could be observed around birth. It was shown that myosins from embryonic “fast” or newborn “fast” muscles and adult fast muscles differ in several properties. Embryonic or newborn myosin contains smaller amounts of LC, (Sreter et al., 1975; Takahashi & Tonomura,, 1975; Takahashi, 1976; Dabrowska et al., 1977) and has lower ATPase activity, when compared with adult myosin (Srkter et ul., 1975; Takahashi & Tonomura, 1975; Trayer & Perry, 1966). Embryonic myosin does not contain 3-methylhistidine, while adult myosin does (Trayer et a/., 1968). In some of its properties, such as immunochemical differences (Perry, 1970) and changes in the pattern of polypeptides released by tryptic digestion (SrCter er al., 1975). embryonic myosin resembles the myosin extracted from slow adult muscle, however, on exposure to pH 9.2 embryonic myosin is stable similarly as adult fast myosin (SrCter et ul., 1975). The staining pattern of light meromyosin paracrystals prepared from embryonic myosin is distinctly different from either adult fast, slow or cardiac myosin staining pattern (SrCter et al., 1975). The significance of changes in myosin properties during development is thus not clear. The repeatedly observed increase in the amount of LC, during development has never been explained. Based upon the unequal alkali light chains of myosin distribution
(Lowey
& Risby,
1971;
Sarkar,
MATERIAL AND METHODS Newborn and adult rats and rabbit embryos (20. 26 and 30 day old) and adult rabbits were used. In 20-day-old rabbit embryos the muscles of the back, in other cases longissimusdorsi muscles were used for myosin preparation. Previously published procedures were used for the preparation and purification of myosin (Syrovj & Gutmann, 1977) including the use of fi-mercaptoethanol. Ca’+-activated ATPase of myosin was measured in a medium containing 0.05 M Tris-HCI, pH 7.5, IOmM CaCI,, 0.025 M KC]. 5 mM ATP and 0.25 mg of myosin/ml (reaction vol 2 ml. 6 min incubation at 25 C). SDS gel electrophoresis was carried out as reported earlier (Syrovi & Gutmann, 1977). Gels were scanned on a Perkin-Elmer microdensitometer. The areas of the peaks (magnified about 4 x ) were measured by planimetry. The results are the average of 5 readings. RESULTS
The Ca *+-ATPase activity of myosin from newborn rats is lower than that of adult rats and there is also sharp increase of ATPase activity during rabbit development from embryos to adulthood (Table 1). Light chains of embryonic or newborn myosin differ from those of adult myosin in the amount of LC, and also LC,. Figure. 1 shows the much smaller content of LC3 in myosin from newborn rats as compared to adult rats. Changes in the amount and pro-
1972;
Abbreviations: LC, light chain 1; LC,, light chain 2; LC,, light chain 3: and SDS, sodium dodecyl sulfate. LC, and LC, light chains have also been termed by some authors as alkali I and alkali 2 light chains respectively. 223
1. SYROV+
224
I. Ca’ ’ -ATPase
Table
activity
of myosin (P,, {cmole mg _ ’ min
Rat (3 Embryonic Embryonic Embryonic
experiments)
Rabbit
(20-day-old) (26-old-day)
0.18 0.29
(30-day-old)
Newborn Adult
0.33 0.69 0.67 0.72 0.86 0.84 0.90
of LC, . LC, and LC, can be seen in Fig. 2 and Table 2. In the rat and also in the rabbit the relative amount of LC, decreases, that of LC, increases and that of LC, remains unchanged during development. In rabbits, where myosin was isolated from muscles during early stages of embryonic development. the differences are much more pronounced and LC, was not detected at all in 2O-day-old embryos. portion
0.64
Table 2. Quantitative evaluation of light chains from electrophoresed myosin from rat and rabbit in the course of development Rat
The present results. showing that the relative amount of LC, is decreased and that LCJ is increased
B
C
D
LC3
LC*
LC1
Fig. 2. Densitometry of light chains of rabbit myosin. (a) Embryonic, 20-day-old: (b) embryonic. 26-day-old: (c) embryonic. 30-day-old and (d) adult.
Newborn
Adult
(“J
(“J
39 56 5
30 54 I6
f-c, LC, LC,
DISCCWTION
A -AIL
‘)
20
LC, LC, LC,
Rabbit Embryonic 26 30-daysold I”,,)
56 44
46 53
0
I
38 55
7
Adult (“
Gels were scanned and the areas of the peaks were measured by planimetry. The
_
total area of LC,,
LC2 and LC, in each myosin sample was taken as IOO”,,.
at the same time during development, provide evidence, that LC, is synthesized later than LC, during development. Recent studies have pointed out (Holt & Lowey, 1977; Trayer et al., 1977; Yagi et u/., 1977) the presence of two homodimers of myosin in adult fast skeletal muscle--one containing LC, and LC2 and the other with LC2 and LC,. But the significance of the existence of two different myosin homodimers has not been explained. In our paper we found that in 20-day-old rabbit embryos LC, and LC2 are present in the ratio of approximately 1 :I; in adult rabbit the ratio is similar to that found by Lowey & Risby (1971) which corresponds to the molar ratio of LC, :LC,: LC, as 0.8:2.2: 1.0. The results suggest that at early developmental stages only the homodimer containing LC, and LC2 is present. This idea is supported by the finding that LC,, isolated from adult myosin is not able to bind to fetal myosin (Takahashi. 1976). The existence of solely one homodimer of myosin during early development could be proved by fractionation of native myosin during development and by measuring ATPase activity of isolated fractions. containing either LC, and LC, or LC, and Lc’, homodimer which has not so far been performed. The present study shows that myosin from ?O-dayold rabbit embryos, which does not contain any LC,, is able to split ATP and that LCJ is therefore not absolutely essential for myosin ATPase activity. The increase of both myosin ATPase activity and the amount of LC, during development suggest that if
N Fig. 1. SDS polyacrylamide (IO”,,) gel electrophoresis of light chains from newborn and adult rat skeletal myosin. Amount of each protein applied. 25 ;ig. Key: N, Newborn; A, adult myosin.
22.5
Changes
in light chains
the homodimer containing LCI and LC3 is really synthesized later, it is enzymatically more active than the other. Other differences in myosin during development (SrCter et ul., 1975; Trayer rt ul., 1968; Perry, 1970) remain unexplained but they may concern different properties of myosin isoenzymes. A~linoM,lrdgrmrnr--The author Hnik for his help in preparation
wishes to thank of the manuscript.
Dr
P.
REFERENCES DABROWSKA R.. SOSINSKI J. & DRABIKOWSKI W. (1977) Changes in the composition of the myofibrillar fraction during development of the rabbit. FEBS Lrtt. 79, 295-300. HOLT J. C. & LOWEY S. (1977) Distribution of alkali light chains in myosin. Isolation of isoenzymes. Biochrmistr!: 16, 4398-4402. L~WEY S. & RISBY D. (1971) Light chains from fast and slow muscle myosins. Noturr, Land. 234, 81-85. MARGRETH A. (1975) In Recent Adrunces in Myoloyy. Proceedinys of the 3rd Internutiontrl Congress on Muscle Diseases. Newcastle upon Tyne, 1974. (Edited by BRADLEY
W. G.. GARI)NER-MEIIWIN D. & WALTON J. N.), pp. 307-309. Excerpta Medica. Amsterdam. PELLONI-MUELLER G.. ERMINI M. & JENNY E. (1976a) Changes in myosin light and heavy chain stoichiometry during development or rabbit fast, slow and cardiac muscle. FEBS Left. 70, 1 l3- 117. PELLONI-MUELLER G.. ERMINI M. & JENNY E. (1976b) Myosin light chains of developing fast and slow rabbit skeletal muscle. FEBS Left. 67, 68-74. PERRY S. V. (1970) In Physiology und Biochemistry of Muscle as u Food. (Edited by BRISKEY E. J., CASSENS R. G. & MARCH B. B.), pp. 537.-553. The University of Wisconsin Press. RUBINSTEIN N. A. 8~ KELLY A. M. (1978) Myogenic and
of myosin
227
neurogenic contributions to the development of fast and slow twitch muscles in rat. Deul. Biol. 62, 473-85. SARKAR S. (1972) Stoichiometry and sequential removal of light chains of myosin. Cold Spring Harh. Sgmp. 37, 14-17. SRBTER F. A., BALINT M. & GERGELY J. (1975) Structural and functional changes of myosin during development. Comparison with adult fast, slow and cardiac myosin. &cl. Biol. 46. 317-325. SYROV? I. (1977) Myosin-characteristics of the semi-tendinosus and extensor digitorum longus muscle of newborn and adult rabbits. Inr. .I. Biochcm. 8, 847-848. SYROV+ I. & GUTMANN E. (1977) Differentiation of myosin in soleus and extensor digitorum longus muscle in different animal species during animal development. Pfliiyers Arch. ges. Physiol. 369, 85-89. TAKAHASHI M. & TONOMURA Y. (1975) Developmental changes in the structure and kinetic properties of myosin adenosinetriphosphatase of rabbit skeletal fast muscle. J. Biochem. 78, 1123-l 133. TAKAHASHI M. (1976) Inability of the smallest light chain bind to fetal fast muscle myosin. J. Biochem. 80, 621-624. TRAYER I. P. 8~ PERRY S. V. (1966) The myosin of developing skeletal muscle. Biochem. Z. 345, 87-100. TRAYER I. P., HARRIS C. 1. & PERRY S. V. (1968) 3-methyl histidine and adult and foetal forms of skeletal muscle myosin. Nature, Lond. 217, 452-453. TRAYER R. H., WINSTANLEY M. A. & TRAYER 1. P. (1977) The separation of heavy meromyosin isoenzymes by differential acting binding. FEBS Left. 83, 141-144. WEEDS A. G. & TAYLOR R. S. (1975) Separation of subfragment 1 isoenzymes from rabbit skeletal musle myosin. Nature, Lond. 257, 54-56. WEEDS A. G., HALL R. & SPURWAY N. C. S. (1975) Characteriazation of myosin light chains from histochemically identical fibres of rabbit psoas muscle. FEBS Left. 49, 32&324. YAGI K., MATSUDA S. & KUWAYAMA H. (1977) Separation of two different heavy meromyosins. Evidence for the presence of myosin isozymes in rabbit skeletal muscle. J. Biochem. 82, 1463-1467.
Institute
IN LIGHT CHAINS OF MYOSIN ANIMAL DEVELOPMENT
of Physiology.
Czechoslovak Academy of Sciences, BudEjovickB 1083. Czechoslovakia (Rrceid
22 Srprrmher
DURING
14220 Prague
4.
1978)
Abstract-l. CaZt-ATPase activity of myosin. prepared from the longissimus dorsi muscle of adult rat or rabbit is higher than that of embryonic ones. 2. The relative amount of LC, of myosin decreases and that of LC, increases during development. 3. It is suggested that at early developmental stages only the homodimer of myosin containing LC, and LCI-is present.
INTRODUCTION It is now clearly
established
that
fast and slow
Weeds ef u/., 1975) three myosin isoenzymes were predicted : two homodimers and a heterodimer. It was shown that subfragment I, heavy meromyosin and myosin can be resolved into two fractions. one rich in alkali 1 and the other rich in alkali 2 light chains (Holt & Lowey, 1977; Trayer et (II., 1977; Yagi et al., 1977; Weeds & Taylor, 1975) and this fractionation has provided direct evidence for the existence of two isoenzymic populations in adult vertebrate skeletal myosin. As the amount of LC, increases during development, it can be speculated whether the ratio of the two myosin isoenzymes does not alter during development. It this is the case, then the amount of LC, should decrease at the same time. In this report we present evidence of the decrease of LC, of myosin from logissimus dorsi of the rat and rabbit during development.
skeletal
muscles differ considerably, as far as changes in myosin during development are concerned. Mammalian muscles, which are in adult animals slow, contain both fast and slow types of myosin before and after birth; during subsequent development the fast type of myosin is eliminated (Margreth, 1975; PelloniMueller et ul., 1976a,b; Syrovjr, 1977; Syrovjl & Gutmann. 1977; Rubinstein & Kelly, 1978). When the isolated from mammalian properties of myosin. muscles which are fast in adult animals, were studied during development, no such heterogeneity of myosin could be observed around birth. It was shown that myosins from embryonic “fast” or newborn “fast” muscles and adult fast muscles differ in several properties. Embryonic or newborn myosin contains smaller amounts of LC, (Sreter et al., 1975; Takahashi & Tonomura,, 1975; Takahashi, 1976; Dabrowska et al., 1977) and has lower ATPase activity, when compared with adult myosin (Srkter et ul., 1975; Takahashi & Tonomura, 1975; Trayer & Perry, 1966). Embryonic myosin does not contain 3-methylhistidine, while adult myosin does (Trayer et a/., 1968). In some of its properties, such as immunochemical differences (Perry, 1970) and changes in the pattern of polypeptides released by tryptic digestion (SrCter er al., 1975). embryonic myosin resembles the myosin extracted from slow adult muscle, however, on exposure to pH 9.2 embryonic myosin is stable similarly as adult fast myosin (SrCter et ul., 1975). The staining pattern of light meromyosin paracrystals prepared from embryonic myosin is distinctly different from either adult fast, slow or cardiac myosin staining pattern (SrCter et al., 1975). The significance of changes in myosin properties during development is thus not clear. The repeatedly observed increase in the amount of LC, during development has never been explained. Based upon the unequal alkali light chains of myosin distribution
(Lowey
& Risby,
1971;
Sarkar,
MATERIAL AND METHODS Newborn and adult rats and rabbit embryos (20. 26 and 30 day old) and adult rabbits were used. In 20-day-old rabbit embryos the muscles of the back, in other cases longissimusdorsi muscles were used for myosin preparation. Previously published procedures were used for the preparation and purification of myosin (Syrovj & Gutmann, 1977) including the use of fi-mercaptoethanol. Ca’+-activated ATPase of myosin was measured in a medium containing 0.05 M Tris-HCI, pH 7.5, IOmM CaCI,, 0.025 M KC]. 5 mM ATP and 0.25 mg of myosin/ml (reaction vol 2 ml. 6 min incubation at 25 C). SDS gel electrophoresis was carried out as reported earlier (Syrovi & Gutmann, 1977). Gels were scanned on a Perkin-Elmer microdensitometer. The areas of the peaks (magnified about 4 x ) were measured by planimetry. The results are the average of 5 readings. RESULTS
The Ca *+-ATPase activity of myosin from newborn rats is lower than that of adult rats and there is also sharp increase of ATPase activity during rabbit development from embryos to adulthood (Table 1). Light chains of embryonic or newborn myosin differ from those of adult myosin in the amount of LC, and also LC,. Figure. 1 shows the much smaller content of LC3 in myosin from newborn rats as compared to adult rats. Changes in the amount and pro-
1972;
Abbreviations: LC, light chain 1; LC,, light chain 2; LC,, light chain 3: and SDS, sodium dodecyl sulfate. LC, and LC, light chains have also been termed by some authors as alkali I and alkali 2 light chains respectively. 223
1. SYROV+
224
I. Ca’ ’ -ATPase
Table
activity
of myosin (P,, {cmole mg _ ’ min
Rat (3 Embryonic Embryonic Embryonic
experiments)
Rabbit
(20-day-old) (26-old-day)
0.18 0.29
(30-day-old)
Newborn Adult
0.33 0.69 0.67 0.72 0.86 0.84 0.90
of LC, . LC, and LC, can be seen in Fig. 2 and Table 2. In the rat and also in the rabbit the relative amount of LC, decreases, that of LC, increases and that of LC, remains unchanged during development. In rabbits, where myosin was isolated from muscles during early stages of embryonic development. the differences are much more pronounced and LC, was not detected at all in 2O-day-old embryos. portion
0.64
Table 2. Quantitative evaluation of light chains from electrophoresed myosin from rat and rabbit in the course of development Rat
The present results. showing that the relative amount of LC, is decreased and that LCJ is increased
B
C
D
LC3
LC*
LC1
Fig. 2. Densitometry of light chains of rabbit myosin. (a) Embryonic, 20-day-old: (b) embryonic. 26-day-old: (c) embryonic. 30-day-old and (d) adult.
Newborn
Adult
(“J
(“J
39 56 5
30 54 I6
f-c, LC, LC,
DISCCWTION
A -AIL
‘)
20
LC, LC, LC,
Rabbit Embryonic 26 30-daysold I”,,)
56 44
46 53
0
I
38 55
7
Adult (“
Gels were scanned and the areas of the peaks were measured by planimetry. The
_
total area of LC,,
LC2 and LC, in each myosin sample was taken as IOO”,,.
at the same time during development, provide evidence, that LC, is synthesized later than LC, during development. Recent studies have pointed out (Holt & Lowey, 1977; Trayer et al., 1977; Yagi et u/., 1977) the presence of two homodimers of myosin in adult fast skeletal muscle--one containing LC, and LC2 and the other with LC2 and LC,. But the significance of the existence of two different myosin homodimers has not been explained. In our paper we found that in 20-day-old rabbit embryos LC, and LC2 are present in the ratio of approximately 1 :I; in adult rabbit the ratio is similar to that found by Lowey & Risby (1971) which corresponds to the molar ratio of LC, :LC,: LC, as 0.8:2.2: 1.0. The results suggest that at early developmental stages only the homodimer containing LC, and LC2 is present. This idea is supported by the finding that LC,, isolated from adult myosin is not able to bind to fetal myosin (Takahashi. 1976). The existence of solely one homodimer of myosin during early development could be proved by fractionation of native myosin during development and by measuring ATPase activity of isolated fractions. containing either LC, and LC, or LC, and Lc’, homodimer which has not so far been performed. The present study shows that myosin from ?O-dayold rabbit embryos, which does not contain any LC,, is able to split ATP and that LCJ is therefore not absolutely essential for myosin ATPase activity. The increase of both myosin ATPase activity and the amount of LC, during development suggest that if
N Fig. 1. SDS polyacrylamide (IO”,,) gel electrophoresis of light chains from newborn and adult rat skeletal myosin. Amount of each protein applied. 25 ;ig. Key: N, Newborn; A, adult myosin.
22.5
Changes
in light chains
the homodimer containing LCI and LC3 is really synthesized later, it is enzymatically more active than the other. Other differences in myosin during development (SrCter et ul., 1975; Trayer rt ul., 1968; Perry, 1970) remain unexplained but they may concern different properties of myosin isoenzymes. A~linoM,lrdgrmrnr--The author Hnik for his help in preparation
wishes to thank of the manuscript.
Dr
P.
REFERENCES DABROWSKA R.. SOSINSKI J. & DRABIKOWSKI W. (1977) Changes in the composition of the myofibrillar fraction during development of the rabbit. FEBS Lrtt. 79, 295-300. HOLT J. C. & LOWEY S. (1977) Distribution of alkali light chains in myosin. Isolation of isoenzymes. Biochrmistr!: 16, 4398-4402. L~WEY S. & RISBY D. (1971) Light chains from fast and slow muscle myosins. Noturr, Land. 234, 81-85. MARGRETH A. (1975) In Recent Adrunces in Myoloyy. Proceedinys of the 3rd Internutiontrl Congress on Muscle Diseases. Newcastle upon Tyne, 1974. (Edited by BRADLEY
W. G.. GARI)NER-MEIIWIN D. & WALTON J. N.), pp. 307-309. Excerpta Medica. Amsterdam. PELLONI-MUELLER G.. ERMINI M. & JENNY E. (1976a) Changes in myosin light and heavy chain stoichiometry during development or rabbit fast, slow and cardiac muscle. FEBS Left. 70, 1 l3- 117. PELLONI-MUELLER G.. ERMINI M. & JENNY E. (1976b) Myosin light chains of developing fast and slow rabbit skeletal muscle. FEBS Left. 67, 68-74. PERRY S. V. (1970) In Physiology und Biochemistry of Muscle as u Food. (Edited by BRISKEY E. J., CASSENS R. G. & MARCH B. B.), pp. 537.-553. The University of Wisconsin Press. RUBINSTEIN N. A. 8~ KELLY A. M. (1978) Myogenic and
of myosin
227
neurogenic contributions to the development of fast and slow twitch muscles in rat. Deul. Biol. 62, 473-85. SARKAR S. (1972) Stoichiometry and sequential removal of light chains of myosin. Cold Spring Harh. Sgmp. 37, 14-17. SRBTER F. A., BALINT M. & GERGELY J. (1975) Structural and functional changes of myosin during development. Comparison with adult fast, slow and cardiac myosin. &cl. Biol. 46. 317-325. SYROV? I. (1977) Myosin-characteristics of the semi-tendinosus and extensor digitorum longus muscle of newborn and adult rabbits. Inr. .I. Biochcm. 8, 847-848. SYROV+ I. & GUTMANN E. (1977) Differentiation of myosin in soleus and extensor digitorum longus muscle in different animal species during animal development. Pfliiyers Arch. ges. Physiol. 369, 85-89. TAKAHASHI M. & TONOMURA Y. (1975) Developmental changes in the structure and kinetic properties of myosin adenosinetriphosphatase of rabbit skeletal fast muscle. J. Biochem. 78, 1123-l 133. TAKAHASHI M. (1976) Inability of the smallest light chain bind to fetal fast muscle myosin. J. Biochem. 80, 621-624. TRAYER I. P. 8~ PERRY S. V. (1966) The myosin of developing skeletal muscle. Biochem. Z. 345, 87-100. TRAYER I. P., HARRIS C. 1. & PERRY S. V. (1968) 3-methyl histidine and adult and foetal forms of skeletal muscle myosin. Nature, Lond. 217, 452-453. TRAYER R. H., WINSTANLEY M. A. & TRAYER 1. P. (1977) The separation of heavy meromyosin isoenzymes by differential acting binding. FEBS Left. 83, 141-144. WEEDS A. G. & TAYLOR R. S. (1975) Separation of subfragment 1 isoenzymes from rabbit skeletal musle myosin. Nature, Lond. 257, 54-56. WEEDS A. G., HALL R. & SPURWAY N. C. S. (1975) Characteriazation of myosin light chains from histochemically identical fibres of rabbit psoas muscle. FEBS Left. 49, 32&324. YAGI K., MATSUDA S. & KUWAYAMA H. (1977) Separation of two different heavy meromyosins. Evidence for the presence of myosin isozymes in rabbit skeletal muscle. J. Biochem. 82, 1463-1467.