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Major contractile proteins of mollusc: tissue polymorphism of actin, tropomyosin and myosin light chains is absent
Comp. Biochem. Physiol. Vol. 72B, pp. 473 to 476, 1982
0305-0491/82/030473-04503.00/0 ©1982 Pergamon Press Ltd
Printed in Great Britain.
MAJOR CONTRACTILE PROTEINS OF MOLLUSC: TISSUE POLYMORPHISM OF ACTIN, TROPOMYOSIN AND MYOSIN LIGHT CHAINS IS ABSENT B. A. MARGULIS, K. I. GALAKTIONOV, 0. I. PODGORNAYA* and G. P. PINAEV Department of Cell Cultures, Institute of Cytology of the Academy of Sciences of U.S.S.R., Leningrad-121 and *Institute of Marine Biology, Far-East Scientific Center of the Academy of Sciences of U.S.S.R., Vladivostok-22
(Received 21 October 1981) A b s t r a c ~ l . Four muscle tissues, adductor, mantle, leg elevator and heart of bivalve mollusc were investigated in order to get information about their contractile proteins. 2. The proteins studied were actin, tropomyosin and myosin light chains. Two electrophoretic methods and a combination of electrophoresis with isoelectric focusing were used. 3. Isoforms of these proteins were shown to have identical molecular masses, mass-charge ratios and isoelectric points irrespective of Spisula tissue type. 4. The results are discussed in the light of newer data on differential gene expression.
INTRODUCTION
The function of muscle contractile systems from different tissues and organisms is associated with cooperative activity of 20--25 contractile proteins (Etlinger et al., 1976; Korn, 1978). In phylogenesis and ontogenesis the structure and function of contractile apparatus undergo alterations followed by the composition and properties variations of the proteins considered (Gzent-GyiSrgyi, 1975; Devlin & Emerson, 1978). The three principal proteins (according to their high content in muscle cells) are actin, myosin and tropomyosin which will be signified in the text as major contractile proteins (MCP). The use of electrophoresis and isoelectric focusing (IEF) techniques has allowed to detect tissue specific forms of every M C P in Vertebrae (Zechel & Weber, 1978; Mikawa et al., 1981). The degree of tissue-specificity of M C P has been shown to exceed that of species-specificity (Vanderkerckhove & Weber, 1978; Gabbiani et al., 198l). The distribution of M C P forms in different Vertebrae tissues and cells is not random (Young & Davey, 1981) and the expression of corresponding genes is regulated in a similar way (Devlin & Emerson, 1978). For vertebrates investigations of M C P tissue-specificity have been carried out only for Drosophila tissues which contain three actin isoelectric types (Horovitch et al., 1979) and for nematode (Schachat et al., 1978). The tissues of the latter incorporate 3 myosin forms. At the same time we have shown that tropomyosin and myosin from bivalve molluscs are characterized by a considerable species polymorphism (Margulis & Pinaev, 1976). For invertebrates the quantity of M C P isoforms should be lower than that for vertebrates (Vandekerckhove & Webber, 1980). To our mind this fact might simplify the observation of incorporation of M C P isoforms in definite tissues of an individual organism. This consideration has determined the purpose of the present work: screening of the properties of M C P from various tissues of bivalve mollusc Spisula sachalinensis which had been investigated earlier in our laboratory. ('.R.P. 723B J
473
We have used different electrophoretic methods and a combination of electrophoresis with isoelectric focusing and have shown that the homological proteins, which are present in tissues of different morphology and functional status are identical. MATERIALS AND METHODS
The molluscs were collected on the shore of the Japan Sea. Hearts and leg elevator muscles were carefully separated from adjacent tissues and jointly with adductor and mantle were stored at -10°-15°C in the solution: 50% glycerol, 40 mM NaCI, 10 mM Tris-HCl, pH 7.0. Myofibril-like preparations (MF) were isolated as described earlier (Margulis & Pinaev, 1976). Smooth muscle actin (fl-,),-forms) used as a marker was isolated from protein band after electrophoresis of chicken gizzard MF. We also used the rabbit skeletal muscle actin (~-form) and scallop actin kindly supplied by S. Yu. Khaitlina. Slab electrophoresis was carried out in the presence of sodium dodecylsulphate (SDS) according to the Laemmli protocol (Laemmli, 1970). For the urea-electrophoresis (8 M urea in gel and sample) we used 0.4 × 14 cm glass tubes (Perrie & Perry, 1970). Isoelectric focusing was executed in gels containing 5% acrylamide; 0.2% methylene-(bis)acrylamide; 8M urea; 2% Nonidet P-40 and Ampholine (2% pH 3.5 10 or 1.5% pH 5-7; 0.5% pH 3.5-10). In addition we used a combination of methods: urea-electrophoresis/IEF, i.e. after the electrophoresis the sections corresponding to the stained identifed bands were excised from unstained gels and transferred onto the IEFgel surface. This permitted us to avoid the complicated procedure of protein purification. Before the comparison of two or more homological proteins from various sources their mixture was electrophoresed in the same tube so that the resulting section for consequent IEF should contain all the fractions. The staining of the gels after IEF proceeded as follows: fixation in 3 changes of 45% methanol (each one for 6 hr), staining in 0.2% Coomassie brilliant blue R, 45% methanol, 10% acetic acid and destaining in 25% ethanol, 10% acetic acid. All the reagents were of analytical grade or specially purified for electrophoresis. Ampholine carrier-ampholytes were purchased from LKB, Sweden. The electrophoretic runs were performed on simplified apparatus, designed for a large amount of samples.
474
B.A.
MARGULIS
et al. RESULTS
MHC
The comparison of overall protein composition was fulfilled by SDS-disc-electrophoresis. It was shown that M F of all the tissues studied contain myosin with its heavy and light chains (MHC and MLC), actin and tropomyosin (TM). The data on the mobilities of homological M C P from different tissues display equality of their molecular masses. The relative content of some proteins in particular myosin and actin, is tissue-dependent• Besides the proteins mentioned above and identified earlier (Margulis & Pinaev, 1976) the adductor M F contain in a marked amount the protein with molecular mass equal to that of paramyosin. Heart M F incorporate the protein with molecular mass smaller than that of paramyosin, Fig. 1, In order to determine the subtle peculiarities of M F proteins under investigation, i.e. charge variety, posttranslational modifications, we used urea electrophoresis. The electrophoregrams for M F from different tissues are presented in Fig. 2, which testifies to the coincidence of relative mobilities of actin, TM, EDTA- and SH-MLC from various muscles. These data are confirmed by the results of electrophoretic runs for mixtures of M F in the same gel. It should be noted that in this electrophoretic system (pH 8.6) the Spisula and smooth muscle actins have equal mobilities differing from that of skeletal muscle actin. IEF of a wide range of pH 3.5 10 was used for the separation of TM and MLC types from Spisula tissues. All the TM were shown to have the same pl value which was confirmed by the investigation of their mixtures in the same tube. Fig. 3. The technique of band cutting and their transferring onto IEF gels was employed for SH- and EDTA-MLC analysis. Irrespective of tissue, the homologous MLC are characterized by the same pl-value. It is noteworthy that
--
pM m
i
A--Ore TM--
o
b
Fig. 1. SDS-electrophoregrams of myofibril-like preparations isolated from the adductor muscle (a) and heart (b) of Spisula s. MHC--myosin heavy chains, PM--paramyosin, A--actin, TH--tropomyosin. Electrophoresis in PAG was made according Laemmli, 1970.
I
I
--A
0
I
I
II
--TM
"-I D
--
-
w
I
EDTA -MLC
i
-- SH -- MLC
o
b
c
d
e
f
Fig. 2. Urea-electrophoresis of myofibril-like preparations from the following Spisula tissues: [a) adductor; (b) heart; (c) mantle; (d) leg elevator; (e) mixture of the adductor and leg myofibrils: (f) mixture of the adductor and heart myofibrils. Electrophoresis in PAG was made according Perrie & Perry, 1970. A--actin, TM--tropomyosin. EDTA-MLC and SH-MLC-EDTA and SH myosin light chains.
Major contractile proteins of mollusc
475
I
o
a
b
c
d
a
b
c
d
B Fig. 3. Isoelectric focusing in 5~o PAG, 2~o Ampholine LKB pH 3, 5-10. After urea electrophoresis protein bands were excised from the gels and transferred onto the surface of IEF gels. (A) IEF of tropomyosins: (a) adductor, (b) heart, (c) the mixture of adductor and mantle, (d) the mixture of heart and leg elevator. Into each tube trace amount of rabbit actin was added. (B) IEF of SH myosin light chains: (a) adductor, (b) heart, (c) mantle, (d) the mixture of adductor and leg elevator.
/LT-actins from gel slices, focused at the range of pH 5-7, occupied a fixed position relative to skeletal a-form, Fig. 4. Spisula actin of all 4 muscles was concentrated just under the band of/~-actin. At the same time the former was coincidental with Patinopecten actin offering a case of species specificity of actin isotypes in molluscs. DISCUSSION
As mentioned above all the electrophoretic techniques used in the present work permit to separate contractile proteins with high resolution in the following parameters: molecular mass (SDS-disc-electrophoresis), charge: mass ratio (urea-electrophoresis) and isoelectric point (IEF). Contrary to vertebrates in the case of molluscs, these methods turned out to be ineffective in the detection of protein polymorphism. Although one cannot definitely assert that mollusc protein isoforms are identical, at least two facts may support this statement: (1) only one isotype of each MCP was observed; (2) irrespective of separation technique no difference between all homologous proteins was revealed.
The absence of tissue specificity of mollusc MCP may be due to one of the following factors: (1) only one structural gene exists for each MCP; (2) only one "major" gene copy is selected from the family of closely related genes. The analysis of actin isoforms and the corresponding genes of Dictyostelium discoideum confirmed the validity of the second models with groups of "silent" genes (McLeod et al., 1980). The same way of expression may be proposed for MCP-coding genes of Spisula. Thinking so we can suggest that expression of "silent" genes is feasible in some specialized cells of mollusc or that it takes place at certain stages of development. Our preliminary data show that even in non-muscle tissues of mollusc and the sea star only fl-like form of actin is present (unpublished results). The four tissues of mollusc studied (heart, obliquely-striated of adductor, smooth of leg elevator and mantle) have different morphological and physiological properties, but high-resolution electrophoretic analysis allowed us to detect only one isotype for every MCP. It should be emphasized that the ultrastructural features of contractile apparatus are not neccessarily determined by the presence of any given
476
B.A. MARGULISet al.
7"
a
I
It
11
b
c
d
°
6
•
f
-
g
$
h
Fig. 4. Isoelectric focusing in 5'~o PAG, 1, 5'!o Ampholine LKB pH 5 7, 0,5",, Ampholine pH 3,5 10. The actins from bands were focused. (a) rabbit skeletal ct-actin, (b) adductor, (c) leg elevator, (d) heart, (e) mantle, (f) adductor plus Patinopecten adductor actin, (g) Patinopecten actin, lh) the mixture of rabbit ~-actin and fl,7-actins from chicken gizzard. Rabbit actin added in tubes (b), (c) and {d) in various proportions. M C P isoform but may be due to changes in content of certain M C P in tissues (Margulis et al., 1979) and/or to the presence of some minor, regulatory proteins. REFERENCES DE COUET H. G., MAZANDER K.-D. & GROSCHEL-STEWART U. (1980) A study of invertebrate actins by isoelectric focusing and immunodiffusion. Experientia 36, 40~405. DEVLIN R. B. & EMERSON C. P. (1978) Coordinate regulation of contractile protein synthesis during myoblast differentiation. Cell 13, 599-612. ETLINGER J. D., ZAK R. & FISHMAND. A. (1976) Compositional studies of myofibrils from rabbit skeletal muscle. J. Cell Biol. 68, 123-141. GABBIANI G., SCHMID E., WINTER S., CHAPONNIER C., DE CHASTONEY C., VANDEKERCKHOVEJ. & WEBER K. (19811 Vascular smooth muscle cells differ from other smooth muscle cells: predominance of vimentin filaments and a specific a-type actin. Proc. natn. Acad. Sci. U.S.A. 78, 298-302. HOROVITCH S. J., STORTI R. V., RICH A. & PARDUE M. L. (1979) Multiple actins in Drosophila melanogaster. J. Cell Biol. 82, 86-92. KORN E. D. (1978) Biochemistry of actomyosin-dependent cell motility. Proc. hath. Acad. Sci. U.S.A. 75, 588-599. LAEMMLIU. K. (1970) Clevage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-685. MARGULIS B. A. & PINAEV G. P. (1976) The species specificity of the contractile protein composition of the bivalve molluscs. Comp. Biochem. Physiol. 55B, 189-194.
MARGULIS B. A., BOBROVA I. F., MASHANSKI V. F. & PINAEV G. P. (1979) Major myofibular protein content and the structure of mollusc adductor contractile apparatus. Comp. Biochem. Physiol. 64A, 291 298. McLE,OD C., F1RTEL R A. & PAPKOFFJ. (1980) Regulation of actin gene expression during spore germination in Dictyostelium discoideum. Develop. Biol. 76, 263 274. MIKAWA T., TAKEDA S., SHIMIZU T. & KITAURAZ. (1981) Gene expression of myofibular proteins in single muscle fibers of adult chicken: micro two-dimensional gel electrophoresis analysis. J. Biochem. (Tokyo) 89, 1951-1962. PERRIE W. T. ~,~ PERRY S. V. (1970) An electrophoretic study of the low-molecular-weight components of myosin. Biochem. J. 119, 31 38. SCHACHAT F.. GARCEA R. L. & EPSTEIN H. F. (1978) Myosin exist as homodimers of heavy chains: demonstration with specific antibody purified by nematode mutant myosin affinity chromatography. Cell 15, 405-411. SZENT-GY(SRGYI A. G. (1975) Calcium regulation of muscle contraction. Biophys. J. 15, 707 723. VANDEKERCKHOVEJ. • WEBER K. (1978) At least six different actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino-terminal tryptic peptides. J. molec. Biol. 126, 783-802. VANDEKERCKHOVEJ. & WEBER K. (1980) Vegetative Dictiostetium cells containing 17 actin genes express a single major actin. Nature, Lond. 284, 475 477. YOUNG O. A. & DAVEYC. L. (1981) Electrophoretic analysis of proteins from single bovine muscle fibers. Biochem. J. 195, 317 327. ZECHEL K. & WEBER K. (1978) Actins from mammals, bird, fish and slime mold characterized by isoelectric focusing in polyacrylamide gels. Eur. J. Bioehem. 89, 105 112.
0305-0491/82/030473-04503.00/0 ©1982 Pergamon Press Ltd
Printed in Great Britain.
MAJOR CONTRACTILE PROTEINS OF MOLLUSC: TISSUE POLYMORPHISM OF ACTIN, TROPOMYOSIN AND MYOSIN LIGHT CHAINS IS ABSENT B. A. MARGULIS, K. I. GALAKTIONOV, 0. I. PODGORNAYA* and G. P. PINAEV Department of Cell Cultures, Institute of Cytology of the Academy of Sciences of U.S.S.R., Leningrad-121 and *Institute of Marine Biology, Far-East Scientific Center of the Academy of Sciences of U.S.S.R., Vladivostok-22
(Received 21 October 1981) A b s t r a c ~ l . Four muscle tissues, adductor, mantle, leg elevator and heart of bivalve mollusc were investigated in order to get information about their contractile proteins. 2. The proteins studied were actin, tropomyosin and myosin light chains. Two electrophoretic methods and a combination of electrophoresis with isoelectric focusing were used. 3. Isoforms of these proteins were shown to have identical molecular masses, mass-charge ratios and isoelectric points irrespective of Spisula tissue type. 4. The results are discussed in the light of newer data on differential gene expression.
INTRODUCTION
The function of muscle contractile systems from different tissues and organisms is associated with cooperative activity of 20--25 contractile proteins (Etlinger et al., 1976; Korn, 1978). In phylogenesis and ontogenesis the structure and function of contractile apparatus undergo alterations followed by the composition and properties variations of the proteins considered (Gzent-GyiSrgyi, 1975; Devlin & Emerson, 1978). The three principal proteins (according to their high content in muscle cells) are actin, myosin and tropomyosin which will be signified in the text as major contractile proteins (MCP). The use of electrophoresis and isoelectric focusing (IEF) techniques has allowed to detect tissue specific forms of every M C P in Vertebrae (Zechel & Weber, 1978; Mikawa et al., 1981). The degree of tissue-specificity of M C P has been shown to exceed that of species-specificity (Vanderkerckhove & Weber, 1978; Gabbiani et al., 198l). The distribution of M C P forms in different Vertebrae tissues and cells is not random (Young & Davey, 1981) and the expression of corresponding genes is regulated in a similar way (Devlin & Emerson, 1978). For vertebrates investigations of M C P tissue-specificity have been carried out only for Drosophila tissues which contain three actin isoelectric types (Horovitch et al., 1979) and for nematode (Schachat et al., 1978). The tissues of the latter incorporate 3 myosin forms. At the same time we have shown that tropomyosin and myosin from bivalve molluscs are characterized by a considerable species polymorphism (Margulis & Pinaev, 1976). For invertebrates the quantity of M C P isoforms should be lower than that for vertebrates (Vandekerckhove & Webber, 1980). To our mind this fact might simplify the observation of incorporation of M C P isoforms in definite tissues of an individual organism. This consideration has determined the purpose of the present work: screening of the properties of M C P from various tissues of bivalve mollusc Spisula sachalinensis which had been investigated earlier in our laboratory. ('.R.P. 723B J
473
We have used different electrophoretic methods and a combination of electrophoresis with isoelectric focusing and have shown that the homological proteins, which are present in tissues of different morphology and functional status are identical. MATERIALS AND METHODS
The molluscs were collected on the shore of the Japan Sea. Hearts and leg elevator muscles were carefully separated from adjacent tissues and jointly with adductor and mantle were stored at -10°-15°C in the solution: 50% glycerol, 40 mM NaCI, 10 mM Tris-HCl, pH 7.0. Myofibril-like preparations (MF) were isolated as described earlier (Margulis & Pinaev, 1976). Smooth muscle actin (fl-,),-forms) used as a marker was isolated from protein band after electrophoresis of chicken gizzard MF. We also used the rabbit skeletal muscle actin (~-form) and scallop actin kindly supplied by S. Yu. Khaitlina. Slab electrophoresis was carried out in the presence of sodium dodecylsulphate (SDS) according to the Laemmli protocol (Laemmli, 1970). For the urea-electrophoresis (8 M urea in gel and sample) we used 0.4 × 14 cm glass tubes (Perrie & Perry, 1970). Isoelectric focusing was executed in gels containing 5% acrylamide; 0.2% methylene-(bis)acrylamide; 8M urea; 2% Nonidet P-40 and Ampholine (2% pH 3.5 10 or 1.5% pH 5-7; 0.5% pH 3.5-10). In addition we used a combination of methods: urea-electrophoresis/IEF, i.e. after the electrophoresis the sections corresponding to the stained identifed bands were excised from unstained gels and transferred onto the IEFgel surface. This permitted us to avoid the complicated procedure of protein purification. Before the comparison of two or more homological proteins from various sources their mixture was electrophoresed in the same tube so that the resulting section for consequent IEF should contain all the fractions. The staining of the gels after IEF proceeded as follows: fixation in 3 changes of 45% methanol (each one for 6 hr), staining in 0.2% Coomassie brilliant blue R, 45% methanol, 10% acetic acid and destaining in 25% ethanol, 10% acetic acid. All the reagents were of analytical grade or specially purified for electrophoresis. Ampholine carrier-ampholytes were purchased from LKB, Sweden. The electrophoretic runs were performed on simplified apparatus, designed for a large amount of samples.
474
B.A.
MARGULIS
et al. RESULTS
MHC
The comparison of overall protein composition was fulfilled by SDS-disc-electrophoresis. It was shown that M F of all the tissues studied contain myosin with its heavy and light chains (MHC and MLC), actin and tropomyosin (TM). The data on the mobilities of homological M C P from different tissues display equality of their molecular masses. The relative content of some proteins in particular myosin and actin, is tissue-dependent• Besides the proteins mentioned above and identified earlier (Margulis & Pinaev, 1976) the adductor M F contain in a marked amount the protein with molecular mass equal to that of paramyosin. Heart M F incorporate the protein with molecular mass smaller than that of paramyosin, Fig. 1, In order to determine the subtle peculiarities of M F proteins under investigation, i.e. charge variety, posttranslational modifications, we used urea electrophoresis. The electrophoregrams for M F from different tissues are presented in Fig. 2, which testifies to the coincidence of relative mobilities of actin, TM, EDTA- and SH-MLC from various muscles. These data are confirmed by the results of electrophoretic runs for mixtures of M F in the same gel. It should be noted that in this electrophoretic system (pH 8.6) the Spisula and smooth muscle actins have equal mobilities differing from that of skeletal muscle actin. IEF of a wide range of pH 3.5 10 was used for the separation of TM and MLC types from Spisula tissues. All the TM were shown to have the same pl value which was confirmed by the investigation of their mixtures in the same tube. Fig. 3. The technique of band cutting and their transferring onto IEF gels was employed for SH- and EDTA-MLC analysis. Irrespective of tissue, the homologous MLC are characterized by the same pl-value. It is noteworthy that
--
pM m
i
A--Ore TM--
o
b
Fig. 1. SDS-electrophoregrams of myofibril-like preparations isolated from the adductor muscle (a) and heart (b) of Spisula s. MHC--myosin heavy chains, PM--paramyosin, A--actin, TH--tropomyosin. Electrophoresis in PAG was made according Laemmli, 1970.
I
I
--A
0
I
I
II
--TM
"-I D
--
-
w
I
EDTA -MLC
i
-- SH -- MLC
o
b
c
d
e
f
Fig. 2. Urea-electrophoresis of myofibril-like preparations from the following Spisula tissues: [a) adductor; (b) heart; (c) mantle; (d) leg elevator; (e) mixture of the adductor and leg myofibrils: (f) mixture of the adductor and heart myofibrils. Electrophoresis in PAG was made according Perrie & Perry, 1970. A--actin, TM--tropomyosin. EDTA-MLC and SH-MLC-EDTA and SH myosin light chains.
Major contractile proteins of mollusc
475
I
o
a
b
c
d
a
b
c
d
B Fig. 3. Isoelectric focusing in 5~o PAG, 2~o Ampholine LKB pH 3, 5-10. After urea electrophoresis protein bands were excised from the gels and transferred onto the surface of IEF gels. (A) IEF of tropomyosins: (a) adductor, (b) heart, (c) the mixture of adductor and mantle, (d) the mixture of heart and leg elevator. Into each tube trace amount of rabbit actin was added. (B) IEF of SH myosin light chains: (a) adductor, (b) heart, (c) mantle, (d) the mixture of adductor and leg elevator.
/LT-actins from gel slices, focused at the range of pH 5-7, occupied a fixed position relative to skeletal a-form, Fig. 4. Spisula actin of all 4 muscles was concentrated just under the band of/~-actin. At the same time the former was coincidental with Patinopecten actin offering a case of species specificity of actin isotypes in molluscs. DISCUSSION
As mentioned above all the electrophoretic techniques used in the present work permit to separate contractile proteins with high resolution in the following parameters: molecular mass (SDS-disc-electrophoresis), charge: mass ratio (urea-electrophoresis) and isoelectric point (IEF). Contrary to vertebrates in the case of molluscs, these methods turned out to be ineffective in the detection of protein polymorphism. Although one cannot definitely assert that mollusc protein isoforms are identical, at least two facts may support this statement: (1) only one isotype of each MCP was observed; (2) irrespective of separation technique no difference between all homologous proteins was revealed.
The absence of tissue specificity of mollusc MCP may be due to one of the following factors: (1) only one structural gene exists for each MCP; (2) only one "major" gene copy is selected from the family of closely related genes. The analysis of actin isoforms and the corresponding genes of Dictyostelium discoideum confirmed the validity of the second models with groups of "silent" genes (McLeod et al., 1980). The same way of expression may be proposed for MCP-coding genes of Spisula. Thinking so we can suggest that expression of "silent" genes is feasible in some specialized cells of mollusc or that it takes place at certain stages of development. Our preliminary data show that even in non-muscle tissues of mollusc and the sea star only fl-like form of actin is present (unpublished results). The four tissues of mollusc studied (heart, obliquely-striated of adductor, smooth of leg elevator and mantle) have different morphological and physiological properties, but high-resolution electrophoretic analysis allowed us to detect only one isotype for every MCP. It should be emphasized that the ultrastructural features of contractile apparatus are not neccessarily determined by the presence of any given
476
B.A. MARGULISet al.
7"
a
I
It
11
b
c
d
°
6
•
f
-
g
$
h
Fig. 4. Isoelectric focusing in 5'~o PAG, 1, 5'!o Ampholine LKB pH 5 7, 0,5",, Ampholine pH 3,5 10. The actins from bands were focused. (a) rabbit skeletal ct-actin, (b) adductor, (c) leg elevator, (d) heart, (e) mantle, (f) adductor plus Patinopecten adductor actin, (g) Patinopecten actin, lh) the mixture of rabbit ~-actin and fl,7-actins from chicken gizzard. Rabbit actin added in tubes (b), (c) and {d) in various proportions. M C P isoform but may be due to changes in content of certain M C P in tissues (Margulis et al., 1979) and/or to the presence of some minor, regulatory proteins. REFERENCES DE COUET H. G., MAZANDER K.-D. & GROSCHEL-STEWART U. (1980) A study of invertebrate actins by isoelectric focusing and immunodiffusion. Experientia 36, 40~405. DEVLIN R. B. & EMERSON C. P. (1978) Coordinate regulation of contractile protein synthesis during myoblast differentiation. Cell 13, 599-612. ETLINGER J. D., ZAK R. & FISHMAND. A. (1976) Compositional studies of myofibrils from rabbit skeletal muscle. J. Cell Biol. 68, 123-141. GABBIANI G., SCHMID E., WINTER S., CHAPONNIER C., DE CHASTONEY C., VANDEKERCKHOVEJ. & WEBER K. (19811 Vascular smooth muscle cells differ from other smooth muscle cells: predominance of vimentin filaments and a specific a-type actin. Proc. natn. Acad. Sci. U.S.A. 78, 298-302. HOROVITCH S. J., STORTI R. V., RICH A. & PARDUE M. L. (1979) Multiple actins in Drosophila melanogaster. J. Cell Biol. 82, 86-92. KORN E. D. (1978) Biochemistry of actomyosin-dependent cell motility. Proc. hath. Acad. Sci. U.S.A. 75, 588-599. LAEMMLIU. K. (1970) Clevage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-685. MARGULIS B. A. & PINAEV G. P. (1976) The species specificity of the contractile protein composition of the bivalve molluscs. Comp. Biochem. Physiol. 55B, 189-194.
MARGULIS B. A., BOBROVA I. F., MASHANSKI V. F. & PINAEV G. P. (1979) Major myofibular protein content and the structure of mollusc adductor contractile apparatus. Comp. Biochem. Physiol. 64A, 291 298. McLE,OD C., F1RTEL R A. & PAPKOFFJ. (1980) Regulation of actin gene expression during spore germination in Dictyostelium discoideum. Develop. Biol. 76, 263 274. MIKAWA T., TAKEDA S., SHIMIZU T. & KITAURAZ. (1981) Gene expression of myofibular proteins in single muscle fibers of adult chicken: micro two-dimensional gel electrophoresis analysis. J. Biochem. (Tokyo) 89, 1951-1962. PERRIE W. T. ~,~ PERRY S. V. (1970) An electrophoretic study of the low-molecular-weight components of myosin. Biochem. J. 119, 31 38. SCHACHAT F.. GARCEA R. L. & EPSTEIN H. F. (1978) Myosin exist as homodimers of heavy chains: demonstration with specific antibody purified by nematode mutant myosin affinity chromatography. Cell 15, 405-411. SZENT-GY(SRGYI A. G. (1975) Calcium regulation of muscle contraction. Biophys. J. 15, 707 723. VANDEKERCKHOVEJ. • WEBER K. (1978) At least six different actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino-terminal tryptic peptides. J. molec. Biol. 126, 783-802. VANDEKERCKHOVEJ. & WEBER K. (1980) Vegetative Dictiostetium cells containing 17 actin genes express a single major actin. Nature, Lond. 284, 475 477. YOUNG O. A. & DAVEYC. L. (1981) Electrophoretic analysis of proteins from single bovine muscle fibers. Biochem. J. 195, 317 327. ZECHEL K. & WEBER K. (1978) Actins from mammals, bird, fish and slime mold characterized by isoelectric focusing in polyacrylamide gels. Eur. J. Bioehem. 89, 105 112.