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[35] Cytoplasmic tropomyosins
[35]
CYTOPLASMICTROPOMYOSINS
373
cle actin in SDS-polyacrylamide with an apparent molecular weight of 48,000. The amino acid composition of brain actin is similar though not identical to that of skeletal muscle actin, r Cytoplasmic actins, like their skeletal muscle counterpart, have been shown to contain the amino acid N-methylhistidine. 8 The actins, muscle and cytoplasmic, will polymerize under the appropriate ionic conditions (i.e., in 2 mM MgC12 or in 0.1 M KC1) to form fibrous actin, F-actin, a 7-nm wide, 35-nm half-pitch, double-stranded helix of actin monomers. The actins will activate the Mg2+-ATPase of myosin. Actin is a protein found in most, if not all, eukaryotic cells. It is well characterized as part of the highly organized contractile apparatus in mammalian skeletal muscle. Cytoplasmic actin is less well characterized, and its function in nonmuscle cells is not as clearly defined. 9 Although actin from skeletal muscle can be purified in high yield by extraction from acetone powder, 2 this procedure has not been as successful when used with nonmuscle cells, except in the purification of embryonic chick brain actin.'° We have used a gentle purification procedure similar to that used by Gordon et al.l~ for the purification of Acanthamoeba actin to isolate actin from porcine brain. 7 7 j. p. Weir and D. W. Frederiksen, Arch. Biochem. Biophys. 203, 1 (1980). M. Clarke and J. A. Spudich, Annu. Rev. Biochem. 46, 797 (1977). 9 H. G. M a n n h e r z and R. S. Goody, Annu. Rev. Biochem. 45, 427 (1976). ~0 j. D. Pardee and J. R. B a m b u r g , Biochemistry 18, 2245 (1979). H D. J. Gordon, E. Eisenberg, and E. D. K o r n , J . Biol. Chem. 251, 4778 (1976).
[35] Cytoplasmic Tropomyosins By
ISAAC C O H E N
After the isolation of tropomyosin from human blood platelets,' several other cytoplasmic tropomyosins were purified from a variety of nonmuscle tissues, such as brain, pancreas, fibroblasts, z sea urchin eggs, 3 bovine thymus, and SV40 virus-transformed 3T3 tissue culture cells? These all resemble muscle tropomyosin in having a two-chain a-helical coiled-coil structure, in forming paracrystals in the presence of divalent cations, and in the amino acid composition. They differ, however, from i I. C o h e n a n d C. C o h e n , J. Mol. Biol. 68, 383 (1972). R. E. Fine a n d A. L. Blitz, J. Mol. Biol. 95, 447 (1975). a T. Ishimoda-Takagi, J. Biochem. (Tokyo) 83, 1757 (1978). 4 A. B r e t s c h e r a n d K. Weber, FEBS Left. 85, 145 (1978).
METHODS IN ENZYMOLOGY.VOL. 85
Copyright © 1982by Academic Press, lnc. All rights of reproduction in any form reserved. ISBN 0-12-181985-X
374
MOTILITY APPARATUS IN NONMUSCLE CELLS
[35]
their muscle counterpart by their size and amino acid sequence. These differences have prompted Fine et al. 5 to distinguish two classes of tropomyosins, a muscle type and a nonmuscle type. Purification The purification procedure for platelet tropomyosin 1 has been used for the isolation of most nonmuscle tropomyosins. The unusual resistance of tropomyosin and other a-helical fibrous proteins to organic solvents and heat treatment 6 has provided the basis for the isolation procedure of cytoplasmic tropomyosins. The tissue of interest is first freed from any vascular or contaminating red cell material by either dissection or differential centrifugation. Then it is homogenized in a Waring blender with 20 volumes of absolute ethanol at 0°. After centrifugation the precipitate is resuspended in 20 volumes of anhydrous ethyl ether at 0°. The precipitate collected by centrifugation is then treated once more with ethanol and ether and air-dried. Dithiothreitol (2 mM) should be added in the subsequent steps. The dried residue is extracted overnight at 4 ° in 1 M KC1 adjusted to pH 7.0. The insoluble material is removed by centrifugation, and the extract is placed in a boiling water bath for 10 min. All the following steps are carried out at 2°. After centrifugation at 100,000 g for 1 hr, the supernatant solution is further purified by isoelectric precipitation, adjusting the pH to 4.1 with 0.1 N HC1. The precipitate is collected by centrifugation and dissolved in 0.2 M KC1, 0.03 M sodium phosphate buffer. An ammonium sulfate fractionation is added as a final step. 5 The pellet from a 40% saturation is discarded, and ammonium sulfate is added to 53% saturation. The resulting precipitate is dissolved in 0.6M KC1, 0.03M sodium phosphate buffer, pH 7.0, and exhaustively dialyzed against the same solvent. About 30 mg of protein, representing 1.5% at the total protein content, is obtained from 1012 human platelets and 0.5 mg per gram of dried brain, corresponding to 0.1% of total brain protein. Properties Purity. The purified cytoplasmic tropomyosins prepared thus far generally show a single band under reducing conditions in a continuous sodium dodecyl sulfate (SDS)-polyacrylamide phosphate buffer electrophoresis system. ~A closely spaced doublet band, possibly representing a and fl polypeptide chains, has been obtained with brain and platelet 5 R. E. Fine, A. L. Blitz, S. E. Hitchcock, and B. K a m i n e r , Nature (London), New Biol. 245, 182 (1973). e K. Bailey, Biochem. J. 43, 271 0948). 7 K. W e b e r and M. O s b o r n , J. Biol. Chem. 244, 4406 (1969).
[35]
CYTOPLASMICTROPOMYOSINS
375
t r o p o m y o s i n s 4,s when applied u n d e r r e d u c e d conditions to discontinuous S D S - p o l y a c r y l a m i d e Tris-HCl electrophoresis sytems.9 T h e relative electrophoretic mobility of t r o p o m y o s i n b a n d s was similar in both systems. Stability. L i k e their muscle c o u n t e r p a r t , c y t o p l a s m i c t r o p o m y o s i n s are v e r y stable. T h e y can be boiled at 100 °, frozen, or freeze-dried without losing their properties. After dissociation in 8 M urea they can be fully renatured after exhaustive dialysis.l Physical a n d Chemical Properties. Like muscle t r o p o m y o s i n s , cytoplasmic t r o p o m y o s i n s are fully a-helical two-chain molecules. T h e y have a subunit molecular weight of 30,000, h o w e v e r , and thus are one-seventh smaller than muscle t r o p o m y o s i n s . T h e molecular weight c o r r e s p o n d s well with the 34.5-nm axial period of t r o p o m y o s i n divalent cation-induced paracrystals. This value, which is close to the length of the molecule, is smaller than the 40-nm axial period of muscle t r o p o m y o s i n paracrystals.10 T h e amino acid composition of nonmuscle t r o p o m y o s i n s is similar to that of muscle t r o p o m y o s i n s , with o v e r 50% of polar residues and the absence of proline. 1"2"5The ratio of L y s to Arg is 1.3, similar to that of some invertebrate t r o p o m y o s i n s ? ~ The peptide maps show at least 10 peptides with mobilities different from those found in maps of muscle t r o p o m y o s i n , z T h e NH2- and C O O H - t e r m i n a l sequences of platelet t r o p o m y o s i n show some differences from skeletal t r o p o m y o s i n s , s Since nonmuscle t r o p o m y o s i n s are six-sevenths the size of muscle tropomyosin, their c o m p l e t e amino acid sequence m a y reveal six similar regions of 42 residues, each which would span six instead of seven actins. 12 These considerations m a y account for the w e a k binding to actin and t r o p o n i n ? 'la Indirect immunofluorescence studies of fibroblasts show, h o w e v e r , that most actin and t r o p o m y o s i n fibers have similar patterns, suggesting a cytoskeletal association of these two molecules.t4 Cytoplasmic t r o p o m y o s i n s can substitute for their muscle c o u n t e r p a r t in conferring Ca z+ sensitivity to a t r o p o n i n - a c t o m y o s i n ATPase system, 5 suggesting the possibility that they m a y h a v e a calcium regulatory role ~ in addition to their cytoskeletal role. Acknowledgments The author is grateful to Dr. Carolyn Cohen (Brandeis University, Waltham, Massachusetts) for careful review of the manuscript. a G. P. C6t6, W. G. Lewis, and L. B. Smillie, FEBS Lett. 91, 237 (1978). 9 U. K. Laemmli, Nature (London), 227, 680 (1970). lo C. Cohen and W. Longley, Science 152, 794 (1966). 11G. R, Millward and E. F. Woods, J. Mol. Biol. 52, 585 (1970). 12A. D. McLachlan and M. Stewart, J. Mol. Biol. 103, 271 (1976). la G. P. C6te, W. G. Lewis, M. D. Pato, and L. B. Smillie, FEBS Lett. 94, 131 (1978). ~4E. Lazarides, J. Cell Biol. 68, 202 (1976). 15I. Cohen, E. Kaminski, and A. deVries, FEBS Lett. 34, 315 (1973).
CYTOPLASMICTROPOMYOSINS
373
cle actin in SDS-polyacrylamide with an apparent molecular weight of 48,000. The amino acid composition of brain actin is similar though not identical to that of skeletal muscle actin, r Cytoplasmic actins, like their skeletal muscle counterpart, have been shown to contain the amino acid N-methylhistidine. 8 The actins, muscle and cytoplasmic, will polymerize under the appropriate ionic conditions (i.e., in 2 mM MgC12 or in 0.1 M KC1) to form fibrous actin, F-actin, a 7-nm wide, 35-nm half-pitch, double-stranded helix of actin monomers. The actins will activate the Mg2+-ATPase of myosin. Actin is a protein found in most, if not all, eukaryotic cells. It is well characterized as part of the highly organized contractile apparatus in mammalian skeletal muscle. Cytoplasmic actin is less well characterized, and its function in nonmuscle cells is not as clearly defined. 9 Although actin from skeletal muscle can be purified in high yield by extraction from acetone powder, 2 this procedure has not been as successful when used with nonmuscle cells, except in the purification of embryonic chick brain actin.'° We have used a gentle purification procedure similar to that used by Gordon et al.l~ for the purification of Acanthamoeba actin to isolate actin from porcine brain. 7 7 j. p. Weir and D. W. Frederiksen, Arch. Biochem. Biophys. 203, 1 (1980). M. Clarke and J. A. Spudich, Annu. Rev. Biochem. 46, 797 (1977). 9 H. G. M a n n h e r z and R. S. Goody, Annu. Rev. Biochem. 45, 427 (1976). ~0 j. D. Pardee and J. R. B a m b u r g , Biochemistry 18, 2245 (1979). H D. J. Gordon, E. Eisenberg, and E. D. K o r n , J . Biol. Chem. 251, 4778 (1976).
[35] Cytoplasmic Tropomyosins By
ISAAC C O H E N
After the isolation of tropomyosin from human blood platelets,' several other cytoplasmic tropomyosins were purified from a variety of nonmuscle tissues, such as brain, pancreas, fibroblasts, z sea urchin eggs, 3 bovine thymus, and SV40 virus-transformed 3T3 tissue culture cells? These all resemble muscle tropomyosin in having a two-chain a-helical coiled-coil structure, in forming paracrystals in the presence of divalent cations, and in the amino acid composition. They differ, however, from i I. C o h e n a n d C. C o h e n , J. Mol. Biol. 68, 383 (1972). R. E. Fine a n d A. L. Blitz, J. Mol. Biol. 95, 447 (1975). a T. Ishimoda-Takagi, J. Biochem. (Tokyo) 83, 1757 (1978). 4 A. B r e t s c h e r a n d K. Weber, FEBS Left. 85, 145 (1978).
METHODS IN ENZYMOLOGY.VOL. 85
Copyright © 1982by Academic Press, lnc. All rights of reproduction in any form reserved. ISBN 0-12-181985-X
374
MOTILITY APPARATUS IN NONMUSCLE CELLS
[35]
their muscle counterpart by their size and amino acid sequence. These differences have prompted Fine et al. 5 to distinguish two classes of tropomyosins, a muscle type and a nonmuscle type. Purification The purification procedure for platelet tropomyosin 1 has been used for the isolation of most nonmuscle tropomyosins. The unusual resistance of tropomyosin and other a-helical fibrous proteins to organic solvents and heat treatment 6 has provided the basis for the isolation procedure of cytoplasmic tropomyosins. The tissue of interest is first freed from any vascular or contaminating red cell material by either dissection or differential centrifugation. Then it is homogenized in a Waring blender with 20 volumes of absolute ethanol at 0°. After centrifugation the precipitate is resuspended in 20 volumes of anhydrous ethyl ether at 0°. The precipitate collected by centrifugation is then treated once more with ethanol and ether and air-dried. Dithiothreitol (2 mM) should be added in the subsequent steps. The dried residue is extracted overnight at 4 ° in 1 M KC1 adjusted to pH 7.0. The insoluble material is removed by centrifugation, and the extract is placed in a boiling water bath for 10 min. All the following steps are carried out at 2°. After centrifugation at 100,000 g for 1 hr, the supernatant solution is further purified by isoelectric precipitation, adjusting the pH to 4.1 with 0.1 N HC1. The precipitate is collected by centrifugation and dissolved in 0.2 M KC1, 0.03 M sodium phosphate buffer. An ammonium sulfate fractionation is added as a final step. 5 The pellet from a 40% saturation is discarded, and ammonium sulfate is added to 53% saturation. The resulting precipitate is dissolved in 0.6M KC1, 0.03M sodium phosphate buffer, pH 7.0, and exhaustively dialyzed against the same solvent. About 30 mg of protein, representing 1.5% at the total protein content, is obtained from 1012 human platelets and 0.5 mg per gram of dried brain, corresponding to 0.1% of total brain protein. Properties Purity. The purified cytoplasmic tropomyosins prepared thus far generally show a single band under reducing conditions in a continuous sodium dodecyl sulfate (SDS)-polyacrylamide phosphate buffer electrophoresis system. ~A closely spaced doublet band, possibly representing a and fl polypeptide chains, has been obtained with brain and platelet 5 R. E. Fine, A. L. Blitz, S. E. Hitchcock, and B. K a m i n e r , Nature (London), New Biol. 245, 182 (1973). e K. Bailey, Biochem. J. 43, 271 0948). 7 K. W e b e r and M. O s b o r n , J. Biol. Chem. 244, 4406 (1969).
[35]
CYTOPLASMICTROPOMYOSINS
375
t r o p o m y o s i n s 4,s when applied u n d e r r e d u c e d conditions to discontinuous S D S - p o l y a c r y l a m i d e Tris-HCl electrophoresis sytems.9 T h e relative electrophoretic mobility of t r o p o m y o s i n b a n d s was similar in both systems. Stability. L i k e their muscle c o u n t e r p a r t , c y t o p l a s m i c t r o p o m y o s i n s are v e r y stable. T h e y can be boiled at 100 °, frozen, or freeze-dried without losing their properties. After dissociation in 8 M urea they can be fully renatured after exhaustive dialysis.l Physical a n d Chemical Properties. Like muscle t r o p o m y o s i n s , cytoplasmic t r o p o m y o s i n s are fully a-helical two-chain molecules. T h e y have a subunit molecular weight of 30,000, h o w e v e r , and thus are one-seventh smaller than muscle t r o p o m y o s i n s . T h e molecular weight c o r r e s p o n d s well with the 34.5-nm axial period of t r o p o m y o s i n divalent cation-induced paracrystals. This value, which is close to the length of the molecule, is smaller than the 40-nm axial period of muscle t r o p o m y o s i n paracrystals.10 T h e amino acid composition of nonmuscle t r o p o m y o s i n s is similar to that of muscle t r o p o m y o s i n s , with o v e r 50% of polar residues and the absence of proline. 1"2"5The ratio of L y s to Arg is 1.3, similar to that of some invertebrate t r o p o m y o s i n s ? ~ The peptide maps show at least 10 peptides with mobilities different from those found in maps of muscle t r o p o m y o s i n , z T h e NH2- and C O O H - t e r m i n a l sequences of platelet t r o p o m y o s i n show some differences from skeletal t r o p o m y o s i n s , s Since nonmuscle t r o p o m y o s i n s are six-sevenths the size of muscle tropomyosin, their c o m p l e t e amino acid sequence m a y reveal six similar regions of 42 residues, each which would span six instead of seven actins. 12 These considerations m a y account for the w e a k binding to actin and t r o p o n i n ? 'la Indirect immunofluorescence studies of fibroblasts show, h o w e v e r , that most actin and t r o p o m y o s i n fibers have similar patterns, suggesting a cytoskeletal association of these two molecules.t4 Cytoplasmic t r o p o m y o s i n s can substitute for their muscle c o u n t e r p a r t in conferring Ca z+ sensitivity to a t r o p o n i n - a c t o m y o s i n ATPase system, 5 suggesting the possibility that they m a y h a v e a calcium regulatory role ~ in addition to their cytoskeletal role. Acknowledgments The author is grateful to Dr. Carolyn Cohen (Brandeis University, Waltham, Massachusetts) for careful review of the manuscript. a G. P. C6t6, W. G. Lewis, and L. B. Smillie, FEBS Lett. 91, 237 (1978). 9 U. K. Laemmli, Nature (London), 227, 680 (1970). lo C. Cohen and W. Longley, Science 152, 794 (1966). 11G. R, Millward and E. F. Woods, J. Mol. Biol. 52, 585 (1970). 12A. D. McLachlan and M. Stewart, J. Mol. Biol. 103, 271 (1976). la G. P. C6te, W. G. Lewis, M. D. Pato, and L. B. Smillie, FEBS Lett. 94, 131 (1978). ~4E. Lazarides, J. Cell Biol. 68, 202 (1976). 15I. Cohen, E. Kaminski, and A. deVries, FEBS Lett. 34, 315 (1973).