- Email: [email protected]
[17] Preparation problems unique to Mercenaria paramyosin
160
STRIATED MUSCLE CHEMISTRY
[17]
species. 5 Similarly, a drop o f paracrystals on a glass slide can be stained with anti-paramyosin by the indirect fluorescent antibody technique 5'za and will appear under dark-field microscopy as brilliant yellow-green needles. Controls for both types o f staining include use of anti-paramyosin previously absorbed with paramyosin and use o f antibody raised against a different protein. 5~a Acknowledgments W e thank H. King and B. Gilfillanfor technical assistance and Phyllis Fliegelman for secretarial help. The work described in this chapter was supported by U S P H S Grants GM21956 and HL15835 to the Pennsylvania Muscle Institute.
23R. J. C. Levine, M. M. Dewey, and G. deVillafranca, J. Cell Biol. 55, 221 (1972).
[1 7] P r e p a r a t i o n
Problems
Unique
to Mercenaria
Paramyosin
B y SONJA KRAUSE and NANCY LETKO MUNSON
As mentioned several times in this volume [16], paramyosin prepared from Mercenaria mercenaria is easily degraded during the extraction procedure, so that great care must be taken to obtain the native protein. Although technique 3 from this volume [16] can be used to prepare the native molecule, a-paramyosin, 1 yields are very small. If technique 3 is nevertheless used, it is suggested that not only 10 m M EDTA, but also 0.5 m M dithiothreitol (DTT), be added to all the solutions used during the extraction. The D T T serves not only to keep the sulfhydryl residues in the reduced state as they are presumed to be in the native molecule, 1 but also to prevent degradation. ,As a matter of fact, evidence exists that the addition o f 0.5 m M D T T to the solutions without any EDTA at all prevents the degradation of paramyosin all by itself, z Increased yields of native paramyosin can be obtained by suitable modification of the acid extraction procedure o f Hodge, a referred to as technique 1 in this volume [16]. In this case, the resulting paramyosin has been called acid-paramyosin, even though it, too, is assumed to be the native molecule, o~-Paramyosin and acid-paramyosin have the same moI W. F. Stafford and D. A. Yphantis, Biochem. Biophys. Res. Commun. 49, 848 (1972). 2 B. D. Gaylinn, Ph.D. Dissertation, Rensselaer Polytechnic Institute, Troy; New York, 1980. 3 A. J. Hodge, Proc. Natl. Acad. Sci. U.S.A. 38, 850 (1952).
METHODS IN ENZYMOLOGY, VOL. 85
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181985-X
[ 17]
Mercenaria PARAMYOSIN
161
lecular weight, using SDS-gel electrophoresis, and similar though not quite identical physical properties, a These slight differences in physical properties such as ultraviolet circular dichroism spectra and solubility 4 are probably due to differences in ion adsorption of paramyosin when it is prepared using different methods and solutions? For most purposes, however, a-paramyosin and acid-paramyosin are identical. The method of extraction detailed below is essentially the method of Hodge a as modified by Edwards et al., 6 but with 0.5 mM DTT included in all solutions to ensure that all sulfhydryl groups in the protein are in the reduced state. 7 Note that, in contrast to the procedures used in this volume [16], the muscles (usually the adductor muscles) of the clams are first homogenized in an acid (pH 3.4) solution instead of in an essentially neutral solution.
Solutions (all made up using distilled, deionized water) Solution A: Aqueous solution (M/6) in citric acid and 0.5 mM in DTT adjusted to pH 3.4 using concentrated KOH. Solution B: Aqueous solution, (M/6) in citric acid, 0.5 mM in DTT, and 0.1 M in KC1, adjusted to pH 3.4 using concentrated KOH. Solution C: Aqueous solution, 0.5 mM in DTT, containing 1 ml of glacial acetic acid per 250 ml of solution. Solution D: Low ionic strength buffer. Aqueous solution, 1 mM in K2HPO4, 9 mM in KH2PO4, 10 mM in KsEDTA, and 0.5 mM in DTI', adjusted to pH 6.0 by addition of concentrated KOH or HCI. This solution has contained 3 mM sodium azide in the past, but this appears to have no major effect on the paramyosin preparation. Solution E: High ionic strength buffer. Aqueous solution, 0.6M in KC1, 9 mM in K2HPO4, 1 mM in KHzPO4, 10 mM in K2EDTA, and 0.5 mM in DTT. The pH should be between 7.0 and 7.5. This solution has contained 3 mM sodium azide in the past, but this appears to have no major effect on the paramyosin preparation.
Preparation of Dialysis Tubing The dialysis tubing is boiled twice in distilled deionized water; during this procedure, the tubing should rise to the surface of the container. The tubing is then boiled twice in 10 mM K2EDTA, after which it is again boiled twice in distilled deionized water. 4 L. B. Cooley, Ph.D. Dissertation, Rensselaer Polytechnic Institute Troy, New York, 1978. 5 N. Letko Munson, work in our laboratory (1980). H. H. Edwards, W. H. Johnson, and J. P. Merrick, Biochemistry 16, 2255 (1977). 7 W. F. Stafford, Ph.D. Dissertation, University of Connecticut, Storrs, 1973.
162
STRIATED MUSCLE CHEMISTRY
[17]
Temperature All extraction procedures including centrifugation are done at 4 °.
Special Notes 1. Seemingly minor changes in the time used for a resuspension of centrifuged pellet or for a dialysis can affect some physical properties of the resulting paramyosin. Addition of sodium azide to solution D and E can have the same effect. Physical properties that appear to be affected included solubility, 5 electro-optic properties, 5 and, quite probably, titration curves. 8 Although evidence exists that paramyosin may be covalently phosphorylated, a'l° there is also much evidence that the molecule can adsorb considerable phosphate TM and that changes in the number of adsorbed phosphates can change the solubility of paramyosin? '11 2. Centrifugation speeds given are for a Sorvall Superspeed RC 2-B preparative ultracentrifuge. For day 1, the speeds are given for a GSA rotor that holds 250-ml bottles, while for subsequent days, the speeds are given for a SS-34 rotor that holds 50-ml centrifuge tubes.
Day 1 The muscles are dissected away from the rest of the animals and weighed (a dozen clams yield 60-85 g of adductor muscle, both transparent and opaque). The muscles are then placed in a blender with 6 ml of solution A per gram of muscle, alternating between "blend" and " c h o p " for I min; the blend has a milky light pink color. After centrifugation for 20 min at 6000 rpm, the supernatant is discarded and the pellet is blended as before, for 1 min, with 6 ml of solution B per gram of muscle. After stirring for about 3 hr, the blend is centrifuged as before, and the supernatant is again discarded. The pellet is now a light buff color. After blending and chopping the pellet with 4 ml of solution C per gram of muscle for a total of 10 sec, the solution is stirred overnight.
Day 2 The now quite viscous solution is centrifuged at 18,000 rpm for 20 min, and the supernatant is poured into a dialysis tube. The supernatant is dialyzed against 5 times its volume of solution D for 2 hr and then against a L. a R. 10 L. 11 L.
B. Cooley and S. Krause, Biophys. J. 32, 755 (1980). K. Achazi, Eur. J. Physiol. 379, 197 (1979). Radlick and W. H. Johnson, private communication. B. Cooley, W. H. Johnson, and S. Krause, J. Biol. Chem. 254, 2195 (1979).
[ 17]
Mercenaria PARAMYOSIN
163
the same volume of fresh solution D for another 2 hr. The solution inside the dialysis tubing will have a gel-like texture, and this "gel" is then centrifuged at 12,000 rpm for 20 min, and the supernatant is discarded if possible (a distinct pellet does not always form). (This is also the return point from day 3 if necessary.) From a total volume of solution E equal to that of both batches of solution D used above, about 3 ml per gram of muscle are added to the pellets and nonseparated material. The pellets and gel are disrupted manually (a plastic spatula may be used) as much as possible, and the suspension is stirred for 1 hr, after which the pH is adjusted using concentrated KOH to the same pH as solution E. After stirring for another hour, the pH is adjusted again if necessary. The suspension is then dialyzed against the remainder of solution E overnight. Day 3
The solution in the dialysis tubes, much less gel-like than on day 2 and containing some precipitate, is centrifuged at 18,000 rpm for 20 min, and the supernatant is dialyzed against five times its volume of solution D. The precipitate is saved, at least until the supernatant has dialyzed for about half an hour. If a large amount of fluffy precipitate appears in the dialysis tubes within half an hour, the old precipitate may be discarded. Whether a large amount of fluffy precipitate appears in the dialysis tubes or not, the dialysis is continued and the dialysis tubes are squeezed every half hour to keep the precipitate from adhering to the tubing. After a total of 2 hr, the dialysis solution is replaced by an equal volume of fresh solution D. Two hours later, the suspension is centrifuged at 12,000 rpm for 20 min. If very little precipitate appeared in the dialysis tubes, it is combined with the precipitate saved from the previous centrifugation and the combined precipitates are broken up into a small volume of solution E (see day. 2--the return point from day 3), and the procedures involving solution E on day 2 must be repeated, including the overnight dialysis. In this case, an appreciable amount of paramyosin was trapped in the earlier precipitate and a second attempt must be made to extract it. When a return to the procedures of day 2 becomes unnecessary, the white precipitate is manually dispersed into 70-80% of the volume of solution E that was used to disperse the pellets on day 2 and the suspension is stirred for 2 hr. After adjusting the pH of the suspension to that of solution E using concentrated KOH, the solution is dialyzed overnight against approximately 10 times the volume of solution E in which the precipitate was dispersed.
164
STRIATED MUSCLE CHEMISTRY
[18]
Day 4 There may be a small amount of flocculent precipitate in the dialysis tubes. After centrifugation at 18,000 rpm for 20 min, the precipitate is discarded and the supernatant is dialyzed for 2 hr against 5 times its volume of solution D; and then for 2 hr more against the same volume of fresh solution D. The resulting milky suspension is centrifuged at 12,000 rpm for 20 min, and the supernatant is discarded. The precipitate is manually dispersed into 10% less solution E than on day 3, and the suspension is stirred for 2 hr. After adjusting the pH of the suspension to that of solution E using concentrated KOH, the suspension is dialyzed overnight against 10 times the volume of solution E used to disperse the precipitate.
Day 5 The material inside the dialysis bag should be a slightly cloudy solution that is centrifuged at 18,000 rpm for 20 min. The precipitate is discarded. The optical density of the clear solution is measured at 280 nm and at 260 nm. If OD28o: OD2e0 < 2.0, the procedure of day 4 is repeated. In a typical preparation in which the procedure of day 4 does not need to be repeated, 12 large clams yield 84 g of adductor muscle, which yield about 900 mg of paramyosin.
[18] P u r i f i c a t i o n o f M u s c l e A c t i n
By JOEL D. PARDEE and JAMES A. SPUDICH The opportunity to study the molecular events responsible for muscle contraction and cell motility was made possible in the early 1940s by Banga and Szent-Gy/irgyi I and by Straub, ~ who discovered myosin and actin in the extracts of rabbit skeletal muscle. Actin was first isolated when Straub separated the viscous protein from an actomyosin preparation. 2 Subsequent work 3 revealed that actin could be obtained in a nonviscous state (G-actin) by extracting muscle with a low ionic strength buffer, and that addition of salt induced conversion to a viscous form called F-actin (Fig. 1). An improved procedure by Straub and his col1I.
B a n g a a n d A. S z e n t - G y 6 r g y i , Studies from the Inst. Med. Chem., Univ. Szeged 1, 5 (1941). 2 F. B. S t r a u b , Studies from the Inst. Med. Chem., Univ. Szeged 2, 3 (1942). 3 F. B. S t r a u b , Studies from the Inst. Med. Chem., Univ. Szeged 3, 23 (1943).
METHODS IN ENZYMOLOGY, VOL. 85
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181985-X
STRIATED MUSCLE CHEMISTRY
[17]
species. 5 Similarly, a drop o f paracrystals on a glass slide can be stained with anti-paramyosin by the indirect fluorescent antibody technique 5'za and will appear under dark-field microscopy as brilliant yellow-green needles. Controls for both types o f staining include use of anti-paramyosin previously absorbed with paramyosin and use o f antibody raised against a different protein. 5~a Acknowledgments W e thank H. King and B. Gilfillanfor technical assistance and Phyllis Fliegelman for secretarial help. The work described in this chapter was supported by U S P H S Grants GM21956 and HL15835 to the Pennsylvania Muscle Institute.
23R. J. C. Levine, M. M. Dewey, and G. deVillafranca, J. Cell Biol. 55, 221 (1972).
[1 7] P r e p a r a t i o n
Problems
Unique
to Mercenaria
Paramyosin
B y SONJA KRAUSE and NANCY LETKO MUNSON
As mentioned several times in this volume [16], paramyosin prepared from Mercenaria mercenaria is easily degraded during the extraction procedure, so that great care must be taken to obtain the native protein. Although technique 3 from this volume [16] can be used to prepare the native molecule, a-paramyosin, 1 yields are very small. If technique 3 is nevertheless used, it is suggested that not only 10 m M EDTA, but also 0.5 m M dithiothreitol (DTT), be added to all the solutions used during the extraction. The D T T serves not only to keep the sulfhydryl residues in the reduced state as they are presumed to be in the native molecule, 1 but also to prevent degradation. ,As a matter of fact, evidence exists that the addition o f 0.5 m M D T T to the solutions without any EDTA at all prevents the degradation of paramyosin all by itself, z Increased yields of native paramyosin can be obtained by suitable modification of the acid extraction procedure o f Hodge, a referred to as technique 1 in this volume [16]. In this case, the resulting paramyosin has been called acid-paramyosin, even though it, too, is assumed to be the native molecule, o~-Paramyosin and acid-paramyosin have the same moI W. F. Stafford and D. A. Yphantis, Biochem. Biophys. Res. Commun. 49, 848 (1972). 2 B. D. Gaylinn, Ph.D. Dissertation, Rensselaer Polytechnic Institute, Troy; New York, 1980. 3 A. J. Hodge, Proc. Natl. Acad. Sci. U.S.A. 38, 850 (1952).
METHODS IN ENZYMOLOGY, VOL. 85
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181985-X
[ 17]
Mercenaria PARAMYOSIN
161
lecular weight, using SDS-gel electrophoresis, and similar though not quite identical physical properties, a These slight differences in physical properties such as ultraviolet circular dichroism spectra and solubility 4 are probably due to differences in ion adsorption of paramyosin when it is prepared using different methods and solutions? For most purposes, however, a-paramyosin and acid-paramyosin are identical. The method of extraction detailed below is essentially the method of Hodge a as modified by Edwards et al., 6 but with 0.5 mM DTT included in all solutions to ensure that all sulfhydryl groups in the protein are in the reduced state. 7 Note that, in contrast to the procedures used in this volume [16], the muscles (usually the adductor muscles) of the clams are first homogenized in an acid (pH 3.4) solution instead of in an essentially neutral solution.
Solutions (all made up using distilled, deionized water) Solution A: Aqueous solution (M/6) in citric acid and 0.5 mM in DTT adjusted to pH 3.4 using concentrated KOH. Solution B: Aqueous solution, (M/6) in citric acid, 0.5 mM in DTT, and 0.1 M in KC1, adjusted to pH 3.4 using concentrated KOH. Solution C: Aqueous solution, 0.5 mM in DTT, containing 1 ml of glacial acetic acid per 250 ml of solution. Solution D: Low ionic strength buffer. Aqueous solution, 1 mM in K2HPO4, 9 mM in KH2PO4, 10 mM in KsEDTA, and 0.5 mM in DTI', adjusted to pH 6.0 by addition of concentrated KOH or HCI. This solution has contained 3 mM sodium azide in the past, but this appears to have no major effect on the paramyosin preparation. Solution E: High ionic strength buffer. Aqueous solution, 0.6M in KC1, 9 mM in K2HPO4, 1 mM in KHzPO4, 10 mM in K2EDTA, and 0.5 mM in DTT. The pH should be between 7.0 and 7.5. This solution has contained 3 mM sodium azide in the past, but this appears to have no major effect on the paramyosin preparation.
Preparation of Dialysis Tubing The dialysis tubing is boiled twice in distilled deionized water; during this procedure, the tubing should rise to the surface of the container. The tubing is then boiled twice in 10 mM K2EDTA, after which it is again boiled twice in distilled deionized water. 4 L. B. Cooley, Ph.D. Dissertation, Rensselaer Polytechnic Institute Troy, New York, 1978. 5 N. Letko Munson, work in our laboratory (1980). H. H. Edwards, W. H. Johnson, and J. P. Merrick, Biochemistry 16, 2255 (1977). 7 W. F. Stafford, Ph.D. Dissertation, University of Connecticut, Storrs, 1973.
162
STRIATED MUSCLE CHEMISTRY
[17]
Temperature All extraction procedures including centrifugation are done at 4 °.
Special Notes 1. Seemingly minor changes in the time used for a resuspension of centrifuged pellet or for a dialysis can affect some physical properties of the resulting paramyosin. Addition of sodium azide to solution D and E can have the same effect. Physical properties that appear to be affected included solubility, 5 electro-optic properties, 5 and, quite probably, titration curves. 8 Although evidence exists that paramyosin may be covalently phosphorylated, a'l° there is also much evidence that the molecule can adsorb considerable phosphate TM and that changes in the number of adsorbed phosphates can change the solubility of paramyosin? '11 2. Centrifugation speeds given are for a Sorvall Superspeed RC 2-B preparative ultracentrifuge. For day 1, the speeds are given for a GSA rotor that holds 250-ml bottles, while for subsequent days, the speeds are given for a SS-34 rotor that holds 50-ml centrifuge tubes.
Day 1 The muscles are dissected away from the rest of the animals and weighed (a dozen clams yield 60-85 g of adductor muscle, both transparent and opaque). The muscles are then placed in a blender with 6 ml of solution A per gram of muscle, alternating between "blend" and " c h o p " for I min; the blend has a milky light pink color. After centrifugation for 20 min at 6000 rpm, the supernatant is discarded and the pellet is blended as before, for 1 min, with 6 ml of solution B per gram of muscle. After stirring for about 3 hr, the blend is centrifuged as before, and the supernatant is again discarded. The pellet is now a light buff color. After blending and chopping the pellet with 4 ml of solution C per gram of muscle for a total of 10 sec, the solution is stirred overnight.
Day 2 The now quite viscous solution is centrifuged at 18,000 rpm for 20 min, and the supernatant is poured into a dialysis tube. The supernatant is dialyzed against 5 times its volume of solution D for 2 hr and then against a L. a R. 10 L. 11 L.
B. Cooley and S. Krause, Biophys. J. 32, 755 (1980). K. Achazi, Eur. J. Physiol. 379, 197 (1979). Radlick and W. H. Johnson, private communication. B. Cooley, W. H. Johnson, and S. Krause, J. Biol. Chem. 254, 2195 (1979).
[ 17]
Mercenaria PARAMYOSIN
163
the same volume of fresh solution D for another 2 hr. The solution inside the dialysis tubing will have a gel-like texture, and this "gel" is then centrifuged at 12,000 rpm for 20 min, and the supernatant is discarded if possible (a distinct pellet does not always form). (This is also the return point from day 3 if necessary.) From a total volume of solution E equal to that of both batches of solution D used above, about 3 ml per gram of muscle are added to the pellets and nonseparated material. The pellets and gel are disrupted manually (a plastic spatula may be used) as much as possible, and the suspension is stirred for 1 hr, after which the pH is adjusted using concentrated KOH to the same pH as solution E. After stirring for another hour, the pH is adjusted again if necessary. The suspension is then dialyzed against the remainder of solution E overnight. Day 3
The solution in the dialysis tubes, much less gel-like than on day 2 and containing some precipitate, is centrifuged at 18,000 rpm for 20 min, and the supernatant is dialyzed against five times its volume of solution D. The precipitate is saved, at least until the supernatant has dialyzed for about half an hour. If a large amount of fluffy precipitate appears in the dialysis tubes within half an hour, the old precipitate may be discarded. Whether a large amount of fluffy precipitate appears in the dialysis tubes or not, the dialysis is continued and the dialysis tubes are squeezed every half hour to keep the precipitate from adhering to the tubing. After a total of 2 hr, the dialysis solution is replaced by an equal volume of fresh solution D. Two hours later, the suspension is centrifuged at 12,000 rpm for 20 min. If very little precipitate appeared in the dialysis tubes, it is combined with the precipitate saved from the previous centrifugation and the combined precipitates are broken up into a small volume of solution E (see day. 2--the return point from day 3), and the procedures involving solution E on day 2 must be repeated, including the overnight dialysis. In this case, an appreciable amount of paramyosin was trapped in the earlier precipitate and a second attempt must be made to extract it. When a return to the procedures of day 2 becomes unnecessary, the white precipitate is manually dispersed into 70-80% of the volume of solution E that was used to disperse the pellets on day 2 and the suspension is stirred for 2 hr. After adjusting the pH of the suspension to that of solution E using concentrated KOH, the solution is dialyzed overnight against approximately 10 times the volume of solution E in which the precipitate was dispersed.
164
STRIATED MUSCLE CHEMISTRY
[18]
Day 4 There may be a small amount of flocculent precipitate in the dialysis tubes. After centrifugation at 18,000 rpm for 20 min, the precipitate is discarded and the supernatant is dialyzed for 2 hr against 5 times its volume of solution D; and then for 2 hr more against the same volume of fresh solution D. The resulting milky suspension is centrifuged at 12,000 rpm for 20 min, and the supernatant is discarded. The precipitate is manually dispersed into 10% less solution E than on day 3, and the suspension is stirred for 2 hr. After adjusting the pH of the suspension to that of solution E using concentrated KOH, the suspension is dialyzed overnight against 10 times the volume of solution E used to disperse the precipitate.
Day 5 The material inside the dialysis bag should be a slightly cloudy solution that is centrifuged at 18,000 rpm for 20 min. The precipitate is discarded. The optical density of the clear solution is measured at 280 nm and at 260 nm. If OD28o: OD2e0 < 2.0, the procedure of day 4 is repeated. In a typical preparation in which the procedure of day 4 does not need to be repeated, 12 large clams yield 84 g of adductor muscle, which yield about 900 mg of paramyosin.
[18] P u r i f i c a t i o n o f M u s c l e A c t i n
By JOEL D. PARDEE and JAMES A. SPUDICH The opportunity to study the molecular events responsible for muscle contraction and cell motility was made possible in the early 1940s by Banga and Szent-Gy/irgyi I and by Straub, ~ who discovered myosin and actin in the extracts of rabbit skeletal muscle. Actin was first isolated when Straub separated the viscous protein from an actomyosin preparation. 2 Subsequent work 3 revealed that actin could be obtained in a nonviscous state (G-actin) by extracting muscle with a low ionic strength buffer, and that addition of salt induced conversion to a viscous form called F-actin (Fig. 1). An improved procedure by Straub and his col1I.
B a n g a a n d A. S z e n t - G y 6 r g y i , Studies from the Inst. Med. Chem., Univ. Szeged 1, 5 (1941). 2 F. B. S t r a u b , Studies from the Inst. Med. Chem., Univ. Szeged 2, 3 (1942). 3 F. B. S t r a u b , Studies from the Inst. Med. Chem., Univ. Szeged 3, 23 (1943).
METHODS IN ENZYMOLOGY, VOL. 85
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181985-X