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Actomyosin solubility and skeletal muscle cell emptying of horseshoe crab
Comp. Biochem. Physiol., 1970, Vol. 36, pp. 279 to 284. Pergamon Press. Printed in Great Britain
A C T O M Y O S I N S O L U B I L I T Y AND S K E L E T A L MUSCLE CELL E M P T Y I N G OF HORSESHOE CRAB* DAVID W. S T A N L E Y t Department of the Biological Sciences, Smith College, Northampton, Massachusetts (Received 31 March 1970)
A b s t r a c t - - 1 . A method has been adapted to produce sarcolemmae from
horseshoe crab muscle which is thought to depend on the association of contractile proteins to form a water-soluble complex. This can be extracted from cell segments leaving the membrane behind. 2. The solubility of aetomyosin is dependent on the degree of purity, ionic strength, concentration and the presence of substances such as divalent cations and ATP. Impure preparations of actomyosin exhibit elevated solubilities at the low ionic strength and concentration used in cell membrane extraction. INTRODUCTION THE GREAT "membrane revolution" in recent years has spurred research in preparation of cell and organdie membranes. Whereas with other cell types the preparation of a reasonably pure membrane fraction requires only choosing the proper disruption and isolation techniques (for example, see Warren et al., 1966), the muscle cell has proven to be a more complex system. A method was developed by Hultin & Westort (1969) in which cell segments of avian breast muscle were incubated several hours post-mortem at 0-5 °. Following homogenization, washing several times in dilute saline and the addition of a weak buffer, it was observed that the contents of cells would spontaneously solubilize and move out of the broken ends. In an attempt to elucidate the mechanism of the emptying reaction, Stanley & Hultin (1968) found a highly significant correlation between actomyosin content, as determined by salting out the protein, and proportion of chicken breast muscle cells that emptied. As much as 70 per cent of the salt extractable protein may be in the form of actomyosin (Stanley, unpublished data). It was postulated that the production of empty muscle cell segments is dependent on the conversion of contractile proteins to a water soluble, hence, extractable state. A puzzling aspect is the finding that the cellular extract is essentially water soluble although purified actomyosin is characterized by its insolubility in water and solubility in high salt solutions (Szent-Gyorgyi, 1945). The present communication shows that actomyosin solubility is dependent not only upon purity and ionic strength but also upon concentration. *Contribution No. 22 from the Smith College Department of the Biological Sciences. tPresent address: Faculty of Food Sciences, University of Toronto, Toronto 181, Ontario, Canada. 279
280
DAVID W. STANLEY
MATERIALS AND METHODS Muscle was removed from aU parts of freshly killed horseshoe crab (Limulus polyphemus), shipped from the Marine Biological Laboratory, Woods Hole, Massachusetts. All chemicals were reagent grade and solutions were made with deionized-distilled water. ATP was purchased from the P-L laboratories and used without further purification. The procedure used for production of empty cell segments was that of Westort & Hultin (1966) with modifications as noted in the text. Protein content was estimated by the biuret procedure. RESULTS
Horseshoe crab sarcolemmae were prepared using a method that consisted of homogenizing 20 g of minced fresh tissue in 200 ml of 0-5 mM CaCla with a Polytron Model BEW cutter (Will Corporation) for 5 see at 0-5°C, straining the resultant suspension through cheesecloth, and centrifuging the cell suspension 10 min at 750g (0-5°C). Cell segments were gathered by decantation and transferred to 50 ml plastic centrifuge tubes in which they were washed four times with 10 m M E D T A (ethylenediaminetetraacetic acid)-I mM histidine, pH 7.5; the cells were recovered after each wash by centrifuging in a clinical centrifuge at top speed for 10 sec. After the final salt wash, segments were washed once with Tris (trishydroxymethylaminomethane)-buffered water (water buffered to pH 7-5 with 1 M Tris, final concentration approximately 1 mM) and emptied by resuspending the cells in buffered water, shaking vigorously, and diluting with a large volume of Tris water-about 1 1/20 gm of original tissue. This procedure routinely gave emptying in the range of 90-95 per cent. Aging muscle 4 hr postmortem did not significantly increase the percentage of empty cells in the final preparation. Washing was necessary for emptying to occur. This effect was not specific; E D T A or 25 m M NaCI-2.5 m M histidine proved just as effective. Even when Ca 2+ ions were not added during homogenization, washing was necessary for emptying. Incubating cell segments with the buffered chloride salts of various cations at 10 m M for 10 min at room temperature prior to extraction resulted in Na + giving normal emptying while Mg ~+, Mn ~+, and Ca ~+ completely inhibited the process. In order to ascertain the role of actomyosin solubility in emptying, the solubility of a pure actomyosin fraction was investigated. By a method previously reported (de Villafranca & Naumann, 1964) actomyosin was extracted from fresh horseshoe crab muscle and subsequently purified. The solubility of this preparation as a function of ionic strength was obtained by diluting the protein to a concentration of 4-0 mg/ml. Samples of this preparation were then dialyzed against varying strengths of KCI solution (pH 7.5) for 6 hr at 0-5 °, the material centrifuged in a Spinco Model L at 100,000 g for 15 min, the supernatant protein measured and taken to represent the soluble fraction. The results are shown in Fig. 1 and are consistant with the solubility reported for other actomyosin preparations (for example, see Szent-Gyorgyi, 1945); properly purified actomyosin at this concentration undergoes a transition from virtually insoluble to high solubility between an ionic strength of 0.2 and 0.3.
281
ACTOMYOSIN SOLUBILITY IN HORSESHOE CRAB
These results, however, fail to explain the solubility characteristics of actomyosin extracted during emptying of muscle cell segments. One factor to be considered is that in preparation of sarcolemmae the last extraction is made into a large volume of Tris-buffered water. A final protein concentration of about 0.4 mg/ml is usually obtained. It was determined to test the effect of concentration 100
\
\
\
60
.S
~
40
2o
0
0.6
0.5
0.4
0-3
0.2
o'q
0
Ionic strengfh
FIG. 1. The solubility of purified horseshoe crab actomyosin as a function of ionic strength. Protein concentration = 4-0 mg/ml. not only on pure actomyosin but also upon a crude, once-precipitated actomyosin fraction and to employ an ionic strength of 0.001, that used in extraction of cell segments. Figure 2 shows typical results. These data indicate the phenomenon previously encountered, namely that decreasing protein concentration increased solubility at low ionic strengths. This holds not only for crude actomyosin but also with purified preparations, however it appears that in purifying some constituent is removed which has a solubilizing effect under these experimental conditions.
\
%
2
\
Protein concentration,
\
rag/mr
3
o Crude octomyosm - - ~ Purified octomyostn
FIG. 2. T h e solubility of horseshoe crab actomyosin as a function of protein concentration. Ionic strength -- 0"001.
20 L
(~ 40
c:
~-
60--
80
IOO
4
0.1
J
0'4
J ~
_
J
0'5
FIC. 3. T h e effect o f A T P a n d d i v a l e n t c a t i o n s o n actomyosin solubility of an unpurified preparation. Protein concentration--0.65mg]ml. Final concentrat i o n s , A T P = 5 r a M , C a 2+ -- M g *+ = 1 r a M .
0
_
0"2 0.3 Ionic strength
ATP l
ATp +.~lgZ+
Y 2oJ
,~ 4o
~o
.
c o
°"° 6C
8(:
I00
e~
),
o0 t,o
ACTOMYOSIN S O L U B I L I T Y I N HORSESHOE CRAB
283
Using the technique of Weber (1956) the effect of ATP treatment on the crude actomyosin preparation was examined. Impure protein was diluted to a concentration of 0.65 mg/ml with buffered KC1 solution to give the desired ionic strength and additions of MgC12 or CaC12 (final concentrations 1 raM), ATP (5 mM) or combinations of these were made. The resulting mixtures were centrifuged in the Spinco Model L at 100,000 g for 2 hr and the percentage of soluble protein assayed as before. These data are presented as a function of ionic strength in Fig. 3. It may be seen that the loss in solubility due to addition of either divalent cation follows the decrease in emptying of cells when these ions are added just prior to extraction. While ATP increased solubility considerably, ATP plus Mg 2+ gave an even higher dissociation. Neither of these two treatments seem dependent upon ionic strength. DISCUSSION Results with avian muscle which show that aging is helpful in promoting emptying might partially reflect a loss of ATP which promotes association of actin and myosin. This does not seem to be necessary for Limulus muscle. The observation by de Villafranca (1959, 1968) that actin and myosin are very firmly combined in this primitive animal might explain the ease and high degree of emptying associated with this preparation and also why aging is without effect. It appears that at least one function of the salt-chelator washing is to remove added Ca ~+ and also possibly the indigenous cations from the segments. It was found by de Villafranca (1968) that in 0-6M KC1 purified Limulus actomyosin exhibited the characteristic dissociation phenomenon in the presence of ATP. While Mg 2+ was not necessary to inhibit ATP hydrolysis, it aided considerably in recovery of myosin and actin after dissociation. In this work presumably the same effect was seen. The effect of Ca ~+ and Mg 2+ appears only at low ionic strength. Perhaps the presence of high levels of a monovalent cation increases the concentration of divalent cations required for precipitation as suggested by Straub (1942) and Szent-Gyorgyi (1945). It is evident that the solubility characteristics of crude and purified actomyosin preparations are quite different. The protein extracted from cell segments is the true 'natural' actomyosin; it is in combination with other factors, pr¢sumably proteins such as tropomyosin, which confer upon it distinctive solubility behaviour. These data suggest that at least four factors are responsible for determining the solubility of actomyosin, viz.-degree of purity, ionic strength, concentration, and presence of substances such as Ca 2+, Mg 2+, ATP and perhaps other, as yet unidentified, components. Acknowledgements--Elizabeth H. Foran's excellent help is acknowledged with gratitude. The writer wishes to thank Professor G. W. de Villafranca for his help and encouragement in the appraisal of the manuscript.
284
DAVID W. STANLEY
REFERENCES HULTIN H. O. & WESTORT C. (1969) Sarcolemmae from chicken skeletal muscle--1. Preparation. ~. Food 8ci. 34, 165-171. STAnLeY D. W. & HULTIN H. O. (1968) Mechanism of emptying of skeletal muscle cell segments. ~t. Cell. Biol. 39, C1-C4. ST~UB F. B. (1942) Actin. Studies Inst. Med. Chem. Univ. Szeged 2, 3-16. SZ~T-GYoRGYt A. (1945) Studies on muscle. Actaphys. stand. 9, Suppl. 25. DE VILLAFRANCAG. W., SC~INBLUM T. S. & PHILPOTT D. E. (1959) A study on the localization of contractile proteins in the muscle of the horseshoe crab (Limulus polyphemus). Biochim. biophys. Acta 34, 147-157. DE VILL~a~gAa~CAG. W. & NAUMANND. C. (1964) Some properties of the myosin B ATPase from Limulus. Comp. Biochem. Physiol. 12, 143-156. D~ VILL~aANCA G. W. (1968) Some physico-chemical properties of myosin B from the horseshoe crab, Limulus polyphemus. Comp. Biochem. Physiol. 2,6, 443-454. WARRENL., GLtCK M. C. & NASS M. K. (1966) Membranes of animal cells.--I. Methods of isolation of the surface membrane. ~t. cell. Physiol. 68, 269-288. Wm~ga A. (1956) The ultracentrifugal separation of L-myosin and actin in an actomyosin sol under the influence of ATP. Biochim. biophys. Acta 19, 345-351. W~TOaT C. & HULTIN H. O. (1966) A procedure for the preparation of empty cell segments from skeletal muscle using solutions of low ionic strength. Anal. Biochem. 16, 314-319.
Key Word Index---Muscle; horseshoe crab; actomyosin; solubility; membranes; sarcolemma; protein.
A C T O M Y O S I N S O L U B I L I T Y AND S K E L E T A L MUSCLE CELL E M P T Y I N G OF HORSESHOE CRAB* DAVID W. S T A N L E Y t Department of the Biological Sciences, Smith College, Northampton, Massachusetts (Received 31 March 1970)
A b s t r a c t - - 1 . A method has been adapted to produce sarcolemmae from
horseshoe crab muscle which is thought to depend on the association of contractile proteins to form a water-soluble complex. This can be extracted from cell segments leaving the membrane behind. 2. The solubility of aetomyosin is dependent on the degree of purity, ionic strength, concentration and the presence of substances such as divalent cations and ATP. Impure preparations of actomyosin exhibit elevated solubilities at the low ionic strength and concentration used in cell membrane extraction. INTRODUCTION THE GREAT "membrane revolution" in recent years has spurred research in preparation of cell and organdie membranes. Whereas with other cell types the preparation of a reasonably pure membrane fraction requires only choosing the proper disruption and isolation techniques (for example, see Warren et al., 1966), the muscle cell has proven to be a more complex system. A method was developed by Hultin & Westort (1969) in which cell segments of avian breast muscle were incubated several hours post-mortem at 0-5 °. Following homogenization, washing several times in dilute saline and the addition of a weak buffer, it was observed that the contents of cells would spontaneously solubilize and move out of the broken ends. In an attempt to elucidate the mechanism of the emptying reaction, Stanley & Hultin (1968) found a highly significant correlation between actomyosin content, as determined by salting out the protein, and proportion of chicken breast muscle cells that emptied. As much as 70 per cent of the salt extractable protein may be in the form of actomyosin (Stanley, unpublished data). It was postulated that the production of empty muscle cell segments is dependent on the conversion of contractile proteins to a water soluble, hence, extractable state. A puzzling aspect is the finding that the cellular extract is essentially water soluble although purified actomyosin is characterized by its insolubility in water and solubility in high salt solutions (Szent-Gyorgyi, 1945). The present communication shows that actomyosin solubility is dependent not only upon purity and ionic strength but also upon concentration. *Contribution No. 22 from the Smith College Department of the Biological Sciences. tPresent address: Faculty of Food Sciences, University of Toronto, Toronto 181, Ontario, Canada. 279
280
DAVID W. STANLEY
MATERIALS AND METHODS Muscle was removed from aU parts of freshly killed horseshoe crab (Limulus polyphemus), shipped from the Marine Biological Laboratory, Woods Hole, Massachusetts. All chemicals were reagent grade and solutions were made with deionized-distilled water. ATP was purchased from the P-L laboratories and used without further purification. The procedure used for production of empty cell segments was that of Westort & Hultin (1966) with modifications as noted in the text. Protein content was estimated by the biuret procedure. RESULTS
Horseshoe crab sarcolemmae were prepared using a method that consisted of homogenizing 20 g of minced fresh tissue in 200 ml of 0-5 mM CaCla with a Polytron Model BEW cutter (Will Corporation) for 5 see at 0-5°C, straining the resultant suspension through cheesecloth, and centrifuging the cell suspension 10 min at 750g (0-5°C). Cell segments were gathered by decantation and transferred to 50 ml plastic centrifuge tubes in which they were washed four times with 10 m M E D T A (ethylenediaminetetraacetic acid)-I mM histidine, pH 7.5; the cells were recovered after each wash by centrifuging in a clinical centrifuge at top speed for 10 sec. After the final salt wash, segments were washed once with Tris (trishydroxymethylaminomethane)-buffered water (water buffered to pH 7-5 with 1 M Tris, final concentration approximately 1 mM) and emptied by resuspending the cells in buffered water, shaking vigorously, and diluting with a large volume of Tris water-about 1 1/20 gm of original tissue. This procedure routinely gave emptying in the range of 90-95 per cent. Aging muscle 4 hr postmortem did not significantly increase the percentage of empty cells in the final preparation. Washing was necessary for emptying to occur. This effect was not specific; E D T A or 25 m M NaCI-2.5 m M histidine proved just as effective. Even when Ca 2+ ions were not added during homogenization, washing was necessary for emptying. Incubating cell segments with the buffered chloride salts of various cations at 10 m M for 10 min at room temperature prior to extraction resulted in Na + giving normal emptying while Mg ~+, Mn ~+, and Ca ~+ completely inhibited the process. In order to ascertain the role of actomyosin solubility in emptying, the solubility of a pure actomyosin fraction was investigated. By a method previously reported (de Villafranca & Naumann, 1964) actomyosin was extracted from fresh horseshoe crab muscle and subsequently purified. The solubility of this preparation as a function of ionic strength was obtained by diluting the protein to a concentration of 4-0 mg/ml. Samples of this preparation were then dialyzed against varying strengths of KCI solution (pH 7.5) for 6 hr at 0-5 °, the material centrifuged in a Spinco Model L at 100,000 g for 15 min, the supernatant protein measured and taken to represent the soluble fraction. The results are shown in Fig. 1 and are consistant with the solubility reported for other actomyosin preparations (for example, see Szent-Gyorgyi, 1945); properly purified actomyosin at this concentration undergoes a transition from virtually insoluble to high solubility between an ionic strength of 0.2 and 0.3.
281
ACTOMYOSIN SOLUBILITY IN HORSESHOE CRAB
These results, however, fail to explain the solubility characteristics of actomyosin extracted during emptying of muscle cell segments. One factor to be considered is that in preparation of sarcolemmae the last extraction is made into a large volume of Tris-buffered water. A final protein concentration of about 0.4 mg/ml is usually obtained. It was determined to test the effect of concentration 100
\
\
\
60
.S
~
40
2o
0
0.6
0.5
0.4
0-3
0.2
o'q
0
Ionic strengfh
FIG. 1. The solubility of purified horseshoe crab actomyosin as a function of ionic strength. Protein concentration = 4-0 mg/ml. not only on pure actomyosin but also upon a crude, once-precipitated actomyosin fraction and to employ an ionic strength of 0.001, that used in extraction of cell segments. Figure 2 shows typical results. These data indicate the phenomenon previously encountered, namely that decreasing protein concentration increased solubility at low ionic strengths. This holds not only for crude actomyosin but also with purified preparations, however it appears that in purifying some constituent is removed which has a solubilizing effect under these experimental conditions.
\
%
2
\
Protein concentration,
\
rag/mr
3
o Crude octomyosm - - ~ Purified octomyostn
FIG. 2. T h e solubility of horseshoe crab actomyosin as a function of protein concentration. Ionic strength -- 0"001.
20 L
(~ 40
c:
~-
60--
80
IOO
4
0.1
J
0'4
J ~
_
J
0'5
FIC. 3. T h e effect o f A T P a n d d i v a l e n t c a t i o n s o n actomyosin solubility of an unpurified preparation. Protein concentration--0.65mg]ml. Final concentrat i o n s , A T P = 5 r a M , C a 2+ -- M g *+ = 1 r a M .
0
_
0"2 0.3 Ionic strength
ATP l
ATp +.~lgZ+
Y 2oJ
,~ 4o
~o
.
c o
°"° 6C
8(:
I00
e~
),
o0 t,o
ACTOMYOSIN S O L U B I L I T Y I N HORSESHOE CRAB
283
Using the technique of Weber (1956) the effect of ATP treatment on the crude actomyosin preparation was examined. Impure protein was diluted to a concentration of 0.65 mg/ml with buffered KC1 solution to give the desired ionic strength and additions of MgC12 or CaC12 (final concentrations 1 raM), ATP (5 mM) or combinations of these were made. The resulting mixtures were centrifuged in the Spinco Model L at 100,000 g for 2 hr and the percentage of soluble protein assayed as before. These data are presented as a function of ionic strength in Fig. 3. It may be seen that the loss in solubility due to addition of either divalent cation follows the decrease in emptying of cells when these ions are added just prior to extraction. While ATP increased solubility considerably, ATP plus Mg 2+ gave an even higher dissociation. Neither of these two treatments seem dependent upon ionic strength. DISCUSSION Results with avian muscle which show that aging is helpful in promoting emptying might partially reflect a loss of ATP which promotes association of actin and myosin. This does not seem to be necessary for Limulus muscle. The observation by de Villafranca (1959, 1968) that actin and myosin are very firmly combined in this primitive animal might explain the ease and high degree of emptying associated with this preparation and also why aging is without effect. It appears that at least one function of the salt-chelator washing is to remove added Ca ~+ and also possibly the indigenous cations from the segments. It was found by de Villafranca (1968) that in 0-6M KC1 purified Limulus actomyosin exhibited the characteristic dissociation phenomenon in the presence of ATP. While Mg 2+ was not necessary to inhibit ATP hydrolysis, it aided considerably in recovery of myosin and actin after dissociation. In this work presumably the same effect was seen. The effect of Ca ~+ and Mg 2+ appears only at low ionic strength. Perhaps the presence of high levels of a monovalent cation increases the concentration of divalent cations required for precipitation as suggested by Straub (1942) and Szent-Gyorgyi (1945). It is evident that the solubility characteristics of crude and purified actomyosin preparations are quite different. The protein extracted from cell segments is the true 'natural' actomyosin; it is in combination with other factors, pr¢sumably proteins such as tropomyosin, which confer upon it distinctive solubility behaviour. These data suggest that at least four factors are responsible for determining the solubility of actomyosin, viz.-degree of purity, ionic strength, concentration, and presence of substances such as Ca 2+, Mg 2+, ATP and perhaps other, as yet unidentified, components. Acknowledgements--Elizabeth H. Foran's excellent help is acknowledged with gratitude. The writer wishes to thank Professor G. W. de Villafranca for his help and encouragement in the appraisal of the manuscript.
284
DAVID W. STANLEY
REFERENCES HULTIN H. O. & WESTORT C. (1969) Sarcolemmae from chicken skeletal muscle--1. Preparation. ~. Food 8ci. 34, 165-171. STAnLeY D. W. & HULTIN H. O. (1968) Mechanism of emptying of skeletal muscle cell segments. ~t. Cell. Biol. 39, C1-C4. ST~UB F. B. (1942) Actin. Studies Inst. Med. Chem. Univ. Szeged 2, 3-16. SZ~T-GYoRGYt A. (1945) Studies on muscle. Actaphys. stand. 9, Suppl. 25. DE VILLAFRANCAG. W., SC~INBLUM T. S. & PHILPOTT D. E. (1959) A study on the localization of contractile proteins in the muscle of the horseshoe crab (Limulus polyphemus). Biochim. biophys. Acta 34, 147-157. DE VILL~a~gAa~CAG. W. & NAUMANND. C. (1964) Some properties of the myosin B ATPase from Limulus. Comp. Biochem. Physiol. 12, 143-156. D~ VILL~aANCA G. W. (1968) Some physico-chemical properties of myosin B from the horseshoe crab, Limulus polyphemus. Comp. Biochem. Physiol. 2,6, 443-454. WARRENL., GLtCK M. C. & NASS M. K. (1966) Membranes of animal cells.--I. Methods of isolation of the surface membrane. ~t. cell. Physiol. 68, 269-288. Wm~ga A. (1956) The ultracentrifugal separation of L-myosin and actin in an actomyosin sol under the influence of ATP. Biochim. biophys. Acta 19, 345-351. W~TOaT C. & HULTIN H. O. (1966) A procedure for the preparation of empty cell segments from skeletal muscle using solutions of low ionic strength. Anal. Biochem. 16, 314-319.
Key Word Index---Muscle; horseshoe crab; actomyosin; solubility; membranes; sarcolemma; protein.