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[44] Preparation of spectrin
[44]
PREPARATION OF SPECTRIN
475
[44] P r e p a r a t i o n o f S p e c t r i n
By W. B. GRATZER Spectrin is the major component of the protein network that covers the cytoplasmic surface of the vertebrate erythrocyte membrane. So far as is known at present, it is unique to the erythrocyte. It is a protein of high molecular weight, comprising two chains associated with one another in the form of a heterodimer unit; their molecular weights are about 230,000 and 250,000. Contrary to early reports, spectrin does not resemble myosin in any important respect. There seems to be no doubt that spectrin exists in the cell as a tetramer, made up of two heterodimers, oriented head-tohead. This elongated element of the cytoskeleton (some 200 nm extended length, as seen in the electron microscope after shadowing 1) is associated with actin and a protein referred to as 4.1 in the standard numbering system based on relative migration rates in sodium dodecyl sulfate (SDS)-polyacrylamide gels, 2 and possibly one more minor constituent. The primary mode of attachment of the cytoskeleton to the membrane is by way of sites on the spectrin, which associate with high affinity with an integral membrane protein, termed 2.1 or ankyrin. 3 The association becomes weak at low ionic strength, and the spectrin is then dissociated. This is the basis for its preparation from membranes. In dealing with spectrin it should be kept in mind that it is sensitive to proteolytic degradation and that it becomes insoluble in the vicinity of its isoelectric point (about pH 5). 4 Thus, insufficient buffering of a spectrin solution can lead to precipitation. Two procedures for the preparation of spectrin from human erythrocytes are available: extraction by dialysis in the cold and extraction at elevated temperature (35°). The first takes at least overnight and yields spectrin tetramer; the second takes only minutes and yields dimer. In either case the spectrin makes up some 70-80% of the total extracted protein, the remainder being almost entirely actin and 4.1. A variable part of the spectrin, moreover, depending evidently on the metabolic state of the cells, ~ appears in the form of an oligomeric complex with the other two D. M. Shotton, B. Burke, and D. Branton, J. Mol. Biol. 131, 303 (1979). 2 G. Fairbanks, T. L. Steck, and D. F. H. Wallach, Biochemistry 10, 2606 (1971). 3 V. Bennett, J. Biol. Chem. 253, 2292 (1978). 4 W. B. Gratzer and G. H. Beaven, Eur. J. Biochem. 58, 403 (1975). S. E. Lux, K. M. John, and M. J. Karnovsky, J. Clin. Invest. 58, 995 (1976).
METHODS IN ENZYMOLOGY, VOL. 85
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181985-X
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components. This cannot be readily dissociated, and is eliminated by gel filtration through a medium such as Sepharose 4B. The relation between the tetramer and dimer is complicated by metastability at low temperatureS: concentration-dependent interconversion is slow at low temperature (say 25° and below), so that at room temperature and below the system is essentially frozen. Thus at low ionic strength, at which the equilibrium strongly favors the dimer, tetramer is nevertheless extracted if the temperature is kept sufficiently low throughout. On warming, dissociation proceeds rapidly. Again, at higher ionic strengths, depending on the protein concentration, the equilibrium balance of dimer and tetramer may be established by warming the solution. It is not in practice possible to attain a concentration at which there is total conversion of dimer to tetramer. Thus if tetramer is required, either the equilibrium mixture may be chromatographed in the cold, or better, the initial extraction is performed in the cold, when dissociation to dimer is suppressed. Method of Preparation The following are the steps involved in the preparation of spectrin. 1. Cells, best from blood that is fresh or not more than a few days old, are washed with isotonic buffer with careful removal of white cells. 2. The cells are lysed, and the membranes are washed to remove hemoglobin. 3. The spectrin is extracted by dialysis overnight or warming at 35° for a brief period. 4. The membrane vesicles remaining after release of spectrin are removed by ultracentrifugation. 5. If necessary, the spectrin is concentrated by precipitation with ammonium sulfate, dialysis against polyethylene glycol or Ficoll, or vacuum dialysis. 6. For purification the solution is chromatographed on Sepharose 4B or its equivalent. Cells
The yield of spectrin from stored cells, tends to be poor. 5 Such cells can be incubated with metabolites required for the resynthesis of ATP, ~ but the usual practice is to use fresh, or relatively fresh, blood. Blood banks will frequently have available "short units," in which for some reason, such as the collapse of the doner's vein, the bag was not filled. 6 E. Ungewickelland W. B. Gratzer,Eur. J. Biochem. 88, 379 (1978).
[44]
PREPARATION OF SPECTRIN
477
Such samples (or those from patients with polycythemia or a history of malaria, etc.) are not used for transfusion. From fresh cells a total yield of spectrin approaching 1.5 mg per milliliter of packed cells (say 2 ml of blood) can be expected. A convenient scale of preparation starts with, say, 50 ml of blood. Wash and lysis buffers, which are respectively 0.15 M sodium chloride, with 20 mM Tris (or 5 mM phosphate) buffer, pH 7.6, and 5 mM Tris, (or again phosphate), pH 7.6, are prepared; about 500 ml and 1 liter, respectively, will be needed. They can be prepared by dilution of stock solutions of 10 times the concentration and are allowed to cool in ice. The blood (50 ml) is distributed between four 40-ml centrifuge tubes (e.g., transparent polycarbonate tubes (DuPont) for use with the Sorvall SS34 rotor). They are made up to volume with the isotonic buffer and mixed by inversion, using Parafilm. The tubes are spun, preferably at this stage in a swing-out rotor (Sorvall HB4) at 5000 rpm for 5 min. The supernatant is removed with a Pasteur pipette attached to a suction pump, and the "buffy coat" layer of white cells is carefully removed. (The volume of blood given allows for generous wastage in the interest of complete removal of the white cells, which are otherwise a damaging source of proteases). The cells are then resuspended by repeated, but not too vigorous, inversion (to avoid lysis) or stirring with a glass rod, and the centrifugation is repeated. Two further washes are desirable; for the last one the cells can be bulked in a single tube. The hemolysis step should then be done without too much delay. (If the cells are left in the packed state overnight, for example, they will tend to clump together during hemolysis.)
Preparation of Ghosts Two milliliters of packed cells are sampled into each of up to eight 40-ml centrifuge tubes to fill the SS34 rotor. An automatic pipette with the tip shortened to give an enlarged orifice can conveniently be used. The tubes are then filled with the chilled lysis buffer, and each is immediately and thoroughly mixed by inversion. They are centrifuged at 20,000 rpm for 5-10 min, gently removed from the rotor, and placed in an ice-bucket. The supernatant is removed with a Pasteur pipette attached to an aspirator. At this stage a light behind the tube is helpful, since the hemolysate is dark red and the pellet, which is not firmly packed down, may be hard to see and is easily disturbed. Its volume is generally several milliliters. Ice-cold hemolysis buffer is then again added, and the centrifugation is repeated. The supernatant is again removed by aspiration. At this stage (or after the first spin) a small pellet of sticky consistency, much more firmly packed, can be seen below the loose membrane layer at the bottom
478
MOTILITY APPARATUSIN NONMUSCLECELLS
[44]
of the tube. The tube may be rotated to expose this pellet at the top, when it can be cleanly r e m o v e d by aspiration. The washing p r o c e d u r e with the hemolysis buffer is repeated once more. The ghosts should then be almost white. Sometimes, and especially from old cells or many kinds o f abnormal, fragile cells, the hemoglobin is less readily dislodged and the ghosts remain reddish. For optimal extraction o f spectrin it has been found advantageous to ensure that the ionic strength is efficiently reduced. This can be achieved b y bulking the ghosts from all the tubes in one or two tubes, making up the volume with cold distilled water, and centrifuging once more. Speed at this stage helps to minimize loss of spectrin.
Extraction of Spectrin Spectrin is extracted from ghosts into a medium o f v e r y low ionic strength at a p H above neutrality; a buffer consisting o f 0.3 m M phosphate, p H 8.0, is suitable, being just sufficient to avoid adventitious drifts of pH. A further precaution is the incorporation o f 0.1 m M E D T A and 0.1 m M phenylmethanesulfonylfluoride to inhibit proteolysis; ghosts are diluted with an equal volume o f this buffer. For preparation ofdimers, the suspension is put into a water bath at 35° and allowed to extract for about 15 min, with periodic swirling. (The limiting factor is probably the rate at which temperature equilibration is reached. At higher temperatures there is a slow onset of irreversible denaturation.) For extraction of tetramers, the suspension is dialyzed against the extraction buffer overnight at 40. 7 The suspension is then centrifuged at 90,000 g for 30 min, when the extracted and vesiculated membranes form a very compact pellet. The supernatant contains some 7 0 - 9 0 % of the total spectrin, together with actin, 4. l, and traces o f other proteins. Extracts from pink ghosts also yield hemoglobin. To determine the concentrations of crude spectrin by spect r o p h o t o m e t r y a correction to the absorbance at 280 nm can be made for hemoglobin, on the basis o f the absorbance at 345 nm. Thus the corrected absorbance at 280 nm is A 2 8 0 - 1.25A345. The specific absorptivity of spectrin at 280 nm is t a k e n to be El~c°m = 10.7. Purification of Spectrin Dimer or T e t r a m e r The m e m b r a n e extract is applied to column o f Sepharose 4B or the equivalent, 2.5 × 90 cm for a total extract on the scale described. The column buffer is conveniently 0.1 M sodium chloride, 0.05 M Tris p H 7.6. 7 It is essential to ensure that the pH remains stable in this barely buffered system. The dialysis tubing can be a source of protons. Equilibration with a sodium bicarbonate solution is the final step of pretreatment (see, e.g., P. McPhie, this series, Vol. 22, p. 23).
[44]
PREPARATION OF SPECTRIN
479
Fractions of 5 ml may be collected. The peak coinciding with the void volume is predominantly a stable complex of spectrin, actin, and 4.1, although some F-actin may also be present. This should be well separated from the tetramer, which follows it, or the dimer. Following the pure spectrin dimer or tetramer two more peaks can generally be seen, the second being hemoglobin, the first mainly denatured actin. The spectrin can be screened for purity by gel electrophoresis in the presence of sodium dodecyl sulfate 8 and should be free of contaminants and contain only equal proportions of the two polypeptide chains with no satellite bands reflecting degradation. The concentration is determined spectrophotometrically ( ~ % = 10.7). Spectrin is best stored in the presence of EDTA (0.1 mM), a reducing agent (0.1 mM dithiothreitol), and sodium azide (20/-~g/ml). Even with azide alone to prevent bacterial contamination the properties of a solution of purified spectrin are in general unchanged after several days at 0°.
Concentration Spectrin is stable at concentrations as high as 50 mg/ml with no or little aggregation. It can be concentrated by precipitation with an equal volume of cold saturated ammonium sulfate, 9 although this sometimes generates some irreversibly aggregated material (and is especially unsatisfactory for the crude protein). It is important that precipitation and resuspension be performed very rapidly. Any of the standard concentration procedures may be applied. Vacuum dialysis and dialysis against Ficoll or polyethylene glycol (Calbiochem Aquacide) are satisfactory. Under all circumstances pH control should be maintained, for, as already remarked, spectrin allowed to drift to its isoelectric pH will precipitate.
Phosphorylation Spectrin is phosphorylated by an endogenous cAMP-independent kinase at four sites, clustered together at the C-terminal end of the smaller subunit.10 One may take advantage of the turnover of these phosphoryl groups to introduce a natural radioactive label into spectrin for binding and other studies. This can be done in either of two ways: extracted spectrin can be treated with labeled ATP and a crude preparation of the U. K. Laemmli, Nature (London) 227, 680 (1970). 9 G. B. Ralston, Aust. J. Biochem. Sci. 28, 259 (1975). ~0 H. W. Harris and S. E. Lux, J. Biol. Chem. 255, 11512 (1980).
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M O T I L I T Y A P P A R A T U S I N N O N M U S C L E CELLS
[45]
red cell kinase (a procedure is given by Pinder e t al. 11), or, more conveniently, the label can be introduced into the spectrin in the intact cell. 1°'1z The procedure is as follows: cells washed as already described are suspended in a medium containing 0.12 M sodium chloride, 5 mM potassium chloride, 20 mM sodium bicarbonate, 2 mM magnesium chloride, 1 mM calcium chloride, 10 mM glucose, 1 mM adenosine, 0.1 mg of streptomycin and 0.1 mg of penicillin G per milliliter, 0.1 mCi [32p]sodium phosphate, pH 7.5, at a hemotocrit of about 20%. The cells are incubated at 37° in a bacteriological water bath, with gentle agitation, for 24 hr. They are then washed with cold isotonic saline, and extraction and purification of spectrin proceeds as before. The spectrin thus prepared contains almost its full complement of phosphoryl groups, with a labeling level of 200-300 dpm per milligram of spectrin. Dephosphorylation can be accomplished by incubation in the same medium, but without glucose, adenosine, or phosphate for a period of some days, or on isolated spectrin, using a high concentration of bacterial alkaline phosphatase. 11 Acknowledgment I am gratefulto JenniferPinder for a critical reading of the manuscript. 11j. C. Pinder, D. Bray, and W. B. Gratzer,Nature (London) 270, 752 (1977). lz j. M. Anderson and J. M. Tyler,J. Biol. Chem. 255, 1259(1980).
[45] M a c r o p h a g e A c t i n - B i n d i n g P r o t e i n By JOHN H. HARTWlG and THOMAS P. STOSSEL High molecular weight proteins capable of binding to and crosslinking actin filaments have been isolated from a variety of cell types (see this volume [30]). The available evidence suggests that this family of large proteins creates and maintains the cortical architecture of cells in a rigid lattice composed of short, branching struts of actin filaments. Formation of a rigid actin filament network depends on a number of factors, which include: (a) the concentration of filaments; (b) the length of the filaments; (c) the functionality of the crosslinker: i.e., how many filaments it crosslinks per molecule; (d) the affinity of the crosslinker for the filaments; and (e) the orientation of the filaments. The theory of gelation is definitive, 1 permitting networks to be characterized quantitatively 1H. L. Yin, K. Zaner, and T. P. Stossel,J. Biol. Chem. 255, 9494 (1980).
METHODS IN ENZYMOLOGY, VOL. 85
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181985-X
PREPARATION OF SPECTRIN
475
[44] P r e p a r a t i o n o f S p e c t r i n
By W. B. GRATZER Spectrin is the major component of the protein network that covers the cytoplasmic surface of the vertebrate erythrocyte membrane. So far as is known at present, it is unique to the erythrocyte. It is a protein of high molecular weight, comprising two chains associated with one another in the form of a heterodimer unit; their molecular weights are about 230,000 and 250,000. Contrary to early reports, spectrin does not resemble myosin in any important respect. There seems to be no doubt that spectrin exists in the cell as a tetramer, made up of two heterodimers, oriented head-tohead. This elongated element of the cytoskeleton (some 200 nm extended length, as seen in the electron microscope after shadowing 1) is associated with actin and a protein referred to as 4.1 in the standard numbering system based on relative migration rates in sodium dodecyl sulfate (SDS)-polyacrylamide gels, 2 and possibly one more minor constituent. The primary mode of attachment of the cytoskeleton to the membrane is by way of sites on the spectrin, which associate with high affinity with an integral membrane protein, termed 2.1 or ankyrin. 3 The association becomes weak at low ionic strength, and the spectrin is then dissociated. This is the basis for its preparation from membranes. In dealing with spectrin it should be kept in mind that it is sensitive to proteolytic degradation and that it becomes insoluble in the vicinity of its isoelectric point (about pH 5). 4 Thus, insufficient buffering of a spectrin solution can lead to precipitation. Two procedures for the preparation of spectrin from human erythrocytes are available: extraction by dialysis in the cold and extraction at elevated temperature (35°). The first takes at least overnight and yields spectrin tetramer; the second takes only minutes and yields dimer. In either case the spectrin makes up some 70-80% of the total extracted protein, the remainder being almost entirely actin and 4.1. A variable part of the spectrin, moreover, depending evidently on the metabolic state of the cells, ~ appears in the form of an oligomeric complex with the other two D. M. Shotton, B. Burke, and D. Branton, J. Mol. Biol. 131, 303 (1979). 2 G. Fairbanks, T. L. Steck, and D. F. H. Wallach, Biochemistry 10, 2606 (1971). 3 V. Bennett, J. Biol. Chem. 253, 2292 (1978). 4 W. B. Gratzer and G. H. Beaven, Eur. J. Biochem. 58, 403 (1975). S. E. Lux, K. M. John, and M. J. Karnovsky, J. Clin. Invest. 58, 995 (1976).
METHODS IN ENZYMOLOGY, VOL. 85
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181985-X
476
M O T I L I T Y A P P A R A T U S IN N O N M U S C L E C E L L S
[44]
components. This cannot be readily dissociated, and is eliminated by gel filtration through a medium such as Sepharose 4B. The relation between the tetramer and dimer is complicated by metastability at low temperatureS: concentration-dependent interconversion is slow at low temperature (say 25° and below), so that at room temperature and below the system is essentially frozen. Thus at low ionic strength, at which the equilibrium strongly favors the dimer, tetramer is nevertheless extracted if the temperature is kept sufficiently low throughout. On warming, dissociation proceeds rapidly. Again, at higher ionic strengths, depending on the protein concentration, the equilibrium balance of dimer and tetramer may be established by warming the solution. It is not in practice possible to attain a concentration at which there is total conversion of dimer to tetramer. Thus if tetramer is required, either the equilibrium mixture may be chromatographed in the cold, or better, the initial extraction is performed in the cold, when dissociation to dimer is suppressed. Method of Preparation The following are the steps involved in the preparation of spectrin. 1. Cells, best from blood that is fresh or not more than a few days old, are washed with isotonic buffer with careful removal of white cells. 2. The cells are lysed, and the membranes are washed to remove hemoglobin. 3. The spectrin is extracted by dialysis overnight or warming at 35° for a brief period. 4. The membrane vesicles remaining after release of spectrin are removed by ultracentrifugation. 5. If necessary, the spectrin is concentrated by precipitation with ammonium sulfate, dialysis against polyethylene glycol or Ficoll, or vacuum dialysis. 6. For purification the solution is chromatographed on Sepharose 4B or its equivalent. Cells
The yield of spectrin from stored cells, tends to be poor. 5 Such cells can be incubated with metabolites required for the resynthesis of ATP, ~ but the usual practice is to use fresh, or relatively fresh, blood. Blood banks will frequently have available "short units," in which for some reason, such as the collapse of the doner's vein, the bag was not filled. 6 E. Ungewickelland W. B. Gratzer,Eur. J. Biochem. 88, 379 (1978).
[44]
PREPARATION OF SPECTRIN
477
Such samples (or those from patients with polycythemia or a history of malaria, etc.) are not used for transfusion. From fresh cells a total yield of spectrin approaching 1.5 mg per milliliter of packed cells (say 2 ml of blood) can be expected. A convenient scale of preparation starts with, say, 50 ml of blood. Wash and lysis buffers, which are respectively 0.15 M sodium chloride, with 20 mM Tris (or 5 mM phosphate) buffer, pH 7.6, and 5 mM Tris, (or again phosphate), pH 7.6, are prepared; about 500 ml and 1 liter, respectively, will be needed. They can be prepared by dilution of stock solutions of 10 times the concentration and are allowed to cool in ice. The blood (50 ml) is distributed between four 40-ml centrifuge tubes (e.g., transparent polycarbonate tubes (DuPont) for use with the Sorvall SS34 rotor). They are made up to volume with the isotonic buffer and mixed by inversion, using Parafilm. The tubes are spun, preferably at this stage in a swing-out rotor (Sorvall HB4) at 5000 rpm for 5 min. The supernatant is removed with a Pasteur pipette attached to a suction pump, and the "buffy coat" layer of white cells is carefully removed. (The volume of blood given allows for generous wastage in the interest of complete removal of the white cells, which are otherwise a damaging source of proteases). The cells are then resuspended by repeated, but not too vigorous, inversion (to avoid lysis) or stirring with a glass rod, and the centrifugation is repeated. Two further washes are desirable; for the last one the cells can be bulked in a single tube. The hemolysis step should then be done without too much delay. (If the cells are left in the packed state overnight, for example, they will tend to clump together during hemolysis.)
Preparation of Ghosts Two milliliters of packed cells are sampled into each of up to eight 40-ml centrifuge tubes to fill the SS34 rotor. An automatic pipette with the tip shortened to give an enlarged orifice can conveniently be used. The tubes are then filled with the chilled lysis buffer, and each is immediately and thoroughly mixed by inversion. They are centrifuged at 20,000 rpm for 5-10 min, gently removed from the rotor, and placed in an ice-bucket. The supernatant is removed with a Pasteur pipette attached to an aspirator. At this stage a light behind the tube is helpful, since the hemolysate is dark red and the pellet, which is not firmly packed down, may be hard to see and is easily disturbed. Its volume is generally several milliliters. Ice-cold hemolysis buffer is then again added, and the centrifugation is repeated. The supernatant is again removed by aspiration. At this stage (or after the first spin) a small pellet of sticky consistency, much more firmly packed, can be seen below the loose membrane layer at the bottom
478
MOTILITY APPARATUSIN NONMUSCLECELLS
[44]
of the tube. The tube may be rotated to expose this pellet at the top, when it can be cleanly r e m o v e d by aspiration. The washing p r o c e d u r e with the hemolysis buffer is repeated once more. The ghosts should then be almost white. Sometimes, and especially from old cells or many kinds o f abnormal, fragile cells, the hemoglobin is less readily dislodged and the ghosts remain reddish. For optimal extraction o f spectrin it has been found advantageous to ensure that the ionic strength is efficiently reduced. This can be achieved b y bulking the ghosts from all the tubes in one or two tubes, making up the volume with cold distilled water, and centrifuging once more. Speed at this stage helps to minimize loss of spectrin.
Extraction of Spectrin Spectrin is extracted from ghosts into a medium o f v e r y low ionic strength at a p H above neutrality; a buffer consisting o f 0.3 m M phosphate, p H 8.0, is suitable, being just sufficient to avoid adventitious drifts of pH. A further precaution is the incorporation o f 0.1 m M E D T A and 0.1 m M phenylmethanesulfonylfluoride to inhibit proteolysis; ghosts are diluted with an equal volume o f this buffer. For preparation ofdimers, the suspension is put into a water bath at 35° and allowed to extract for about 15 min, with periodic swirling. (The limiting factor is probably the rate at which temperature equilibration is reached. At higher temperatures there is a slow onset of irreversible denaturation.) For extraction of tetramers, the suspension is dialyzed against the extraction buffer overnight at 40. 7 The suspension is then centrifuged at 90,000 g for 30 min, when the extracted and vesiculated membranes form a very compact pellet. The supernatant contains some 7 0 - 9 0 % of the total spectrin, together with actin, 4. l, and traces o f other proteins. Extracts from pink ghosts also yield hemoglobin. To determine the concentrations of crude spectrin by spect r o p h o t o m e t r y a correction to the absorbance at 280 nm can be made for hemoglobin, on the basis o f the absorbance at 345 nm. Thus the corrected absorbance at 280 nm is A 2 8 0 - 1.25A345. The specific absorptivity of spectrin at 280 nm is t a k e n to be El~c°m = 10.7. Purification of Spectrin Dimer or T e t r a m e r The m e m b r a n e extract is applied to column o f Sepharose 4B or the equivalent, 2.5 × 90 cm for a total extract on the scale described. The column buffer is conveniently 0.1 M sodium chloride, 0.05 M Tris p H 7.6. 7 It is essential to ensure that the pH remains stable in this barely buffered system. The dialysis tubing can be a source of protons. Equilibration with a sodium bicarbonate solution is the final step of pretreatment (see, e.g., P. McPhie, this series, Vol. 22, p. 23).
[44]
PREPARATION OF SPECTRIN
479
Fractions of 5 ml may be collected. The peak coinciding with the void volume is predominantly a stable complex of spectrin, actin, and 4.1, although some F-actin may also be present. This should be well separated from the tetramer, which follows it, or the dimer. Following the pure spectrin dimer or tetramer two more peaks can generally be seen, the second being hemoglobin, the first mainly denatured actin. The spectrin can be screened for purity by gel electrophoresis in the presence of sodium dodecyl sulfate 8 and should be free of contaminants and contain only equal proportions of the two polypeptide chains with no satellite bands reflecting degradation. The concentration is determined spectrophotometrically ( ~ % = 10.7). Spectrin is best stored in the presence of EDTA (0.1 mM), a reducing agent (0.1 mM dithiothreitol), and sodium azide (20/-~g/ml). Even with azide alone to prevent bacterial contamination the properties of a solution of purified spectrin are in general unchanged after several days at 0°.
Concentration Spectrin is stable at concentrations as high as 50 mg/ml with no or little aggregation. It can be concentrated by precipitation with an equal volume of cold saturated ammonium sulfate, 9 although this sometimes generates some irreversibly aggregated material (and is especially unsatisfactory for the crude protein). It is important that precipitation and resuspension be performed very rapidly. Any of the standard concentration procedures may be applied. Vacuum dialysis and dialysis against Ficoll or polyethylene glycol (Calbiochem Aquacide) are satisfactory. Under all circumstances pH control should be maintained, for, as already remarked, spectrin allowed to drift to its isoelectric pH will precipitate.
Phosphorylation Spectrin is phosphorylated by an endogenous cAMP-independent kinase at four sites, clustered together at the C-terminal end of the smaller subunit.10 One may take advantage of the turnover of these phosphoryl groups to introduce a natural radioactive label into spectrin for binding and other studies. This can be done in either of two ways: extracted spectrin can be treated with labeled ATP and a crude preparation of the U. K. Laemmli, Nature (London) 227, 680 (1970). 9 G. B. Ralston, Aust. J. Biochem. Sci. 28, 259 (1975). ~0 H. W. Harris and S. E. Lux, J. Biol. Chem. 255, 11512 (1980).
480
M O T I L I T Y A P P A R A T U S I N N O N M U S C L E CELLS
[45]
red cell kinase (a procedure is given by Pinder e t al. 11), or, more conveniently, the label can be introduced into the spectrin in the intact cell. 1°'1z The procedure is as follows: cells washed as already described are suspended in a medium containing 0.12 M sodium chloride, 5 mM potassium chloride, 20 mM sodium bicarbonate, 2 mM magnesium chloride, 1 mM calcium chloride, 10 mM glucose, 1 mM adenosine, 0.1 mg of streptomycin and 0.1 mg of penicillin G per milliliter, 0.1 mCi [32p]sodium phosphate, pH 7.5, at a hemotocrit of about 20%. The cells are incubated at 37° in a bacteriological water bath, with gentle agitation, for 24 hr. They are then washed with cold isotonic saline, and extraction and purification of spectrin proceeds as before. The spectrin thus prepared contains almost its full complement of phosphoryl groups, with a labeling level of 200-300 dpm per milligram of spectrin. Dephosphorylation can be accomplished by incubation in the same medium, but without glucose, adenosine, or phosphate for a period of some days, or on isolated spectrin, using a high concentration of bacterial alkaline phosphatase. 11 Acknowledgment I am gratefulto JenniferPinder for a critical reading of the manuscript. 11j. C. Pinder, D. Bray, and W. B. Gratzer,Nature (London) 270, 752 (1977). lz j. M. Anderson and J. M. Tyler,J. Biol. Chem. 255, 1259(1980).
[45] M a c r o p h a g e A c t i n - B i n d i n g P r o t e i n By JOHN H. HARTWlG and THOMAS P. STOSSEL High molecular weight proteins capable of binding to and crosslinking actin filaments have been isolated from a variety of cell types (see this volume [30]). The available evidence suggests that this family of large proteins creates and maintains the cortical architecture of cells in a rigid lattice composed of short, branching struts of actin filaments. Formation of a rigid actin filament network depends on a number of factors, which include: (a) the concentration of filaments; (b) the length of the filaments; (c) the functionality of the crosslinker: i.e., how many filaments it crosslinks per molecule; (d) the affinity of the crosslinker for the filaments; and (e) the orientation of the filaments. The theory of gelation is definitive, 1 permitting networks to be characterized quantitatively 1H. L. Yin, K. Zaner, and T. P. Stossel,J. Biol. Chem. 255, 9494 (1980).
METHODS IN ENZYMOLOGY, VOL. 85
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181985-X