Proc. Natl. Acad. Sci. USA Vol. 76, No. 10, pp. 5192-5196, October 1979
Cell Biology
Purification of two spectrin-binding proteins: Biochemical and electron microscopic evidence for site-specific reassociation between spectrin and bands 2.1 and 4.1 (erythrocyte mbmbrane/cytoskeleton)
JONATHAN M. TYLER, WILLIAM R. HARGREAVES, AND DANIEL BRANTON Cell and Developmental Biology, The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138
Communicated by Lawrence Bogorad, July 9,1979
ABSTRACT Two peripheral proteins of the human erythrocyte membrane that are capable of fonning a stable complex with spectrin have been purified. The proteins, band 2.1 (Mr 210,000) and band 4.1 (M, 82,000), are water soluble and exist as monomers in solution. Both exhibit strong, specific binding to purified spectrin molecules as determined by cosedimentation in sucrose gradients and both enhance binding to spectrin-depleted, inside-out vesicles that have been- stripped of bands 2.1 and 4.1. Rotary replicas of bound material reveal site-specific associations among native, but not heat-denatured, molecules.
The human erythrocyte membrane provides a model system for investigating protein-membrane associations at the molecular level. Of particular interest is the existence of an extensive cytoskeletal network that may control cell shape and deformability (1-3) and the distribution of intramembrane particles (4) and surface markers (5). Interactions between spectrin, the major cytoskeletal protein, and the cytoplasmic surface of the erythrocyte membrane have been studied in detail and partially characterized (6-10). Bennett and Branton (6) have demonstrated the existence of a class of sites to which spectrin binds with high affinity, and recent evidence (8, 11, 12) indicates that band 2.1* and a family of sequence-related polypeptides mediate the association of spectrin with the membrane. However, there is no indication that this is the only class of sites to which spectrin can bind. For spectrin to fdrm a flexible, controlling cytoskeletal meshwork, it must self-associate extensively, or bind to a class of multivalent sites, or atociate with more than one class of monovalent sites. Pure spectrin does not self-associate beyond the tetrameric state (14-16). Band 2.1, a polypeptide that is known to bind spectrin to the membrane, appears to be monovalent (8) because it can be-cleaved to yield one active binding fragment that associates in solution with one spectrin heterodimer. Thus, it appears likely that there is a second class of sites to which spectrin binds. Cytoskeletal "shells" derived from detergent-extracted erythrocyte ghosts characteristically contain bands 4.1 and 5 in addition to spectrin (17-19). A search for spectrin-binding proteins would necessarily include investigations of these polypeptides. We observed that, although bands 2.1 and 4.1 are distinct polypeptides (11, 12), they exhibit some similar properties. Neither can be efficiently removed when spectrin remains bound to the membrane, but they can be eluted simultaneously from spectrin-depleted vesicles under moderate conditions, with concomitant loss of spectrin-binding activity. In addition, crude extracts of spectrin eluted with low ionic
strength buffers at 37"C are invariably contaminated with band 4.1. Hence, we considered the possibility that band 4.1 is also a spectrin-binding component. We therefore purified bands 2.1 and 4.1 separately to investigate the interactions of each of these polypeptides with purified spectrin. MATERIALS AND METHODS Extraction of Bands 2.1 and 4.1. Erythrocyte ghosts from 50 ml of fresh human blood were prepared (20) with 1 mM EDTA present in all phases of ghost preparation. To remove band 6, the ghosts were extracted for 30 min on ice in 30 vol of phosphate-buffered saline (5 mM NaPO4/1 mM EDTA/155 mM NaCl, pH-7.6) and 0.4 mM diisopropyl fluorophosphate (iPr2FP) (0.0075%, wt/vol). The vesicles were pelleted, washed once in phosphate-buffered saline without iPr2FP, and then in 5 mM NaPO4/1 mM EDTA, pH 7.6. Prior to extraction of spectrin and erythrocyte actin, the pellet was washed once in 0.3 mM NaPO4/0.2 mM EDTA, pH 7.6, and resuspended in 30 vol of this buffer with iPr2FP added to 0.2 mM. Incubation of the ghosts for 30 min at 370C removed 2 90% of the spectrin and virtually all of band 5, and these two proteins remained in the supernatant during a 3S-tnin centrifugation at 19,000 rpm (2-C, Sorvall SS-34 rotor) which pelleted the spectrin-depleted vesicles. The vesicles were washed once in 0.3 mM NaPO4/0.2 mM EDTA, pH 7.6, and pooled prior to a final wash in 5 mM NaPO4/1 mM EDTA, pH 7.6. The vesicles, depleted of bands 1, 2, 5, and 6, were suspended in approximately 6 ml of the above buffer and prepared for extraction of bands 2.1 and 4.1 by the addition of iPr2FP and dry KC1, with vortexing, to produce final concentrations of 0.4 mM and 1 M, respectively. The pH was adjusted to 7.6 by titration with 1 M borate buffer at pH 8.5. Incubation for 30 min at 370C released the bulk of bands 2.1 and 4.1, and centrifugation at 225,000 X g for 20 min at 20C pelleted the membranes, leaving a crude extract consisting primarily of bands 2.1 and 4.1 in the supernatant Labeling with 125I Bolton-Hunter Reagent. 125I BoitonHunter reagent (21) (Amersham) (0.1 ml in benzene) was dried onto a borosilicate glass tube under a gentle stream of dry nitrogen; then, 0.5 ml of 1 M borate pH 8.5 buffer was pipetted into the tube, mixed, and immediately removed to a plastic tube containing 5 ml of crude extract. The reaction was allowed to proceed for 1 hr on ice and the material was then desalted on a 1.0 X 10.0 cm Sephadex G-25 column equilibrated with 5 mM NaPO4/1 mM EDTA/20 mM KC1/0.5 mM dithiothreitol, pH 7.6, and 0.5-ml fractions were collected. Abbreviations: iPr2FP, diisopropyl fluorophosphate; IOV, inside-out vesicle. * Nomenclature for human erythrocyte proteins is according to Steck (1). Band 2.1 has been named "ankyrin" (8), but this name has previously been used to describe another protein (13). Band 2.1 and the series of polypeptides related to band 2.1 by sequence homology have been called syndeins (12).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. § 1734 solely to indicate this fact. 5192
Cell Biology:
Tyler et al.
Proc. Nati. Acad. Sci. USA 76 (1979)
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FIG. 1. (A) Sodium dodecyl sulfate/polyacrylamide gels. Lanes: a, human erythrocyte ghosts; b, ghosts from which band 6 had been extracted; spectrin-depleted vesicles (bands 1 and 2 decreased, bands 5 and 6 absent); d, extracted residue; e, crude extract enriched in bands 2.1 and 4. 1; f and g, column-purified bands 2.1 and 4.1 (see text). (B and C) Velocity sedimentation profiles of highly purified bands 2.1 and 4.1 through preparative sucrose gradients. Enzyme markers were cosedimented to provide standards for the estimation of so values: F, fumarase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; cyt. c, cytochrome c. s° was 5.7 S for band 2.1 and 4.1 S for band 4.1. c,
,
,
Purification of Bands 2.1 and 4.1. Peak fractions were loaded onto a 1.6 X 30 cm DEAE-cellulose column and, after a 20-ml wash with 5 mM NaPO4/1 mM EDTA/20 mM KCI/0.5 mM dithiothreitol, pH 7.6, elution was carried out with a 20-400 mM linear KCI gradient in the above buffer. Recovery of bands 2.1 and 4.1 from the column was estimated to be >85% under these conditions. Fractions were analyzed for 125I activity and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Fractions containing pure band 2.1 or 4.1 were separately pooled and dialyzed overnight at 40C against 4 liters of 2 mM NaPO4/1 mM EDTA/20 mM KCI/0.5 mM dithiothreitol, pH 7.6. Aliquots (1 ml) of the purified material were layered onto 12.5 ml of 5-20% linear sucrose gradients in 1 mM NaPO4/1 mM EDTA/20 mM KCI/0.5 mM dithiothreitol, pH 7.6, and centrifuged (20C, 18 hr, 40,000 rpm in a Beckman SW 40 rotor) to obtain fractions free from any low molecular weight proteolytic contaminants and to characterize the material as to sedimentation properties. After centrifugation, 0.4-ml fractions were collected and peak fractions were pooled and dialyzed overnight at 40C against 4 liters of a buffer (1 mM NaPO4/0.5 mM EDTA/20 mM KCI/0.5 mM dithiothreitol, pH 7.6) suitable for binding assays. The absorbance at 280 nm was measured after dialysis, and this was compared with Lowry (22) and ninhydrin protein assays. Typically, the proteins were present at concentrations of approximately 100 mg/ml with specific activities of 4000-16,000 cpm/mg. Preparation of Spectrin Heterodimers and Tetramers. Spectrin was prepared by using well-established methods (6, 9, 10, 14, 15); low ionic strength extraction was followed by either rate zonal centrifugation or column chromatography on Sepharose 4B. Material in peak fractions was equilibrated with 1 mM NaPO4/0.5 mM EDTA/20 mM KCI/0.5 mM dithiothreitol, pH 7.6. When appropriate, 3 mM NaN3 was included. Spectrin was labeled with 32p (6, 10) or 125I (9, 10). Preparation of Selectively Depleted Vesicles. Bands 2.1 and 4.1 were extracted by incubating spectrin-depleted inside-out vesicles (IOVs) (6) in 30 vol of 25 mM NaPO4/25 mM EDTA/1 M KCI/0.4 mM iPr2FP, pH 8.5 at 370C for 30 min. After incubation, the vesicles were pelleted and washed once in buffer without iPr2FP, and then in 1 mM NaPO4/1 mM
EDTA/20 mM KCI/3 mM NaN3. KCI extraction removed >65% of band 2.1 and >75% of band 4.1. Binding Analysis by Rate Zonal Sedimentation. Highly purified spectrin heterodimers 2.1 and 4.1 were assayed for binding activity by measuring states of association among the components as a function of their velocity sedimentation profiles. Protein mixtures were incubated for 90-120 min at 20C prior to layering onto 12.5 ml of 5-20% linear sucrose gradients in 1 mM NaPO4/0.5 mM EDTA/20 mM KCI/0.5 mM dithiothreitol, pH 7.6. After centrifugation (20C, 18 hr, 40,000 rpm in a Beckman SW 40 rotor), 0.4-ml fractions were collected. Gradient fractions were assayed for 125I activity and, when applicable, for 32P activity. In addition, aliquots (50-100
,ul) from peak fractions or from the material initially layered onto the gradient were prepared for low-angle shadowing, and
the remainder was lyophilized for polyacrylamide gel electrophoresis and autoradiography. Analysis of Spectrin Binding to Membrane Preparations.
Binding of 125I-labeled spectrin to IOVs and KCI-extracted IOVs was assayed by a modification of the technique of Bennett and Branton (6). Because we found binding affinities to be 2-3 times greater at physiological ionic strength, a concentrated salt solution was added to bring the final concentrations to 5 mM NaPO4, 1 mM EDTA, 130 mM KCI, 10 mM NaCl, 1 mM dithiothreitol, and 3 mM NaN3 at pH 7.6. 25I-Labeled spectrin behaves identically to metabolically labeled [32P]spectrin with respect to membrane binding.(9, 10). The assay mixture contained '25I-spectrin (0-24 ,g/ml), vesicles (20-25 ,g of band 3 protein per ml, estimated by scanning microdensitometry of Coomassie blue-stained gels) and, when appropriate, purified band 2.1 or 4.1 (40,ug/ml). Purified bands 2.1 and 4.1 were preincubated with spectrin for 30 min on ice prior to the addition of vesicles as described (8). After incubation for not less than 30 min with vesicles, membrane-bound and free spectrin were separated (6). Low-Angle Shadowing of Proteins. Low-angle shadowing was performed by a modification of the method of Shotton et al. (15). The samples of pure protein, the protein mixtures after incubation, or the peak gradient fractions after centrifugation of these proteins were diluted with 1 mM NaPO4/0.5 mM
Cell Biology:
5194
Tyler et al.
Proc. Natl. Acad. Sci. USA 76 (1979) :
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Gradient fraction -top FIG. 2. Rate zonal sedimentation binding analyses. Gradients were fractionated into 400-,ul aliquots and the 32P and 1251 in each fraction were determined. (A) Pure [32P] spectrin; (B) pure 1251-laheled band 2.1 alone (-) or plus nonradiolabeled spectrin (-.---); (C) pure 1251-labeled band 4.1 alone (-) or plus nonradiolabeled spectrin Arrows denote the velocity sedimentation position of purified (.----). tree spectrin (7.6 S). Note that spectrin bound to band 2.1 or 4.1 migrates further in the gradient than does pure spectrin, indicating an increased S value for the complex. (D) Sodium dodecyl sulfate/ polyacrylamide gels and autoradiographs showing the protein com-
position (left, Coomassie blue staining) and radioactivity source (right,
autoradiogram) of each peak.
EDTA/20 mM KCI/0.5 mM dithiothreitol, pH 7.6, gently mixed with glycerol to give final concentrations of 60% glycerol, and sprayed onto mica without removing the nonvolatile solutes by dialysis (15). RESULTS Purification of Bands 2.1 and 4.1. The protocol for purification outlined in this report consistently gave high yields of bands 2.1 and 4.1 with minimal contamination by other proteins or polypeptides. Purity was judged to be >97% in each case as measured by microspectrophotometric scans of both the Coomassie blue staining patterns (Fig. 1A) and the autoradiographic profiles of sodium dodecyl sulfate/polyacrylamide gel electrophoretograms (not shown). The inclusion of EDTA and iPr2FP throughout the elution and subsequent purification stages prevented the rapid and devastating breakdown of bands 2.1 and 4.1 by endogenous protease activity. Proteins protected by these agents during isolation and purification were stable and retained binding activity for at least 4 weeks when stored at 20C. In the absence of protease inhibitors, purified band 2.1 free from detectable contamination by other polypeptides underwent proteolytic degradation when stored at 4VC. A series of bands approximating the relative electrophoretic mobility characteristics of bands 2.2 through 2.6 were produced. Band 4.1 also underwent degradation upon extended storage, giving rise to a polypeptide that comigrated with band 4.2 on electrophoresis. Molecular weights of 210,000 for band 2.1 and 82,000 for 4.1
Spectrin concentration,glml FIG. 3. Effects of bands 2.1 (Left) and 4.1 (Right) on spectrin binding to IOVs and KCl-extracted IOVs. Binding of spectrin, preincubated with (triangles) or without (circles) band 2.1 or 4.1 (40 ALg/ml), to IOVs (open symbols) or KCl-extracted IOVs (solid symbols) is shown. Spectrin bound is expressed as Ag/mg of total membrane protein in IOVs (78 Ag/ml). Equivalent numbers of vesicles were present in the experiments with IOVs and KCl-extracted IOVs.
are assumed from electrophoretic gels run by the methods of Fairbanks et al. (23) and Laemmli (24). These molecular weights are in agreement with published values (1). Globular molecules with molecular weight approximating that of 2.1 typically have s2% ,f values ranging from 9 to 11 S, whereas those similar to band 4.1 have sOW values in the range 4 to 6 S (25). Thus, from its velocity sedimentation profile (Fig. 1 B and C) and chromatographic elution characteristics (not shown) it appears likely that purified band 2.1 exists as an asymmetric monomer in solution; band 4.1 is a relatively globular monomer under these conditions. Amino acid analyses indicate that both polypeptides contain approximately 33% hydrophobic amino acid residues, a value consistent with their solubility in water. Binding Analyses by Rate Zonal Sedimentation. Radioactivity profiles of rate zonal sedimentation binding assays indicated the existence of specific interactions between spectrin and band 2.1 and between spectrin and band 4.1 (Fig. 2). Spectrin was incubated with band 2.1 or 4.1 in a wide range of molar ratios at concentrations of band 2.1 or 4.1 up to 350 gg/ml. That the association states in each case were specific is suggested by the following observations. (i) When band 2.1 or 4.1 was denatured by heating to 65°C for 6 min prior to incubation, neither exhibited measurable binding. (ii) When band 2.1 or 4.1 was incubated with spectrin in a buffer of low ionic strength (comparable to that used for the extraction of spectrin from ghost membranes), binding was decreased to
Cell Biology
Purification of two spectrin-binding proteins: Biochemical and electron microscopic evidence for site-specific reassociation between spectrin and bands 2.1 and 4.1 (erythrocyte mbmbrane/cytoskeleton)
JONATHAN M. TYLER, WILLIAM R. HARGREAVES, AND DANIEL BRANTON Cell and Developmental Biology, The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138
Communicated by Lawrence Bogorad, July 9,1979
ABSTRACT Two peripheral proteins of the human erythrocyte membrane that are capable of fonning a stable complex with spectrin have been purified. The proteins, band 2.1 (Mr 210,000) and band 4.1 (M, 82,000), are water soluble and exist as monomers in solution. Both exhibit strong, specific binding to purified spectrin molecules as determined by cosedimentation in sucrose gradients and both enhance binding to spectrin-depleted, inside-out vesicles that have been- stripped of bands 2.1 and 4.1. Rotary replicas of bound material reveal site-specific associations among native, but not heat-denatured, molecules.
The human erythrocyte membrane provides a model system for investigating protein-membrane associations at the molecular level. Of particular interest is the existence of an extensive cytoskeletal network that may control cell shape and deformability (1-3) and the distribution of intramembrane particles (4) and surface markers (5). Interactions between spectrin, the major cytoskeletal protein, and the cytoplasmic surface of the erythrocyte membrane have been studied in detail and partially characterized (6-10). Bennett and Branton (6) have demonstrated the existence of a class of sites to which spectrin binds with high affinity, and recent evidence (8, 11, 12) indicates that band 2.1* and a family of sequence-related polypeptides mediate the association of spectrin with the membrane. However, there is no indication that this is the only class of sites to which spectrin can bind. For spectrin to fdrm a flexible, controlling cytoskeletal meshwork, it must self-associate extensively, or bind to a class of multivalent sites, or atociate with more than one class of monovalent sites. Pure spectrin does not self-associate beyond the tetrameric state (14-16). Band 2.1, a polypeptide that is known to bind spectrin to the membrane, appears to be monovalent (8) because it can be-cleaved to yield one active binding fragment that associates in solution with one spectrin heterodimer. Thus, it appears likely that there is a second class of sites to which spectrin binds. Cytoskeletal "shells" derived from detergent-extracted erythrocyte ghosts characteristically contain bands 4.1 and 5 in addition to spectrin (17-19). A search for spectrin-binding proteins would necessarily include investigations of these polypeptides. We observed that, although bands 2.1 and 4.1 are distinct polypeptides (11, 12), they exhibit some similar properties. Neither can be efficiently removed when spectrin remains bound to the membrane, but they can be eluted simultaneously from spectrin-depleted vesicles under moderate conditions, with concomitant loss of spectrin-binding activity. In addition, crude extracts of spectrin eluted with low ionic
strength buffers at 37"C are invariably contaminated with band 4.1. Hence, we considered the possibility that band 4.1 is also a spectrin-binding component. We therefore purified bands 2.1 and 4.1 separately to investigate the interactions of each of these polypeptides with purified spectrin. MATERIALS AND METHODS Extraction of Bands 2.1 and 4.1. Erythrocyte ghosts from 50 ml of fresh human blood were prepared (20) with 1 mM EDTA present in all phases of ghost preparation. To remove band 6, the ghosts were extracted for 30 min on ice in 30 vol of phosphate-buffered saline (5 mM NaPO4/1 mM EDTA/155 mM NaCl, pH-7.6) and 0.4 mM diisopropyl fluorophosphate (iPr2FP) (0.0075%, wt/vol). The vesicles were pelleted, washed once in phosphate-buffered saline without iPr2FP, and then in 5 mM NaPO4/1 mM EDTA, pH 7.6. Prior to extraction of spectrin and erythrocyte actin, the pellet was washed once in 0.3 mM NaPO4/0.2 mM EDTA, pH 7.6, and resuspended in 30 vol of this buffer with iPr2FP added to 0.2 mM. Incubation of the ghosts for 30 min at 370C removed 2 90% of the spectrin and virtually all of band 5, and these two proteins remained in the supernatant during a 3S-tnin centrifugation at 19,000 rpm (2-C, Sorvall SS-34 rotor) which pelleted the spectrin-depleted vesicles. The vesicles were washed once in 0.3 mM NaPO4/0.2 mM EDTA, pH 7.6, and pooled prior to a final wash in 5 mM NaPO4/1 mM EDTA, pH 7.6. The vesicles, depleted of bands 1, 2, 5, and 6, were suspended in approximately 6 ml of the above buffer and prepared for extraction of bands 2.1 and 4.1 by the addition of iPr2FP and dry KC1, with vortexing, to produce final concentrations of 0.4 mM and 1 M, respectively. The pH was adjusted to 7.6 by titration with 1 M borate buffer at pH 8.5. Incubation for 30 min at 370C released the bulk of bands 2.1 and 4.1, and centrifugation at 225,000 X g for 20 min at 20C pelleted the membranes, leaving a crude extract consisting primarily of bands 2.1 and 4.1 in the supernatant Labeling with 125I Bolton-Hunter Reagent. 125I BoitonHunter reagent (21) (Amersham) (0.1 ml in benzene) was dried onto a borosilicate glass tube under a gentle stream of dry nitrogen; then, 0.5 ml of 1 M borate pH 8.5 buffer was pipetted into the tube, mixed, and immediately removed to a plastic tube containing 5 ml of crude extract. The reaction was allowed to proceed for 1 hr on ice and the material was then desalted on a 1.0 X 10.0 cm Sephadex G-25 column equilibrated with 5 mM NaPO4/1 mM EDTA/20 mM KC1/0.5 mM dithiothreitol, pH 7.6, and 0.5-ml fractions were collected. Abbreviations: iPr2FP, diisopropyl fluorophosphate; IOV, inside-out vesicle. * Nomenclature for human erythrocyte proteins is according to Steck (1). Band 2.1 has been named "ankyrin" (8), but this name has previously been used to describe another protein (13). Band 2.1 and the series of polypeptides related to band 2.1 by sequence homology have been called syndeins (12).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. § 1734 solely to indicate this fact. 5192
Cell Biology:
Tyler et al.
Proc. Nati. Acad. Sci. USA 76 (1979)
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12 16 20 24 28 32 Gradient fraction -top
FIG. 1. (A) Sodium dodecyl sulfate/polyacrylamide gels. Lanes: a, human erythrocyte ghosts; b, ghosts from which band 6 had been extracted; spectrin-depleted vesicles (bands 1 and 2 decreased, bands 5 and 6 absent); d, extracted residue; e, crude extract enriched in bands 2.1 and 4. 1; f and g, column-purified bands 2.1 and 4.1 (see text). (B and C) Velocity sedimentation profiles of highly purified bands 2.1 and 4.1 through preparative sucrose gradients. Enzyme markers were cosedimented to provide standards for the estimation of so values: F, fumarase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; cyt. c, cytochrome c. s° was 5.7 S for band 2.1 and 4.1 S for band 4.1. c,
,
,
Purification of Bands 2.1 and 4.1. Peak fractions were loaded onto a 1.6 X 30 cm DEAE-cellulose column and, after a 20-ml wash with 5 mM NaPO4/1 mM EDTA/20 mM KCI/0.5 mM dithiothreitol, pH 7.6, elution was carried out with a 20-400 mM linear KCI gradient in the above buffer. Recovery of bands 2.1 and 4.1 from the column was estimated to be >85% under these conditions. Fractions were analyzed for 125I activity and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Fractions containing pure band 2.1 or 4.1 were separately pooled and dialyzed overnight at 40C against 4 liters of 2 mM NaPO4/1 mM EDTA/20 mM KCI/0.5 mM dithiothreitol, pH 7.6. Aliquots (1 ml) of the purified material were layered onto 12.5 ml of 5-20% linear sucrose gradients in 1 mM NaPO4/1 mM EDTA/20 mM KCI/0.5 mM dithiothreitol, pH 7.6, and centrifuged (20C, 18 hr, 40,000 rpm in a Beckman SW 40 rotor) to obtain fractions free from any low molecular weight proteolytic contaminants and to characterize the material as to sedimentation properties. After centrifugation, 0.4-ml fractions were collected and peak fractions were pooled and dialyzed overnight at 40C against 4 liters of a buffer (1 mM NaPO4/0.5 mM EDTA/20 mM KCI/0.5 mM dithiothreitol, pH 7.6) suitable for binding assays. The absorbance at 280 nm was measured after dialysis, and this was compared with Lowry (22) and ninhydrin protein assays. Typically, the proteins were present at concentrations of approximately 100 mg/ml with specific activities of 4000-16,000 cpm/mg. Preparation of Spectrin Heterodimers and Tetramers. Spectrin was prepared by using well-established methods (6, 9, 10, 14, 15); low ionic strength extraction was followed by either rate zonal centrifugation or column chromatography on Sepharose 4B. Material in peak fractions was equilibrated with 1 mM NaPO4/0.5 mM EDTA/20 mM KCI/0.5 mM dithiothreitol, pH 7.6. When appropriate, 3 mM NaN3 was included. Spectrin was labeled with 32p (6, 10) or 125I (9, 10). Preparation of Selectively Depleted Vesicles. Bands 2.1 and 4.1 were extracted by incubating spectrin-depleted inside-out vesicles (IOVs) (6) in 30 vol of 25 mM NaPO4/25 mM EDTA/1 M KCI/0.4 mM iPr2FP, pH 8.5 at 370C for 30 min. After incubation, the vesicles were pelleted and washed once in buffer without iPr2FP, and then in 1 mM NaPO4/1 mM
EDTA/20 mM KCI/3 mM NaN3. KCI extraction removed >65% of band 2.1 and >75% of band 4.1. Binding Analysis by Rate Zonal Sedimentation. Highly purified spectrin heterodimers 2.1 and 4.1 were assayed for binding activity by measuring states of association among the components as a function of their velocity sedimentation profiles. Protein mixtures were incubated for 90-120 min at 20C prior to layering onto 12.5 ml of 5-20% linear sucrose gradients in 1 mM NaPO4/0.5 mM EDTA/20 mM KCI/0.5 mM dithiothreitol, pH 7.6. After centrifugation (20C, 18 hr, 40,000 rpm in a Beckman SW 40 rotor), 0.4-ml fractions were collected. Gradient fractions were assayed for 125I activity and, when applicable, for 32P activity. In addition, aliquots (50-100
,ul) from peak fractions or from the material initially layered onto the gradient were prepared for low-angle shadowing, and
the remainder was lyophilized for polyacrylamide gel electrophoresis and autoradiography. Analysis of Spectrin Binding to Membrane Preparations.
Binding of 125I-labeled spectrin to IOVs and KCI-extracted IOVs was assayed by a modification of the technique of Bennett and Branton (6). Because we found binding affinities to be 2-3 times greater at physiological ionic strength, a concentrated salt solution was added to bring the final concentrations to 5 mM NaPO4, 1 mM EDTA, 130 mM KCI, 10 mM NaCl, 1 mM dithiothreitol, and 3 mM NaN3 at pH 7.6. 25I-Labeled spectrin behaves identically to metabolically labeled [32P]spectrin with respect to membrane binding.(9, 10). The assay mixture contained '25I-spectrin (0-24 ,g/ml), vesicles (20-25 ,g of band 3 protein per ml, estimated by scanning microdensitometry of Coomassie blue-stained gels) and, when appropriate, purified band 2.1 or 4.1 (40,ug/ml). Purified bands 2.1 and 4.1 were preincubated with spectrin for 30 min on ice prior to the addition of vesicles as described (8). After incubation for not less than 30 min with vesicles, membrane-bound and free spectrin were separated (6). Low-Angle Shadowing of Proteins. Low-angle shadowing was performed by a modification of the method of Shotton et al. (15). The samples of pure protein, the protein mixtures after incubation, or the peak gradient fractions after centrifugation of these proteins were diluted with 1 mM NaPO4/0.5 mM
Cell Biology:
5194
Tyler et al.
Proc. Natl. Acad. Sci. USA 76 (1979) :
B ^ 15C x
6
24(
x
E0. a 1 6( CQ ca
E 10C
I
Q
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0 4 8 12 1620 2428 32
Gradient fraction -top
D
2001 1-1 7
150
eI I
x
F. 100
Ir
A
0
50 i
U 4 8
121620242832
Gradient fraction -top FIG. 2. Rate zonal sedimentation binding analyses. Gradients were fractionated into 400-,ul aliquots and the 32P and 1251 in each fraction were determined. (A) Pure [32P] spectrin; (B) pure 1251-laheled band 2.1 alone (-) or plus nonradiolabeled spectrin (-.---); (C) pure 1251-labeled band 4.1 alone (-) or plus nonradiolabeled spectrin Arrows denote the velocity sedimentation position of purified (.----). tree spectrin (7.6 S). Note that spectrin bound to band 2.1 or 4.1 migrates further in the gradient than does pure spectrin, indicating an increased S value for the complex. (D) Sodium dodecyl sulfate/ polyacrylamide gels and autoradiographs showing the protein com-
position (left, Coomassie blue staining) and radioactivity source (right,
autoradiogram) of each peak.
EDTA/20 mM KCI/0.5 mM dithiothreitol, pH 7.6, gently mixed with glycerol to give final concentrations of 60% glycerol, and sprayed onto mica without removing the nonvolatile solutes by dialysis (15). RESULTS Purification of Bands 2.1 and 4.1. The protocol for purification outlined in this report consistently gave high yields of bands 2.1 and 4.1 with minimal contamination by other proteins or polypeptides. Purity was judged to be >97% in each case as measured by microspectrophotometric scans of both the Coomassie blue staining patterns (Fig. 1A) and the autoradiographic profiles of sodium dodecyl sulfate/polyacrylamide gel electrophoretograms (not shown). The inclusion of EDTA and iPr2FP throughout the elution and subsequent purification stages prevented the rapid and devastating breakdown of bands 2.1 and 4.1 by endogenous protease activity. Proteins protected by these agents during isolation and purification were stable and retained binding activity for at least 4 weeks when stored at 20C. In the absence of protease inhibitors, purified band 2.1 free from detectable contamination by other polypeptides underwent proteolytic degradation when stored at 4VC. A series of bands approximating the relative electrophoretic mobility characteristics of bands 2.2 through 2.6 were produced. Band 4.1 also underwent degradation upon extended storage, giving rise to a polypeptide that comigrated with band 4.2 on electrophoresis. Molecular weights of 210,000 for band 2.1 and 82,000 for 4.1
Spectrin concentration,glml FIG. 3. Effects of bands 2.1 (Left) and 4.1 (Right) on spectrin binding to IOVs and KCl-extracted IOVs. Binding of spectrin, preincubated with (triangles) or without (circles) band 2.1 or 4.1 (40 ALg/ml), to IOVs (open symbols) or KCl-extracted IOVs (solid symbols) is shown. Spectrin bound is expressed as Ag/mg of total membrane protein in IOVs (78 Ag/ml). Equivalent numbers of vesicles were present in the experiments with IOVs and KCl-extracted IOVs.
are assumed from electrophoretic gels run by the methods of Fairbanks et al. (23) and Laemmli (24). These molecular weights are in agreement with published values (1). Globular molecules with molecular weight approximating that of 2.1 typically have s2% ,f values ranging from 9 to 11 S, whereas those similar to band 4.1 have sOW values in the range 4 to 6 S (25). Thus, from its velocity sedimentation profile (Fig. 1 B and C) and chromatographic elution characteristics (not shown) it appears likely that purified band 2.1 exists as an asymmetric monomer in solution; band 4.1 is a relatively globular monomer under these conditions. Amino acid analyses indicate that both polypeptides contain approximately 33% hydrophobic amino acid residues, a value consistent with their solubility in water. Binding Analyses by Rate Zonal Sedimentation. Radioactivity profiles of rate zonal sedimentation binding assays indicated the existence of specific interactions between spectrin and band 2.1 and between spectrin and band 4.1 (Fig. 2). Spectrin was incubated with band 2.1 or 4.1 in a wide range of molar ratios at concentrations of band 2.1 or 4.1 up to 350 gg/ml. That the association states in each case were specific is suggested by the following observations. (i) When band 2.1 or 4.1 was denatured by heating to 65°C for 6 min prior to incubation, neither exhibited measurable binding. (ii) When band 2.1 or 4.1 was incubated with spectrin in a buffer of low ionic strength (comparable to that used for the extraction of spectrin from ghost membranes), binding was decreased to
