Proc. Nati. Acad. Sci. USA
Vol. 76, No. 2, pp. 935-938, February 1979
Medical Sciences
Comparison of structure and function of human erythrocyte and human muscle actin (deformability/membrane) K. NAKASHIMA* AND E. BEUTLERt* *Third Department of Internal Medicine, Yamaguchi University School of Medicine, 1144 Kogushi, Ube-Shi, Yamaguchi-Ken, 755 Japan; and tDepartment of Hematology, City of Hope Medical Center, 1500 E. Duarte Road, Duarte, California 91010
Contributed by Ernest Beutler, October 30, 1978
ABSTRACT Human erythrocyte actin and human skeletal muscle actin were purified by acetone powder extraction and gel filtration. Pure human erythrocyte actin resembles muscle actin in its polymerization and depolymerization by phalloidin, cytochalasin B, and DNase I, in its peptide mapping pattern, and in the amino acid composition of corresponding peptides. Isoelectric focusing gel ana ysis showed that human erythrocyte actin exists in the j8/ form, but muscle actin is in the a form. Abnormal deformability of resealed erythrocyte membranes was observed after incorporation of the actin-specific agents, phalloidin and DNase I, suggesting that erythrocyte actin might function as a membrane structural element to maintain erythrocyte membrane deformability. Because the erythrocyte is extremely deformable and has the capacity to change readily in shape from discocyte to echinocyte, it has been suggested that it contains actomyosin-like proteins (1). About eight major polypeptides are separated when erythrocyte membranes are subjected to sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis (2). Components 1 and 2 are subunits of spectrin, which crossreacts with muscle myosin antibody but has a higher molecular weight than muscle myosin (3, 4). Component 5 is similar to actin in molecular weight, ability to polymerize into filaments with a double-helical structure, its decoration with heavy meromyosin, and stimulation of myosin ATPase activity (5). For these reasons it is commonly referred to as erythrocyte actin. Unlike muscle and most tissue actins, erythrocyte actin is believed to exist in the cell in nonfilamentous form (5). It has been suggested that erythrocyte actin and spectrin function in restricting the lateral movement of membrane-penetrating particles, thereby regulating erythrocyte shape and deformability. However, most studies of interaction of spectrin and actin have been carried out with muscle actin (5, 6), since erythrocyte actin has not previously been purified to a completely homogeneous state and during even partial purification the yield is poor. In an effort to determine the structure of erythrocyte actin and its function in the cell, we purified erythrocyte and muscle actin to homogeneity and carried out comparative studies, including peptide mapping, amino acid analysis of some of the peptides, isoelectric focusing, and reactivity with the actinspecific agents DNase I, phalloidin, and cytochalasin B. MATERIALS AND METHODS Purification of Erythrocyte Actin. A modification of the method of Sheetz et al. (4) was used. Fresh blood from normal human donors was drawn into bags containing citrate/phosphate/dextrose solution. The bags were centrifuged at 1500 X g for 10 min to remove platelet-rich plasma. The packed 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.
erythrocytes were centrifuged at 3000 X g for 10 min and the buffy coat was removed. Approximately 300 ml of packed cells was suspended in an equal volume of 145 mM NaCl containing 10 mM Tris-HCl (pH 7.4 at 25°C) and passed through a cellulose column to remove leukocytes and platelets (7). The erythrocytes were washed twice with the same isotonic buffer. The packed cells were suspended in 20 times their volume of 10 mM Tris-HCl (pH 7.4) and stirred for 30 min. The hemolysate was centrifuged at 23,000 X g for 20 min. The membranes were washed three times in the buffer, centrifuged at 39,000 X g for 15 min, and then washed twice with 0.5 M NaCl containing 10 mM Tris-HCl (pH 7.4) and twice with distilled water. The supernatant 0.5 M NaCl solutions were used for preparation of protein kinase needed for phosphorylation of spectrin. Now free of hemoglobin, the ghosts were mixed with 2 vol of cold acetone, centrifuged at 27,000 X g for 15 min, and washed twice with acetone. The pellet was dried in a desiccator at 4°C overnight. The acetone powder was extracted twice with 20 ml of buffer A (2.0 mM Tris-HCI, pH 8.0/0.2 mM ATP/0.2 mM CaCI2/0.5 mM 2-mercaptoethanol) at 0°C for 30 min. The extracts were pooled and concentrated by Amicon filtration. After centrifugation at 80,000 X g for 3 hr, the supematant was applied to a Sephadex G-100 column (2.5 X 90 cm) equilibrated with buffer A. A preparation representing the second protein peak was pooled and concentrated. The final product was entirely homogeneous on NaDodSO4/polyacrylamide electrophoresis, occupying the position of component 5. Purification of Human Muscle Actin. Muscle actin was purified by the method of Spudich and Watt (8) from human muscle obtained during an autopsy. In addition, a Sephadex G-100 gel filtration was performed on a column equilibrated with buffer A. Fractions with peaks identical to that of erythrocyte actin were pooled and concentrated. The preparation was subjected to NaDodSO4/polyacrylamide gel electrophoresis to confirm homogeneity of the final product. Polyacrylamide Gel Electrofocusing. Isoelectric focusing was performed as described by O'Farrell (9) in a polyacrylamide gel containing 1.6% pH 5-7 Ampholine, 0.4% pH 3.5-10 Ampholine, 55% urea, and 2% Nonidet P-40. After the gels were run for 60 min, the samples were applied and focusing was carried out at 400 V for 12 hr and then at 800 V for 1 additional hr. The gel was fixed with 10% trichloroacetic acid and stained with Coomassie blue R 25. Peptide Mapping. Actin aggregates easily to form a trypsin-resistant core during S-aminoethylation and trypsin digestion when the pH is lowered and the preparation is lyophilized. Therefore, an alkaline pH was maintained and lyophilization was not carried out until digestion was complete. Eight milligrams of actin in buffer A was mixed with 4 g of urea, 0.2 ml of 0.2 M EDTA, and 2 ml of I M Tris-HCl (pH 8.6). Abbreviation: NaDodSO4, sodium dodecyl sulfate. To whom reprint requests should be addressed.
t
935
Proc. Nati. Acad. Sci. USA 76 (1979)
Medical Sciences: Nakashima and Beutler
936
Water was added to give a final volume of 8 ml. Two hundred microliters of 2-mercaptoethanol was added and the mixture was stirred under N2 for 4 hr at room temperature. A 0.6-ml volume of ethyleneimine was added and stirring was continued for 30 min under N2. After addition of 100 pAl of 2-mercaptoethanol, the mixture was dialyzed against a change of 440 vol each of 0.05% ammonium hydroxide at 4VC for 48 hr. The pH was adjusted to 9.2 with acetic acid, 100 1Ag of trypsin was added, and the mixture was stirred at room temperature for 8 hr. Another 100 ,tg of trypsin was added and the mixture was digested for 16 hr in the presence of 5 ,l of toluene. The digest was centrifuged at 80,000 X g for 60 min and the supernatant was lyophilized. The trypsin-resistant core represented only 1.4% of the total weight in this procedure. Peptide mapping was performed by the method of Katz et al. (10). Initially, paper chromatography was carried out with n-butanol/acetic acid/water 4:1:5 (vol/vol) for 17 hr. This was followed by paper electrophoresis at about 30 V/cm for 70 min with pyridine/acetic acid/water 1:10:290 (vol/vol, pH 6.3) as buffer and staining with ninhydrin. Three corresponding peptide spots of erythrocyte actin and muscle actin were eluted and hydrolyzed, and amino acid compositions were determined with an automatic amino acid analyzer. Polymerization and Depolymerization Studies. Erythrocyte actin and muscle actin were diluted with buffer A to a concentration of 0.6 mg/ml. After addition of 50 mM KCl and 2 mM MgCl2, the relative viscosity was measured in a capillary glass-tube viscometer both in the absence and the presence of 10 1Ag of phalloidin per ml and of 2 mM cytochalasin B. Depolymerization was reflected by the decrease in viscosity of polymerized actin after addition of DNase 1 (0.2 mg/ml). The effect of phosphorylated and dephosphorylated human erythrocyte spectrin on the viscosity of human erythrocyte and muscle actin and on commercially available (Worthington) rabbit muscle actin was studied by the methods described by Pinder et al. (6). Deformability of Resealed Erythrocyte Membrane. Fresh blood was anticoagulated with heparin, and erythrocytes were washed three times with 100 mM NaCI to remove the buffy coat. One volume of erythrocytes was mixed with 2 vol of water
pH 7
or phalloidin (1 mg/mi). After 15 min at 00C, sufficient 4 M NaCl was added to the final concentration of 150 mM; the samples were mixed well and were incubated at 370C for 10 min. The resealed erythrocyte membranes were washed twice with 154 mM NaCl. Erythrocyte membranes were resealed to contain DNase I by the method of Dale et at. (11), with smallpore dialysis membranes (3500 molecular weight cutoff). Deformability of the resealed erythrocyte membranes was measured in an ektacytometer with a 0.566-poise (56.6 mPa-sec) dextran solution (12, 13). The elongation of the membranes under shear stress was estimated by photographing and measuring the diffraction pattern. The relative width of the pattern was used as the control length and given an arbitrary value of 1.00.
Phalloidin, cytochalasin B, and DNase I were products of Sigma.
RESULTS On NaDodSO4 electrophoresis, erythrocyte actin and muscle actin each appeared as a single band with identical mobility (Fig. 1 left). On isoelectric focusing, however, the main components of erythrocyte and muscle actin had different isoelectric points (Fig. 1 right). Whereas, as reported by Whalen et al. (14), muscle actin was an a form, the isoelectric focusing pattern of erythrocyte actin resembled the ,3 and oy forms, which are characteristic of platelet and other tissue actins (15). In spite of this difference in isoelectric point, the peptide mapping patterns of erythrocyte and muscle actin were very similar (Fig. 2). Three corresponding peptides were eluted. All had very similar amino acid compositions (Table 1). One of the three spots apparently represented overlapping peptides, since at least four equivalents of arginine, a trypsin-cleavage site, were present for each equivalent of some of the other amino acids. Phalloidin promoted polymerization of erythrocyte actin at a rate comparable to that observed with muscle actin (16). Similarly, the extent to which cytochalasin B inhibited polymerization (17) and to which DNase I produced depolymerization of actin (18) was the same in erythrocyte as in muscle actin (Fig. 3). Both forms of phalloidin-treated actin were similarly resistant to the depolymerizing effect of DNase I (16). Heated DNase was inactive. Neither phosphorylated nor nonphosphorylated spectrin had any effect on human erythrocyte, human muscle, or rabbit muscle actin. Only when phosphorylated spectrin containing crude protein kinase in 10 mM Tris-HCI/100 mM NaCl/5 mM MgCl2 (6) was used without dialysis or, indeed, when this buffer was used without
.-. / and ;. ..
f
,
a
t
._
C)
0
E
0
pH 5 M
E
.-)
f
M M E +
Fic. 1. (Left) NaDodSO4/polyacrylamide gel electrophoresis. M, purified muscle actin; E, purified erythrocyte actin. (Right) Stained isoelectric focusing gels of purified actin. M, purified muscle actin; M + E, mixture of muscle actin and erythrocyte actin; E, purified erythrocyte actin. Only the middle part of the gel is shown.
L
(.4,4*
4
_..
.
I*i 9-4.
Electrophoresis
Fic(.
2. Tryptic peptide map of human erythrocyte actin (Right) and human muscle actin (Left).
Medical Sciences: Nakashima and Beutler
Proc. Nati. Acad. Sci. USA 76 (1979)
937
Table 1. Amino acid composition of peptides eluted from peptide map 1.6
Amino acid
Peptide 1 E M
Lys Arg Asp Thr Ser Glu Gly Ala Val Ile Leu Pro
7.4
9.7
7.5
11.0
15.2 14.8 12.8 10.2 9.2
Peptide 2 M E
11.4 10.8 11.0
19.0 20.8 15.3 12.3 10.3
Peptide 3 M E
12.6 12.3 14.1
21.6 22.8 16.2 14.0 14.6
27.2 28.8 20.0 18.3 14.1
aChn 1.4
+1 -
26.0
29.0
6.6
5.6
EX/ */
00
it .2
a:
14.8
18.0
9.4 11.6 20.8 8.0 6.6
8.0 11.4 21.4 7.4 13.4
_ a~~~~~~~~~~~
1.0
1
,
.
.
200
. 400
600
800
Values are in nmol. M, peptides from muscle actin; E, peptides from erythrocyte actin. a)1.6
spectrin, was an increase in the viscosity of actin observed. The effect of the incorporation of phalloidin and DNase I on the deformability of resealed erythrocyte membranes was investigated in the ektacytometer. Resealed membranes containing heated DNase I served as controls. Phalloidin, which accelerates polymerization of actin, reduced deformability of the erythrocyte membranes; DNase I, which depolymerizes actin, produced increased deformability of the membranes (Fig. 4). Membranes that were resealed by three slightly different techniques showed the same results.
r
-
03~~~~~~~~
1.4 aC 03
1.0
~//
-
600
800
FI.a 4. Variation of resealed erythrocyte membrane length as a function of applied shear stress (1 dyne/cm2 = 0.1 Pa). *, Control with water; 03, phalloidin; 0, DNase I; *, control with heated DNase I.
0"_0 ~~~~~~~~~~~0
U,
0~~~~~~~~~~~~ 03 03C >~
0
> 1.0
-
0
0
0
0.~~~~~~~~~
0
0~~~~~ 31.2
400
Shear stress, dyne/cm2 0
> 1.4
/~~
0/0
200 1.6
i Ho
I!
00
5
,C 20 1
5
15
D,20
Time, min
FI(c. 3. Polymerization and depolymerization of muscle actin (Upper) and erythrocyte actin (Lower). Depolymerization (B and D) by DNase I was studied by addition of DNase I to the actin polymerized (A and C) in 50 mM KCl and 2 mM MgCl2. 0-0, Only actin; 0-0, actin plus phalloidin; 3-3, actin plus cytochalasin B; o ----- 0, actin plus heated DNase I.
DISCUSSION Although the results of our isoelectric focusing studies (Fig. 1 right) suggest that erythrocyte actin is not identical to muscle actin, the results of peptide mapping and similarities in physical properties indicate that only minor differences in the structure of these actins exist. Elzinga et al. (1-9) and Storti and Rich (20) have shown that actin from sarcomeric tissues and actin from nonmuscle cells are products of different genes. Whalen et al. (14), using isoelectric focusing, described the existence of three isozymic forms of actin, a, A, and y. Although erythrocyte actin is believed to exist in a nonfilamentous form, in contrast to the filamentous form of muscle actin, the physical properties of erythrocyte actin were very similar to those of muscle actin. Our studies are consistent with the view that erythrocyte actin associated with spectrin plays an important role in regulating erythrocyte shape and deformability Phalloidin, an agent that promotes actin polymerization, rendered the membrane less deformable, and DNase I, a depolymerizing agent, increased deformability of membranes (Fig. 4). Our results with phalloidin may be analogous to those reported by Wehland et al. (21) in tissue culture cells. In their studies, injection of phalloidin into cells resulted in aggregation of actin into islands and in disturbed cell locomotion. Although much remains to be learned about the function of actin in erythrocytes, our studies indicate that erythrocyte membrane protein component 5 may confidently be regarded as a bona fide tissue actin. We gratefully acknowledge the help of Dr. Akira Yoshida with the amino acid analyses. This work was supported in part by Grant HL
938
Medical Sciences: Nakashima and Beutler
07449 from the National Institutes of Health. K.N. was a U.S. Public Health Service International Research Fellow (1 F05 TW02503-01). 1. Onishi, T. (1962) J. Biochem. 52,307-308. 2. Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971) Biochemistry 10, 2602-2617. 3. Kirkpatrick, F. H. (1976) Life Sci. 19, 1-18. 4. Sheetz, M. P., Painter, R. G. & Singer, S. J. (1976) Biochemistry 15,4486-4492. 5. Tilney, L. G. & Detmers, P. (1975) J. Cell Biol. 66,508-520. 6. Pinder, J. C., Bray, D. & Gratzer, W. B. (1977) Nature (London)
270,752-754. 7. Beutler, E., West, C. & Blume, K. G. (1976) J. Lab. Clin. Med. 88,328-333. 8. Spudich, J. A. & Watt, S. (1971) J. Biol. Chem. 246, 48664871. 9. O'Farrell, P. H. (1975) J. Biol. Chem. 250,4007-4021. 10. Katz, A. M., Dreyer, W. J. & Anfinsen, C. B. (1959) J. Biol. Chem.
234,2897-2900.
Proc. Nati. Acad. Sci. USA 76 (1d79) 11. Dale, G. L., Villacorte, D. G. & Beutler, E. (1977) Biochem. Med. 18, 220-225. 12. Bessis, M. & Mohandas, N. (1975) Blood Cells 1, 307-313. 13. Nakashima, K. & Beutler, E. (1978) Proc. Nati. Acad. Sci. USA 75,3823-3825. 14. Whalen, R. G., Butler-Browne, G. S. & Gros, F. (1976) Proc. Nati. Acad. Sci. USA 73,2018-2022. 15. London, F., Huc, C., Thome, F., Oriol, C. & Olomucki, A. (1977) Eur. J. Biochem. 81, 571-577. 16. Wieland, T. (1977) Naturwissenschaften 64,303-309. 17. Spudich, J. A. (1973) Cold Spring Harbor Symp. Quant. Biol. 37,585-593. 18. Hitchcock, S. E., Carlsson, L. & Lindberg, U. (1976) Cell 7, 531-542. 19. Elzinga, M., Maron, B. J. & Adelstein, R. S. (1976) Science 191, 94-95. 20. Storti, R. V. & Rich, A. (1976) Proc. Natl. Acad. Sci. USA 73, 2346-2350. 21. Wehland, J., Osborn, M. & Weber, K. (1977) Proc. NatI. Acad. Sci. USA 74,5613-5617.
Vol. 76, No. 2, pp. 935-938, February 1979
Medical Sciences
Comparison of structure and function of human erythrocyte and human muscle actin (deformability/membrane) K. NAKASHIMA* AND E. BEUTLERt* *Third Department of Internal Medicine, Yamaguchi University School of Medicine, 1144 Kogushi, Ube-Shi, Yamaguchi-Ken, 755 Japan; and tDepartment of Hematology, City of Hope Medical Center, 1500 E. Duarte Road, Duarte, California 91010
Contributed by Ernest Beutler, October 30, 1978
ABSTRACT Human erythrocyte actin and human skeletal muscle actin were purified by acetone powder extraction and gel filtration. Pure human erythrocyte actin resembles muscle actin in its polymerization and depolymerization by phalloidin, cytochalasin B, and DNase I, in its peptide mapping pattern, and in the amino acid composition of corresponding peptides. Isoelectric focusing gel ana ysis showed that human erythrocyte actin exists in the j8/ form, but muscle actin is in the a form. Abnormal deformability of resealed erythrocyte membranes was observed after incorporation of the actin-specific agents, phalloidin and DNase I, suggesting that erythrocyte actin might function as a membrane structural element to maintain erythrocyte membrane deformability. Because the erythrocyte is extremely deformable and has the capacity to change readily in shape from discocyte to echinocyte, it has been suggested that it contains actomyosin-like proteins (1). About eight major polypeptides are separated when erythrocyte membranes are subjected to sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis (2). Components 1 and 2 are subunits of spectrin, which crossreacts with muscle myosin antibody but has a higher molecular weight than muscle myosin (3, 4). Component 5 is similar to actin in molecular weight, ability to polymerize into filaments with a double-helical structure, its decoration with heavy meromyosin, and stimulation of myosin ATPase activity (5). For these reasons it is commonly referred to as erythrocyte actin. Unlike muscle and most tissue actins, erythrocyte actin is believed to exist in the cell in nonfilamentous form (5). It has been suggested that erythrocyte actin and spectrin function in restricting the lateral movement of membrane-penetrating particles, thereby regulating erythrocyte shape and deformability. However, most studies of interaction of spectrin and actin have been carried out with muscle actin (5, 6), since erythrocyte actin has not previously been purified to a completely homogeneous state and during even partial purification the yield is poor. In an effort to determine the structure of erythrocyte actin and its function in the cell, we purified erythrocyte and muscle actin to homogeneity and carried out comparative studies, including peptide mapping, amino acid analysis of some of the peptides, isoelectric focusing, and reactivity with the actinspecific agents DNase I, phalloidin, and cytochalasin B. MATERIALS AND METHODS Purification of Erythrocyte Actin. A modification of the method of Sheetz et al. (4) was used. Fresh blood from normal human donors was drawn into bags containing citrate/phosphate/dextrose solution. The bags were centrifuged at 1500 X g for 10 min to remove platelet-rich plasma. The packed 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.
erythrocytes were centrifuged at 3000 X g for 10 min and the buffy coat was removed. Approximately 300 ml of packed cells was suspended in an equal volume of 145 mM NaCl containing 10 mM Tris-HCl (pH 7.4 at 25°C) and passed through a cellulose column to remove leukocytes and platelets (7). The erythrocytes were washed twice with the same isotonic buffer. The packed cells were suspended in 20 times their volume of 10 mM Tris-HCl (pH 7.4) and stirred for 30 min. The hemolysate was centrifuged at 23,000 X g for 20 min. The membranes were washed three times in the buffer, centrifuged at 39,000 X g for 15 min, and then washed twice with 0.5 M NaCl containing 10 mM Tris-HCl (pH 7.4) and twice with distilled water. The supernatant 0.5 M NaCl solutions were used for preparation of protein kinase needed for phosphorylation of spectrin. Now free of hemoglobin, the ghosts were mixed with 2 vol of cold acetone, centrifuged at 27,000 X g for 15 min, and washed twice with acetone. The pellet was dried in a desiccator at 4°C overnight. The acetone powder was extracted twice with 20 ml of buffer A (2.0 mM Tris-HCI, pH 8.0/0.2 mM ATP/0.2 mM CaCI2/0.5 mM 2-mercaptoethanol) at 0°C for 30 min. The extracts were pooled and concentrated by Amicon filtration. After centrifugation at 80,000 X g for 3 hr, the supematant was applied to a Sephadex G-100 column (2.5 X 90 cm) equilibrated with buffer A. A preparation representing the second protein peak was pooled and concentrated. The final product was entirely homogeneous on NaDodSO4/polyacrylamide electrophoresis, occupying the position of component 5. Purification of Human Muscle Actin. Muscle actin was purified by the method of Spudich and Watt (8) from human muscle obtained during an autopsy. In addition, a Sephadex G-100 gel filtration was performed on a column equilibrated with buffer A. Fractions with peaks identical to that of erythrocyte actin were pooled and concentrated. The preparation was subjected to NaDodSO4/polyacrylamide gel electrophoresis to confirm homogeneity of the final product. Polyacrylamide Gel Electrofocusing. Isoelectric focusing was performed as described by O'Farrell (9) in a polyacrylamide gel containing 1.6% pH 5-7 Ampholine, 0.4% pH 3.5-10 Ampholine, 55% urea, and 2% Nonidet P-40. After the gels were run for 60 min, the samples were applied and focusing was carried out at 400 V for 12 hr and then at 800 V for 1 additional hr. The gel was fixed with 10% trichloroacetic acid and stained with Coomassie blue R 25. Peptide Mapping. Actin aggregates easily to form a trypsin-resistant core during S-aminoethylation and trypsin digestion when the pH is lowered and the preparation is lyophilized. Therefore, an alkaline pH was maintained and lyophilization was not carried out until digestion was complete. Eight milligrams of actin in buffer A was mixed with 4 g of urea, 0.2 ml of 0.2 M EDTA, and 2 ml of I M Tris-HCl (pH 8.6). Abbreviation: NaDodSO4, sodium dodecyl sulfate. To whom reprint requests should be addressed.
t
935
Proc. Nati. Acad. Sci. USA 76 (1979)
Medical Sciences: Nakashima and Beutler
936
Water was added to give a final volume of 8 ml. Two hundred microliters of 2-mercaptoethanol was added and the mixture was stirred under N2 for 4 hr at room temperature. A 0.6-ml volume of ethyleneimine was added and stirring was continued for 30 min under N2. After addition of 100 pAl of 2-mercaptoethanol, the mixture was dialyzed against a change of 440 vol each of 0.05% ammonium hydroxide at 4VC for 48 hr. The pH was adjusted to 9.2 with acetic acid, 100 1Ag of trypsin was added, and the mixture was stirred at room temperature for 8 hr. Another 100 ,tg of trypsin was added and the mixture was digested for 16 hr in the presence of 5 ,l of toluene. The digest was centrifuged at 80,000 X g for 60 min and the supernatant was lyophilized. The trypsin-resistant core represented only 1.4% of the total weight in this procedure. Peptide mapping was performed by the method of Katz et al. (10). Initially, paper chromatography was carried out with n-butanol/acetic acid/water 4:1:5 (vol/vol) for 17 hr. This was followed by paper electrophoresis at about 30 V/cm for 70 min with pyridine/acetic acid/water 1:10:290 (vol/vol, pH 6.3) as buffer and staining with ninhydrin. Three corresponding peptide spots of erythrocyte actin and muscle actin were eluted and hydrolyzed, and amino acid compositions were determined with an automatic amino acid analyzer. Polymerization and Depolymerization Studies. Erythrocyte actin and muscle actin were diluted with buffer A to a concentration of 0.6 mg/ml. After addition of 50 mM KCl and 2 mM MgCl2, the relative viscosity was measured in a capillary glass-tube viscometer both in the absence and the presence of 10 1Ag of phalloidin per ml and of 2 mM cytochalasin B. Depolymerization was reflected by the decrease in viscosity of polymerized actin after addition of DNase 1 (0.2 mg/ml). The effect of phosphorylated and dephosphorylated human erythrocyte spectrin on the viscosity of human erythrocyte and muscle actin and on commercially available (Worthington) rabbit muscle actin was studied by the methods described by Pinder et al. (6). Deformability of Resealed Erythrocyte Membrane. Fresh blood was anticoagulated with heparin, and erythrocytes were washed three times with 100 mM NaCI to remove the buffy coat. One volume of erythrocytes was mixed with 2 vol of water
pH 7
or phalloidin (1 mg/mi). After 15 min at 00C, sufficient 4 M NaCl was added to the final concentration of 150 mM; the samples were mixed well and were incubated at 370C for 10 min. The resealed erythrocyte membranes were washed twice with 154 mM NaCl. Erythrocyte membranes were resealed to contain DNase I by the method of Dale et at. (11), with smallpore dialysis membranes (3500 molecular weight cutoff). Deformability of the resealed erythrocyte membranes was measured in an ektacytometer with a 0.566-poise (56.6 mPa-sec) dextran solution (12, 13). The elongation of the membranes under shear stress was estimated by photographing and measuring the diffraction pattern. The relative width of the pattern was used as the control length and given an arbitrary value of 1.00.
Phalloidin, cytochalasin B, and DNase I were products of Sigma.
RESULTS On NaDodSO4 electrophoresis, erythrocyte actin and muscle actin each appeared as a single band with identical mobility (Fig. 1 left). On isoelectric focusing, however, the main components of erythrocyte and muscle actin had different isoelectric points (Fig. 1 right). Whereas, as reported by Whalen et al. (14), muscle actin was an a form, the isoelectric focusing pattern of erythrocyte actin resembled the ,3 and oy forms, which are characteristic of platelet and other tissue actins (15). In spite of this difference in isoelectric point, the peptide mapping patterns of erythrocyte and muscle actin were very similar (Fig. 2). Three corresponding peptides were eluted. All had very similar amino acid compositions (Table 1). One of the three spots apparently represented overlapping peptides, since at least four equivalents of arginine, a trypsin-cleavage site, were present for each equivalent of some of the other amino acids. Phalloidin promoted polymerization of erythrocyte actin at a rate comparable to that observed with muscle actin (16). Similarly, the extent to which cytochalasin B inhibited polymerization (17) and to which DNase I produced depolymerization of actin (18) was the same in erythrocyte as in muscle actin (Fig. 3). Both forms of phalloidin-treated actin were similarly resistant to the depolymerizing effect of DNase I (16). Heated DNase was inactive. Neither phosphorylated nor nonphosphorylated spectrin had any effect on human erythrocyte, human muscle, or rabbit muscle actin. Only when phosphorylated spectrin containing crude protein kinase in 10 mM Tris-HCI/100 mM NaCl/5 mM MgCl2 (6) was used without dialysis or, indeed, when this buffer was used without
.-. / and ;. ..
f
,
a
t
._
C)
0
E
0
pH 5 M
E
.-)
f
M M E +
Fic. 1. (Left) NaDodSO4/polyacrylamide gel electrophoresis. M, purified muscle actin; E, purified erythrocyte actin. (Right) Stained isoelectric focusing gels of purified actin. M, purified muscle actin; M + E, mixture of muscle actin and erythrocyte actin; E, purified erythrocyte actin. Only the middle part of the gel is shown.
L
(.4,4*
4
_..
.
I*i 9-4.
Electrophoresis
Fic(.
2. Tryptic peptide map of human erythrocyte actin (Right) and human muscle actin (Left).
Medical Sciences: Nakashima and Beutler
Proc. Nati. Acad. Sci. USA 76 (1979)
937
Table 1. Amino acid composition of peptides eluted from peptide map 1.6
Amino acid
Peptide 1 E M
Lys Arg Asp Thr Ser Glu Gly Ala Val Ile Leu Pro
7.4
9.7
7.5
11.0
15.2 14.8 12.8 10.2 9.2
Peptide 2 M E
11.4 10.8 11.0
19.0 20.8 15.3 12.3 10.3
Peptide 3 M E
12.6 12.3 14.1
21.6 22.8 16.2 14.0 14.6
27.2 28.8 20.0 18.3 14.1
aChn 1.4
+1 -
26.0
29.0
6.6
5.6
EX/ */
00
it .2
a:
14.8
18.0
9.4 11.6 20.8 8.0 6.6
8.0 11.4 21.4 7.4 13.4
_ a~~~~~~~~~~~
1.0
1
,
.
.
200
. 400
600
800
Values are in nmol. M, peptides from muscle actin; E, peptides from erythrocyte actin. a)1.6
spectrin, was an increase in the viscosity of actin observed. The effect of the incorporation of phalloidin and DNase I on the deformability of resealed erythrocyte membranes was investigated in the ektacytometer. Resealed membranes containing heated DNase I served as controls. Phalloidin, which accelerates polymerization of actin, reduced deformability of the erythrocyte membranes; DNase I, which depolymerizes actin, produced increased deformability of the membranes (Fig. 4). Membranes that were resealed by three slightly different techniques showed the same results.
r
-
03~~~~~~~~
1.4 aC 03
1.0
~//
-
600
800
FI.a 4. Variation of resealed erythrocyte membrane length as a function of applied shear stress (1 dyne/cm2 = 0.1 Pa). *, Control with water; 03, phalloidin; 0, DNase I; *, control with heated DNase I.
0"_0 ~~~~~~~~~~~0
U,
0~~~~~~~~~~~~ 03 03C >~
0
> 1.0
-
0
0
0
0.~~~~~~~~~
0
0~~~~~ 31.2
400
Shear stress, dyne/cm2 0
> 1.4
/~~
0/0
200 1.6
i Ho
I!
00
5
,C 20 1
5
15
D,20
Time, min
FI(c. 3. Polymerization and depolymerization of muscle actin (Upper) and erythrocyte actin (Lower). Depolymerization (B and D) by DNase I was studied by addition of DNase I to the actin polymerized (A and C) in 50 mM KCl and 2 mM MgCl2. 0-0, Only actin; 0-0, actin plus phalloidin; 3-3, actin plus cytochalasin B; o ----- 0, actin plus heated DNase I.
DISCUSSION Although the results of our isoelectric focusing studies (Fig. 1 right) suggest that erythrocyte actin is not identical to muscle actin, the results of peptide mapping and similarities in physical properties indicate that only minor differences in the structure of these actins exist. Elzinga et al. (1-9) and Storti and Rich (20) have shown that actin from sarcomeric tissues and actin from nonmuscle cells are products of different genes. Whalen et al. (14), using isoelectric focusing, described the existence of three isozymic forms of actin, a, A, and y. Although erythrocyte actin is believed to exist in a nonfilamentous form, in contrast to the filamentous form of muscle actin, the physical properties of erythrocyte actin were very similar to those of muscle actin. Our studies are consistent with the view that erythrocyte actin associated with spectrin plays an important role in regulating erythrocyte shape and deformability Phalloidin, an agent that promotes actin polymerization, rendered the membrane less deformable, and DNase I, a depolymerizing agent, increased deformability of membranes (Fig. 4). Our results with phalloidin may be analogous to those reported by Wehland et al. (21) in tissue culture cells. In their studies, injection of phalloidin into cells resulted in aggregation of actin into islands and in disturbed cell locomotion. Although much remains to be learned about the function of actin in erythrocytes, our studies indicate that erythrocyte membrane protein component 5 may confidently be regarded as a bona fide tissue actin. We gratefully acknowledge the help of Dr. Akira Yoshida with the amino acid analyses. This work was supported in part by Grant HL
938
Medical Sciences: Nakashima and Beutler
07449 from the National Institutes of Health. K.N. was a U.S. Public Health Service International Research Fellow (1 F05 TW02503-01). 1. Onishi, T. (1962) J. Biochem. 52,307-308. 2. Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971) Biochemistry 10, 2602-2617. 3. Kirkpatrick, F. H. (1976) Life Sci. 19, 1-18. 4. Sheetz, M. P., Painter, R. G. & Singer, S. J. (1976) Biochemistry 15,4486-4492. 5. Tilney, L. G. & Detmers, P. (1975) J. Cell Biol. 66,508-520. 6. Pinder, J. C., Bray, D. & Gratzer, W. B. (1977) Nature (London)
270,752-754. 7. Beutler, E., West, C. & Blume, K. G. (1976) J. Lab. Clin. Med. 88,328-333. 8. Spudich, J. A. & Watt, S. (1971) J. Biol. Chem. 246, 48664871. 9. O'Farrell, P. H. (1975) J. Biol. Chem. 250,4007-4021. 10. Katz, A. M., Dreyer, W. J. & Anfinsen, C. B. (1959) J. Biol. Chem.
234,2897-2900.
Proc. Nati. Acad. Sci. USA 76 (1d79) 11. Dale, G. L., Villacorte, D. G. & Beutler, E. (1977) Biochem. Med. 18, 220-225. 12. Bessis, M. & Mohandas, N. (1975) Blood Cells 1, 307-313. 13. Nakashima, K. & Beutler, E. (1978) Proc. Nati. Acad. Sci. USA 75,3823-3825. 14. Whalen, R. G., Butler-Browne, G. S. & Gros, F. (1976) Proc. Nati. Acad. Sci. USA 73,2018-2022. 15. London, F., Huc, C., Thome, F., Oriol, C. & Olomucki, A. (1977) Eur. J. Biochem. 81, 571-577. 16. Wieland, T. (1977) Naturwissenschaften 64,303-309. 17. Spudich, J. A. (1973) Cold Spring Harbor Symp. Quant. Biol. 37,585-593. 18. Hitchcock, S. E., Carlsson, L. & Lindberg, U. (1976) Cell 7, 531-542. 19. Elzinga, M., Maron, B. J. & Adelstein, R. S. (1976) Science 191, 94-95. 20. Storti, R. V. & Rich, A. (1976) Proc. Natl. Acad. Sci. USA 73, 2346-2350. 21. Wehland, J., Osborn, M. & Weber, K. (1977) Proc. NatI. Acad. Sci. USA 74,5613-5617.
