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[25] Isolation of spectrin from erythrocyte membranes
[25]
SPECTRIN FROM ERYTHROCYTE MEMBRANES
[25] Isolation of Spectrin from Erythrocyte
275
Membranes
B y VINCENT T. MARCHESI
Erythrocyte membranes contain high molecular weight polypeptides which are easily extracted by exposing ghosts to low ionic strength media containing chelating agents 1 or to media made cation-free by deionization. 2 These polypeptides, which appear to be in the 200,000 molecular weight range, make up approximately 20% of the total membrane protein and represent the predominant species of the so-called peripheral membrane proteins. When isolated and purified by the procedures described below, this protein, which is called spectrin, 1 does not contain significant amounts of lipid or carbohydrate. After treatment with divalent cations some spectrin preparations polymerize in the form of fibrous strands, '3 which are similar to those seen along the inner surfaces of red cell ghosts when the latter are examined by thin sectioning techniques and electron microscopy. 4 Since these fibrous structures are no longer evident on ghosts examined after spectrin is extracted from the membranes, it was suggested that spectrin might exist as a fibrous network along the inner surfaces of red cell membranes, ~ an idea which has since been amply confirmed by experiments with ferritin-conjugated antibodies 6 and nonpenetrating labeling reagents. 7-9 Procedure Ghost membranes are prepared from freshly drawn blood by osmotic lysis in either dilute phosphate buffer 1° or Tris. HC1 buffer? The procedure first developed for the isolation of spectrin involved dialyzing ghost membranes against large volumes of ATP or E D T A in the cold for periods ranging from 18 to 24 hours, followed by centrifugation, ammonium sulfate precipitation, and gel filtration. Relatively small amounts of blood were 1V. T. Marchesi and E. Steers, Science 159, 203 (1968). 2M. Clarke, Biochem. Biophys. Res. Commun. 45, 1063 (1971). 'E. Steers and V. T. Marchesi, J. Gen. Physiol. 54, 655 (1969). *V. T. Marchesi and G. E. Palade, J. Cell Biol. 35, 385 (1967). V. T. Marchesi, E. Steers, T. W. Tillack, and S. L. Marchesi, in "Red Cell Membrane, Structure and Function" (G. A. Jamieson and T. J. Greenwalt, eds.), p. 117. Lippincott, Philadelphia, Pennsylvania, 1969. ~G. L. Nicolson, V. T. Marchesi, and S. J. Singer, J. Cell Biol. 51, 265 (1971). H. C. Berg, Biochem. Biophys. Acta 183, 65 (1969). 8D. R. Phillips and M. Morrison, Biochem. Biophys. Res. Commun. 40, 284 (1970). 9M. S. Bretscher, J. Mol. Biol. 58, 775 (1971). l"J. T. Dodge, C. Mitchell, and D. J. Hanahan, Arch. Biochem. Biophys. 100, 119 (1963).
276
ISOLATIONOF SELECTED MEMBRANE COMPONENTS
[25]
processed in these initial studies and great care was taken to minimize the amount of leukocyte and bacterial contamination. However, we and others have since found that this procedure is difficult to use for the large-scale extraction of ghost membranes, and artifactual degradation of the high molecular weight polypeptides frequently occurs if strict precautions are not observed. 11 Recently a modification of this method has been suggested lz which involves a shorter incubation in EDTA, and this greatly reduces the risk of proteolytic degradation and is clearly the method of choice for the preparation of spectrin at the present time. Preparation of Ghost Membranes Freshly drawn blood of compatible ABO type is collected in acid citrate dextrose and washed three times by suspension in phosphate-saline (pH 7.2) and centrifugation at 2500 rpm (Sorvall RC-3) for 15 minutes. Each time the "huffy-coat" material is aspirated off along with a generous amount of underlying red ceils. It is important to realize that it is probably not possible to remove all the leukocytes by this procedure although the amount of such contamination can be substantially decreased. The leukocytes in "old" blood are more unstable and tend to aggregate and are more difficult to remove by this method. Washed cells are lysed in either Tris.HCl (10 m M , pH 7.4) or in phosphate buffer ( 1 5 - 2 0 mOs, pH 7.1) at a ratio of approximately 1 volume of packed red cells to 30 volumes of lysing medium maintained at 5 - 1 0 ° . If ratios greater than 1:40 are used, some of the ghosts may fragment, and it will be difficult to collect them by continuous-flow centrifugation. Ghosts collected after a single lysis are grossly pink since they still contain substantial amounts of hemoglobin. Most of this contaminating hemoglobin can be removed by a second lysis, but since this may also result in a considerable loss of spectrin, we prefer to extract pink membranes and remove the hemoglobin during the gel filtration step described below. Most of our large-scale work in the past has been carried out using a Szent-GySrgyi-Blum-type continuous flow apparatus in a Sorvall RC-2B centrifuge (20,000 rpm, 5 °) for collection of the ghost membranes. When using this device it is important to remove the ghost membranes from the steel centrifuge tubes without dislodging the pellets of leukocyte nuclei and lysosomal granules. Recently we have begun using a JCF-Z rotor in a Beckman J-21 centrifuge (I 8,000 rpm, 5°). We have been able to achieve ~1H. R. Trayer, Y. Nozaki, J. A. Reynolds, and C. Tanford, J. Biol. Chem. 246, 4485 (1971). 12G. Fairbanks, T. L. Steck, and D. F. H. Wallach, Biochemistry 10, 2606 (1971).
[26]
ISOLATION OF (Na ÷ + K+)-ATPase
277
greater flow rates with the latter instrument, permitting shorter lysing times, and, in addition, a sucrose "cushion" can also be pumped into this rotor during the run which allows us to separate contaminating leukocyte fragments from the ghost membranes. Membranes are pumped out of this rotor and resuspended in either dilute Tris-HCl (10 mM) or distilled water, pelleted by centrifugation, then either stored frozen or extracted immediately. Extraction of Spectrin Freshly prepared ghosts (at 10-20 mg dry weight per milliliter) are suspended in 10 volumes of 0.1 mM EDTA (pH 8.0) and incubated at 37 ° for 10-15 minutes as described previously.TM The ghosts fragment into small vesicles during this treatment, and the spectrin which is released in the medium can be separated from membrane fragments by centrifugation (approximately 100,000 g, 60-90 minutes, 4 °) and then concentrated by pressure dialysis. Spectrin can also be precipitated by the addition of an equal volume of saturated ammonium sulfate, and then redissolved by dialysis against 1 mM EDTA, but this is less satisfactory. Freeze-drying should be avoided. Spectrin has been purified free of small molecular weight contaminants by gel filtration on Sephadex G-200 ~ and, more recently, on Bio-Gel P-300 in the presence of imidazole glycyl glycine, 1 mM, pH 7.3. 2 '~ S. L. Marchesi, E. Steers, V. T. Marchesi, and T. W. Tillack, Biochemistry 9, 50 (1969).
[26] I s o l a t i o n of ( N a + + K + ) - A T P a s e
By
PETER LETH J~iRGENSEN
Since the discovery of (Na + + K+)-ATPase, 1 intensive studies have been carried out to elucidate its rote in the active transport of sodium and potassium.2 4 The enzyme has not been purified to a homogeneous state, but recently some progress in this direction has been made and informa-
a J. C. Skou, Biochim. Biophys. Acta 23, 394 (1957). ~J. C. Skou, Physiol. Rev. 45, 596 (1965). 3 R. L. Post, S. Kume, T. Tobin, B. Orcutt, and A. K. Sen, J. Gen. Physiol. 54, 306 (1969). ' J . C. Skou, Curr. Top. Bioenerg. 4, 357 (1971).
SPECTRIN FROM ERYTHROCYTE MEMBRANES
[25] Isolation of Spectrin from Erythrocyte
275
Membranes
B y VINCENT T. MARCHESI
Erythrocyte membranes contain high molecular weight polypeptides which are easily extracted by exposing ghosts to low ionic strength media containing chelating agents 1 or to media made cation-free by deionization. 2 These polypeptides, which appear to be in the 200,000 molecular weight range, make up approximately 20% of the total membrane protein and represent the predominant species of the so-called peripheral membrane proteins. When isolated and purified by the procedures described below, this protein, which is called spectrin, 1 does not contain significant amounts of lipid or carbohydrate. After treatment with divalent cations some spectrin preparations polymerize in the form of fibrous strands, '3 which are similar to those seen along the inner surfaces of red cell ghosts when the latter are examined by thin sectioning techniques and electron microscopy. 4 Since these fibrous structures are no longer evident on ghosts examined after spectrin is extracted from the membranes, it was suggested that spectrin might exist as a fibrous network along the inner surfaces of red cell membranes, ~ an idea which has since been amply confirmed by experiments with ferritin-conjugated antibodies 6 and nonpenetrating labeling reagents. 7-9 Procedure Ghost membranes are prepared from freshly drawn blood by osmotic lysis in either dilute phosphate buffer 1° or Tris. HC1 buffer? The procedure first developed for the isolation of spectrin involved dialyzing ghost membranes against large volumes of ATP or E D T A in the cold for periods ranging from 18 to 24 hours, followed by centrifugation, ammonium sulfate precipitation, and gel filtration. Relatively small amounts of blood were 1V. T. Marchesi and E. Steers, Science 159, 203 (1968). 2M. Clarke, Biochem. Biophys. Res. Commun. 45, 1063 (1971). 'E. Steers and V. T. Marchesi, J. Gen. Physiol. 54, 655 (1969). *V. T. Marchesi and G. E. Palade, J. Cell Biol. 35, 385 (1967). V. T. Marchesi, E. Steers, T. W. Tillack, and S. L. Marchesi, in "Red Cell Membrane, Structure and Function" (G. A. Jamieson and T. J. Greenwalt, eds.), p. 117. Lippincott, Philadelphia, Pennsylvania, 1969. ~G. L. Nicolson, V. T. Marchesi, and S. J. Singer, J. Cell Biol. 51, 265 (1971). H. C. Berg, Biochem. Biophys. Acta 183, 65 (1969). 8D. R. Phillips and M. Morrison, Biochem. Biophys. Res. Commun. 40, 284 (1970). 9M. S. Bretscher, J. Mol. Biol. 58, 775 (1971). l"J. T. Dodge, C. Mitchell, and D. J. Hanahan, Arch. Biochem. Biophys. 100, 119 (1963).
276
ISOLATIONOF SELECTED MEMBRANE COMPONENTS
[25]
processed in these initial studies and great care was taken to minimize the amount of leukocyte and bacterial contamination. However, we and others have since found that this procedure is difficult to use for the large-scale extraction of ghost membranes, and artifactual degradation of the high molecular weight polypeptides frequently occurs if strict precautions are not observed. 11 Recently a modification of this method has been suggested lz which involves a shorter incubation in EDTA, and this greatly reduces the risk of proteolytic degradation and is clearly the method of choice for the preparation of spectrin at the present time. Preparation of Ghost Membranes Freshly drawn blood of compatible ABO type is collected in acid citrate dextrose and washed three times by suspension in phosphate-saline (pH 7.2) and centrifugation at 2500 rpm (Sorvall RC-3) for 15 minutes. Each time the "huffy-coat" material is aspirated off along with a generous amount of underlying red ceils. It is important to realize that it is probably not possible to remove all the leukocytes by this procedure although the amount of such contamination can be substantially decreased. The leukocytes in "old" blood are more unstable and tend to aggregate and are more difficult to remove by this method. Washed cells are lysed in either Tris.HCl (10 m M , pH 7.4) or in phosphate buffer ( 1 5 - 2 0 mOs, pH 7.1) at a ratio of approximately 1 volume of packed red cells to 30 volumes of lysing medium maintained at 5 - 1 0 ° . If ratios greater than 1:40 are used, some of the ghosts may fragment, and it will be difficult to collect them by continuous-flow centrifugation. Ghosts collected after a single lysis are grossly pink since they still contain substantial amounts of hemoglobin. Most of this contaminating hemoglobin can be removed by a second lysis, but since this may also result in a considerable loss of spectrin, we prefer to extract pink membranes and remove the hemoglobin during the gel filtration step described below. Most of our large-scale work in the past has been carried out using a Szent-GySrgyi-Blum-type continuous flow apparatus in a Sorvall RC-2B centrifuge (20,000 rpm, 5 °) for collection of the ghost membranes. When using this device it is important to remove the ghost membranes from the steel centrifuge tubes without dislodging the pellets of leukocyte nuclei and lysosomal granules. Recently we have begun using a JCF-Z rotor in a Beckman J-21 centrifuge (I 8,000 rpm, 5°). We have been able to achieve ~1H. R. Trayer, Y. Nozaki, J. A. Reynolds, and C. Tanford, J. Biol. Chem. 246, 4485 (1971). 12G. Fairbanks, T. L. Steck, and D. F. H. Wallach, Biochemistry 10, 2606 (1971).
[26]
ISOLATION OF (Na ÷ + K+)-ATPase
277
greater flow rates with the latter instrument, permitting shorter lysing times, and, in addition, a sucrose "cushion" can also be pumped into this rotor during the run which allows us to separate contaminating leukocyte fragments from the ghost membranes. Membranes are pumped out of this rotor and resuspended in either dilute Tris-HCl (10 mM) or distilled water, pelleted by centrifugation, then either stored frozen or extracted immediately. Extraction of Spectrin Freshly prepared ghosts (at 10-20 mg dry weight per milliliter) are suspended in 10 volumes of 0.1 mM EDTA (pH 8.0) and incubated at 37 ° for 10-15 minutes as described previously.TM The ghosts fragment into small vesicles during this treatment, and the spectrin which is released in the medium can be separated from membrane fragments by centrifugation (approximately 100,000 g, 60-90 minutes, 4 °) and then concentrated by pressure dialysis. Spectrin can also be precipitated by the addition of an equal volume of saturated ammonium sulfate, and then redissolved by dialysis against 1 mM EDTA, but this is less satisfactory. Freeze-drying should be avoided. Spectrin has been purified free of small molecular weight contaminants by gel filtration on Sephadex G-200 ~ and, more recently, on Bio-Gel P-300 in the presence of imidazole glycyl glycine, 1 mM, pH 7.3. 2 '~ S. L. Marchesi, E. Steers, V. T. Marchesi, and T. W. Tillack, Biochemistry 9, 50 (1969).
[26] I s o l a t i o n of ( N a + + K + ) - A T P a s e
By
PETER LETH J~iRGENSEN
Since the discovery of (Na + + K+)-ATPase, 1 intensive studies have been carried out to elucidate its rote in the active transport of sodium and potassium.2 4 The enzyme has not been purified to a homogeneous state, but recently some progress in this direction has been made and informa-
a J. C. Skou, Biochim. Biophys. Acta 23, 394 (1957). ~J. C. Skou, Physiol. Rev. 45, 596 (1965). 3 R. L. Post, S. Kume, T. Tobin, B. Orcutt, and A. K. Sen, J. Gen. Physiol. 54, 306 (1969). ' J . C. Skou, Curr. Top. Bioenerg. 4, 357 (1971).