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Spectrin extractability from erythrocyte in duchenne muscular dystrophies and the effect of proteases on erythrocyte ghosts
285
Clinica Chimica Acta, 109 (1981) @ Elsevier/North-Holland
285-293 Biomedical Press
CCA 1625
SPECTRIN EXTRACTABILITY FROM ERYTHROCYTE IN DUCHENNE MUSCULAR DYSTROPHIES AND THE EFFECT OF PROTEASES ON ERYTHROCYTE GHOSTS
YOKO TSUCHIYA *q*, HIDE0 and KAZUTOMO IMAHORI d
SUGITA
b,c, SHOICHI
ISHIURA
c
a Division of Central Laboratory,
Branched Hospital of Department of Medicine, University of Tokyo, Mejirodai, Bunkyo-ku, Tokyo, b Department of Neurology, Institute of Brain Research, Faculty of Medicine, University of Tokyo, c Division of Neuromuscular Research, National Center for Nervous, Mental and Muscular Disorders, and d Department of Biochemistry, Faculty of Medicine, University of Tokyo (Japan) (Received
May 9th, 1980)
Summary We studied the erythrocyte membrane proteins from patients with Duchenne muscular dystrophy (DMD) using SDS-polyacrylamide gel electrophoresis. Our observations were the following: (1) The electrophoretic densitogram of freshly prepared DMD-ghosts was similar to that of controls. After the extraction of spectrin from ghosts with 1 mmol/l EDTA, pH 8.0, the unextractable spectrin remained more firmly bound in the DMD ghost residues than in controls. Extractability of spectrin from DMD ghosts was decreased about 20%. In addition, several minor bands were detected between spectrin and Band 3 in the DMD ghost residues (treated ghosts). (2) Ca’+-activated, neutral protease reacted more effectively with spectrin of DMD ghosts and ATP-depleted ghosts than with that of controls. (3) Erythrocyte actin (Band 5) of DMD ghosts was more fragile than that of controls and of ATPdepleted actin in the EDTA extracts. Gradually, partial degradation of actin was observed for three weeks at 4°C. (4) The intracellular ATP level and the activities of membrane-bound (Mg2’-Ca2+) ATPases in DMD erythrocytes were unchanged. We suggest that spectrin from DMD ghosts as well as actin may be subject to increased degradation.
Introduction Several Duchenne
laboratories have reported a variety of abnormalities in human shapes muscular dystrophy (DMD) erythrocytes, e.g. abnormal
* To whom correspondence should be addressed.
286
[1,2], reduced deformability [3], increased K’ permeability [4], and altered activities of membrane-bound enzymes [ 5,6]. These alterations could result from biochemical or structural abnormalities of the erythrocyte membranes. We have been interested in characterizing the nature of membrane proteins of DMD erythrocytes. Human erythrocyte membranes contain at least seven major proteins [7]. One of the most abundant proteins is spectrin. It appears to be a tetramer in its native form [8,9], composed of two kinds of polypeptides with molecular weights of 240 000 and 220 000, respectively. These are Bands 1 and 2 in SDSpolyacrylamide gels (by the nomenclature of Fairbanks et al. [7]), which can be selectively removed together with erythrocyte actin (Band 5) from the cytoplasmic surface of the membrane. Band 5 has many properties similar to muscle actin e.g. molecular weight, net charge and ability to polymerize into filaments [lO,ll]. Spectrin and actin are important components of the cytoskeletal network that is believed to be a determinant of erythrocyte shape and deformability [ 12,131. In the present study we compared the extractability of spectrin from DMD ghosts with that of controls and we investigated the effects of exogenous protease on ghosts. Materials and methods Materials
We studied six patients with Duchenne muscular dystrophy, aged 9-11 years. The diagnosis of DMD was established by the conventional criteria. Their ability of physical motion is inhibited in the medium grade (grade 6). The control group consisted of five healthy adults, aged 25-30 years. Blood was collected in heparinised syringes. Preparation
of membrane
ghosts
Human erythrocytes, washed three timesin a salt solution composed of 0.13 mol/l NaCl, 5 mmol/l KC1 and 7.5 mmol/l MgS04 at 4°C were hemolyzed in 10 ~01s. of a buffer composed of 5 mmol/l Tris containing 1 mmol/l EDTA, pH 7.4 (buffer I), for 30 min. The lysed cells were centrifuged in a Sorvall centrifuge at 25 000 X g for 30 min. The ghost pellets were suspended in the same vol. of a solution containing 0.5 mol/l NaCl, 50 mmol/l Tris and 1 mmol/l EDTA, pH 7.4 (buffer II), and centrifuged as above for 15 min. The ghosts were then washed three or four times with buffer I until the hemoglobin was completely removed. Extraction
of spectrin
The ghosts were dialyzed against 1000 ~01s. of distilled water containing 1 mmol/l EDTA, pH 8.0, for four days at 4°C and centrifuged at 105 000 X g for 90 min. The supernatants thus obtained were precipitated by the addition of an equal volume of cold saturated (NH&SO+ The precipitate was dissolved in the smallest vol. of 50 mmol/l Tris, pH 7.5, just before the experiment. This spectrin solution was dialyzed overnight against the above buffer.
287
Protein determination Analysis was performed by the modified bovine serum albumin as the standard.
method
of Lowry et al. [ 141, using
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis in the presence of 1% sodium dodecyl sulfate (SDS) was performed following the method of Fairbanks et al. [7]. The concentration of protein in each ghost suspension was determined by the method of Lowry before reduction. About 100 yg of ghost protein was added into the incubation mixture. All samples were reduced with 1% P-mercaptoethanol and incubated at 30°C for 30 min in the presence of 2% SDS. All of the reduced sample was applied on the gel. Electrophoresis was carried out for 3 h in a buffer consisting of 0.025 mol/l Tris, 0.192 mol/l glycine and 0.1% SDS, pH 8.3, at a current of 7 mA for a long gel (5 mm X 12 mm), or 3 mA for a short gel (5 mm X 7 mm). Gels were stained for proteins with Coomassie blue. The spectrin and Band 3 contents of the stained gels were determined by densitometry. Enzymatic treatment of ghosts The Ca’+-activated neutral protease was prepared from chicken skeletal muscle [15]. Erythrocyte ghosts were suspended in 5 mmol/l T&acetic acid buffer, pH 7.5, and the final concentration of proteins was adjusted to 1 mg/ml. The reaction mixtures containing 5 mmol/l Ca2’, 15 mol/l @-mercaptoethanol, 5 mmol/l T&-acetic acid, pH 7.5, ghosts and protease were incubated at 30°C for 30 min or some other designated interval. The amount of enzyme in the reaction mixture was about 30 pg per 1 ml. The digestion was terminated by cooling and by addition of l/10 vol of 20% SDS. ATP-deple ted ghost preparation Fresh red cells collected from 15 ml of blood were washed three times with a solution containing 0.13 mol/l NaCl, 5 mmol/l KC1 and 7.5 mmol/l MgS04 at 4°C. The packed cells were suspended in 25 ml of the above solution and incubated at 30°C for 24 h in the presence of 3 mg/25 ml potassium benzyl penicillin. After incubation, the preparation of ghosts and extraction of spectrin was performed with the methods mentioned above. Results Extractability of spectrin Fig. 1A shows densitograms of erythrocyte ghosts from patients with DMD (broken line) and controls. Electrophoretically there were no differences between them, especially in the relative positions in peaks of spectrin, Band 3 and in the other minor peaks. However, in the pattern of the treated ghosts (i.e. after extraction of spectrin with 1 mmol/l EDTA, pH 8.0) the ratio of the maximum absorbance at 550 nm of spectrin to that of Band 3 of DMD ghosts was higher than that of normal ghosts (Fig. 1B). It seemed that spectrin from DMD ghost membranes was less extractable than that from control ghosts. As shown in Table I, extractability of spectrin from DMD ghosts was decreased
288
----
DMD
-
Normal
----
Patient
ghosts
0 (A)
Origin
6
Migration +
03)
End
TD Migration -
Fig. 1. (A) SDS-gel electrophoresis of erythrocyte ghosts. Experimental details are described in “Materials and methods”. Densitometer scans at 550 nm of Coomassie-blue-stained SDS-polyacrylamide gels of normal ghosts and DMD ghosts. SP represents spectrin. TD stands for the tracking dye which shows the front of electrophoresis. (B) SDS-gel electrophoresis of treated ghosts. After extraction of spectrins, ghost pellet residues (treated ghosts) were suspended in 5 mmol/l barbital buffer pH 7.4 (concentrations were about 1.3-1.6 mg/ml) and stored in the cold room for a week. 0.1-0.08 ml of suspension was used for SDS-gel electrophoresis.
from 72.0% to 52.5%. Extractability of spectrin was calculated from the speckin/Band 3 of both original ghosts and treated,ghosts. The contents of spectrin and Band 3 were determined by the areas in the densitogram. This calculation was performed based on two probable points: (1) Band 3, one of major polypeptides, is not extractable from ghost membrane under the hypotonic conditions in which spectrin can easily be extracted; (2) although we have no evidence that the Band 3 of DMD ghosts is chemically the same as that of the normal controls, Band 3 of DMD ghosts is assumed to be unchanged.
TABLE
I
EXTRACTABILITY Sample of ghosts
OF SPECTRIN Spectrlnlband
Spectrin
3
ghosts
treated
extracted
ghosts
Normal
(n = 4)
1.24-1.52
0.30-0.50
72.0
Patients No. No. No. No.
2 3 5 6
1.41 1.44 1.51 1.37
0.63 0.77 0.79 0.54
55.3 46.5 47.7 60.6 52.6
f 3.2
i 5.8
(%)
289
Effects of proteases on spectrin As seen in Figs. 1A and B, the occurrence of three or four minor peaks between spectrin and Band 3 in DMD ghosts and also in treated DMD ghosts could suggest the degradation of spectrin at the inner face of cell membrane, probably due to the activation of membrane-bound proteases. The Ca”-activated, neutral protease, isolated from the animal skeletal muscle, was recently demonstrated to take part in the catabolic degradation of muscle structural proteins [ 151. This neutral protease may be considered as an important, relatively mild reagent in the investigation of the nature of spectrin. As shown in Fig. 2, this enzyme selectively digested spectrin or proteins existing at the inner face of membrane. Band 3 seemed to be not affected by this enzyme. The remnant of spectrin in ghosts after enzymatic treatment is described in Fig. 3. When ghosts were incubated without the addition of protease to check the effects of their own membrane-bound enzymes, the contents of spectrins in control ghosts were unchanged within 30 min, even in the presence of Ca” (CG in Fig. 3). At the same time we examined ATP-depleted ghosts (AG) as the model of abnormally shaped cells (ethinocytes). The ATPdepleted ghosts (AG) were affected by their own membrane-bound proteases, as shown in Fig. 3, and the rate of degradation in ghosts was similar to that in control ghosts, when incubated with Ca*+-protease (CG + E). The degradation of ATP-depleted ghosts’ spectrin by Ca’+-protease was more accelerated (AG + E). In the enzymatic treatment of DMD ghosts, we observed that the degrada-
Crude
ghosts
-
Control
----
Ca2+-protease
1
Origin Fig. 2. Effects protease-treated
Migration-
0
End
of Ca*+-protease treatment on normal ghosts. Hgb means hemoglobin.
ghosts.
Densitograms
20
Time
of control
40 (min)
1 50
f
ghosts and Ca*+-
Fig. 3. Degradation of spectrins in control ghosts and ATP-depleted ghosts by Ca*+-protease. All four samples contain Ca*+, P-mercap toethanol. The buffer and 1 mg ghost proteins/ml were incubated for the designated intervals at 3OO.Cwith or without enzyme. SDS-gel electrophoresis was performed usually after incubation was stopped. The contents of spectrins in ghosts were estimated at the‘areas of bands 1 and 2 in densitograms. CG, control ghosts; AG. ATP-depleted ghosts; CG + E, control ghosts with enzyme; AG + E. ATP-depleted ghosts with enzyme.
290
tion rate of spectrin in DMD ghosts by Ca2+-protease in ATP-depleted ghosts (AG + E in Fig. 3).
was exactly
similar to that
Fragility of DMD ery throcy te actin We observed that erythrocyte actin in EDTA extracts from DMD ghosts was autodegradative more easily than in that from control and ATP-depleted ghosts (Fig. 4, shown as the mean of 4 patients). In this experiment normal control ghosts and ATPdepleted ghosts were prepared from blood which was stored for several days at 4°C. Normal erythrocyte ghosts and ATP-depleted ghosts were often prepared from recently outdated titrated blood (bank blood). And the stable spectrin and actin were extracted from them. Certainly the degradation curve of actin from freshly drawn blood shifted slightly to the right of that of the control in Fig. 4. Electrophoretically there were no important differences between spectrin and actin extracted from freshly drawn blood and that from blood several days old. DMD ghosts were freshly prepared as soon as blood samples were drawn. Spectrin and actin in a half saturated (NHJ2S04 suspension were stored in the cold room just before each electrophoretic analysis. Contents of Band 5 were demonstrated as the ratio of the area of spectrin to that of Band 5 in the electrophoretical densitogram, because spectrin extracted with EDTA did not degradate for a month. As shown in Fig. 4, a half degradation time was about nine or ten days for DMD Band 5, whereas about three weeks for that of controls and ATPdepleted. There were no changes in the ratio of Band 5 to Band 3 in DMD ghosts compared with controls (0.10 and 0.11, respectively).
Fig. 4. Degradation of Band 5 extracted by EDTA solution. Spectrin and Band 5 were prepared following “Materials and methods”. The (NH4)2S04 precipitates of spectrin and actin were collected just before electrophoretical analysis. Contents of Band 5 were estimated as the ratio of spectrin to Band 5 in the electrophoretogram, 0: control. @ : ATP-depleted, a; DMD.
291 TABLE
II
INTRACELLULAR
ATP LEVEL
OF THE ERYTHROCYTES
Sample of erythrocytes
ATP concentration
Normal (n = 4) DMD-patients No. 1 No. 2 No. 3 No. 4
1.06
* + 0.19
0.62 0.90 1.14 1.87 1.13
Intracellular ATP was determined * Range 1.34-0.79.
TABLE
+ 0.46
by the enzymatic
method.
III
MEMBRANE-BOUND Sample
(mmol/l)
of ghosts
Normal (n = 4) DMD-patients No. 1 No. 2 No. 3 No. 4
ATPase
ACTIVITY
Pi-released @lo1 . mg-1 protein 1.21
* 30
min-l
* * 0.22
1.47 0.89 0.76 1.42 1.14
* 0.30
Reaction mixtures contained: 100 mmol/l NaCl, 10 mmol/l KCl, 5 mmol/l MgCl2.0.5 mmol/l CaC12, and 50 mmol/l HEPES, pH 7.5, 2 mmol/l ATP. After incubation for 30 min at 36’C,.PGreleased protein was determined colorlmetrically by the method of Fiske-Subbarow. Abbreviation: HEPES, N-P-hydroxethylpiperazine-N’-2-ethanesulfonic acid. * Range 1.44-Q.93.
Normal ATP level and normal membrane-bound racy tes
ATPase activity in DMD eryth-
Although a change of the intracellular ATP level with DMD erythrocytes was expected, because of the morphological abnormality [1,2], no significant differences could be found between DMD and control erythrocytes (Table II). Besides, the membrane-bound (Mg”-Ca’+) ATPase activities of DMD ghosts in vitro were almost the same as those of controls (Table III). Discussion We found a significant decrease of spectrin extractability from DMD erythrocyte ghosts. This result appears to be due to a change of membrane permeability and the partial degradation of spectrin which might be susceptible to proteases. Kobayashi et al. [ 161 and Koshi et al. [ 171 reported the normality of phospholipids and proteins in the erythrocyte membrane of DMD patients. Though
292
the components of DMD erythrocyte membrane are probably unchanged, the structural nature of membrane might be altered following a change of permeability. Our enzymic studies suggest that spectrin in DMD ghosts is more degradable than that of controls (Fig. 3). Lux and John [ 181 observed that spectrin extractability with Triton X-100 decreased slowly as ATP levels declined. In our analysis of intracellular ATP levels there was no difference between normal cells and DMD cells. Moreover, (Mg’+-Ca*‘) ATPase activity of DMD ghosts is normal. It appears that the decrease of spectrin extractability is not such a simple phenomenon. We suggest that spectrin as well as actin from DMD ghosts may be subjected to increased degradation and that this degeneration might be structural, not chemical. Surprisingly little direct evidence exists for the interaction of spectrin and actin. Further investigations of erythrocyte actin are needed to clarify this relationship. Acknowledgement We wish to express our gratitude to Dr. T. Fujii and Dr. T. Nakao for their valuable advice, and Miss M. Hokoyama for the tracing of figures. References 1 Matheson, D.W. and Howland, J.L. (1974) Erythrocyte deformation in human muscular dystrophy. Science 184.165-166 2 Miller, S.E., Roses, A.D. and Appel, S.H. (1976) Scanning electron microscopy studies in muscular dystrophy. Arch. Neural. 33, 172-174 3 Percy, A.K. and Miller. M.E. (1975) Reduced deformability of erythrocyte membranes from patients with Duchenne muscular dystrophy. Nature (Land.) 258, 147-148 4 Sha’afi, R.I., Rodan, S.B., Hintz. R.L., Fernandez, S.M. and Rodan. G.A. (1975) Abnormalities in membrane microviscosity and ion transport in genetic muscular dystrophy. Nature (Land.) 254. 525526 5 Roses, A.D., Herbstreith, M.H. and Appel, S.H. (1975) Membrane protein kinase alteration in Duchenne muscular dystrophy. Nature (Land.) 254, 350-351 6 Wacholtz. M.C., Raible, D.G., Jackowski, S., Rodan, S.B., Rodan, G.A. and Sha’afi. R.I. (1979) Adenylate cyclase and ATPase activities in red celI membranes of patients and genetic carriers of Duchenne muscular dystrophy. Clin. Chim. Acta 96, 255-259 7 Fairbanks, G., Steck. T.L. and Wallach, D.F.H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10. 2606-2617 8 Ralston, G.B.. Dunbar. J. and White, M. (1977) The temperature-dependent dissociation of spectrin. Biochim. Biophys. Acta 491, 345-348 9 Ralston, G.B. (1978) The structure of spectrin and the shape of the red blood cell. Trends Biochem. Sciences 3.195-198 10 Tilney, L.G. and Detmers, D. (1975) Actin in erythrocyte ghosts and its association with spectrin: evidence for a non-filamentous form of these two molecules in situ. J. Cell Biol. 66, 508-520 11 Sheetz, M.P., Painter, R.G. and Singer, S.J. (1976) Relationships of the spectrin complex of human erythrocyte membranes to the actomyosins of muscle cells. Biochemistry 15, 4486-4491 12 Sheetz. M.P. and Singer.‘S.J. (1977) On the mechanism of ATP-induced shape changes in human erythrocyte membranes. I. The role of the spectrin complex. J. Cell Biol. 73, 638-646 13 Birchmeier, W. and Singer, S.J. (1977) On the mechanism of ATP-induced shape changes in human erythrocyte membranes. II. The role of ATP. J. Cell Biol. 73, 647-659 14 Lowry. O.H., Rosebrough, N.J.. Farr, A.L. and Randall, R.T. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193. 265-275 15 Ishiura, S.. Murofushi, K.. Suzuki. K. and Imahori. K. (1978) Studies of calcium-activated neutral protease from chicken skeletal muscle. I. Purification and characterization. J. Biochem. (Tokyo), 84, 225-230
293 16 17 18
Kobayashi. T., Mawatari, S. and Kuroiwa, Y. (1978)
Lipids and proteins
of erythrocyte
membrane
in
Duchenne muscular dystrophy. Clin. Chim. Acta 85, 259-266 Koski, L.C., Jungalwala, F. and Kolodny, H.E. (1978) Normality of erythrocyte phospholipids in Duchenne muscular dystrophy. Clin. Chim. Acta 85.295-298 Lux. S.E. and John, K.M. (1977) Evidence that spectrin is a determinant of shape and deformability in the human erythrocyte. In: Cell Shape and Surface Architecture (Revel, J.P., Henning. U. and Fox. C.F., eds.), pp. 487-491, Allan R. Liss Inc., New York
Clinica Chimica Acta, 109 (1981) @ Elsevier/North-Holland
285-293 Biomedical Press
CCA 1625
SPECTRIN EXTRACTABILITY FROM ERYTHROCYTE IN DUCHENNE MUSCULAR DYSTROPHIES AND THE EFFECT OF PROTEASES ON ERYTHROCYTE GHOSTS
YOKO TSUCHIYA *q*, HIDE0 and KAZUTOMO IMAHORI d
SUGITA
b,c, SHOICHI
ISHIURA
c
a Division of Central Laboratory,
Branched Hospital of Department of Medicine, University of Tokyo, Mejirodai, Bunkyo-ku, Tokyo, b Department of Neurology, Institute of Brain Research, Faculty of Medicine, University of Tokyo, c Division of Neuromuscular Research, National Center for Nervous, Mental and Muscular Disorders, and d Department of Biochemistry, Faculty of Medicine, University of Tokyo (Japan) (Received
May 9th, 1980)
Summary We studied the erythrocyte membrane proteins from patients with Duchenne muscular dystrophy (DMD) using SDS-polyacrylamide gel electrophoresis. Our observations were the following: (1) The electrophoretic densitogram of freshly prepared DMD-ghosts was similar to that of controls. After the extraction of spectrin from ghosts with 1 mmol/l EDTA, pH 8.0, the unextractable spectrin remained more firmly bound in the DMD ghost residues than in controls. Extractability of spectrin from DMD ghosts was decreased about 20%. In addition, several minor bands were detected between spectrin and Band 3 in the DMD ghost residues (treated ghosts). (2) Ca’+-activated, neutral protease reacted more effectively with spectrin of DMD ghosts and ATP-depleted ghosts than with that of controls. (3) Erythrocyte actin (Band 5) of DMD ghosts was more fragile than that of controls and of ATPdepleted actin in the EDTA extracts. Gradually, partial degradation of actin was observed for three weeks at 4°C. (4) The intracellular ATP level and the activities of membrane-bound (Mg2’-Ca2+) ATPases in DMD erythrocytes were unchanged. We suggest that spectrin from DMD ghosts as well as actin may be subject to increased degradation.
Introduction Several Duchenne
laboratories have reported a variety of abnormalities in human shapes muscular dystrophy (DMD) erythrocytes, e.g. abnormal
* To whom correspondence should be addressed.
286
[1,2], reduced deformability [3], increased K’ permeability [4], and altered activities of membrane-bound enzymes [ 5,6]. These alterations could result from biochemical or structural abnormalities of the erythrocyte membranes. We have been interested in characterizing the nature of membrane proteins of DMD erythrocytes. Human erythrocyte membranes contain at least seven major proteins [7]. One of the most abundant proteins is spectrin. It appears to be a tetramer in its native form [8,9], composed of two kinds of polypeptides with molecular weights of 240 000 and 220 000, respectively. These are Bands 1 and 2 in SDSpolyacrylamide gels (by the nomenclature of Fairbanks et al. [7]), which can be selectively removed together with erythrocyte actin (Band 5) from the cytoplasmic surface of the membrane. Band 5 has many properties similar to muscle actin e.g. molecular weight, net charge and ability to polymerize into filaments [lO,ll]. Spectrin and actin are important components of the cytoskeletal network that is believed to be a determinant of erythrocyte shape and deformability [ 12,131. In the present study we compared the extractability of spectrin from DMD ghosts with that of controls and we investigated the effects of exogenous protease on ghosts. Materials and methods Materials
We studied six patients with Duchenne muscular dystrophy, aged 9-11 years. The diagnosis of DMD was established by the conventional criteria. Their ability of physical motion is inhibited in the medium grade (grade 6). The control group consisted of five healthy adults, aged 25-30 years. Blood was collected in heparinised syringes. Preparation
of membrane
ghosts
Human erythrocytes, washed three timesin a salt solution composed of 0.13 mol/l NaCl, 5 mmol/l KC1 and 7.5 mmol/l MgS04 at 4°C were hemolyzed in 10 ~01s. of a buffer composed of 5 mmol/l Tris containing 1 mmol/l EDTA, pH 7.4 (buffer I), for 30 min. The lysed cells were centrifuged in a Sorvall centrifuge at 25 000 X g for 30 min. The ghost pellets were suspended in the same vol. of a solution containing 0.5 mol/l NaCl, 50 mmol/l Tris and 1 mmol/l EDTA, pH 7.4 (buffer II), and centrifuged as above for 15 min. The ghosts were then washed three or four times with buffer I until the hemoglobin was completely removed. Extraction
of spectrin
The ghosts were dialyzed against 1000 ~01s. of distilled water containing 1 mmol/l EDTA, pH 8.0, for four days at 4°C and centrifuged at 105 000 X g for 90 min. The supernatants thus obtained were precipitated by the addition of an equal volume of cold saturated (NH&SO+ The precipitate was dissolved in the smallest vol. of 50 mmol/l Tris, pH 7.5, just before the experiment. This spectrin solution was dialyzed overnight against the above buffer.
287
Protein determination Analysis was performed by the modified bovine serum albumin as the standard.
method
of Lowry et al. [ 141, using
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis in the presence of 1% sodium dodecyl sulfate (SDS) was performed following the method of Fairbanks et al. [7]. The concentration of protein in each ghost suspension was determined by the method of Lowry before reduction. About 100 yg of ghost protein was added into the incubation mixture. All samples were reduced with 1% P-mercaptoethanol and incubated at 30°C for 30 min in the presence of 2% SDS. All of the reduced sample was applied on the gel. Electrophoresis was carried out for 3 h in a buffer consisting of 0.025 mol/l Tris, 0.192 mol/l glycine and 0.1% SDS, pH 8.3, at a current of 7 mA for a long gel (5 mm X 12 mm), or 3 mA for a short gel (5 mm X 7 mm). Gels were stained for proteins with Coomassie blue. The spectrin and Band 3 contents of the stained gels were determined by densitometry. Enzymatic treatment of ghosts The Ca’+-activated neutral protease was prepared from chicken skeletal muscle [15]. Erythrocyte ghosts were suspended in 5 mmol/l T&acetic acid buffer, pH 7.5, and the final concentration of proteins was adjusted to 1 mg/ml. The reaction mixtures containing 5 mmol/l Ca2’, 15 mol/l @-mercaptoethanol, 5 mmol/l T&-acetic acid, pH 7.5, ghosts and protease were incubated at 30°C for 30 min or some other designated interval. The amount of enzyme in the reaction mixture was about 30 pg per 1 ml. The digestion was terminated by cooling and by addition of l/10 vol of 20% SDS. ATP-deple ted ghost preparation Fresh red cells collected from 15 ml of blood were washed three times with a solution containing 0.13 mol/l NaCl, 5 mmol/l KC1 and 7.5 mmol/l MgS04 at 4°C. The packed cells were suspended in 25 ml of the above solution and incubated at 30°C for 24 h in the presence of 3 mg/25 ml potassium benzyl penicillin. After incubation, the preparation of ghosts and extraction of spectrin was performed with the methods mentioned above. Results Extractability of spectrin Fig. 1A shows densitograms of erythrocyte ghosts from patients with DMD (broken line) and controls. Electrophoretically there were no differences between them, especially in the relative positions in peaks of spectrin, Band 3 and in the other minor peaks. However, in the pattern of the treated ghosts (i.e. after extraction of spectrin with 1 mmol/l EDTA, pH 8.0) the ratio of the maximum absorbance at 550 nm of spectrin to that of Band 3 of DMD ghosts was higher than that of normal ghosts (Fig. 1B). It seemed that spectrin from DMD ghost membranes was less extractable than that from control ghosts. As shown in Table I, extractability of spectrin from DMD ghosts was decreased
288
----
DMD
-
Normal
----
Patient
ghosts
0 (A)
Origin
6
Migration +
03)
End
TD Migration -
Fig. 1. (A) SDS-gel electrophoresis of erythrocyte ghosts. Experimental details are described in “Materials and methods”. Densitometer scans at 550 nm of Coomassie-blue-stained SDS-polyacrylamide gels of normal ghosts and DMD ghosts. SP represents spectrin. TD stands for the tracking dye which shows the front of electrophoresis. (B) SDS-gel electrophoresis of treated ghosts. After extraction of spectrins, ghost pellet residues (treated ghosts) were suspended in 5 mmol/l barbital buffer pH 7.4 (concentrations were about 1.3-1.6 mg/ml) and stored in the cold room for a week. 0.1-0.08 ml of suspension was used for SDS-gel electrophoresis.
from 72.0% to 52.5%. Extractability of spectrin was calculated from the speckin/Band 3 of both original ghosts and treated,ghosts. The contents of spectrin and Band 3 were determined by the areas in the densitogram. This calculation was performed based on two probable points: (1) Band 3, one of major polypeptides, is not extractable from ghost membrane under the hypotonic conditions in which spectrin can easily be extracted; (2) although we have no evidence that the Band 3 of DMD ghosts is chemically the same as that of the normal controls, Band 3 of DMD ghosts is assumed to be unchanged.
TABLE
I
EXTRACTABILITY Sample of ghosts
OF SPECTRIN Spectrlnlband
Spectrin
3
ghosts
treated
extracted
ghosts
Normal
(n = 4)
1.24-1.52
0.30-0.50
72.0
Patients No. No. No. No.
2 3 5 6
1.41 1.44 1.51 1.37
0.63 0.77 0.79 0.54
55.3 46.5 47.7 60.6 52.6
f 3.2
i 5.8
(%)
289
Effects of proteases on spectrin As seen in Figs. 1A and B, the occurrence of three or four minor peaks between spectrin and Band 3 in DMD ghosts and also in treated DMD ghosts could suggest the degradation of spectrin at the inner face of cell membrane, probably due to the activation of membrane-bound proteases. The Ca”-activated, neutral protease, isolated from the animal skeletal muscle, was recently demonstrated to take part in the catabolic degradation of muscle structural proteins [ 151. This neutral protease may be considered as an important, relatively mild reagent in the investigation of the nature of spectrin. As shown in Fig. 2, this enzyme selectively digested spectrin or proteins existing at the inner face of membrane. Band 3 seemed to be not affected by this enzyme. The remnant of spectrin in ghosts after enzymatic treatment is described in Fig. 3. When ghosts were incubated without the addition of protease to check the effects of their own membrane-bound enzymes, the contents of spectrins in control ghosts were unchanged within 30 min, even in the presence of Ca” (CG in Fig. 3). At the same time we examined ATP-depleted ghosts (AG) as the model of abnormally shaped cells (ethinocytes). The ATPdepleted ghosts (AG) were affected by their own membrane-bound proteases, as shown in Fig. 3, and the rate of degradation in ghosts was similar to that in control ghosts, when incubated with Ca*+-protease (CG + E). The degradation of ATP-depleted ghosts’ spectrin by Ca’+-protease was more accelerated (AG + E). In the enzymatic treatment of DMD ghosts, we observed that the degrada-
Crude
ghosts
-
Control
----
Ca2+-protease
1
Origin Fig. 2. Effects protease-treated
Migration-
0
End
of Ca*+-protease treatment on normal ghosts. Hgb means hemoglobin.
ghosts.
Densitograms
20
Time
of control
40 (min)
1 50
f
ghosts and Ca*+-
Fig. 3. Degradation of spectrins in control ghosts and ATP-depleted ghosts by Ca*+-protease. All four samples contain Ca*+, P-mercap toethanol. The buffer and 1 mg ghost proteins/ml were incubated for the designated intervals at 3OO.Cwith or without enzyme. SDS-gel electrophoresis was performed usually after incubation was stopped. The contents of spectrins in ghosts were estimated at the‘areas of bands 1 and 2 in densitograms. CG, control ghosts; AG. ATP-depleted ghosts; CG + E, control ghosts with enzyme; AG + E. ATP-depleted ghosts with enzyme.
290
tion rate of spectrin in DMD ghosts by Ca2+-protease in ATP-depleted ghosts (AG + E in Fig. 3).
was exactly
similar to that
Fragility of DMD ery throcy te actin We observed that erythrocyte actin in EDTA extracts from DMD ghosts was autodegradative more easily than in that from control and ATP-depleted ghosts (Fig. 4, shown as the mean of 4 patients). In this experiment normal control ghosts and ATPdepleted ghosts were prepared from blood which was stored for several days at 4°C. Normal erythrocyte ghosts and ATP-depleted ghosts were often prepared from recently outdated titrated blood (bank blood). And the stable spectrin and actin were extracted from them. Certainly the degradation curve of actin from freshly drawn blood shifted slightly to the right of that of the control in Fig. 4. Electrophoretically there were no important differences between spectrin and actin extracted from freshly drawn blood and that from blood several days old. DMD ghosts were freshly prepared as soon as blood samples were drawn. Spectrin and actin in a half saturated (NHJ2S04 suspension were stored in the cold room just before each electrophoretic analysis. Contents of Band 5 were demonstrated as the ratio of the area of spectrin to that of Band 5 in the electrophoretical densitogram, because spectrin extracted with EDTA did not degradate for a month. As shown in Fig. 4, a half degradation time was about nine or ten days for DMD Band 5, whereas about three weeks for that of controls and ATPdepleted. There were no changes in the ratio of Band 5 to Band 3 in DMD ghosts compared with controls (0.10 and 0.11, respectively).
Fig. 4. Degradation of Band 5 extracted by EDTA solution. Spectrin and Band 5 were prepared following “Materials and methods”. The (NH4)2S04 precipitates of spectrin and actin were collected just before electrophoretical analysis. Contents of Band 5 were estimated as the ratio of spectrin to Band 5 in the electrophoretogram, 0: control. @ : ATP-depleted, a; DMD.
291 TABLE
II
INTRACELLULAR
ATP LEVEL
OF THE ERYTHROCYTES
Sample of erythrocytes
ATP concentration
Normal (n = 4) DMD-patients No. 1 No. 2 No. 3 No. 4
1.06
* + 0.19
0.62 0.90 1.14 1.87 1.13
Intracellular ATP was determined * Range 1.34-0.79.
TABLE
+ 0.46
by the enzymatic
method.
III
MEMBRANE-BOUND Sample
(mmol/l)
of ghosts
Normal (n = 4) DMD-patients No. 1 No. 2 No. 3 No. 4
ATPase
ACTIVITY
Pi-released @lo1 . mg-1 protein 1.21
* 30
min-l
* * 0.22
1.47 0.89 0.76 1.42 1.14
* 0.30
Reaction mixtures contained: 100 mmol/l NaCl, 10 mmol/l KCl, 5 mmol/l MgCl2.0.5 mmol/l CaC12, and 50 mmol/l HEPES, pH 7.5, 2 mmol/l ATP. After incubation for 30 min at 36’C,.PGreleased protein was determined colorlmetrically by the method of Fiske-Subbarow. Abbreviation: HEPES, N-P-hydroxethylpiperazine-N’-2-ethanesulfonic acid. * Range 1.44-Q.93.
Normal ATP level and normal membrane-bound racy tes
ATPase activity in DMD eryth-
Although a change of the intracellular ATP level with DMD erythrocytes was expected, because of the morphological abnormality [1,2], no significant differences could be found between DMD and control erythrocytes (Table II). Besides, the membrane-bound (Mg”-Ca’+) ATPase activities of DMD ghosts in vitro were almost the same as those of controls (Table III). Discussion We found a significant decrease of spectrin extractability from DMD erythrocyte ghosts. This result appears to be due to a change of membrane permeability and the partial degradation of spectrin which might be susceptible to proteases. Kobayashi et al. [ 161 and Koshi et al. [ 171 reported the normality of phospholipids and proteins in the erythrocyte membrane of DMD patients. Though
292
the components of DMD erythrocyte membrane are probably unchanged, the structural nature of membrane might be altered following a change of permeability. Our enzymic studies suggest that spectrin in DMD ghosts is more degradable than that of controls (Fig. 3). Lux and John [ 181 observed that spectrin extractability with Triton X-100 decreased slowly as ATP levels declined. In our analysis of intracellular ATP levels there was no difference between normal cells and DMD cells. Moreover, (Mg’+-Ca*‘) ATPase activity of DMD ghosts is normal. It appears that the decrease of spectrin extractability is not such a simple phenomenon. We suggest that spectrin as well as actin from DMD ghosts may be subjected to increased degradation and that this degeneration might be structural, not chemical. Surprisingly little direct evidence exists for the interaction of spectrin and actin. Further investigations of erythrocyte actin are needed to clarify this relationship. Acknowledgement We wish to express our gratitude to Dr. T. Fujii and Dr. T. Nakao for their valuable advice, and Miss M. Hokoyama for the tracing of figures. References 1 Matheson, D.W. and Howland, J.L. (1974) Erythrocyte deformation in human muscular dystrophy. Science 184.165-166 2 Miller, S.E., Roses, A.D. and Appel, S.H. (1976) Scanning electron microscopy studies in muscular dystrophy. Arch. Neural. 33, 172-174 3 Percy, A.K. and Miller. M.E. (1975) Reduced deformability of erythrocyte membranes from patients with Duchenne muscular dystrophy. Nature (Land.) 258, 147-148 4 Sha’afi, R.I., Rodan, S.B., Hintz. R.L., Fernandez, S.M. and Rodan. G.A. (1975) Abnormalities in membrane microviscosity and ion transport in genetic muscular dystrophy. Nature (Land.) 254. 525526 5 Roses, A.D., Herbstreith, M.H. and Appel, S.H. (1975) Membrane protein kinase alteration in Duchenne muscular dystrophy. Nature (Land.) 254, 350-351 6 Wacholtz. M.C., Raible, D.G., Jackowski, S., Rodan, S.B., Rodan, G.A. and Sha’afi. R.I. (1979) Adenylate cyclase and ATPase activities in red celI membranes of patients and genetic carriers of Duchenne muscular dystrophy. Clin. Chim. Acta 96, 255-259 7 Fairbanks, G., Steck. T.L. and Wallach, D.F.H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10. 2606-2617 8 Ralston, G.B.. Dunbar. J. and White, M. (1977) The temperature-dependent dissociation of spectrin. Biochim. Biophys. Acta 491, 345-348 9 Ralston, G.B. (1978) The structure of spectrin and the shape of the red blood cell. Trends Biochem. Sciences 3.195-198 10 Tilney, L.G. and Detmers, D. (1975) Actin in erythrocyte ghosts and its association with spectrin: evidence for a non-filamentous form of these two molecules in situ. J. Cell Biol. 66, 508-520 11 Sheetz, M.P., Painter, R.G. and Singer, S.J. (1976) Relationships of the spectrin complex of human erythrocyte membranes to the actomyosins of muscle cells. Biochemistry 15, 4486-4491 12 Sheetz. M.P. and Singer.‘S.J. (1977) On the mechanism of ATP-induced shape changes in human erythrocyte membranes. I. The role of the spectrin complex. J. Cell Biol. 73, 638-646 13 Birchmeier, W. and Singer, S.J. (1977) On the mechanism of ATP-induced shape changes in human erythrocyte membranes. II. The role of ATP. J. Cell Biol. 73, 647-659 14 Lowry. O.H., Rosebrough, N.J.. Farr, A.L. and Randall, R.T. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193. 265-275 15 Ishiura, S.. Murofushi, K.. Suzuki. K. and Imahori. K. (1978) Studies of calcium-activated neutral protease from chicken skeletal muscle. I. Purification and characterization. J. Biochem. (Tokyo), 84, 225-230
293 16 17 18
Kobayashi. T., Mawatari, S. and Kuroiwa, Y. (1978)
Lipids and proteins
of erythrocyte
membrane
in
Duchenne muscular dystrophy. Clin. Chim. Acta 85, 259-266 Koski, L.C., Jungalwala, F. and Kolodny, H.E. (1978) Normality of erythrocyte phospholipids in Duchenne muscular dystrophy. Clin. Chim. Acta 85.295-298 Lux. S.E. and John, K.M. (1977) Evidence that spectrin is a determinant of shape and deformability in the human erythrocyte. In: Cell Shape and Surface Architecture (Revel, J.P., Henning. U. and Fox. C.F., eds.), pp. 487-491, Allan R. Liss Inc., New York