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Erythrocyte membrane cation-stimulated ATPase activities in myotonic muscular dystrophy
Journal of the Neurological Sciences, 1982, 53:23-28 Elsevier Biomedical Press
23
ERYTHROCYTE MEMBRANE CATION-STIMULATED ATPase ACTIVITIES IN MYOTONIC MUSCULAR DYSTROPHY
SHIRO MAWATARI 1, YASUNOBU ANTOKU 2 and YOSHIGORO KUROIWA 1
! Department of Neurology, NeurologicalInstitute, Faculty of Medicine, Kyushu University, Fukuoka 812, and 2 National Akasaka Hospital, Chikugo City, Fukuoka-ken (Japan) (Received 9 March, 1981) (Revised, received 20 May, 1981) (Accepted 28 May, 1981)
SUMMARY
The cation-stimulated ATPase activities of erythrocyte membranes from patients with myotonic muscular dystrophy (MyD) were compared with the activities in age- and sex-matched controls. The enzymes included ouabain-sensitive ATPase, Mg2+-ATPase and Ca2÷ + Mg2÷.ATPase. Sampling and processing of the materials from patients with MyD and controls were simultaneously done in each experiment. The enzyme activities were varied with or without EGTA in the reaction medium, or with different temperatures for membrane storage, but no significant differences between MyD and control were observed in any conditions. The present study indicates no specific abnormality of the cation-stimulated ATPase activities of erythrocyte membranes in MyD.
INTRODUCTION
Various abnormalities of erythrocytes (or erythrocyte membranes) have been reported in human myotonic muscular dystrophy (MyD). They included Na + + K +ATPase (Brown et al. 1967; Niebroj-Do bosz 1976; Mishra et al. 1980), Ca2+ + Mg2+ATPase (Luthra et al. 1979; Ruitenbeek 1979), cation transport (Hull and Roses 1976; Plishker et al. 1978; Hobbs et al. 1979), protein kinase (Roses and Appel 1973; Vickers et al. 1979), adenine incorporation into ATP and ADP (Solomons et al.
This work was supported by a Grant of the National Center for Nervous, Mental and Muscular Disorders of the Ministry of Health and Welfare, Japan. Correspondence: Dr. Shiro Mawatari, Department of Neurology, Neurological Institute, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan. 0022-510X/82/0000-0000/$02.75 © Elsevier Biomedical Press
24 1977; Muller et al. 1980), and ATP content of erythrocytes (Danon et al. 1978). It is of interest that these abnormalities are related to ATP. However, there are often discrepancies among these reported results. Therefore, we re-examined ATPase activities of erythrocyte membranes from patients with MyD by comparing with deliberately selected and manipulated control membranes. Additionally, adenylate kinase, which catalyzes the reaction ATP + AMP ~ 2 ADP, was examined in the erythrocyte membranes. MATERIALSAND METHODS Subjects were 8 patients with MyD and 8 patients with pulmonary tuberculosis (controls). Diagnoses of the patients were made by conventional criteria. Age and sex of the controls were matched to those of the patients with MyD. All subjects were in the same institution for more than one month. Samples from the subjects were obtained on the same day at almost the same time and were processed simultaneously in each experiment. Blood was taken into heparinized tubes and immediately put on ice. After removal of plasma and buffy coat, erythrocytes were washed 3 times with isotonic saline by centrifugation at 1,000 × g for 10 min. Buffy coat was removed at each washing. The washed erythrocytes were hemolyzed in about 6 volumes of 10 mM Tris-HC1 (pH. 7.4) and ghosts were collected by centrifugation at 25,000 x g for 20 min. The ghosts were washed 5 times with the same solution by the same centrifugation, and button at the bottom of centrifuge tube (Fairbanks et al. 1971) was removed each time. All procedures were carried out at 2-5 °C. The white ghosts (membranes) were finally suspended in 10 mM Tris-HCl (pH. 7.4) to make protein concentration about 5 mg/ml, and stored at - 8 0 ° C (experiment A) or - 3 0 ° C (experiment B) until use. Enzyme activities were assayed by coupled enzymatic methods using a recording spectrophotometer (Hitachi 200-20) with temperature control (Lauda K-2RD). Experiment A: The erythrocyte membranes stored at -80°C for 2 days were used. Assay mixture for Na + + K +-ATPase plus Mg+-ATPase consisted of, in a totalvolume of 2 ml, 100 mM Tris-HC1, pH 7.4, 1 mM EGTA, 10 mM MgC12, 100 mM NaC1, 15 mM KC1, 1 mM ATP, 1 mM phosphoenol pyruvate (PEP), 0.25 mM NADH, pyruvate kinase (8 U/ml), lactic dehydrogenase (8 U/ml) and erythrocyte membrane protein (300-400 #g). The enzyme activity at 37°C was recorded at 340 nm for at least 4 min. Ouabain-sensitive ATPase activity was measured by adding 0.5 mM (final concentration) ouabain to the reaction mixture. The ouabain-sensitive ATPase activity was calculated by subtracting the activity in the presence of ouabain from the Na + + K +-ATPase plus Mg2+-ATPase activity. For assay of Ca2+ + Mg2+-ATPase, the activity in a reaction mixture (total volume 2 ml) consisting of 100 mM Tris-HC1, pH 7.4, 1 mM EGTA, 10 mM MgCI2, 100 mM KC1, 1 mM ATP, 1 mM PEP, 0.25 mM NADH, pyruvate kinase, lactic dehydrogenase and membrane protein (150-200 g) was initially measured as described, and then CaC12 was added to the reaction mixture. The enzyme was
25 almost maximally stimulated by 0.125 m M CaCI2 in the assay condition, therefore, 0.125 m M CaCI2 (as total calcium concentration) was used in the present study. Ca 2+ + Mg 2+-ATPase activity was calculated as the difference between activity with and without CaCI2. The activity without CaC12 was designated as Mg2+-ATPase. Experiment B: The erythrocyte membranes used in this experiment were stored at - 30 °C for 4 weeks. Methods for ATPase assay were the same as experiment A, but E G T A was omitted from the reaction medium and concentration of ouabain was 0.1 m M when it was used. Adenylate kinase activity (in the direction; A T P + A M P --*2 A D P ) was measured immediately after the recording of ATPase activity in the presence of ouabain by addition of A M P to a final concentration of 1 mM. p1, ps_Di(adenosine.5,)pentaphosphat e (10 lzM), which is known as a specific inhibitor ofadenylate kinase (Lienhard et al. 1973), blocked the activity to the level without AMP. All of the enzyme activities were linear for more than 10 min and proportional to membrane protein concentration up to 700 #g. Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. RESULTS The enzyme activities in the present study were shown in Tables 1 and 2. Specific activities of the total ATPase and the ouabain-sensitive ATPase were much higher in experiment A than in experiment B. Adenylate kinase activity (experiment B) of the erythrocyte membranes was about 4 times higher than that of total ATPase (Table 2). When the membranes from a control stored at - 3 0 °C were further washed with 0.1 m M E D T A according to the method described by Fairbanks et al. (1971), the specific activity of adenylate kinase was decreased to a half of the original membranes. Nevertheless, no significant differences of enzyme activities between M y D and control were observed in any conditions examined. TABLE 1 ERYTHROCYTE MEMBRANE ATPase ACTIVITIES IN MYOTONIC DYSTROPHY AND CONTROL SUBJECTS BY EXPERIMENT A. Enzyme activities; m/~moles/min/mgprotein at 37 °C (mean + SD). Myotonic dystrophy (n = 8)
Control (n = 8)
Na + + K +-ATPase plus Mg2+-ATPase Ouabain-sensitive ATPasea
29.2 + 5.3 8.3 + 2.3
31.1 + 8.2 8.6 + 3.9
Mg2+-ATPase MgZ+-ATPase
22.1 +3.1 18.1 + 4.0
22.5 4- 6.8 16.8 + 4.9
C a 2+ +
a
Ouabain concentration was 0.5 mM.
26 TABLE 2 E R Y T H R O C Y T E M E M B R A N E A T P A S E A N D A D E N Y L A T E K I N A S E ACTIVITIES IN M Y O T O N I C D Y S T R O P H Y A N D C O N T R O L SUBJECTS BY E X P E R I M E N T B Enzyme activities; m # m o l e s / m i n / m g protein at 37 °C (mean ± SD).
Myotonic dystrophy
Control
(n = 5)
(n = 6)
Na + + K + - A T P a s e plus Mg2+-ATPase
9.4±
2.7
9.9±
2.6
Ouabain-sensitive ATPase a
2,7±
0.7
2.8±
0.8
Adenylate kinase
41,1±16.0
39.1±18.8
a Ouabain concentration was 0.1 mM.
DISCUSSION
Several reports have been published on ouabain-sensitive ATPase of erythrocyte membranes in MyD, but the results are contradictory. Brown et al. (1967) described a stimulation of the enzyme by 0.1 mM ouabain in 2 cases of MyD, and Niebroj-Dobosz (1976) reported a reduced susceptibility of the enzyme to 0.1 mM ouabain along with a decrease of total ATPase activity. A decreased ouabain-sensitive Na-effiux rate of erythrocytes was also reported (Hull and Roses 1976). On the other hand, Mishra et al. (1980), who used 1 mM ouabain in the assay, reported an increased ouabain-sensitive ATPase of erythrocyte membranes from patients with MyD. With respect to Ca2+ + MgZ+-ATPase of erythrocyte membranes in MyD, one report (Luthra et al. 1979) indicated a decreased activity and another (Ruitenbeek 1979) found an increased activity. The present study indicates no alteration of ouabain-sensitive ATPase nor of Ca 2+ + MgZ+-ATPase in the erythrocyte membranes of MyD. Presence or absence of EGTA in the reaction medium, different ouabain concentrations and different temperatures for membrane storage disclosed no difference of the enzyme activities between MyD and control. Adenylate kinase may not be a "true" membrane-bound enzyme of erythrocyte (Heller and Hanahan 1972). Therefore, the degree of its removal from erythrocyte membranes may vary with differences in the methodology for preparation of the membranes. The adenylate kinase activity in the present study was also not different between MyD and control. The exact cause(s) of the discrepancies among the reports on erythrocyte membranes from patients with MyD are not certain. Slight differences in the process to final determination of erythrocyte membrane properties may cause different results (Hanahan and Ekholm 1978; Roses 1979; Mawatari et al. 1981). Moreover, difference of age of the blood donors (Kobayashi et al. 1978; Igisu et al. 1979), diet (Farquhar and Ahren 1963; De Gier et al. 1964) and even physical activity (Martin et al. 1977) may induce different properties of erythrocytes. In the present study, patients with MyD and age- and sex-matched controls were in the same
27
institution for more than one month, and all of them were able to walk. Therefore, meals and physical activities were not greatly different between the patients with MyD and controls. Sampling and processing of the materials from patients and controls were simultaneous in each experiment. Although the controls used were not healthy persons, the present results indicate no specific abnormality of erythrocyte membrane ATPase activities in MyD. REFERENCES Brown, H. D., S. K. Chattopadhyay and A. B. Patel (1967) Erythrocyte abnormality in human myopathy, Science, 157: 1577-1578. Danon, M. J., W.E. Marshall, G. Sarpel and A. Ohmachi (1978) Erythrocyte metabolism in muscular dystrophy, Arch. Neurol. (Chic.), 35: 592-595. De Gier, J. and L.M. Van Deenen (1964) A dietary investigation of the variations in phospholipid characteristics of red cell membranes, Biochim. biophys. Acta, 84: 294-304. Fairbanks, G., T. L. Steck and D. F. H. Wallach (197 I) Electrophoretic analysis of the human erythrocyte membrane, Biochemistry, 10: 2606-2616. Farquhar, J.W. and E. H. Ahrens, Jr. (1963) Effects of dietary fats on human erythrocyte ,fatty acid patterns, J. clin. lnvest., 42: 675-685. Hanahan, D.J. and J.E. Ekholm (1978) The expression of optimum ATPase activities in human e r y t h r o c y t e s - A comparison of different lytic procedures, Arch. Biochem. Biophys., 187: 170-179. Heller, M. and D.J. Hanahan (1972) Erythrocyte membrane-bound enzymes - - ATPase, phosphatase and adenylate kinase in human, bovine and porcine erythrocytes, Biochim. biophys. Acta, 255: 239-250. Hobbs, A. S., R. A. Brumback and B. Festoff (1979) Monovalent cation transport in myotonic dystrophy, J. neurol. Sci., 41 : 299-306. Hull, Jr., K.H. and A.D. Roses (1976) Stoichiometry of sodium and potassium transport in erythrocytes from patients with myotonic muscular dystrophy, J. Physiol. (Lond.), 254: 169-181. Igisu, H., S. Mawatari and Y. Kuroiwa (1979) Erythrocyte ATP in Duchenne dystrophy - - Effects of ouabain and propranolol, Neurology (NY), 29:992-995. Kobayashi, T., S. Mawatari and Y. Kuroiwa (1978) Lipids and proteins of erythrocyte membrane in Duchenne muscular dystrophy, Clin. chim. Acta, 85: 259-266. Lienhard, G.E. and I.I. Secemski (1973) P,P-Di(adenosine-5)pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase, J. biol. Chem., 248:1121-1123. Lowry, O. H., N.J. Rosebrough, A. L. Farr and R.J. Randall (1951) Protein measurement with Folin phenol reagent, J. biol. Chem., 193: 265-275. Luthra, M.G., L.Z. Stern and H.D. Kim (1979) (Ca ++ + Mg+÷)-ATPase of red cells in Duchenne and myotonic dystrophy - - Effect of soluble cytoplasmic activator, Neurology (Minneap.), 29: 83~841. Martin, R. P., W. L. Haskell and P. D. Wood (1977) Blood chemistry and lipid profiles of elite distance runners, Ann. N. Y. Acad. Sci., 301 : 346-360. Mawatari, S., H. Igisu, Y. Kuroiwa and S. Miyoshino (1981) Na + + K+-ATPase of erythrocyte membranes in Duchenne muscular dystrophy, Neurology (Minneap.), 31: 293-297. Mishra, S.K., M. Hobson and D. Desaiah (1980) Erythrocyte membrane abnormalities in human myotonic dystrophy, J. neurol. Sci., 46: 333-340. Muller, M.M., R. Kuzmits, M. Frass and B. Mamoli (1980) Purine metabolism of erythrocytes in myotonic dystrophy, J. Neurol., 223: 59-66. Niebroj-Dobosz, L. (1976) Erythrocyte ghosts ( N a + + K+)ATPase activity in Duchenne's dystrophy and myotonia, J. Neurol., 214: 61-69. Plishker, G.A., H.J. Gitelman and A.H. Appel (1978) Myotonic muscular dystrophy - - Altered calcium transport in erythrocytes, Science, 200: 323-325. Roses, A.D. (1979) Erythrocyte membrane autophosphorylation in Duchenne muscular dystrophy - Effects of two methods of erythrocyte ghosts preparation on results, Clin. chim. Acta, 95: 69-73. Roses, A.D. and S.H. Appel (1973) Protein kinase activity in erythrocyte ghosts of patients with myotonic muscular dystrophy, Proc. nat. Acad. Sci. (U.S.A.), 70: 1855-1859.
28 Ruitenbeek, W. (1979) Membrane-bound enzymes of erythrocytes in human muscular dystrophy, J. neurol. Sci., 41 : 71-80. Solomons, C.C., S.P. Ringel, E.I. Nwuke and H. Suga (1977) Abnormal adenine metabolism of erythrocytes in Duchenne and myotonic muscular dystrophy, Nature (Lond.), 268: 55-56. Vickers, J. D., A. J. McComas and M.P. Rathbone (1979) Myotonic muscular dystrophy - - Abnormal temperature response of membrane phosphorylation in erythrocyte membranes, Neurology (NY), 29:791 796.
23
ERYTHROCYTE MEMBRANE CATION-STIMULATED ATPase ACTIVITIES IN MYOTONIC MUSCULAR DYSTROPHY
SHIRO MAWATARI 1, YASUNOBU ANTOKU 2 and YOSHIGORO KUROIWA 1
! Department of Neurology, NeurologicalInstitute, Faculty of Medicine, Kyushu University, Fukuoka 812, and 2 National Akasaka Hospital, Chikugo City, Fukuoka-ken (Japan) (Received 9 March, 1981) (Revised, received 20 May, 1981) (Accepted 28 May, 1981)
SUMMARY
The cation-stimulated ATPase activities of erythrocyte membranes from patients with myotonic muscular dystrophy (MyD) were compared with the activities in age- and sex-matched controls. The enzymes included ouabain-sensitive ATPase, Mg2+-ATPase and Ca2÷ + Mg2÷.ATPase. Sampling and processing of the materials from patients with MyD and controls were simultaneously done in each experiment. The enzyme activities were varied with or without EGTA in the reaction medium, or with different temperatures for membrane storage, but no significant differences between MyD and control were observed in any conditions. The present study indicates no specific abnormality of the cation-stimulated ATPase activities of erythrocyte membranes in MyD.
INTRODUCTION
Various abnormalities of erythrocytes (or erythrocyte membranes) have been reported in human myotonic muscular dystrophy (MyD). They included Na + + K +ATPase (Brown et al. 1967; Niebroj-Do bosz 1976; Mishra et al. 1980), Ca2+ + Mg2+ATPase (Luthra et al. 1979; Ruitenbeek 1979), cation transport (Hull and Roses 1976; Plishker et al. 1978; Hobbs et al. 1979), protein kinase (Roses and Appel 1973; Vickers et al. 1979), adenine incorporation into ATP and ADP (Solomons et al.
This work was supported by a Grant of the National Center for Nervous, Mental and Muscular Disorders of the Ministry of Health and Welfare, Japan. Correspondence: Dr. Shiro Mawatari, Department of Neurology, Neurological Institute, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan. 0022-510X/82/0000-0000/$02.75 © Elsevier Biomedical Press
24 1977; Muller et al. 1980), and ATP content of erythrocytes (Danon et al. 1978). It is of interest that these abnormalities are related to ATP. However, there are often discrepancies among these reported results. Therefore, we re-examined ATPase activities of erythrocyte membranes from patients with MyD by comparing with deliberately selected and manipulated control membranes. Additionally, adenylate kinase, which catalyzes the reaction ATP + AMP ~ 2 ADP, was examined in the erythrocyte membranes. MATERIALSAND METHODS Subjects were 8 patients with MyD and 8 patients with pulmonary tuberculosis (controls). Diagnoses of the patients were made by conventional criteria. Age and sex of the controls were matched to those of the patients with MyD. All subjects were in the same institution for more than one month. Samples from the subjects were obtained on the same day at almost the same time and were processed simultaneously in each experiment. Blood was taken into heparinized tubes and immediately put on ice. After removal of plasma and buffy coat, erythrocytes were washed 3 times with isotonic saline by centrifugation at 1,000 × g for 10 min. Buffy coat was removed at each washing. The washed erythrocytes were hemolyzed in about 6 volumes of 10 mM Tris-HC1 (pH. 7.4) and ghosts were collected by centrifugation at 25,000 x g for 20 min. The ghosts were washed 5 times with the same solution by the same centrifugation, and button at the bottom of centrifuge tube (Fairbanks et al. 1971) was removed each time. All procedures were carried out at 2-5 °C. The white ghosts (membranes) were finally suspended in 10 mM Tris-HCl (pH. 7.4) to make protein concentration about 5 mg/ml, and stored at - 8 0 ° C (experiment A) or - 3 0 ° C (experiment B) until use. Enzyme activities were assayed by coupled enzymatic methods using a recording spectrophotometer (Hitachi 200-20) with temperature control (Lauda K-2RD). Experiment A: The erythrocyte membranes stored at -80°C for 2 days were used. Assay mixture for Na + + K +-ATPase plus Mg+-ATPase consisted of, in a totalvolume of 2 ml, 100 mM Tris-HC1, pH 7.4, 1 mM EGTA, 10 mM MgC12, 100 mM NaC1, 15 mM KC1, 1 mM ATP, 1 mM phosphoenol pyruvate (PEP), 0.25 mM NADH, pyruvate kinase (8 U/ml), lactic dehydrogenase (8 U/ml) and erythrocyte membrane protein (300-400 #g). The enzyme activity at 37°C was recorded at 340 nm for at least 4 min. Ouabain-sensitive ATPase activity was measured by adding 0.5 mM (final concentration) ouabain to the reaction mixture. The ouabain-sensitive ATPase activity was calculated by subtracting the activity in the presence of ouabain from the Na + + K +-ATPase plus Mg2+-ATPase activity. For assay of Ca2+ + Mg2+-ATPase, the activity in a reaction mixture (total volume 2 ml) consisting of 100 mM Tris-HC1, pH 7.4, 1 mM EGTA, 10 mM MgCI2, 100 mM KC1, 1 mM ATP, 1 mM PEP, 0.25 mM NADH, pyruvate kinase, lactic dehydrogenase and membrane protein (150-200 g) was initially measured as described, and then CaC12 was added to the reaction mixture. The enzyme was
25 almost maximally stimulated by 0.125 m M CaCI2 in the assay condition, therefore, 0.125 m M CaCI2 (as total calcium concentration) was used in the present study. Ca 2+ + Mg 2+-ATPase activity was calculated as the difference between activity with and without CaCI2. The activity without CaC12 was designated as Mg2+-ATPase. Experiment B: The erythrocyte membranes used in this experiment were stored at - 30 °C for 4 weeks. Methods for ATPase assay were the same as experiment A, but E G T A was omitted from the reaction medium and concentration of ouabain was 0.1 m M when it was used. Adenylate kinase activity (in the direction; A T P + A M P --*2 A D P ) was measured immediately after the recording of ATPase activity in the presence of ouabain by addition of A M P to a final concentration of 1 mM. p1, ps_Di(adenosine.5,)pentaphosphat e (10 lzM), which is known as a specific inhibitor ofadenylate kinase (Lienhard et al. 1973), blocked the activity to the level without AMP. All of the enzyme activities were linear for more than 10 min and proportional to membrane protein concentration up to 700 #g. Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. RESULTS The enzyme activities in the present study were shown in Tables 1 and 2. Specific activities of the total ATPase and the ouabain-sensitive ATPase were much higher in experiment A than in experiment B. Adenylate kinase activity (experiment B) of the erythrocyte membranes was about 4 times higher than that of total ATPase (Table 2). When the membranes from a control stored at - 3 0 °C were further washed with 0.1 m M E D T A according to the method described by Fairbanks et al. (1971), the specific activity of adenylate kinase was decreased to a half of the original membranes. Nevertheless, no significant differences of enzyme activities between M y D and control were observed in any conditions examined. TABLE 1 ERYTHROCYTE MEMBRANE ATPase ACTIVITIES IN MYOTONIC DYSTROPHY AND CONTROL SUBJECTS BY EXPERIMENT A. Enzyme activities; m/~moles/min/mgprotein at 37 °C (mean + SD). Myotonic dystrophy (n = 8)
Control (n = 8)
Na + + K +-ATPase plus Mg2+-ATPase Ouabain-sensitive ATPasea
29.2 + 5.3 8.3 + 2.3
31.1 + 8.2 8.6 + 3.9
Mg2+-ATPase MgZ+-ATPase
22.1 +3.1 18.1 + 4.0
22.5 4- 6.8 16.8 + 4.9
C a 2+ +
a
Ouabain concentration was 0.5 mM.
26 TABLE 2 E R Y T H R O C Y T E M E M B R A N E A T P A S E A N D A D E N Y L A T E K I N A S E ACTIVITIES IN M Y O T O N I C D Y S T R O P H Y A N D C O N T R O L SUBJECTS BY E X P E R I M E N T B Enzyme activities; m # m o l e s / m i n / m g protein at 37 °C (mean ± SD).
Myotonic dystrophy
Control
(n = 5)
(n = 6)
Na + + K + - A T P a s e plus Mg2+-ATPase
9.4±
2.7
9.9±
2.6
Ouabain-sensitive ATPase a
2,7±
0.7
2.8±
0.8
Adenylate kinase
41,1±16.0
39.1±18.8
a Ouabain concentration was 0.1 mM.
DISCUSSION
Several reports have been published on ouabain-sensitive ATPase of erythrocyte membranes in MyD, but the results are contradictory. Brown et al. (1967) described a stimulation of the enzyme by 0.1 mM ouabain in 2 cases of MyD, and Niebroj-Dobosz (1976) reported a reduced susceptibility of the enzyme to 0.1 mM ouabain along with a decrease of total ATPase activity. A decreased ouabain-sensitive Na-effiux rate of erythrocytes was also reported (Hull and Roses 1976). On the other hand, Mishra et al. (1980), who used 1 mM ouabain in the assay, reported an increased ouabain-sensitive ATPase of erythrocyte membranes from patients with MyD. With respect to Ca2+ + MgZ+-ATPase of erythrocyte membranes in MyD, one report (Luthra et al. 1979) indicated a decreased activity and another (Ruitenbeek 1979) found an increased activity. The present study indicates no alteration of ouabain-sensitive ATPase nor of Ca 2+ + MgZ+-ATPase in the erythrocyte membranes of MyD. Presence or absence of EGTA in the reaction medium, different ouabain concentrations and different temperatures for membrane storage disclosed no difference of the enzyme activities between MyD and control. Adenylate kinase may not be a "true" membrane-bound enzyme of erythrocyte (Heller and Hanahan 1972). Therefore, the degree of its removal from erythrocyte membranes may vary with differences in the methodology for preparation of the membranes. The adenylate kinase activity in the present study was also not different between MyD and control. The exact cause(s) of the discrepancies among the reports on erythrocyte membranes from patients with MyD are not certain. Slight differences in the process to final determination of erythrocyte membrane properties may cause different results (Hanahan and Ekholm 1978; Roses 1979; Mawatari et al. 1981). Moreover, difference of age of the blood donors (Kobayashi et al. 1978; Igisu et al. 1979), diet (Farquhar and Ahren 1963; De Gier et al. 1964) and even physical activity (Martin et al. 1977) may induce different properties of erythrocytes. In the present study, patients with MyD and age- and sex-matched controls were in the same
27
institution for more than one month, and all of them were able to walk. Therefore, meals and physical activities were not greatly different between the patients with MyD and controls. Sampling and processing of the materials from patients and controls were simultaneous in each experiment. Although the controls used were not healthy persons, the present results indicate no specific abnormality of erythrocyte membrane ATPase activities in MyD. REFERENCES Brown, H. D., S. K. Chattopadhyay and A. B. Patel (1967) Erythrocyte abnormality in human myopathy, Science, 157: 1577-1578. Danon, M. J., W.E. Marshall, G. Sarpel and A. Ohmachi (1978) Erythrocyte metabolism in muscular dystrophy, Arch. Neurol. (Chic.), 35: 592-595. De Gier, J. and L.M. Van Deenen (1964) A dietary investigation of the variations in phospholipid characteristics of red cell membranes, Biochim. biophys. Acta, 84: 294-304. Fairbanks, G., T. L. Steck and D. F. H. Wallach (197 I) Electrophoretic analysis of the human erythrocyte membrane, Biochemistry, 10: 2606-2616. Farquhar, J.W. and E. H. Ahrens, Jr. (1963) Effects of dietary fats on human erythrocyte ,fatty acid patterns, J. clin. lnvest., 42: 675-685. Hanahan, D.J. and J.E. Ekholm (1978) The expression of optimum ATPase activities in human e r y t h r o c y t e s - A comparison of different lytic procedures, Arch. Biochem. Biophys., 187: 170-179. Heller, M. and D.J. Hanahan (1972) Erythrocyte membrane-bound enzymes - - ATPase, phosphatase and adenylate kinase in human, bovine and porcine erythrocytes, Biochim. biophys. Acta, 255: 239-250. Hobbs, A. S., R. A. Brumback and B. Festoff (1979) Monovalent cation transport in myotonic dystrophy, J. neurol. Sci., 41 : 299-306. Hull, Jr., K.H. and A.D. Roses (1976) Stoichiometry of sodium and potassium transport in erythrocytes from patients with myotonic muscular dystrophy, J. Physiol. (Lond.), 254: 169-181. Igisu, H., S. Mawatari and Y. Kuroiwa (1979) Erythrocyte ATP in Duchenne dystrophy - - Effects of ouabain and propranolol, Neurology (NY), 29:992-995. Kobayashi, T., S. Mawatari and Y. Kuroiwa (1978) Lipids and proteins of erythrocyte membrane in Duchenne muscular dystrophy, Clin. chim. Acta, 85: 259-266. Lienhard, G.E. and I.I. Secemski (1973) P,P-Di(adenosine-5)pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase, J. biol. Chem., 248:1121-1123. Lowry, O. H., N.J. Rosebrough, A. L. Farr and R.J. Randall (1951) Protein measurement with Folin phenol reagent, J. biol. Chem., 193: 265-275. Luthra, M.G., L.Z. Stern and H.D. Kim (1979) (Ca ++ + Mg+÷)-ATPase of red cells in Duchenne and myotonic dystrophy - - Effect of soluble cytoplasmic activator, Neurology (Minneap.), 29: 83~841. Martin, R. P., W. L. Haskell and P. D. Wood (1977) Blood chemistry and lipid profiles of elite distance runners, Ann. N. Y. Acad. Sci., 301 : 346-360. Mawatari, S., H. Igisu, Y. Kuroiwa and S. Miyoshino (1981) Na + + K+-ATPase of erythrocyte membranes in Duchenne muscular dystrophy, Neurology (Minneap.), 31: 293-297. Mishra, S.K., M. Hobson and D. Desaiah (1980) Erythrocyte membrane abnormalities in human myotonic dystrophy, J. neurol. Sci., 46: 333-340. Muller, M.M., R. Kuzmits, M. Frass and B. Mamoli (1980) Purine metabolism of erythrocytes in myotonic dystrophy, J. Neurol., 223: 59-66. Niebroj-Dobosz, L. (1976) Erythrocyte ghosts ( N a + + K+)ATPase activity in Duchenne's dystrophy and myotonia, J. Neurol., 214: 61-69. Plishker, G.A., H.J. Gitelman and A.H. Appel (1978) Myotonic muscular dystrophy - - Altered calcium transport in erythrocytes, Science, 200: 323-325. Roses, A.D. (1979) Erythrocyte membrane autophosphorylation in Duchenne muscular dystrophy - Effects of two methods of erythrocyte ghosts preparation on results, Clin. chim. Acta, 95: 69-73. Roses, A.D. and S.H. Appel (1973) Protein kinase activity in erythrocyte ghosts of patients with myotonic muscular dystrophy, Proc. nat. Acad. Sci. (U.S.A.), 70: 1855-1859.
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