Journal of the Neurological Sciences, 1977, 32: 361-369
361
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
E R Y T H R O C Y T E CATION-ACTIVATED A D E N O S I N E TRIPHOSPHATASES IN D U C H E N N E M U S C U L A R D Y S T R O P H Y
ANDREW HODSON and DAVID PLEASURE Children's Hospital of Philadelphia, Philadelphia, Pa. 19104 (U.S.A.)
(Received 20 November, 1976)
SUMMARY The cation-stimulated adenosine triphosphatase (ATPase) activities of erythrocyte ghosts and erythrocyte ghost plasma membrane fragments of patients with Duchenne muscular dystrophy (DMD) were compared with activities in age-matched normal male controls. D M D Mg++-stimulated ATPase activity was within the normal range. The specific activity of D M D erythrocyte ghost Na +, K+-stimulated, Mg ++dependent ATPase was also normal, and was inhibited by 10-4 M ouabain to an extent comparable with controls. Ca++-stimulated, Mg++-dependent ATPase activity of D M D erythrocyte ghost plasma membrane fragments, assayed at 0.5 m M free Ca ++, was 21 ~ above that in age-matched male controls (n = 22, 2-tailed paired t-test, P < 0.01). Kinetic studies indicated that the D M D erythrocyte Ca++-stimulated, Mg++-dependent ATPase has greater affinity for MgATP than the enzyme in control erythrocytes.
INTRODUCTION Duchenne muscular dystrophy (DMD) is an inherited, sex-linked disease characterized by progressive degeneration of skeletal muscle (Walton 1974). Several lines of evidence suggest that D M D may be the expression of an inherited defect in the structure and function of skeletal muscle plasma membrane (sarcolemma). Levels of creatine kinase and other high molecular weight intramuscular constituents are elevated in plasma very early in the disease, before weakness is demonstrable, and clinically unaffected carrier females may also have elevated plasma creatine kinase This investigation was supported by funds from the Muscular Dystrophy Associations of America, and by NIH Grants NS-08075, HD-08536, and R-00240. A. Hodson is a Muscular Dystrophy Associations of America Postdoctoral Fellow, and D. Pleasure was supported, in part, by a Travelling Fellowship of the Royal Society of Medicine.
362 levels (Pearce, Pennington and Walton 1964). Examination of muscle from patients with DMD demonstrates defects in the continuity of sarcolemma as the earliest morphological lesion in affected muscle fibers (Mokri and Engel 1975). Stimulation of DMD skeletal muscle sarcolemmal adenylate cyclase by epinephrine is reduced (Mawatari, Takagi and Rowland 1974), and the specific activities of DMD sarcolemmal Mg++-stimulated adenosine triphosphatase (Mg++-ATPase) and Ca-~ ~-stimulated, Mg++-dependent adenosine triphosphatase (Ca+~-ATPase) are elevated (Dhalla, McNamara, Balasubramanian, Greenlaw and Tucker 1973). It is difficult to ascertain whether these sarcolemmal abnormalities are important in the pathogenesis of DMD, or secondary to the progressive myopathy. A number of studies suggest, however, that there are structural and functional changes in the membranes of DMD erythrocytes. DMD and DMD-carrier erythrocytes have been reported to have increased numbers of spicular malformations (Matheson and Howland 1974), reduced deformability (Percey and Miller 1975), increased electrophoretic mobility (Bosmann, Gersten, Griggs, Howland, Hudecki, Katyare and McLaughlin 1976), increased K + permeability (Howland 1974; Sha'afi, Rodan, Hintz, Fernandez and Rodan 1975), and an altered phospholipid fatty acid composition (Kunze, Reichmann, Egger, Leuschner and Eckhardt 1973). DMD erythrocyte ghost Na +, K ~stimulated, Mg++-dependent adenosine triphosphatase (Na +, K+-ATPase) has been noted to be stimulated, not inhibited, by 10-4 M ouabain (Brown, Chattopadhyay and Patel 1967; Peter, Worsfold and Pearson 1969; Araki and Mawatari 1971). Stimulation of DMD erythrocyte ghost adenylate cyclase by catecholamines is reduced (Mawatari, Schonberg and Olarte 1976), while the Mg++-dependent protein kinase activity of DMD and DM D-carrier erythrocyte ghosts has been reported to be elevated (Roses, Roses, Miller, Hull and Appel 1976). To delineate further possible plasma membrane alterations in DMD, we have investigated the Mg++-ATPase, Na +, K+-ATPase and Ca++-stimulated, Mg+~-dependent ATPase (Ca+
361
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
E R Y T H R O C Y T E CATION-ACTIVATED A D E N O S I N E TRIPHOSPHATASES IN D U C H E N N E M U S C U L A R D Y S T R O P H Y
ANDREW HODSON and DAVID PLEASURE Children's Hospital of Philadelphia, Philadelphia, Pa. 19104 (U.S.A.)
(Received 20 November, 1976)
SUMMARY The cation-stimulated adenosine triphosphatase (ATPase) activities of erythrocyte ghosts and erythrocyte ghost plasma membrane fragments of patients with Duchenne muscular dystrophy (DMD) were compared with activities in age-matched normal male controls. D M D Mg++-stimulated ATPase activity was within the normal range. The specific activity of D M D erythrocyte ghost Na +, K+-stimulated, Mg ++dependent ATPase was also normal, and was inhibited by 10-4 M ouabain to an extent comparable with controls. Ca++-stimulated, Mg++-dependent ATPase activity of D M D erythrocyte ghost plasma membrane fragments, assayed at 0.5 m M free Ca ++, was 21 ~ above that in age-matched male controls (n = 22, 2-tailed paired t-test, P < 0.01). Kinetic studies indicated that the D M D erythrocyte Ca++-stimulated, Mg++-dependent ATPase has greater affinity for MgATP than the enzyme in control erythrocytes.
INTRODUCTION Duchenne muscular dystrophy (DMD) is an inherited, sex-linked disease characterized by progressive degeneration of skeletal muscle (Walton 1974). Several lines of evidence suggest that D M D may be the expression of an inherited defect in the structure and function of skeletal muscle plasma membrane (sarcolemma). Levels of creatine kinase and other high molecular weight intramuscular constituents are elevated in plasma very early in the disease, before weakness is demonstrable, and clinically unaffected carrier females may also have elevated plasma creatine kinase This investigation was supported by funds from the Muscular Dystrophy Associations of America, and by NIH Grants NS-08075, HD-08536, and R-00240. A. Hodson is a Muscular Dystrophy Associations of America Postdoctoral Fellow, and D. Pleasure was supported, in part, by a Travelling Fellowship of the Royal Society of Medicine.
362 levels (Pearce, Pennington and Walton 1964). Examination of muscle from patients with DMD demonstrates defects in the continuity of sarcolemma as the earliest morphological lesion in affected muscle fibers (Mokri and Engel 1975). Stimulation of DMD skeletal muscle sarcolemmal adenylate cyclase by epinephrine is reduced (Mawatari, Takagi and Rowland 1974), and the specific activities of DMD sarcolemmal Mg++-stimulated adenosine triphosphatase (Mg++-ATPase) and Ca-~ ~-stimulated, Mg++-dependent adenosine triphosphatase (Ca+~-ATPase) are elevated (Dhalla, McNamara, Balasubramanian, Greenlaw and Tucker 1973). It is difficult to ascertain whether these sarcolemmal abnormalities are important in the pathogenesis of DMD, or secondary to the progressive myopathy. A number of studies suggest, however, that there are structural and functional changes in the membranes of DMD erythrocytes. DMD and DMD-carrier erythrocytes have been reported to have increased numbers of spicular malformations (Matheson and Howland 1974), reduced deformability (Percey and Miller 1975), increased electrophoretic mobility (Bosmann, Gersten, Griggs, Howland, Hudecki, Katyare and McLaughlin 1976), increased K + permeability (Howland 1974; Sha'afi, Rodan, Hintz, Fernandez and Rodan 1975), and an altered phospholipid fatty acid composition (Kunze, Reichmann, Egger, Leuschner and Eckhardt 1973). DMD erythrocyte ghost Na +, K ~stimulated, Mg++-dependent adenosine triphosphatase (Na +, K+-ATPase) has been noted to be stimulated, not inhibited, by 10-4 M ouabain (Brown, Chattopadhyay and Patel 1967; Peter, Worsfold and Pearson 1969; Araki and Mawatari 1971). Stimulation of DMD erythrocyte ghost adenylate cyclase by catecholamines is reduced (Mawatari, Schonberg and Olarte 1976), while the Mg++-dependent protein kinase activity of DMD and DM D-carrier erythrocyte ghosts has been reported to be elevated (Roses, Roses, Miller, Hull and Appel 1976). To delineate further possible plasma membrane alterations in DMD, we have investigated the Mg++-ATPase, Na +, K+-ATPase and Ca++-stimulated, Mg+~-dependent ATPase (Ca+
