1973), vascular (Hathway et al., 1970) and autoimmune (Jasmin & Bokdawala, 1970) mechanisms has been put forward. Attempts to find a mutant protein have ...
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Stenflo, J., Fernlund, P., Egan, W. & Roepstofl, P. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2730-2733 Tiselius, A., HjCrten, S. & Lewin, 6.(1956) Arch. Biochem. Biophys. 65, 132-155 Tishkoff, G. H., Williams, L. C. & Brown, D. M. (1968) J. Biol. Chem. 243,4151-4167 Tokiwa, F. & Imamura, T. (1969) J. Am. Oil Chem. SOC.46,280-284
Apparent Alterations of Erythrocyte Acetylcholinesterase and other Membrane Proteins in Duchenne Muscular Dystrophy: a further example of a Generalized Membrane Defect Associated with Hereditary Muscular Dystrophy P. K. DAS,* D. GRAESSLINt and H. W. GOEDDE* *Institute of Human Genetics and ?Department of Clinical and Experimental Endocrinology, University of Hamburg, Hamburg, Federal Republic of Germany The primary cause of genetically determined muscular dystrophy remains obscure. Evidence in favour of purely myopathic (Walton, 1969), neoronal (Gallup & Dubowitz, 1973), vascular (Hathway et al., 1970) and autoimmune (Jasmin & Bokdawala, 1970) mechanisms has been put forward. Attempts to find a mutant protein have not yet produced any substantial results. The increase that occurs in certain intracellular enzymes, such as creatine kinase, in the serum of patients with Duchenne muscular dystrophy (Pennington, 1969) may only indicate that the organization of plasma membrane with respect to its permeability is altered. A postulation, based on various observations (Das et al., 1971a,b; Rodan et al., 1974; Roses et al., 1975), strongly points to a generalized membrane defect as the fundamental problem in this disorder. To assimilate the different concepts on the lesion underlying this disease, we chose t o investigate a functional protein that may be involved in maintaining normal neuromuscular activity. Acetylcholinesterase plays an important role in neuromuscular transmission (Nachmansohn, 1959). Since erythrocyte acetylcholinesterase appears to be similar (at least with respect to its substrate specificity) to the acetylcholinesterase at the neuromuscular junction and because the material is easily available, we have previously compared some properties of the enzyme in normal and diseased mice (Das et al., 1971a,b). In this presentation, we report further evidence confirming earlier observations on the altered properties of Triton X-100-solubilized erythrocyte acetylcholinesterase, which indicates membrane abnormality in Duchenne muscular dystrophy. Other erythrocyte-membrane enzymes (Brown et al., 1967; Roses et al., 1975) have also been reported to be altered in this disease. The possibility remains that the membrane defect in Duchenne muscular dystrophy can also be demonstrated when membrane constituents other than those already reported are investigated. For example, in spite of the reported similar protein pattern on sodium dodecyl sulphate/polyacrylamide-geldisc electrophoresis (Roses et at., 1975), we have observed different protein patterns of Triton-solubilized erythrocyte ‘ghosts’ from patients with Duchenne muscular dystrophy, on slab gel electrofocusing. P. K. D. is indebted to the Muscular Dystrophy Group, U.K., the Hong Kong University Research Grant no. 158/229for financial support and to Alexander von Humboldt-Stiftungfor the award of Senior Fellowship, to continue this work. We thank Professor W. J. Bradeley, Professor Bethlem, Dr. J. O’Brian, Professor R. Beckmann, Professor D. Seitz, for supplying the samples.
Brown, H. D., Chattapodhya, S. K. & Patel, A. B. (1967) Science 57, 1577-1578 Das, P. K., Watts, R. L. & Watts, D. C. (1971~)Biochem. J. 123.24~-25~ Das, P. K., Watts, D. C. & Coles, H. M. T. (1971b) Abstr. FEBS Meet. 7th, 307 Gallup, B. & Dubowitz, V. (1973) Nature (Landon)243, 287-289 Hathway, P. W., Engel, W. K. & Zwellger, H. (1970) Arch. Neurol. 22, 365-378 Jasmin, G. & Bokdawala, F. (1970) Rev. Can. Biol. 29,197-201
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Nachmansohn, D. (1 959) Chemical and Molecular Basis of Nerve Acfiuity, Academic Press, New York and London Pennington, R. T. (1969) Disorders of VolunfuryMuscle, p. 385, Churchill, London Rodan, S. B., Hintz, R. L.,Sha’afi, R. I. &Rodan,G. A. (1974) Nuture(London)252,589-591 Roses, A. D., Herbstreith, M. H. & Appel, S. H. (1975) Nature (London)254,350-351 Walton, J. N. (1969) Br. Med. J . i, 1271-1274
Assay of Enzyme Activity by Polarography P. D. J. WEITZMAN Department of Biochemistry, School of Biological Sciences, University of Leicester, Leicester LE1 I R H , U.K.
Polarography is a powerful electroanalytical technique, but, except for measurements made with the oxygen electrode, it has not been fully exploited in biochemical studies. Polarographic equipment is rarely encountered in biochemical laboratories and the technique is frequently viewed as requiring considerable skill and experience for the interpretation of results. Although there are, as with other techniques, problems that may beset the newcomer, a working familiarity with the method and the ability to make reliable measurements may readily be acquired. It is the purpose of this communication to show that polarography may usefully and advantageously be applied to the determination of the activities of a variety of enzymes. The technique relies on examination of the current produced at a polarizable microelectrode as a function of the applied potential. The circuit is completed with a nonpolarizable reference electrode (both electrodes being in contact with the test solution) and a sensitive instrument for applying the potential and measuring the small currents. The micro-electrode generally used in polarographic studies is the dropping mercury electrode, which consists of a very fine capillary tube from which mercury emerges regularly as tiny drops; a saturated calomel electrode usually serves as the reference electrode. Reduction or oxidation of substances at the micro-electrode give rise respectively to cathodic or anodic currents. If the solution contains an electroactive substance, the dependence of current on applied potential is characteristically of the form shown in Fig. 1. As the potential is varied a limiting current is reached at which the substance is electrolysed as fast as it arrives at the electrode surface by diffusion, and this limiting current is therefore directly proportional to the concentration of the substance. Continuous and automatic recording of the change in limiting current at an appropriate fixed potential provides a measure of the rate of formation or consumption of an electroactive species. Many enzyme-catalysed reactions involve substrates or products which are electroactive and such enzymes should lend themselves to polarographic assay. I previously reported that citrate synthase and malate synthase may be assayed polarographically and described a suitable apparatus and procedure (Weitzman, 1966,1969).The technique of polarographic assay has now been extended to a number of enzymes of different reaction types. The assays for citrate synthase and malate synthase are based on the fact that CoA, but not its S-acyl derivatives, gives an anodic polarographic wave whose magnitude is proportional to CoA concentration. The cleavage of acetyl-CoA to CoA by the citrate synthase or malate synthase reactions may thus be followed by monitoring the limiting current at -0.2V to -0.3V. For malate synthase, the polarographic method is particularly advantageous as the chromogenic thiol-specific reagent 5,5’-dithiobis-(2-nitrobenzoate), which might in principle be used for the continuous spectrophotomctric measurement of CoA production, inactivates the enzyme. Some citrate synthases are also inactivated or desensitized to regulatory effectors by 5,5’-dithiobis-(2-nitrobenzoate) (Weitzman & Danson, 1976). In all these cases, polarographic measurement of activities does not affect the enzymes. 1976
