Research on molecular mechanisms of McArdle's disease (muscle glycogen phosphorylase deficiency). Use of new protein mapping and immunological techniques.

McArdle's disease is due to the lack of activity of muscle glycogen phosphorylase. We investigated the presence of an inactive protein by two techniques: (a) Bidimensional protein maps, using a modification of the original O'Farrell technique allowing location of phosphorylase. (b) Purification of enzyme from crude muscle extracts, using an immunoaffinity microchromatographic procedure. Protein maps of three patients were obtained. No protein was detected at the normal (97 K) position of phosphorylase but 70 and 60 K spots were visible. Results of enzyme purificaton by immunoaffinity were negative for one patient, whereas a small band of phosphorylase-like material was detected in the other. Our results confirm the molecular heterogeneity of the disease. We think such methods might be useful for investigating other genetic diseases.

Calmodulin ligands. The interaction of muscle phosphorylase kinase with phosphodiesterase. Comparison of calmodulin ligands in muscle extracts from normal and phosphorylase kinase-deficient mice.

Interactions between phosphorylase kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38) and calmodulin were studied with pure preparations of muscle phosphorylase kinase, and with crude extracts from muscles of control (C57 Black) and deficient (ICR/IAn) mice, which lack muscle phosphorylase kinase activity. Calmodulin was determined by its ability to stimulate a calmodulin-dependent phosphodiesterase. The amount of calmodulin bound to phosphorylase kinase in muscle extract was estimated to a maximum of 30% of the total amount of calmodulin. In the muscle of the deficient strain a decrease of 35% in the total amount of calmodulin was observed. This correlates with the absence of the calmodulin fraction specifically bound to phosphorylase kinase. From sucrose gradient studies we demonstrated that in the presence of Ca2+ the amount of calmodulin bound to phosphorylase kinase was enhanced, compared to the control in the presence of EGTA. This observation was made both in crude extracts and in pure phosphorylase kinase preparations. Sucrose gradient also showed that muscle phosphorylase kinase can be dissociated to low molecular species when extracts are made in the presence of Ca+; this dissociation was found to be related to a Ca2+-dependent proteolytic effect.

Phosphorylase kinase isoenzymes in deficient ICR/IAn mice.

ICR/IAn mice present a deficiency in phosphorylase kinase activity; the extent of this deficiency is less in some tissues [Lyon, S.B. Biochem. Genet. 4, 169--185 (1970)] than in skeletal muscle, where enzyme activity is 0.3% of normal [Cohen, P.T. W & Cohen, P. FEBS Lett. 29, 113--115 (1973)]. New-born mice of this strain were also reported (Lyon, 1970) to reveal a small amount of skeletal muscle enzyme activity. The properties of these residual phosphorylase kinases were compared to those of control C57 BL mice, with reference to control muscle and liver enzymes which were shown to be of different molecular species [Daegelen-Proux et al. Biochim. Biophys Acta, 452, 398--405 (1976)]. The properties investigated were the immunological reactivity against an antiserum raised against muscle phosphorylase kinase, the thermal stability and the Ca2+ dependency. The results suggest that the muscle enzyme from the new-born ICR/IAn mice and the heart enzyme from adult deficient mice are different to the muscle enzyme from adult normal mice, but they have properties in common with normal adult liver enzyme. These results lead to the conclusion that there exists in the muscle of I strain a "foetal form" of phosphorylase kinase, the activity of which decreases progressively after birth. Out work also confirmed the observations made by Cohen et al. [Eur. J. Biochem. 66, 347--356 (1976)] which showed that there is no evidence for the existence of a cross-reacting material in the muscle of adult deficient mice.

Molecular heterogeneity of rabbit heart phosphorylase kinase.

Phosphorylase kinase (ATP: phosphorylase-b phosphotransferase, EC 2.7.1.38) from rabbit heart, when submitted to electrophoresis on Pevikon, separates into two discrete peaks A and B. The two peaks have been analyzed using reelectrophoresis, chromatography on DEAE-cellulose, thermal stability, inactivation by EGTA (ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) and reaction with an anti-muscle phosphorylase kinase antiserum. It can be concluded that rabbit heart extracts contain two isozymes of phosphorylase kinase. The more negatively charged isozyme seems to be identical with the muscle enzyme. The other isozyme resembles the liver enzyme but differs from the major fraction of the latter by its charge. It is likely that there exist at least three molecular types of phosphorylase kinase.