Characteristics of skeletal muscle ecto-ATPase in situ

Abstract

The properties of a cell surface nucleoside 5′-triphosphatase have been studied in small, intact, frog skeletal muscles, as a means of distinguishing the enzyme from other adenosine 5′-triphosphatases and of understanding its behaviour in the muscle membrane. The ectoenzyme in situ was shown to be a Ca2+- or Mg2+-activated ATPase liberating 7.5 ± 0.4 (mean ± SEM, n = 30) μmol of inorganic phosphate/g of muscle per 20 min, when the muscle was exposed to 2 mM ATP and 2 mM Ca2+ in Ringer's solution. The apparent Km for Mg2+ was 0.74 mM and for Ca2+ was 0.23 mM. A residual ATPase activity (20%) was found in the complete absence of divalent cations suggesting the existence of two ATPase types. A broad specificity toward nucleoside 5′-triphosphates was exhibited by the ecto-ATPase, but there was no nonspecific phosphatase activity. The enzyme was inhibited by La3+ and Cd2+, but was insensitive to ouabain, 2,4-dinitrophenol, oligomycin, and ruthenium red. Thus the ectoenzyme was not a Na+,K+-transport ATPase, was not an ATPase of mitochondrial origin, or a Ca2+-transport enzyme. Insulin had no effect. Inhibition by mersalyl, carbodiimide, and polar and cross-linking nonpolar nitrobenzene derivatives suggested that, for maximum activity, this membrane-bound enzyme required free sulfhydryl groups, certain free carboxyls, and an appreciable degree of hydrophobicity in its microenvironment.