The inactivation by concanavalin A of the ecto-ATPase and ectoacetylcholinesterase of intact skeletal muscles

Abstract

The (Ca2+ or Mg2+)-activated ectophosphohydrolase of intact frog muscle liberates, in situ, about 37 μmol inorganic phosphate/g muscle in 20 min at 20 °C with 10 mM ATP. Pretreatment with concanavalin A (ConA) at 4 °C for 18 h caused ectoenzyme inactivation which plateaued at 35–40% of the control rate. The inhibition was concentration dependent, being maximal at about 500 μg ConA/mL Ringer's solution. The lectin mediated its effect via the membrane glycoproteins since the inhibition was specifically prevented by α-methyl D-mannopyranoside. As the temperature increased from 10 to 40 °C, the ectoenzyme activity of untreated muscles increased linearly between 10 and 35 °C, with a "break point" and a clear change in slope at 35 °C. When treated with ConA the activity increased linearly from 10 to 40 °C, eliminating the transition temperature. The findings suggested that a phase transition toward fluidity in the lipid bilayer may have occurred at 35 °C and that this was abolished by the lectin binding. Hence we perturbed the surface membrane phospholipids of muscle pretreated with the lectin. Phospholipase C increased the activation by the lectin; phospholipase D had no effect, but phospholipase A2 completely prevented it. The lectin may require the more fluid fatty acyl chains of membrane lipids to achieve inhibition of this ecto-ATPase. Ectoacetylcholinesterase, in situ, and its inactivation by ConA were measured directly on whole, intact skeletal muscles.