ADP-insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca2+-ATPase has a compact conformation resistant to proteinase K, V8 protease and trypsin

Sarcoplasmic reticulum Ca2+-ATPase was digested with proteinase K, V8 prote ase and trypsin in the absence of Ca2+. Unphosphorylated enzyme was rapidly degraded. In contrast, ADP-insensitive phosphoenzyme formed with Pi and ph osphorylated state analogues produced by the binding of F- or orthovanadate , were almost completely resistant to the proteolysis except for tryptic cl eavage at the T1 site (Arg(505)). The results indicate that the phosphoenzy me and its analogues have a very compact form in the cytoplasmic region, be ing consistent with large domain motions (gathering of three cytoplasmic do mains). Results further show that the structure of the enzyme with bound de cavanadate is very similar to ADP-insensitive phosphoenzyme. Thapsigargin d id not affect the changes in digestion time course induced by the formation of the phosphorylated state analogues. (C) 2001 Federation of European Bio chemical Societies. Published by Elsevier Science B.V. All rights reserved.