Structural aspects of skeletal muscle G-actin molecule as studied by proteolytic digestion: effect of nucleotide

Abstract

Trypsin and chymotrypsin were used as probes of conformation of G-actin molecule. The pattern of fragments produced has been analyzed by sodium dodecyl sulfate gel electrophoresis. G-actin is known to be nonrefractory to proteolysis [Jacobson, G.R., and Rosenbusch, J.P. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2742-2746]. It is really true that G-actin is cut easily into a 33-kDa fragment by trypsin or chymotrypsin, but only when free ATP is present in the medium. After the removal of free ATP from the medium, G-actin became more refractory to proteolysis. The amounts of degradation of G-actin depended on the ATP concentration in the medium with saturating at about 0.5 mM. epsilon-ADP also had the effect and its fluorescence spectrum was changed on the addition of G-actin. After the removal of free ATP, G-actin still bound 1 mol/mol of ATP. So, the present results suggest the presence of a second ATP interaction site on G-actin and that ATP interaction at this site induces conformational changes in G-actin molecule.