Discrimination of assembled and disassembled forms of gizzard myosin by papain.
Chicken gizzard myosin in 0.15 M or 0.5 M NaCl was cleaved at two sites of heavy chain with 2-10 micrograms/ml papain. MgATP inhibited these cleavages of myosin in 0.15 M NaCl but not in 0.5 M NaCl. The protective effect of ATP was observed at concentrations as low as 10 microM and increased in proportion to ATP concentration to a maximum at 1 mM. ADP was as effective as ATP, while adenosine 5'-[beta, gamma-imido]triphosphate, an unhydrolyzable ATP analogue, was less effective than ATP or ADP. AMP had no protective effect on the digestion of myosin and GTP inhibited slightly the digestion. When the papain-insensitive myosin in 0.15 M NaCl and 2.5 mM MgATP was phosphorylated by Ca2+/calmodulin-dependent myosin light-chain kinase, the myosin restored the vulnerability to papain. However, the two papain-susceptible forms, nonphosphorylated form in the absence of MgATP and phosphorylated form in the presence of MgATP, yielded very similar but distinct proteolytic fragments upon the digestion. When the extent of myosin assembly was estimated by the turbidimetry of myosin suspension in 0.15 M NaCl, nonphosphorylated myosin in the absence and presence of MgATP was assembled and disassembled, respectively, and phosphorylated myosin in the presence of MgATP was assembled. These results suggest that, at physiological ionic strength, papain as a probe distinguishes disassembled myosin and assembled myosin as papain-insensitive and papain-sensitive forms, respectively.
D000255:Adenosine Triphosphate; D000818:Animals; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian / Q000032:analysis*; D009218:Myosins / Q000302:isolation & purification*; D010206:Papain; D010766:Phosphorylation; D011487:Protein Conformation; D012965:Sodium Chloride
D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D010206:Papain; D009218:Myosins