LC 20 and kinetics of gizzard myosin subfragment‐1: Digestion with papain vs. S. aureus protease

Previous reports have shown that papain‐digested gizzard subfragment‐1 (PAP‐S1) has a cleaved regulatory light chain (LC 20 ), and V max similar to phosphorylated heavy meromyosin (HMM) Greene et al., Biochemistry 22:530–535, 1983; Sellers et al., J. Biol. Chem. 257:13880‐13883, 1982; Umemoto et al., [ J. Biol. Chem. 264:1431–1436, 1989], while S. aureus protease‐digested S‐1 (SAP‐S1) has intact LC 20 , but V max closer to that of unphosphorylated HMM [Ikebe and Hartshorne, 1985]. To determine whether intact LC 20 inhibits ATPase activity for subfragment‐ 1 (S1), we compared the kinetic properties and structures of unphosphorylated PAP‐S1 and SAP‐S1. SDS‐PAGE showed that SAP‐S1 had 68 and 24 KDa heavy chain and 20 and 17 KDa light chain components. PAP‐S1 (15 minutes digestion at 20°C) also had 68 and 17 KDa bands, but the single 24 KDa band (24HC) was replaced by a group of 22–24 KDa fragments and LC 20 was cleaved to a 16 KDa fragment. At 13 mM ionic strength, both PAP‐S1 and SAP‐S1 had V max similar to phosphorylated HMM (1.1–1.5 s −1 ). SAP‐S1 had the same K ATPase as phosphorylated HMM (38 μM actin). but K ATPase for PAP‐S1 was 3‐fold stronger (11 μM actin). Subsequent digestion of SAP‐S1 with papain did not significantly change V max , but as LC 20 and 24HC were cleaved, both K ATPase and K binding strengthened 3‐ to 5‐fold. Thus, intact LC 20 did not inhibit, and cleavage of LC 20 did not increase V max for S1. Rather, papain cleavage of LC 20 and 24HC was associated with strengthened actin binding. © 1992 Wiley‐Liss, Inc.