LC20 AND KINETICS OF GIZZARD MYOSIN SUBFRAGMENT-1 - DIGESTION WITH PAPAIN VS STAPHYLOCOCCUS-AUREUS PROTEASE

Previous reports have shown that papain-digested gizzard subfragment-1 (PAP-S1) has a cleaved regulatory light chain (LC20), 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 LC20, but V(max) closer to that of unphosphorylated HMM [Ikebe and Hartshorne, 1985]. To determine whether intact LC20 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-degrees-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 LC20 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 muM actin), but K(ATPase) for PAP-S1 was 3-fold stronger (11 muM actin). Subsequent digestion of SAP-S1 with papain did not significantly change V(max), but as LC20 and 24HC were cleaved, both K(ATPase) and K(binding) Strengthened 3- to 5-fold. Thus, intact LC20 did not inhibit, and cleavage of LC20 did not increase V(max) for S1. Rather, papain cleavage of LC20 and 24HC was associated with strengthened actin binding.