LC20 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 (LC20), and Vmax 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 Vmax 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 Vmax similar to phosphorylated HMM (1.1-1.5 s-1). SAP-S1 had the same KATPase as phosphorylated HMM (38 microM actin), but KATPase for PAP-S1 was 3-fold stronger (11 microM actin). Subsequent digestion of SAP-S1 with papain did not significantly change Vmax, but as LC20 and 24HC were cleaved, both KATPase and Kbinding strengthened 3- to 5-fold. Thus, intact LC20 did not inhibit, and cleavage of LC20 did not increase Vmax for S1. Rather, papain cleavage of LC20 and 24HC was associated with strengthened actin binding.
Drew
J S
JS
Department of Medicine, SUNY, Stony Brook 11794-8661.
White
M P
MP
Moos
C
C
Stein
L A
LA
D000199:Actins / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D010450:Endopeptidases; D005895:Gizzard, Avian / Q000737:chemistry*; D007700:Kinetics; D015879:Myosin Subfragments / Q000737:chemistry*; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis; D014422:Turkeys
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D010450:Endopeptidases; D010206:Papain; D017301:Ca(2+) Mg(2+)-ATPase