FIGURE 4.
Expression of FLAG-tagged HMM II-C isoforms and actin-activated MgATPase activities. Actin-activated MgATPase activities of HMM II-C0 (A), HMM II-C1 (B), HMM II-C2 (C), and HMM II-C1C2 (D) were measured at 25 °C with various concentrations of actin before (open symbol, dotted line) and after (closed symbol, solid line) phosphorylation by MLCK. Data sets were fitted to a hyperbolic equation to determine the kinetic constants, Vmax and KATPase (see Table 1). The data shown are from a single representative preparation of each HMM. UnP, unphosphorylated; P, phosphorylated. Of note is that in contrast to HMM II-C0 or C1, both HMM II-C2 and C1C2 have similar MgATPase activities in the phosphorylated and unphosphorylated states. D, inset, Coomassie Blue-stained gel of purified HMM II-C proteins. C0, HMM with no insert; C1, HMM with C1 insert; C2, HMM with C2 insert; C1C2, HMM with C1 and C2 inserts. The spurious, slower migration of MLC20 in the SDS gel is because of the buffer system and the particular markers used in this gel (Invitrogen SeeBlue® Plus2 Pre-stained Standards).