Abstract
Besides driving contraction of various types of muscle tissue, conventional (class II) myosins serve essential cellular functions and are ubiquitously expressed in eukaryotic cells. Three different isoforms in the human myosin complement have been identified as non-muscle class II myosins. Here we report the kinetic characterization of a human non-muscle myosin IIB subfragment-1 construct produced in the baculovirus expression system. Transient kinetic data show that most steps of the actomyosin ATPase cycle are slowed down compared with other class II myosins. The ADP affinity of subfragment-1 is unusually high even in the presence of actin filaments, and the rate of ADP release is close to the steady-state ATPase rate. Thus, non-muscle myosin IIB subfragment-1 spends a significantly higher proportion of its kinetic cycle strongly attached to actin than do the muscle myosins. This feature is even more pronounced at slightly elevated ADP levels, and it may be important in carrying out the cellular functions of this isoform working in small filamentous assemblies.
Highlights
Edly enriched in NMIIB [8], whereas platelets contain only NMIIA [9]
We show that NMIIB subfragment-1 (S1) has generally slower kinetics at most steps than those reported for other myosin II isoforms
The detailed kinetic properties of human NMIIB S1 were investigated in this study to unravel the functional adaptations this conventional myosin has undergone to exert its cellular function
Summary
Expression, and Purification of the NMIIB S1 Protein— cDNA for human NMIIB was truncated at amino acid 843 to create an S1-like fragment and subcloned into baculovirus transfer vector pVL1392 (Invitrogen). Stopped-flow Experiments—Unless stated otherwise, all stoppedflow measurements were done in an SF-2001 stopped-flow apparatus (KinTek Corp., Austin, TX) at 25 °C in buffer comprising 25 mM MOPS (pH 7.0), 5 mM MgCl2, 100 mM KCl, and 0.1 mM EGTA. Labeled actin was excited at 365 nm, and the emitted light was selected using a 400-nm long-pass cutoff filter. Mant-ATP and mant-ADP were excited via energy transfer from tryptophan (295 nm excitation), and the emitted light was selected using a 400-nm long-pass cutoff filter. After aging in the delay line, reactions were stopped by mixing with a solution containing 22% trichloroacetic acid and 1 mM KH2PO4 (to approximately one-third of the total volume). ADP contamination was removed from NMIIB S1 samples by incubation with 0.01/ml apyrase (Sigma) at 25 °C for 30 min. Means Ϯ S.D. reported for the kinetic constants are those of three to four separate rounds of experiment
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