Characterization of Blebbistatin Inhibition of Smooth Muscle Myosin and Nonmuscle Myosin‑2

Blebbistatin is a potent and specific inhibitor of the motor functions of class II myosins, including striated muscle myosin and nonmuscle myosin-2 (NM2). However, the blebbistatin inhibition of NM2c has not been assessed and remains controversial with respect to its efficacy with smooth muscle myos...

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Published in:Biochemistry (Easton) 2017-08, Vol.56 (32), p.4235-4243
Main Authors: Zhang, Hai-Man, Ji, Huan-Hong, Ni, Tong, Ma, Rong-Na, Wang, Aibing, Li, Xiang-dong
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Animals
Avian Proteins - antagonists & inhibitors
Avian Proteins - chemistry
Avian Proteins - genetics
Avian Proteins - metabolism
Chickens
Heterocyclic Compounds, 4 or More Rings - chemistry
Humans
Mice
Mutation, Missense
Nonmuscle Myosin Type IIB - antagonists & inhibitors
Nonmuscle Myosin Type IIB - chemistry
Nonmuscle Myosin Type IIB - genetics
Nonmuscle Myosin Type IIB - metabolism
Smooth Muscle Myosins - antagonists & inhibitors
Smooth Muscle Myosins - chemistry
Smooth Muscle Myosins - genetics
Smooth Muscle Myosins - metabolism
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Summary:Blebbistatin is a potent and specific inhibitor of the motor functions of class II myosins, including striated muscle myosin and nonmuscle myosin-2 (NM2). However, the blebbistatin inhibition of NM2c has not been assessed and remains controversial with respect to its efficacy with smooth muscle myosin (SmM), which is highly homologous to NM2. To clarify these issues, we analyzed the effects of blebbistatin on the motor activities of recombinant SmM and three NM2s (NM2a, -2b, and -2c). We found that blebbistatin potently inhibits the actin-activated ATPase activities of SmM and NM2s with following IC50 values: 6.47 μM for SmM, 3.58 μM for NM2a, 2.30 μM for NM2b, and 1.57 μM for NM2c. To identify the blebbistatin-resistant myosin-2 mutant, we performed mutagenesis analysis of the conserved residues in the blebbistatin-binding site of SmM and NM2s. We found that the A456F mutation renders SmM and NM2s resistant to blebbistatin without greatly altering their motor activities or phosphorylation-dependent regulation, making A456F a useful mutant for investigating the cellular function of NM2s.
ISSN:0006-2960
1520-4995
DOI:10.1021/acs.biochem.7b00311