Differential Impact of Temperature and Magnesium on Myosin V and Myosin II

Abstract

We examined the impact of temperature and free magnesium concentration on monomeric FlAsH labeled myosin V (MV FlAsH), dimeric myosin V (MV HMM), and dimeric fast skeletal muscle myosin II (SK HMM) using ATPase and motility assays. Our results indicate that MV HMM and SK HMM both have a linear dependence on temperature that is similar in both ATPase and motility assays. However, MV FlAsH contains a different temperature dependence in ATPase and motility assays suggesting its short lever arm may impart a high strain dependence in the motility assay. MV HMM and MV FlAsH are inhibited by high concentrations of magnesium in both ATPase and motility assays. The rate-limiting step in myosin V is known to be ADP release, which we demonstrate correlates well with the magnesium and temperature dependence of ATPase and motility assays. Interestingly, SK HMM exhibits magnesium inhibition in ATPase assays, but only a slight decrease is observed in the motility assay. In SK HMM the rate-limiting step in ATPase assays is thought to be attachment to actin or phosphate release, while in motility assays it is controversial. Our results indicate that SK HMM is better described by an attachment limited model in the motility assay. Magnesium may reduce the duty ratio of SK HMM which alters ATPase activity but not velocity in the motility assay. Future experiments will determine if magnesium alters the actin binding and/or the product release steps in myosin V and skeletal muscle myosin. Myosin V contains a tyrosine (residue 439) in the switch II region, which is an alanine at the corresponding position in myosin II, suggesting this residue may play a key role in differentially altering magnesium coordination in the active site of myosins.