Actin and Light Chain Isoform Dependence of Myosin V Kinetics

• Published in: Biochemistry (Easton) Vol. 39; no. 46; pp. 14196 - 14202
• Main Authors: De La Cruz, Enrique M, Wells, Amber L, Sweeney, H. Lee, Ostap, E. Michael
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.11.2000
• Subjects: Actins - metabolism; Actins - physiology; Adenosine Diphosphate - analogs & derivatives; Adenosine Diphosphate - metabolism; Adenosine Triphosphate - metabolism; Adenosine Triphosphate - pharmacology; Amino Acid Motifs; Animals; Calmodulin-Binding Proteins - metabolism; Calmodulin-Binding Proteins - physiology; Chickens; Kinetics; Muscle, Skeletal - metabolism; Muscle, Skeletal - physiology; Myosin Light Chains - metabolism; Myosin Light Chains - physiology; Myosin Type V; Myosins - metabolism; Nerve Tissue Proteins - metabolism; Nerve Tissue Proteins - physiology; ortho-Aminobenzoates - metabolism; Protein Binding; Protein Isoforms - metabolism; Protein Isoforms - physiology; Rabbits; Spectrometry, Fluorescence; Tryptophan
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi001701b

Recent studies on myosin V report a number of kinetic differences that may be attributed to the different heavy chain (chicken vs mouse) and light chain (essential light chains vs calmodulin) isoforms used. Understanding the extent to which individual light chain isoforms contribute to the kinetic behavior of myosin V is of critical importance, since it is unclear which light chains are bound to myosin V in cells. In addition, all studies to date have used α-skeletal muscle actin, whereas myosin V is in nonmuscle cells expressing β- and γ-actin. Therefore, we characterized the actin and light chain dependence of single-headed myosin V kinetics. The maximum actin-activated steady-state ATPase rate (V max) of a myosin V construct consisting of the motor domain and first light chain binding domain is the same when either of two essential light chain isoforms or calmodulin is bound. However, with bound calmodulin, the K ATPase is significantly higher and there is a reduction in the rate and equilibrium constants for ATP hydrolysis, indicating that the essential light chain favors formation of the M·ADP·Pi state. No kinetic parameters of myosin V are strongly influenced by the actin isoform. ADP release from the actin−myosin complex is the rate-limiting step in the ATPase cycle with all actin and light chain isoforms. We postulate that although there are significant light-chain-dependent alterations in the kinetics that could affect myosin V processivity in in vitro assays, these differences likely are minimized under physiological conditions.