Biochemical studies of myosin.
This article describes methods for expressing and obtaining purified smooth muscle myosin subfragments using the baculovirus/insect cell expression system, as well as methods for purifying whole myosin from tissue. Protocols for several gel assays that are routinely used with myosin are given, including gels to monitor light chain phosphorylation state and native gels to determine protein homogeneity. Steady-state myosin ATPase and actin-activated ATPase determinations are described, as are some of the more basic transient-state kinetic parameters that can be measured. The in vitro motility assay, in which the rate of actin movement over myosin or its subfragments is quantified, is also presented.
Trybus
K M
KM
Department of Molecular Physiology and Biophysics, Given E205, University of Vermont, Burlington, Vermont 05405, USA. trybus@salus.med.uvm.edu
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002645:Chickens; D005895:Gizzard, Avian; D007700:Kinetics; D020409:Molecular Motor Proteins / Q000235:genetics / Q000302:isolation & purification*; D009068:Movement / Q000502:physiology; D009130:Muscle, Smooth; D015879:Myosin Subfragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D014422:Turkeys
D000199:Actins; D020409:Molecular Motor Proteins; D015879:Myosin Subfragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate