A novel stopped-flow method for measuring the affinity of actin for myosin head fragments using microgram quantities of protein.
The dissociation constant for actin binding to myosin and its subfragments (S1 & HMM) is <<1 microM at physiological ionic strength. Many of the methods used to measure such affinities are unreliable for a Kd below 0.1 microM. We show here that the use of phalloidin to stablise F-actin and fluorescently labelled proteins allows the affinity of actin for myosin S1 to be measured in a simple transient kinetic assay. The method can be used for Kd's as low as 10 nM and we demonstrate that the Kd's can be estimated using only microgram quantities of material. Furthermore we suggest how this method may be adapted for ng quantities of protein. This will allow the affinity of actin for myosin fragments to be estimated for proteins which are difficult to obtain in large quantities i.e. from biopsy material or from proteins expressed in baculovirus.
Kurzawa
S E
SE
Max-Planck-Institut für Molekulare Physiologie, Dortmund, Germany.
Geeves
M A
MA
D000199:Actins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D000205:Actomyosin / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D005470:Fluorometry; D015879:Myosin Subfragments / Q000378:metabolism / Q000494:pharmacology; D009218:Myosins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010590:Phalloidine / Q000494:pharmacology; D011721:Pyrenes / Q000494:pharmacology; D011817:Rabbits
D000199:Actins; D015879:Myosin Subfragments; D011721:Pyrenes; D010590:Phalloidine; D000255:Adenosine Triphosphate; D000205:Actomyosin; C030984:pyrene; D009218:Myosins