Quantitation of Cap Z in conventional actin preparations and methods for further purification of actin.
Gel-filtration is commonly used to remove contaminants from conventional actin prepared by the method of Spudich and Watt. It has been shown that this procedure removes the majority of a factor that reduces the low-shear viscosity of actin. We have previously reported that this factor is Cap Z, a barbed end capping protein. We now establish that, even after gel-filtration, enough Cap Z can be present in conventionally prepared actin to affect events occurring at the barbed ends of actin filaments. We also demonstrate that the concentration of Cap Z can be reduced to more than a log below the KD for binding of Cap Z to actin by either 1) immunoabsorbtion of conventionally prepared actin with anti-Cap Z antibodies, or 2) an additional cycle of polymerization/depolymerization followed by repeat gel-filtration.
Casella
J F
JF
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Barron-Casella
E A
EA
Torres
M A
MA
D000199:Actins / Q000302:isolation & purification*; D001696:Biomechanical Phenomena; D001704:Biopolymers; D015153:Blotting, Western; D051347:CapZ Actin Capping Protein; D002846:Chromatography, Affinity; D004355:Drug Stability; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D008840:Microfilament Proteins; D009124:Muscle Proteins / Q000032:analysis*
D000199:Actins; D001704:Biopolymers; D051347:CapZ Actin Capping Protein; D008840:Microfilament Proteins; D009124:Muscle Proteins