Effects of CapZ, an actin capping protein of muscle, on the polymerization of actin.
We have studied the interaction of CapZ, a barbed-end actin capping protein from the Z line of skeletal muscle, with actin. CapZ blocks actin polymerization and depolymerization (i.e., it "caps") at the barbed end with a Kd of approximately 0.5-1 nM or less, measured by three different assays. CapZ inhibits the polymerization of ATP-actin onto filament ends with ATP subunits slightly less than onto ends with ADP subunits, and onto ends with ADP-BeF3- subunits about as much as ends with ADP subunits. No effect of CapZ is seen at the pointed end by measurements either of polymerization from acrosomal processes or of the critical concentration for polymerization at steady state. CapZ has no measureable ability to sever actin filaments in a filament dilution assay. CapZ nucleates actin polymerization at a rate proportional to the first power of the CapZ concentration and the 2.5 power of the actin concentration. No significant binding is observed between CapZ and rhodamine-labeled actin monomers by fluorescence photobleaching recovery. These new experiments are consistent with but do not distinguish between three models for nucleation proposed previously (Cooper & Pollard, 1985). As a prelude to the functional studies, the purification protocol for CapZ was refined to yield 2 mg/kg of chicken breast muscle in 1 week. The activity is stable in solution and can be lyophilized. The native molecular weight is 59,600 +/- 2000 by equilibrium ultracentrifugation, and the extinction coefficient is 1.25 mL mg-1 cm-1 by interference optics. Polymorphism of the alpha and beta subunits has been detected by isoelectric focusing and reverse-phase chromatography. CapZ contains no phosphate (less than 0.1 mol/mol).
Caldwell
J E
JE
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Heiss
S G
SG
Mermall
V
V
Cooper
J A
JA
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001704:Biopolymers; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D018260:Gelsolin; D007700:Kinetics; D008840:Microfilament Proteins / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism*
D000199:Actins; D001704:Biopolymers; D002135:Calcium-Binding Proteins; C052696:Cap Z(36-32) muscle protein; D018260:Gelsolin; D008840:Microfilament Proteins; D009124:Muscle Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate