ACTIN FILAMENT BARBED-END CAPPING ACTIVITY IN NEUTROPHIL LYSATES - THE ROLE OF CAPPING PROTEIN-BETA(2)

A barbed-end capping activity was found in high speed supernates of ne utrophils lysed in submicromolar calcium. In dilute supernate (greater than or equal to 100-fold dilution of cytoplasm), this activity accou nted for most of the inhibition of barbed-end elongation of pyrenyl-G- actin from spectrin-F-actin seeds. Pointed-end elongation from gelsoli n-capped F-actin seeds was not inhibited at comparable concentrations of supernate, thus excluding actin monomer sequestration as a cause of the observed inhibition. Most of the capping activity was due to capp ing protein-beta(2) (a homologue of cap Z). Thus, while immunoadsorpti on of greater than or equal to 95% of the gelsolin in the supernate di d not decrease capping activity, immunoadsorption of capping protein-b eta(2) reduced capping activity proportionally to the amount of cappin g protein-beta(2) adsorbed. Depletion of >90% of capping protein-beta( 2) from the supernate removed 90% of its capping activity. The functio nal properties of the capping activity were defined. The dissociation constant for binding to barbed ends (determined by steady state and ki netic analyses) was similar to 1-2 nM; the on-rate of capping was betw een 7 x 10(5) and 5 x 10(6) M(-1) s(-1) ; and the off-rate was similar to 2 10(-3) s(-1). The concentration of capper free in the intact cel l (determined by adsorption of supernate with spectrin-actin seeds) wa s estimated to be similar to 1-2 mu M. Thus, there appeared to be enou gh high affinity capper to cap all the barbed ends in vivo. Neverthele ss, immediately after lysis with detergent, neutrophils contained site s that nucleate barbed-end elongation of pyrenyl-G-actin. These barbed ends subsequently become capped with a time course and concentration dependence similar to that of spectrin-F-actin seeds in high speed sup ernates. These observations suggest that, despite the excess of high a ffinity capper, some ends either are not capped in vivo or are transie ntly uncapped upon lysis and dilution.