PURIFICATION AND PROPERTIES OF A CA2-INDEPENDENT BARBED-END ACTIN FILAMENT CAPPING PROTEIN, CAPZ, FROM HUMAN POLYMORPHONUCLEAR LEUKOCYTES()

In human polymorphonuclear leukocytes (PMN), changes in the actin arch itecture are critical for the shape changes required for chemotaxis an d phagocytosis. Barbed-end capping proteins are likely to regulate act in assembly in PMN. The previously identified barbed-end blocking prot eins in PMN, gelsolin and CapG, require Ca2+ to initiate capping of ac tin filaments. Because chemoattractants can stimulate PMN actin assemb ly by a calcium-independent signal transduction pathway, we sought to purify a calcium-independent barbed-end capping activity from PMN cyto plasmic extracts. A Ca2+-insensitive actin polymerization inhibitory a ctivity was partially purified from human PMN [Southwick & Stossel (19 81) J. Biol. Chem 256, 3030]. Using five column chromatography steps, we purified the protein to homogeneity as assessed by silver staining. Purification was associated with an increase in specific activity of greater than 40 X. Western blot analysis identified the protein as the nonmuscle isoform of the heterodimeric capping protein capZ. Human PM N capZ has an apparent disassociation constant of 3 nM for capping in the presence or absence of micromolar Ca2+, as assessed by both pyreny lactin elongation and depolymerization assays. Similar to the the acti vity reported for the actin polymerization inhibitor, activity of PMN capZ was inhibited by increasing the KCl concentration from 0.1 M to 0 .6 M, The capping function was also inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2) micelles, with half-maximal inhibition occurri ng at 5.5 mu g mL(-1), PMN capZ did not nucleate actin assembly, seque ster actin monomers, or sever actin filaments. Quantitative Western bl ot analysis revealed that capZ levels corresponded to 0.7-1.0% of the total human PMN cytoplasmic protein. Given its abundance and high affi nity for barbed filament ends, capZ is likely to play an important rol e in the calcium-independent regulation of actin filament assembly ass ociated with PMN chemotaxis.