Actin-regulating activities in cultured BHK cells.

Abstract

A large proportion (60-85%) of the actin present in lysates of cultured baby hamster kidney (BHK) cells is monomeric at low temperature. All this G-actin is accounted for in the fractions obtained by high-speed centrifugation of the lysate in the presence or absence of non-ionic detergent; 98 (+/- 7)% of its reacts as monomer in the deoxyribonuclease assay. The fractions nevertheless possess actin polymerization nucleating activity, as assayed by the fluorescence of extraneous pyrene-conjugated actin, reflecting the presence of capping proteins and their complexes with oligomeric actin, which dissociate during the deoxyribonuclease assay. Despite the enhanced nucleation, the total proportion of actin polymerized at equilibrium is reduced, relative to that of actin in the absence of cell extract, presumably because of the presence of 'barbed'-end capping protein(s). With increasing concentrations of the supernatant material, the proportion of polymerized actin was progressively reduced and the presence of inhibitory activity against polymerization became apparent. On addition of micromolar concentrations of free calcium, nucleation by the supernatant fractions is greatly accelerated, but the extent of polymerization undergoes a further reduction, due to the elevated critical monomer concentration resulting from capping. Inhibition of polymerization by the supernatant fraction in the presence of calcium is much greater than by saturating concentrations of cytochalasin E. These observations are most simply interpreted as showing that the cytoplasm additionally contains a 'pointed'-end capping protein. The plasma membranes were found to have nucleating activity largely residing in the Triton-insoluble fractions. Actin cross-linking activity was assayed by adding small proportions of either the supernatant or membrane fractions to a large excess of F-actin and analysing the pellet resulting from a low-speed centrifugation. Supernatant fraction, containing Triton-soluble membrane-derived material, possessed much greater cross-linking capacity than the membrane-free supernatant prepared in the absence of detergent. The membrane fractions had practically no cross-linking activity on their own. Nevertheless, a non-ionic detergent extract of the membrane enhanced the cross-linking activity of the supernatant, indicating the participation of more than one species in a cross-linking process.