Kinetics of polymerization and ATP hydrolysis by covalently crosslinked actin dimer.

Actin dimer covalently cross-linked by NP-p-phenylenebismaleimide is a single species of molecular weight 85, 000 that shares properties of both monomeric and polymeric actin. The dimer, in a nonpolymerizing buffer, resembles F-actin in its ability to bind 3Hcytochalasin B and its inability to interact with profilin. Dimer resembles monomer in that the fluorescence spectra of monomer and dimer labeled with 7-chloro-4nitrobenzeno-2-oxa-l, 3-diazole (NBD) are identical and both undergo a 2-fold increase in fluorescence upon polymerization. Cross-linked actin dimer contains two nucleotide binding sites, one that rapidly exchanges bound ATP for ATP free in solution and one that exchanges much more slowly. The enhancement in fluorescence of NBD-dimer was used to compare the time course of polymerization to the time course of hydrolysis of dimer-bound Y-~'PATP. In 50 m~ KC1 or 1 m~ MgClz at 25 "C, polymerization was complete within 2-3 min, while ATP hydrolysis at the rapidly exchangeable site required 6-10 min. ATP hydrolysis at the slowly exchangeable site occurred much more slowly. Polymerization was complete when only about 15% of the total dimer-bound ATP was hydrolyzed. Thus, ATP hydrolysis and polymerization were uncoupled and filament-bound ATP, rather than dimer-bound ATP, was hydrolyzed. Dimer polymerization showed a slight lag phase in MgClz but not in KC1. The lag was not due to a nucleation phase because it could be eliminated by replacing dimer-bound Ca2+ with M+ before initiating polymerization. In contrast, polymerization of NBDphenylenebismaleimide-actin monomer had a nucleation phase and was tightly coupled to ATP hydrolysis. Dimer could nucleate the polymerization of monomer and elongation occurred at both ends of fragments of glutaraldehyde-fixed filaments of dimer decorated with myosin subfragment-1. Monomeric G-actin polymerizes to polymeric F-actin by the addition of subunits to both ends of growing filaments and, in the presence of ATP, with hydrolysis of approximately one molecule of ATP for every molecule of G-actin converted to F-actin (for review see Ref. 1). ATP continues to be hydrolyzed when polymerization has reached steady state. From theoretical considerations, it has been proposed (2) that slow ATPase cycles are catalyzed by monomeric actin alone and filament ends alone. The accelerated ATPase activity that occurs when both monomer and polymer are present would be due to the coupling of the complementary, fast steps of the two separate cycles. There is experimental evidence for a * The costs of