We have recently reported on the characterization of β-actin carrying the mutation S14C in one of the phosphate-binding loops. The present paper describes the attachment of the adenosine 5′-[gamma-thio]-triphosphate (ATPγS) to actin containing this mutation. Treatment of S14C-actin with ATPγS blocked further nucleotide exchange and raised the thermal stability of the protein, suggesting the formation of a covalent bond between the sulfhydryl on the terminal phosphate of ATPγS and cysteine-14 of the mutant actin. The affinity of the derivatized G-actin for DNase I as compared to wild-type ATP-actin was lowered to a similar extent as that of ADP.AlF4-actin. The derivatized actin polymerized slower than ATP-actin but faster than ADP-actin. Under these conditions the bound ATPγS was hydrolyzed, suggesting the formation of a state corresponding to the transient ADP.Pi-state. ATPγS-actin interacted normally with profilin, whereas the interaction with actin depolymerizing factor (ADF) was disturbed, as judged on the effects of these proteins on actin polymerization.
