Investigation of the actin-deoxyribonuclease I interaction using a pyrene-conjugated actin derivative.
The interaction of deoxyribonuclease I with muscle actin was studied with the aid of a pyrenyl derivative of the actin [Kouyama, T., & Mihashi, K. (1981) Eur. J. Biochem. 114, 33-38] that increases its quantum yield by an order of magnitude on polymerization. It is shown that this derivative copolymerizes with unlabeled G-actin in a random manner and will also bind to deoxyribonuclease with inhibition of enzymic activity. The derivative affords a highly sensitive means of following nucleated polymerization. Preincubation of F-actin with deoxyribonuclease at a concentration of 5% or less of that of total subunits causes inhibition of polymerization of additional G-actin onto the filaments. In red cell membranes that contain stabilized short filaments of actin such that the concentration of filament ends is large relative to monomers, complete inhibition of nucleated polymerization of G-actin is achieved by preincubation with deoxyribonuclease. The results indicate that binding of DNase occurs at the "plus" ends of the actin filaments. Competition with cytochalasin E, which is known to have a high affinity for the plus or preferentially growing ends of F-actin, can be observed. Whereas the activity of deoxyribonuclease in the 1:1 complex with G-actin is inhibited, the enzyme attached to the ends of filaments appears to be fully active. This causes a reduction in the inhibition of enzymic activity with increasing F-actin concentration, presumably by reason of a change in the partition of the enzyme between monomers and filament ends. The degree of inhibition increases with time, however, as the actin depolymerizes. Implications for measurements of actin monomer concentrations by the deoxyribonuclease assay procedure are considered.
D000199:Actins / Q000097:blood / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002645:Chickens; D003850:Deoxyribonuclease I; D004305:Dose-Response Relationship, Drug; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors*; D004910:Erythrocyte Membrane; D006801:Humans; D046911:Macromolecular Substances; D008433:Mathematics; D010179:Pancreas / Q000201:enzymology; D011721:Pyrenes
D000199:Actins; D046911:Macromolecular Substances; D011721:Pyrenes; C030984:pyrene; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I