Modulation of gelsolin-induced actin-filament severing by caldesmon and tropomyosin and the effect of these proteins on the actin activation of myosin Mg(2+)-ATPase activity.
We have investigated the cumulative effects of three smooth-muscle actin-binding proteins, gelsolin, caldesmon and tropomyosin, on actin activation of myosin Mg(2+)-ATPase activity under low-ionic-strength conditions. A combination of tropomyosin (at a stoicheiometric ratio to actin) and gelsolin (at a molar ratio to actin of up to 1:100) showed essentially additive stimulatory effects that were counteracted by caldesmon. Suppression of the gelsolin-induced activation of the ATPase by caldesmon was higher in the presence of tropomyosin although it was not complete even at stoicheiometric amounts of both proteins to actin. Since activation of actin-activated ATPase activity of myosin by gelsolin is related to its severing action, it is concluded that caldesmon and tropomyosin cannot fully protect actin filaments against the severing activity of gelsolin. Direct analysis of the actin-severing activity of gelsolin by a fluorimetric assay using pyrene-labelled actin confirmed this conclusion. Tropomyosin and caldesmon in saturating amounts relative to actin inhibited the activity of gelsolin by between 21 and 40% and 25 and 48% respectively, depending on the molar ratio of gelsolin to actin. The inhibitory effect was increased with a combination of both (up to 67%) although it was evident that even under these conditions the actin filaments were not fully protected from being severed by gelsolin. These findings were corroborated by electron-microscopic investigation of actin filaments with or without tropomyosin and caldesmon after the addition of gelsolin.
Dabrowska
R
R
Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Hinssen
H
H
Gałazkiewicz
B
B
Nowak
E
E
D000199:Actins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000494:pharmacology*; D002645:Chickens; D004789:Enzyme Activation / Q000187:drug effects; D018260:Gelsolin / Q000494:pharmacology*; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D009218:Myosins / Q000378:metabolism*; D011817:Rabbits; D013552:Swine; D014335:Tropomyosin / Q000494:pharmacology*
D000199:Actins; D002148:Calmodulin-Binding Proteins; D018260:Gelsolin; D014335:Tropomyosin; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins