A novel actin label: a fluorescent probe at glutamine-41 and its consequences.
By peptide isolation and analysis, it has been shown that the dansyl fluorophore of dansylcadaverine [N-(5-aminopentyl)-5-(dimethylamino)naphthalene-1-sulfonamide] transfers to Gln-41 of actin from rabbit skeletal muscle when the reaction is catalyzed by guinea pig liver transglutaminase. As a function of time, the degree of labeling asymptotically approaches 1 mol of dansyl/l mol of actin. About 80-85% of the attached dansyl fluorophore was found at Gln-41. Such labeled G-actin polymerizes to the same extent as control actin, but the polymerization rate is greater and the critical concentration is less than for control actin. Complete polymerization is accompanied by a 1.5-2.0-fold increase in the emission intensity of the attached fluorophore. Labeled F-actin thus obtained activates myosin subfragment 1 (S-1) Mg2+-ATPase activity with the same Kapp, and to the same Vmax, as control actin; moreover, when such labeled F-actin is cross-linked to S-1 by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, the resulting superactivation of Mg2+-ATPase is the same as that attained with control actin. The attributes of this label thus make it an ideal reporter of events in the N-terminal 10-kilodalton region of actin, and a new topological point for proximity mapping.
D000199:Actins / Q000378:metabolism; D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002103:Cadaverine / Q000031:analogs & derivatives*; D003488:Cyanogen Bromide; D003959:Diamines; D004229:Dithiothreitol; D005973:Glutamine; D007460:Iodoacetamide; D010446:Peptide Fragments / Q000032:analysis; D013050:Spectrometry, Fluorescence; D011503:Transglutaminases / Q000378:metabolism
D000199:Actins; D000596:Amino Acids; D003959:Diamines; D010446:Peptide Fragments; D005973:Glutamine; D011503:Transglutaminases; D017301:Ca(2+) Mg(2+)-ATPase; C008542:monodansylcadaverine; D002103:Cadaverine; D003488:Cyanogen Bromide; D004229:Dithiothreitol; D007460:Iodoacetamide