Functional characterization of skeletal F-actin labeled on the NH2-terminal segment of residues 1-28.
Rabbit skeletal alpha-actin was covalently labeled in the filamentous state by the fluorescent nucleophile, N-(5-sulfo-1-naphthyl)ethylenediamine (EDANS) in the presence of the carboxyl group activator 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide (EDC). The coupling reaction was continued until the incorporation of nearly 1 mol EDANS/mol actin. After limited proteolytic digestion of the labeled protein and chromatographic identification of the EDANS-peptides, about 80% of the attached fluorophore was found on the actin segment of residues 1-28, most probably within the N-terminal acidic region of residues 1-7. A minor labeling site was located on the segment that consists of residues 40-113. No label was incorporated into the COOH-terminal moiety consisting of residues 113-375. The isolated EDANS-G-actin undergoes polymerization in the presence of salts but at a rate significantly greater than unlabeled actin. The EDANS-F-actin could be complexed to skeletal chymotryptic myosin subfragment 1 (S-1) and to tropomyosin. The complex formed between EDANS-F-actin and S-1 could not be further crosslinked by EDC but the two proteins were readily joined by glutaraldehyde as observed for native actin-S-1, suggesting that the EDANS-substituted carboxyl site is also involved in the EDC crosslinking of native actin to S-1. Moreover, the EDANS labeling of F-actin resulted in a 20-fold increase in the Km of the actin-activated Mg2+.ATPase of S-1. Thus, this labeling, while it did not much affect the rigor actin-S-1 interaction, changes the actin binding to the S-1-nucleotide complexes significantly. The selective introduction of a variety of spectral probes, like EDANS, or other classes of fluorophores, on the N-terminal region of actin, through the reported carbodiimide coupling reaction, would provide several different derivatives valuable for assessing the functional role of the negatively charged N-terminus of actin during its interaction with myosin and other actin-binding proteins.
Bertrand
R
R
Centre de Recherches de Biochimie Macromoléculaire du CNRS, INSERM Unité 249, Université de Montpellier I, France.
Chaussepied
P
P
Audemard
E
E
Kassab
R
R
D000199:Actins / Q000032:analysis* / Q000494:pharmacology; D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D001704:Biopolymers / Q000032:analysis; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007700:Kinetics; D009132:Muscles / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases; D011817:Rabbits; D013329:Structure-Activity Relationship
D000199:Actins; D000345:Affinity Labels; D000596:Amino Acids; D001704:Biopolymers; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D010447:Peptide Hydrolases; D017301:Ca(2+) Mg(2+)-ATPase