7-Chloro-4-nitrobenzeno-2-oxa-1,3-diazole actin as a probe for actin polymerization.
Lysine 372 of N-ethylmaleimide actin was specifically (60%) labeled by 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole chloride (NBD-Cl), which also reacted with lysines on cyanogen bromide fragment 17 (20%) and other undetermined residues (20%). Isolation of N-ethylmaleimide peptides and two-dimensional peptide mapping demonstrated that 90% of bound N-ethylmaleimide was attached to an adjacent residue, cysteine 373, independent of the polymerization state of actin during the labeling reaction. Formation of NBD cysteine severely inhibited lysine modification. After N-ethylmaleimide blockage of cysteine 373, lysine labeling with NBD was greatly accelerated. The kinetics of formation of fluorescent compounds were biphasic, with fluorescence decreasing upon prolonged incubation of actin in NBD-Cl. Lysine 372 of purified NBD actin reproducibly responded to polymerization by a 2.2- to 2.3-fold enhancement of fluorescence. By contrast, interaction of NBD actin with several actin-binding proteins caused only very small or undetectable changes in fluorescence intensity: 10% enhancement on myosin subfragment 1 binding, about 6% quenching by DNase I, and no change at all by tropomyosin-troponin. Despite its sensitivity to polymerization the probe did not affect it. Native and modified actin polymerized randomly indicating that the rate constants for polymerization remained the same. Labeling actin with NBD did not diminish its cofactor activity for myosin ATPase activity. Contrary to previous reports we observed that myosin subfragment 1 (single myosin heads) caused actin polymerization in the absence of salt.
D009327:4-Chloro-7-nitrobenzofurazan; D000199:Actins; D000818:Animals; D005033:Ethylmaleimide; D007700:Kinetics; D046911:Macromolecular Substances; D009132:Muscles; D015879:Myosin Subfragments; D010069:Oxadiazoles; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D000199:Actins; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D010069:Oxadiazoles; D009327:4-Chloro-7-nitrobenzofurazan; D005033:Ethylmaleimide