Selective fluorescent labeling of the 50-, 26-, and 20-kilodalton heavy chain segments of myosin ATPase.
7-Diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin (CPI), rhodamine B isothiocyanate (RITC), and 4-bromomethyl-6,7-dimethoxycoumarin (BDMC), fluorescent reagents that can react covalently with amino or sulfhydryl groups, have been used to label myosin subfragment-1 (S-1) ATPase. The conditions under which CPI, RITC, and BDMC selectively label the 50-, 26-, and 20-kDa segments of the S-1 heavy chain, respectively, are described. CPI and RITC labeling little affects the ATPase activities of S-1 in the presence and absence of actin. BDMC labeling activates the Ca2+- and Mg2+-ATPases of S-1, and abolishes the K+-EDTA-ATPase. The three S-1 derivatives fluoresce strongly even under acidic conditions, suggesting the wide applicability of these fluorescent reagents as selective labels for the three segments of the S-1 heavy chain.
D000251:Adenosine Triphosphatases / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D003374:Coumarins; D005456:Fluorescent Dyes; D017879:Isothiocyanates; D008970:Molecular Weight; D015879:Myosin Subfragments; D009218:Myosins / Q000032:analysis* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D011817:Rabbits; D012235:Rhodamines; D013050:Spectrometry, Fluorescence
D003374:Coumarins; D005456:Fluorescent Dyes; D017879:Isothiocyanates; D015879:Myosin Subfragments; D010446:Peptide Fragments; D012235:Rhodamines; C053768:7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; C053769:4-bromomethyl-6,7-dimethoxycoumarin; C027755:rhodamine isothiocyanate; D000251:Adenosine Triphosphatases; D009218:Myosins