Production and properties of skeletal myosin subfragment 1 selectively labeled with fluorescein at lysine-553 proximal to the strong actin-binding site.
We describe, for the first time, the reaction of skeletal myosin subfragment 1 (S-1) with the succinimido ester of 6-[fluorescein-5(and 6)-carboxamido]hexanoic acid (FHS), which takes place at pH 7.0, 20 degrees C, within a 15 min period, in the presence of 1.5-1.8-fold molar excess of reagent over protein. As a result, 0.9-1.0 mol of fluorescyl group/mol of S-1 was covalently incorporated exclusively into the 95 kDa heavy chain as monitored by spectroscopic measurements. The central 50 kDa segment included the main site of fluorescence attachment as assessed by gel electrophoresis. The extent of S-1--FHS conjugation is strongly sensitive to F-actin binding but not to the interaction of nucleotides. The formation of the rigor F-actin--S-1 complex decreased the level of S-1 labeling to 20% without any competition between actin and S-1 for FHS binding. The derivatization of S-1 did not alter the K(+)-ATPase activity, but it enhanced the Ca(2+)-ATPase and Mg(2+)-ATPase to 150% and 225%, respectively, whereas it lowered the actin-activated ATPase to only 75% of the original activity. A double-reciprocal plot of the ATPase rate against actin concentration indicated a 2-fold decrease of the Vmax value for modified S-1, while the Km for actin was unchanged. Cosedimentation experiments did not reveal disruption of the rigor acto-S-1 interaction by the bound fluorophore. The labeled S-1 heavy chain was isolated, and its total tryptic digest was fractionated by reverse-phase HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)
Bertrand
R
R
Centre de Recherches de Biochimie Macromoléculaire du CNRS, INSERM U 249, Université de Montpellier I, France.
Derancourt
J
J
Kassab
R
R
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002208:Caproates; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000378:metabolism*; D015879:Myosin Subfragments / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014357:Trypsin
C115825:6-(fluorescein-5-carboxamido)hexanoic acid succimidyl ester; D000199:Actins; D002208:Caproates; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D010446:Peptide Fragments; C094938:succinimido 6-(fluorescein-6-carboxamido)hexanoate; D014357:Trypsin; D009218:Myosins; D000252:Calcium-Transporting ATPases