Incorporation of 6-carboxyfluorescein into myosin subfragment 1.
We describe for the first time the introduction of a label into the "50K" domain of myosin subfragment 1 (S-1), and we investigate the properties of this fluorescent modification in relation to the ATPase and actin-binding activities, both residing in the myosin head. The labeling consists of a major incorporation of 6-carboxyfluorescein into the "50K" domain of S-1. Using different conditions for tryptic digestion that allowed a fragmentation of the "50K" domain with a loss of 5 kilodaltons (kDa) leading to a final product of 45 kDa, we have shown that the fluorescent dye remains in the 45-kDa final product. By studying cross-linking as a function of time, we have demonstrated that the "50K" domain and the 45-kDa fluorescent peptide are equally cross-linkable to actin. We have also investigated the K+EDTA-, Ca2+-, Mg2+-, and actin-activated ATPase activities of this modified S-1 and after purification observed no enzymatic changes.
D000199:Actins / Q000302:isolation & purification; D000818:Animals; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide; D005452:Fluoresceins / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits
D000199:Actins; D005452:Fluoresceins; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D010446:Peptide Fragments; C024098:6-carboxyfluorescein; D009218:Myosins; D005022:Ethyldimethylaminopropyl Carbodiimide