Incorporation of 6-carboxyfluorescein into myosin subfragment 1

• Published in: Biochemistry (Easton) Vol. 24; no. 4; pp. 840 - 846
• Main Authors: Mornet, Dominique, Ue, Kathleen
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.02.1985
• Subjects: Actins - isolation & purification; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; Contractile proteins; Electrophoresis, Polyacrylamide Gel; Ethyldimethylaminopropyl Carbodiimide; Fluoresceins - metabolism; Fundamental and applied biological sciences. Psychology; Holoproteins; Kinetics; Macromolecular Substances; Molecular Weight; Muscles - metabolism; myosin; Myosin Subfragments; Myosins - isolation & purification; Myosins - metabolism; Peptide Fragments - metabolism; Protein Binding; Proteins; Rabbits; Incorporation; ATPase; Actins; Rabbit; Contractile protein; Molecular interaction; Subfragment 1; Lagomorpha; Vertebrata; Mammalia; Fluorescent labelling; Myosin; Muscle
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00325a005

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.