Mapping myosin light chains by immunoelectron microscopy. Use of anti-fluorescyl antibodies as structural probes.
The two classes of light chains in vertebrate fast muscle myosin have been selectively labeled with the thiol specific reagent 5-(iodoacetamido) fluorescein to determine their location in the myosin head. The alkali light chains (A1 and A2) were labeled at a single cysteine residue near the COOH terminus, whereas the regulatory light chain (LC2) was reacted at either cysteine 125 or 154. The two cysteines of LC2 appear to be near each other in the tertiary structure as evidenced by the ease of formation of an intramolecular disulfide bond. Besides having favorable spectral properties, fluorescein is a potent haptenic immunogen for raising high affinity antibodies. When anti-fluorescyl antibodies were added to the fluorescein-labeled light chains, the fluorescence was quenched by greater than 90%, thereby providing a simple method for determining an association constant. The interaction with antibody was the same for light chains exchanged into myosin as for free light chains. Complexes of antibody bound to light chain could be visualized in the electron microscope by rotary shadowing with platinum. By this approach we have shown that the COOH-terminal regions of the two classes of light chains are widely separated in myosin: the cysteine residues of LC2 lie close to the head/rod junction, whereas the single cysteine of A1 or A2 is located approximately 90 A distal to the junction. These sites correspond to the positions of the NH2 termini of the light chains mapped in earlier studies (Winkelmann, D. A., and S. Lowey. 1986. J. Mol. Biol. 188:595-612; Tokunaga, M., M. Suzuki, K. Saeki, and T. Wakabayashi. 1987b. J. Mol. Biol. 194:245-255). We conclude that the two classes of light chains do not lie in a simple colinear arrangement, but instead have a more complex organization in distinct regions of the myosin head.
Katoh
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Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254.
Lowey
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S
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002645:Chickens; D000939:Epitopes / Q000032:analysis; D005452:Fluoresceins; D007700:Kinetics; D008854:Microscopy, Electron; D008961:Models, Structural; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism / Q000648:ultrastructure*; D009218:Myosins / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000906:Antibodies; D000936:Antigen-Antibody Complex; D000939:Epitopes; D005452:Fluoresceins; D015879:Myosin Subfragments; C015296:5-iodoacetamidofluorescein; D009218:Myosins