The effects of various nucleotides on the structure of actin-attached myosin subfragment-1 studied by quick-freeze deep-etch electron microscopy.
Stereo electron microscopy of negatively stained images showed that myosin heads in acto-subfragment-1 (S1) covalently cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide were predominantly short and round when ATP was added, in contrast to their uniform tilted appearance in the rigor state. As an attempt to exclude molecules which were actually dissociated but still tethered to actin by artificial cross-links, quick-freeze deep-etch electron microscopy was coupled with the mica flake method to observe uncross-linked native acto-S1 in the presence of ATP. To maintain the low affinity S1 associated to actin in the presence of ATP, a high concentration of acto-S1 was applied to mica flakes whose absorption had been chemically modified. The image of acto-S1 with added ATP agreed well with the expected time-course of reversible dissociation and reassociation, confirming the applicability of this approach to examination of the structural changes of acto-S1. S1 molecules attached to F-actin under rigor conditions or in the presence of ADP were elongated, with the long axis tilted to F-actin. Actin-attached S1 became short and round upon addition of ATP or ADP-inorganic vanadate. Adenyl-5'-yl imidodiphosphate and inorganic pyrophosphate each partially dissociated S1 from actin, as expected.
Katayama
E
E
Department of Pharmacology, Faculty of Medicine, University of Tokyo.
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000502:physiology*; D000538:Aluminum Silicates; D003432:Cross-Linking Reagents; D005613:Freeze Etching; D006868:Hydrolysis; D008854:Microscopy, Electron; D015879:Myosin Subfragments / Q000648:ultrastructure; D011485:Protein Binding; D013194:Staining and Labeling; D013499:Surface Properties; D014638:Vanadates
D000199:Actins; D000538:Aluminum Silicates; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D014638:Vanadates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C011934:mica