Effect of mild heat treatment on actin and nucleotide binding of myosin subfragment 1.
Chymotryptic subfragment 1 (S-1) prepared from rabbit skeletal myosin has lost its ATPase activity upon incubation at 35 degrees C for 3 h. The loss in ATPase activity was accompanied by the perturbation of the structure of the 50K domain as indicated by a dramatic increase in the tryptic susceptibility of this domain without any change in the susceptibility of the other domains of S-1. The perturbation starts at the C-terminal region of the domain as suggested by the appearance of a 29K intermediate protein band in the tryptic peptide pattern of the heat-treated S-1. The heat-treated molecule essentially retained its actin and polyphosphate binding ability, and the actin binding was still sensitive to the presence of ATP or pyrophosphate. However, as opposed to native S-1, in heat-treated S-1 the addition of ATP does not induce an increase in tryptophan fluorescence, and, in the case of the treated species, the fluorescence of 1,N6-ethenoadenosine 5'-diphosphate added to the mixture is quenchable by acrylamide. This latter observation suggests that the binding of the adenine ring of the nucleotide has been altered following the heat treatment. The results indicate that the actin and polyphosphate binding sites of S-1 are distinct and that they are relatively independent of the adenine ring binding site.
Setton
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Department of Oral Biology, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.
Dan-Goor
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Muhlrad
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D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002918:Chymotrypsin; D004795:Enzyme Stability; D005456:Fluorescent Dyes; D006358:Hot Temperature; D007700:Kinetics; D009132:Muscles / Q000378:metabolism*; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D010446:Peptide Fragments; C015195:1,N(6)-ethenoadenosine diphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002918:Chymotrypsin; D000251:Adenosine Triphosphatases; D009218:Myosins