On the mechanism of detergent modification of myosin structure and function.
The concentration dependences of the activation of myosin subfragment-1 (S1) Mg-ATPase by the detergents CHAPS and C12E8 were determined at 23 degrees C in 25 mM Tris (pH 7.0), 250 microM EDTA, 5 mM MgCl2, and 100 microM ATP. At detergent concentrations expected to bind hydrophobic S1 surface areas equally, C12E8 caused an 8.5-fold greater increase in activity than CHAPS, which suggests that detergent binding to the surface of S1 is not the mechanism of activation. At detergent concentrations above their critical micelle concentrations, C12E8 was also much more effective than CHAPS, suggesting that micelles are not involved. A series of n-alcohols (which do not form micelles) with from 3 to 10 carbons all increased S1 Mg-ATPase activity as much or more than C12E8. The largest increase (5.7-fold) was caused by n-hexanol. The more hydrophobic alcohols activated S1 at lower concentrations. A linear plot of the alcohol concentration that caused 50% of maximum activity versus the number of carbons in the alcohol, indicated the apparent free energy of binding per CH2-group was -0.60 +/- 0.03 kcal/mol. There were two indications that alcohol binding caused an S1 conformational change. The intrinsic fluorescence increase of S1 during steady-state activity was reduced from 17.5 to 12.8%, and the apparent hydrodynamic rotational mobility of fluorescently labeled S1 was decreased 25% by the present of n-hexanol. The data suggest that S1 activation by C12E8 and by n-alcohols is due to hydrophobic binding to S1 at non-surface sites, which causes an S1 structural change.
Highsmith
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Department of Biochemistry, University of the Pacific School of Dentistry, San Francisco, CA 94115.
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002793:Cholic Acids / Q000493:pharmacokinetics; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D000441:Hexanols / Q000493:pharmacokinetics; D015879:Myosin Subfragments / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000493:pharmacokinetics; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D013329:Structure-Activity Relationship
D002793:Cholic Acids; D000441:Hexanols; D015879:Myosin Subfragments; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D017830:Octoxynol; D017301:Ca(2+) Mg(2+)-ATPase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate