Abstract
It has been possible to specifically label rabbit skeletal muscle actin at Lys-237 with 2,4-pentanedione, producing an enamine. This reaction can be reversed with hydroxylamine. The modification can be carried out with actin in either the G- or F-forms and does not affect polymerization-depolymerization. The modification does affect, however, the interaction of tropomyosin (Tm) with the modified F-actin. In the absence of Ca2+ and Mg2+ (mu = 0.12), Tm failed to bind to the modified F-actin whereas it did bind to unmodified F-actin (1 Tm:7 actins). Tm binding could be restored under these conditions by the addition of either troponin (Tn), Mg2+, or Mg2+ and Ca2+. Under certain conditions, Tm alone has been shown to inhibit actin-activated heavy meromyosin (HMM)-Mg2+-ATPase. This inhibition did not occur with the modified F-actin even though Tm was bound (approximately 1 Tm:7 actins). Even when Tn was added to this system (in the absence of Ca2+), no inhibition of ATPase could be observed. Thus, this modification appears to prevent F-actin X Tm from assuming the "blocking" inhibitory position (conformation). In addition, Tn appears to enhance the activation of heavy meromyosin-Mg2+-ATPase by the modified F-actin X Tm complex whether Ca2+ is present or not. This state may be analogous to the potentiated state (Murray, J. M., Knox, M. K., Trueblood, C. E., and Weber, A. (1982) Biochemistry 27, 906-915) seen with myosin subfragment 1-saturated actin at low ATP levels. Thus, using modified and unmodified F-actin, it is possible to produce three Tm X actin states: off (F-actin X Tm), on (modified F-actin X Tm), and "potentiated" (modified F-actin X Tm X Tn).
Highlights
From the $Sectionof Contractile Proteins, Department of Pharmacology and Cell Biophysics, Universityof Cincinnati College of Medicine, Cincinnati, Ohio 45267 and the TDepartmentof Chemistry and College of Osteopathic Medicine, Ohio University, Athens, Ohio 45701
It hasbeen possible to label rabbit skel- Tn’ together with T m in the thin filament form a regulated etal muscle actin at Lys-237 with 2,4-pentanedione, actin thin filament ata molar ratio of actin:Tm:Tn of7:1:1, producing an enamine
The modifi- through Tn thatCa2+ releasedfrow the sarcoplasmic reticucation does affect, the interaction of tropo- lum into the sarcoplasmregulates contraction by controlling myosin (Tm) with themodified F-actin
Summary
A Functionally Critical Residu(eLys-237)in Actin filament, forming the weak state of the regulated actin which binds weakly to both S-l.ADP and S-l.ATP In this form, the release of Pi and the associated change of S-1 from the 90" state to the45" state is inhibited by Tm and thusoccurs very slowly, causing muscle to relax. The protein mixture was allowed to studies have directly implicated His-40 [17] and Tyr-53 [18] in actin polymerization and Arg-95 in Tm-binding [19], and a variety of other studies have examined the availability of various actin amino acid residues, notably cysteine [20,21,22,23] and lysine [23,24,25,26], to chemical modification in the presence incubate for 3 h at 22"C after which it was treated with 2,4-pentanedione at 200-fold molar excess of the reagent to all lysine amino groups in the complex for 10 h.
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