Absence Of Myosin Research Articles

Calcium binds cooperatively to the regulatory sites of the cardiac thin filament.

To investigate the relationship between thin filament Ca2+ binding and activation of the MgATPase rate of myosin subfragment 1, native cardiac thin filaments were isolated and characterized. Direct measurements of 45Ca binding to the thin filament were consistent with non-cooperative binding to two high affinity sites (Ka 7.3 +/- 0.8 x 10(6) M-1) and either cooperative or non-cooperative binding to one low affinity site (Ka 4 +/- 2 x 10(5) M-1) per troponin at 25 degrees C, 30 mM ionic strength, pH 7.06. Addition of a low concentration of myosin subfragment 1 to the native thin filaments produced a Ca2+-regulated MgATPase activity with Kapp (2.5 +/- 1.3 x 10(5) M-1), matching the low affinity Ca2+ site. The MgATPase rate was cooperatively activated by Ca2+ (Hill coefficient 1.8). To determine whether Ca2+ binding to the low affinity sites was cooperative, native thin filament troponin was exchanged with troponin labeled on troponin C with 2-(4'-iodoacetamidanilo)naphthalene-6-sulfonic acid. From the Ca2+-sensitive fluorescence of this complex, Ca2+ binding was cooperative with a Hill coefficient of 1.7-2.0. Using the troponin-exchanged thin filaments, myosin subfragment 1 MgATPase rate activation was also cooperative and closely proportional to Ca2+ thin filament binding. Reconstitution of the thin filament from its components raised the Ca2+ affinity by a factor of 2 (compared with native thin filaments) and incorporation of fluorescently modified troponin raised the Ca2+ affinity by another factor of 2. Stoichiometrically reconstituted thin filaments produced non-cooperative MgATPase rate activation, contrasting with cooperative activation with native thin filaments, troponin-exchanged thin filaments and thin filaments reconstituted with a stoichiometric excess of troponin. The Ca2+-induced fluorescence transition of stoichiometrically reconstituted thin filaments was non-cooperative. These results suggest that Ca2+ binds cooperatively to the regulatory sites of the cardiac thin filament, even in the absence of myosin, and even though cardiac troponin C has only one Ca2+-specific binding site. A theoretical model for these observations is described and related to the experimental data. Well-known interactions between neighboring troponin-tropomyosin complexes are the proposed source of cooperativity and also influence the overall Ka. The data indicate that Ca2+ is four times more likely to elongate a sequence of troponin-tropomyosin units already binding Ca2+ than to bind to a site interior to a sequence of units without Ca2+.

Effect of taxol on the interaction of tubulin with myosin filaments.

The interaction between polymerized tubulin from porcine brain and myosin from rabbit skeletal muscle was examined. The addition of myosin to the solution of tubulin polymerized by taxol resulted in a remarkable increase in turbidity within a few minutes at 37 degrees C, and a dense and stable precipitate was formed. The maximal molar ratio of tubulin bound to myosin was calculated to be about 4, while the value was about 2 when 6S tubulin was used. Both podophyllotoxin and colchicine suppressed the taxol-dependent increase of the binding of tubulin to myosin, but only when they were preincubated with tubulin prior to addition of taxol. 6S tubulin inhibited with actin-activated Mg2+-ATPase activity of myosin, and polymerized tubulin inhibited the Mg-ATPase more than 6S tubulin. Dense precipitates of tubulin and myosin were observed by thin-section electron microscopy. Microtubules were observed to be entangled in myosin filaments and single microtubules were occasionally surrounded by five myosin filaments in a cross section, similar to actin-myosin arrays in muscle. After incubation of tubulin with myosin, taxol was able to induce tubulin polymerization in the same way as it polymerized microtubules in the absence of myosin.

Cooperative binding to the Ca2+-specific sites of troponin C in regulated actin and actomyosin.

The Ca2+-binding component of troponin (TnC) and its proteolytic fragments containing Ca2+-binding sites I-III (TH1) or sites III and IV (TR2C) have been labeled with the fluorescent probes dansylaziridine (DANZ) at methionine 25 or 5-(iodoacetamidoethyl)amino-naphthalene-1-sulfonic acid (AEDANS) at cysteine-98. These probes report binding of Ca2+ to the low and high affinity sites, respectively. Fluorescence changes as a function of [Ca2+] were measured for the free peptides, their complexes with troponin I + troponin T, and these complexes bound to actin-tropomyosin in the presence of Mg2+ and ATP with and without myosin. An apparent Hill coefficient of 1.0-1.1 has been obtained for the Ca2+-induced fluorescence changes in TnC, its fragments, and their ternary complexes regardless of the label used. When a ternary complex containing appropriately labeled TnC or its fragment is bound to the actin-tropomyosin complex, the Hill coefficient for the titration of the low affinity sites increases to 1.5-1.6 and further increases to greater than 2 in the presence of myosin. To interpret the apparent Hill coefficients, we used a model containing two binding sites and a single reporter of the conformational change. Hill coefficients between 1.0 and 1.2 can be obtained for the fluorescence change without true cooperativity in metal binding, depending on the mechanism of the fluorescence change; i.e. the contribution of the singly or doubly occupied species to the fluorescence change. A Hill coefficient between 1.2 and 2, however, always indicates cooperativity in binding independently of the mechanism. Thus, our finding that fluorescence titrations of Ca2+ binding to TnCDANZ bound to actin-tropomyosin exhibit a Hill coefficient of 1.5 in the absence of myosin and 2.4 in its presence indicates the existence of true positive cooperativity in metal binding to sites I and II. No cooperativity was observed for AEDANS-labeled complexes that reflect Ca2+-binding to the high affinity sites. Plots of the Ca2+ dependence of myosin ATPase activity activated by actin-tropomyosin in the presence of any of the troponin complexes used had apparent Hill coefficients of approximately 4. The higher value suggests cooperative interactions in the activation of ATPase beyond those involved in Ca2+-binding to the Ca2+-specific sites.

Effect of bound-nucleotide substitution on the properties of F-actin

F-actins have been prepared in which the bound adenosine nucleotide has been replaced by inosine or cytidine nucleotide, and their properties have been studied. The intrinsic viscocity of IDP-containing F-actin was the same as that of ADP-containing F-actin, but the intrinsic viscosity of CDP-containing F-actin was about half this value. There were no differences in the myosin-binding affinities of the three types of F-actin or in their ability to activate the magnesium-ATPase of myosin. On the other hand, the exchangeability of the F-actin-bound nucleotide, both in the presence and in the absence of myosin, was dependent on the type of bound nucleotide in the actin, increasing in the order IDP<ADP<CDP. Furthermore, under conditions of partial polymerization, actin containing bound inosine nucleotide polymerized faster and to a greater extent than actin containing bound adenosine nucleotide. These results suggest that the bound nucleotide may influence the interaction between monomers in the F-actin polymer.

The removal of the bound ADP of F-actin

F-actin essentially free of bound nucleotide has been prepared by replacing bound ADP with AMP under sonic vibration and subsequently removing bound AMP by charcoal treatment. A comparison of nucleotide-deficient and normal F-actin revealed no differences between the two types in reversible depoly-merization, viscosity, combination with myosin at high ionic strength, stimulation of Mg2+ -ATPase activity of myosin at low ionic strength or superprecipitation with myosin. Nucleotide-deficient F-actin bound ATP stoichiometrically. ATP, on combining with this F-actin, was hydrolyzed into inorganic phosphate and F-actin-bound ADP. ATP was bound to nucleotide-deficient F-actin under various conditions, both in the presence and absence of myosin, and whether or not the actomyosin was superprecipitated, e.g. in the presence of Mg2+ , EDTA or Ca2+. Nucleotide-deficient F-actin did not appreciably bind deoxyATP or CTP either in the presence or absence of myosin, but stimulated the Mg2+ -deoxy-ATPase and Mg2+ -CTPase activity of myosin several-fold as normal F-actin does. Mg2+-deoxyATP and Mg2+ -CTP caused superprecipitation of myosin with nucleotide-deficient F-actin to the same extent that it did with normal F-actin. It is suggested that the bound ADP of F-actin is not involved in the physiologically important properties of F-actin, namely, the activation of the ATPase activity of myosin and superprecipitation with myosin in the presence of ATP.

Exchange of F-actin-bound nucleotide in the presence and absence of myosin

Exchange of F-actin-bound nucleotide in the presence and absence of myosin