The reactive thiol of the myosin head, SH-1, can be selectively labelled in glycerinated rabbit muscle fibres. This residue has been used as an attachment site for either fluorescent or spectroscopic probes which report on head movements and orientations in various functional states of muscle. We have specifically modified SH-1 in vitro, using purified rabbit myosin and conditions similar to those employed in the labelling of muscle fibres (low ionic strength [40 mM NaCl] at 4 degrees C), with stoichiometric amounts of either [14C]-iodoacetamide, 5-(2[iodoacetyl)amino)ethyl) aminonaphthalene-1- sulphonic acid (IAEDANS), or 4-(2-iodoacetamido-2,2,6,6-tetramethyl piperidinooxyl (IASL). The specificity of modification was determined by measuring the well-defined alterations in the high salt ATPase activities of myosin and by localizing both IAAm and IAEDANS to the 20-kDa C-terminal subfragment 1 (S1) which contains SH-1. The low ionic strength actin-activated Mg2+-ATPase of SH-1-modified rabbit myosin was measured in the presence of the thin filament regulatory, complex, troponin-tropomyosin. A significant increase in this activity in the absence of calcium, concomitant with a decrease in activity in the presence of calcium, was observed as the extent of SH-1 modification was incrementally increased from zero to one mole of label bound per mole of SH-1. The elevated myosin Mg2+-ATPase, which results from SH-1 modification, does not account for the increased actin-activated Mg2+-ATPase in resting conditions (i.e. in the absence of calcium).(ABSTRACT TRUNCATED AT 250 WORDS)

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The exchange of essential light chains (SH-LCs) of scallop myosin was followed with the aid of scallop SH-LC alkylated with 14C-labeled iodoacetate. More than 70% of the SH-LCs were exchanged in myosin preparations that were desensitized by removal of both regulatory light chains (R-LCs) with ethylenediaminetetraacetic acid (EDTA) treatment. Although desensitized myosin solubilized with 0.6 M NaCl or with 10 mM adenosine 5'-triphosphate (ATP) in the absence of salt equilibrated rapidly with SH-LCs even in the cold, exchange in myosin filaments required elevated temperatures. Equilibration of the SH-LCs in desensitized preparations did not depend on ATP or magnesium ions but was significantly accelerated by actin. The desensitized myosin preparations containing alkylated SH-LCs (approximately 1 mol of thiol alkylated/mol of SH-LC) readily recombined with R-LCs. The preparations regained fully the calcium dependence of the actin-activated magnesium adenosinetriphosphatase (Mg-ATPase), contained R-LCs and SH-LCs in equimolar amounts, and had an ATPase activity similar to that of untreated myosin preparations. R-LCs interfered with the equilibration of the SH-LCs. In intact myosin preparations, the exchange of SH-LCs was slow and was frequently associated with the dissociation of the R-LCs. The blocking action of the R-LC on SH-LC exchange agrees with evidence showing that the two light chain types interact and suggests that parts of the SH-LC may lie between the R-LC and the heavy chain of myosin.

1. Clam-foot paramyosin strongly inhibited superprecipitation of clam-foot actomyosin whereas it did not at all affect its ATPase reaction. On the other hand, superprecipitation of rabbit actomyosin and its ATPase reaction were both inhibited by clam foot paramyosin. 2. Clam-foot paramyosin did not at all affect the Mg-ATPase activity of clam-foot myosin alone (with no actin) but inhibited that of rabbit skeletal myosin alone. 3. Clam-foot paramyosin did not affect the ATPase reaction of rabbit acto-clam myosin but inhibited its superprecipitation. In other words, the effect of paramyosin on the ATPase reaction of actomyosin was simply dependent on the myosin source. 4. Clam-foot tropomyosin slightly enhanced superprecipitation of clam-foot actomyosin and scarcely affected its ATPase reaction, but it inhibited the reactions both of skeletal actomyosin and of rabbit acto-clam myosin. 5. Rabbit skeletal light-meromyosin (LMM) inhibited the ATPase and superprecipitation reactions of rabbit skeletal actomyosin, but it scarcely affected the ATPase reaction of rabbit acto-clam myosin while inhibiting its superprecipitation. In other words, rabbit LMM behaved the same way as clam paramyosin did. 6. Speculating on these findings and our previous findings, we suggest that the formation of a certain filamentous structure is the essential link between the ATPase reaction and contraction in molluscan muscles.

1. The ATPase activity of clam foot myosin alone in the presence of 10 mM MgCl2 was activated approximately ten-fold by 10 muM free calcium ions. The calcium activation was observed in various concentrations of KCl (35-600 mM) and ATO (1 muM-1 mM), and at various pHs (pH 6-9.4). 2. The superprecipitation and ATPase activities of clam foot myosin B were studied by conducting experiments in two different ways. In one of these, the ATP concentration was varied at a fixed concentration of MgCl2, and in the other, the MgCl2 concentration was varied at a fixed concentration of ATP. The following was found: (a) The activities responded in a biphasic manner to change in either the ATP or MgCl2 concentration, giving a peak activity around 10 muM ATP or MgCl2. It is thus suggested that Mg-ATP complex is responsible for both activation and inhibition in the biphasic response. (b) When the ATP or MgCl2 concentration was higher than 100-300 muM, practically no superprecipitation occurred in either the presence or absence of calcium, whereas the ATPase activity was still strongly activated by calcium. 3. Similar results to those described above (a, b) were obtained by using rabbit skeletal actoclam foot myosin in place of clam foot myosin B. Moreover, it was found that as the ATP concentration increased from 1 muM to 1 mM, Mg-ATPase activity of clam foot myosin in the presence of calcium increased in a monophasic manner and that it was as active as actomyosin in the presence of calcium when the ATP concentration was higher than approximately 200 muM. In other words, actin-activation of myosin-ATPase was absent in the ATP concentration where no superprecipitation of actomyosin was observed. 4. Clam foot myosin contained two types of light chain subunits: LCl (17,000 daltons) and LC2 (16,000 daltons). Only LC1 was removed upon washing clam myosin with 10 mM EDTA, and removal of LC1 resulted in loss of the calcium sensitivity of actomyosin-ATPase. 5. In our previous report (J. Biochem. 85, 1543-1546, 1979), it was shown that removal of LC1 from clam foot myosin also resulted in loss of the superprecipitation activity of actomyosin reconstituted from "EDTA-washed" myosin. We now provide further evidence that removal of the regulatory light chain (LC1) results in a reversible uncoupling of ATPase reaction from superprecipitation reaction.