Actomyosin Binding Research Articles

Rotational dynamics of actin-bound intermediates of the myosin adenosine triphosphatase cycle in myofibrils

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to measure the microsecond rotational motion of actin-bound myosin heads in spin-labeled myofibrils in the presence of the ATP analogs AMPPNP (5'-adenylylimido-diphosphate) and ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)). AMPPNP and ATP gamma S are believed to trap myosin in two major conformational intermediates of the actomyosin ATPase cycle, respectively known as the weakly bound and strongly bound states. Previous ST-EPR experiments with solutions of acto-S1 have demonstrated that actin-bound myosin heads are rotationally mobile on the microsecond time scale in the presence of ATP gamma S, but not in the presence of AMPPNP. However, it is not clear that results obtained with acto-S1 in solution can be extended to actomyosin constrained within the myofibrillar lattice. Therefore, ST-EPR spectra of spin-labeled myofibrils were analyzed explicitly in terms of the actin-bound component of myosin heads in the presence of AMPPNP and ATP gamma S. The fraction of actin-attached myosin heads was determined biochemically in the spin-labeled myofibrils, using the proteolytic rates actomyosin binding assay. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to be rotationally mobile on the microsecond time scale (tau r = 24 +/- 8 microseconds) in the presence of ATP gamma S, but not AMPPNP. Similar results were obtained at low ionic strength, confirming the acto-S1 solution studies. The microsecond rotational motions of actin-attached myosin heads in the presence of ATP gamma S are similar to those observed for spin-labeled myosin heads during the steady-state cycling of the actomyosin ATPase, both in solution and in an active isometric muscle fiber. These results indicate that weakly bound myosin heads, in the pre-force phase of the ATPase cycle, are rotationally mobile, while strongly bound heads, in the force-generating phase, are rotationally immobile. We propose that force generation involves a transition from a dynamically disordered crossbridge to a rigid and stereospecific one.

Rotational dynamics of actin-bound myosin heads in active myofibrils

We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to measure the submillisecond rotational motions of actin-bound myosin heads in active myofibrils. The cross-bridges were spin-labeled with a maleimide nitroxide derivative (MSL) that has previously been shown to undergo microsecond rotational motions on actin-bound myosin heads in solution during steady-state ATPase activity at low ionic strength [Berger, C. L., Svensson, E. C., & Thomas, D. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8573]. To determine whether this is also true for cross-bridges in the myofibrillar lattice under physiological buffer conditions, we have performed ST-EPR experiments during the brief steady state following photolysis of caged ATP in a suspension of spin-labeled myofibrils. The myofibrils were partially cross-linked with EDC [1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide] to prevent their shortening upon activation. The fraction of actin-attached myosin heads was determined biochemically at physiological ionic strength in the active myofibrils, using the proteolytic rates acto-myosin binding assay [Duong, A. M., & Reisler, E. (1989) Biochemistry 28, 3502]. These data were then used to correct the ST-EPR spectra of active myofibrils for the presence of unattached myosin heads, which were assumed to undergo the same motions as in relaxation. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to have considerable microsecond rotational motion (tau r = 3.5 +/- 1.1 microseconds) in the active myofibrils. Similar results (tau r = 3.2 +/- 0.8 microseconds) were obtained with active myofibrils at low ionic strength (mu = 45 mM), confirming the work done in solution. Thus, under physiological conditions and even within the constraints of the myofibrillar lattice, actively cycling actin-attached myosin heads are rotationally mobile on the microsecond time scale. Since partially EDC-fixed myofibrils are an excellent analog of isometrically contracting muscle fibers in solution, it is likely that these microsecond rotational motions are directly related to the molecular mechanism of muscle contraction in vivo.

Antibody against the amino terminus of α-actin inhibits actomyosin interactions in the presence of ATP

Actomyosin interactions in the presence of ATP were examined by using site-specific antibodies directed against the first seven N-terminal residues on skeletal α-actin. F ab fragments of these antibodies (SαN F ab) inhibited effectively the actin-activated ATPase of myosin subfragment 1 (S-1) at both 5 and 25 °C. Binding experiments carried out in the presence of ATP at 5 °C revealed that the catalytic inhibition was related to the inhibition of S-l binding to actin by F ab. At equimolar ratios of F ab to actin, the binding of S-1 to actin and the activated ATPase were inhibited by 75 and 82%, respectively. These results, when contrasted with the small effect of F ab on rigor actomyosin binding, suggest ATP-induced changes at the interface of actin and myosin.

Binding of myosin to actin in myofibrils during ATP hydrolysis

Measurements of cross-bridge attachment to actin in myofibrils during ATP hydrolysis require prior fixation of myofibrils to prevent their contraction. The optimal cross-linking of myofibrils was achieved by using 10 mM carbodiimide (EDC) under rigor conditions and at 4 degrees C. The fixed myofibrils had elevated MgATPase activity (150%) and could not contract. As judged by chymotryptic digestions and subsequent SDS gel electrophoresis analysis, less than 25% of myosin heads were cross-linked in these myofibrils. The isolated, un-cross-linked myosin heads showed pH-dependent Ca2+- and EDTA(K+)-ATPase activities similar to those of standard intact S-1. For measurements of myosin binding to actin, the modified myofibrils were digested with trypsin at a weight ratio of 1:50 under rigor, relaxed, and active-state conditions. Aliquots of tryptic digestion reactions were then cleaved with chymotrypsin to yield isolated myosin heads and their fragments. Analysis of the decay of myosin heavy-chain bands on SDS gels yielded the rates of myosin cleavage under all conditions and enabled the measurements of actomyosin binding in myofibrils in the presence of MgATP. Using this approach, we detected rigorlike binding of 25 +/- 6% of myosin heads to actin in myofibrils during ATP hydrolysis.

Antibodies directed against N-terminal residues on actin do not block acto-myosin binding.

Several studies using a variety of approaches have suggested a possible role for the amino-terminal residues of skeletal muscle actin in acto-myosin interaction. In order to assess the significance of acto-S-1 contacts involving the N-terminal segment of actin, we have prepared polyclonal antisera against a synthetic peptide corresponding to the seven amino-terminal residues of rabbit skeletal muscle actin (alpha-N-terminal peptide). Affinity-purified immunoglobulin (Ig) G (and Fab) prepared from these antisera reacts strongly and specifically with the amino-terminal segment of both G- and F-actin but not with myosin subfragment 1 (S-1). This specificity was determined by Western blot analysis of actin and its proteolytic fragments and the inhibition of the above reactivity by the alpha-N-terminal peptide. The alpha-N-terminal peptide did not interact with S-1 in solution, affect S-1 and actin-activated S-1 MgATPase, or cause dissociation of the acto-S-1 complex. In separate experiments F-actin could be cosedimented with S-1 and affinity-purified IgG or Fab by using an air-driven ultracentrifuge. Densitometric analysis of sodium dodecyl sulfate/polyacrylamide gels of pellet and supernatant fractions from such experiments demonstrated the binding of both S-1 and IgG or Fab to the same F-actin protomer. Our results suggest that, while the acidic N-terminal amino acids of actin may contact the myosin head, these residues cannot be the main determinants of acto-S-1 interaction.

The binding of myosin subfragment 1 to actin can be measured by proteolytic rates method.

The initial rates of tryptic digestion at the 50/20-kDa junction in myosin subfragment 1 (S-1) were determined for free S-1, acto-S-1, and acto-S-1 in the presence of magnesium adenyl-5'-yl imidodiphosphate (Mg AMP-PNP) and MgATP under ionic strength conditions ranging from 30 to 124 mM. The percentage of S-1 bound to actin in the presence of Mg AMP-PNP and MgATP was calculated from these rates for each set of digestion experiments. Parallel experiments carried out in an Airfuge centrifuge on identical acto-S-1 solutions yielded independent information on the binding of S-1 to actin. The results of binding measurements by these two methods were in excellent agreement in all cases tested, covering the range from 15 to 95% binding of S-1 to actin. Tryptic digestions of synthetic mixtures of S-1 and p-phenylenedimaleimide S-1 in the presence of actin demonstrated that a two-component system of myosin heads with different affinities for actin can be resolved into its constituents by the proteolytic rates method. The results of this work justify applications of the proteolytic rates method to actomyosin binding studies in more complex systems.

Effects of physical training and detraining on intrinsic cardiac control mechanisms.

The effects of a moderate physical training program on the hearts of rats have been studied. The mechanical responses of these hearts are improved. Possible contributing factors in this improvement are increased coronary reserve and capacity to deliver oxygen to the myocardium, increased myocardial glycogen stores and increased turnover of fatty acids through the endogenous triglyceride pool. Myocardial oxidative compounds and high energy phosphate stores are not altered. Major changes are found in the energy utilization pathways. Actomyosin, myosin, and heavy meromyosin ATPase activity and binding activity of isolated sarcoplasmic reticulum are all enhanced. Sulfhydryl control of the active site of myosin ATPase is altered. The biochemical effects of conditioning are short lived when training is decreased or discontinued.

Contractile proteins: Major components of nuclear and chromosome non-histone proteins

Two of the major non-histone proteins from Physarum polycephalum have been isolated under nondenaturing conditions and identified as actin and myosin. A third protein has been purified from crude nuclear actomyosin and from residual nonhistone fractions and found to bind actomyosin in the presence of Mg 2+. In Physarum these proteins are not components of the nuclear membrane. Based on sodium dodecylsulfate-polyacrylamide gel electrophoresis, similar proteins are also present in nuclei of HeLa cells and mouse embryo fibroblasts. Isolated metaphase chromosomes from Physarum show a several-fold enrichment in myosin and an altered ratio of actin to the Mg 2+-dependent actomyosin binding protein as compared to interphase nuclei. When non-proliferative states are induced in any of these cells, the Mg 2+-dependent actomyosin binding protein decreases while actin increases several fold in intranuclear concentration; concomitantly, there is a generalized condensation and inactivation of chromatin. Experiments with added purified radioactive nuclear actomyosin; comparative studies on nuclear protein during stepwise nuclear purification; and studies on isolated metaphase chromosomes indicate that these proteins exist in nuclei in vivo. These observations suggest that contractile proteins may function in the structural interconversions of chromatin and in the regulation of cell proliferation.

Superprecipitation of actomyosin extracted from crayfish skeletal muscle: the effect of heavy metal cations, EDTA and drugs.

AbstractActomyosin from crayfish skeletal muscle will superprecipitate in the presence of ATP without added Ca++ or Mg++. EDTA totally inhibits Ca++‐induced superprecipitation of actomyosin, but in some cases Mg++ will reverse this effect. It is suggested that Mg++ and not Ca++ is a prerequisite for superprecipitation with ATP alone. EDTA only partly inhibits heavy metal‐induced actomyosin superprecipitation.Heavy metal cations induce far greater actomyosin superprecipitation than Ca++, and they can even enhance previously formed Ca++‐induced precipitates. It is suggested that heavy metal cations may be more strongly bound to crayfish actomyosin than Ca++, and that they may displace Ca++ from existing actomyosin binding sites. Heavy metal potentiation of muscular contraction may thus involve direct action at myofibril binding sites.Caffeine and quinine are without effect either on the rate or extent of development of actomyosin precipitates in the presence of ATP, ATP plus Ca++, and ATP plus Zn++. These observations rule out any direct effect of these drugs at the myofibril level during drug‐induced potentiation of muscular contraction.