Rigor Complex Research Articles

The binding of calcium to glycerinated muscle fibers in rigor. The effect of filament overlap

The binding of Ca 2+ to glycerinated rabbit psoas fibers of varying sarcomere length was measured with a double isotope technique and ethyleneglycol-bis-(β-aminoethylether)- N, N'-tetraacetic acid buffers. Experiments were carried out under rigor conditions with fiber bundles pre-set at different lengths prior to extraction with detergent and glycerol. These experiments were designed to test whether rigor complex formation, determined by the degree of filament overlap, enhances Ca 2+-receptor affinity in the intact filament lattice, as it does in reconstituted actomyosin systems. The Ca 2+-receptor affinity, as indicated by the free Ca 2+ concentration at half-saturation and by the slopes of Scatchard plots, was found to be relatively unaffected by variations in filament overlap. However, the maximum bound Ca 2+ was significantly reduced in stretched fibers. With maximum filament overlap the bound Ca 2+ was equivalent to 4 mol per mol troponin. When streched to zero overlap the fibers bound a maximum of 3 mol Ca 2+ per mol troponin. When fibers with maximum overlap were incubated in the presence of 5 mM MgATP there was a reduction in the number of Ca 2+-binding sites equivalent to that caused by stretching the fibers. These findings, taken together with other data in the literature, suggest that in the intact filament lattice at least one of the Ca 2+-binding sites is present only when cross-bridge attachments are formed.

Regulation of actin-myosin interaction.

In the presence of the regulatory protein complex tropomyosin-troponin ATPase activity of vertebrate skeletal muscle actomyosin is either higher or lower than in the absence of tropomyosin-troponin. The actual behavior depends on ionic strength, Ca2+ concentration, ATP concentration (determining the amount of rigor complexes present), and the ratio between actin and myosin. This effect of the myosin-actin ratio implies that under certain conditions cooperativity in actin-myosin interaction can be seen.

Mechanism of inhibition of relaxation by N-ethylmaleimide treatment of myosin.

It has remained unexplained why N-ethylmalaeimide (NEM) treatment of myosin can inhibit relaxation in actomyosin systems from rabbit skeletal muscle which appear to be regulated solely through tropomyosin and troponin. Since rigor complexes between (nucleotide-free) myosin and actin affect the tropinin-tropomyosin system, the possibility was explored that, as a result of NEM treatment, some of the myosin maintains rigor complexes with actin in the presence of ATP which might be responsible for inhibition of relaxation. Evidence is presented indicating that such a mechanism might account for the effects of NEM treatment. First, after exhaustive NEM treatment of heavy meromysin (HMM), acto-HMM complexes were no longer dissociated by ATP. Second, admixture of such NEM-treated, enzymatically inactive HMM or myosin to native regulated actomyosin or acto-HMM inhibited relaxation.

ATPase activity and light scattering of acto-heavy meromyosin: dependence on ATP concentration and on ionic strength.

1. The dependence on ATP concentration of ATPase activity and light scattering decrease of acto-HMM could be described at very low ionic strength by one hyperbolic adsorption isotherm with a dissociation constant of 3 X 10(-6)M. Hence the increase of ATP ase activity was paralleled by a decrease in light scattering. At higher values of ionic strength ATPase activity stopped rising before HMM was completely saturated with ATP. Higher ionic strength prevented ATPase activity from further increasing when the rigor links (links between actin and nucleotide-free myosin), which have formerly protected the ATPase against the suppressing action of higher ionic strength have fallen below a certain amount. This protecting influence of rigor links did not require tropomyosin-troponin. 2. For complete activation of ATPase activity by actin less actin was needed when HMM was incompletely saturated with ATP than when it was completely saturated with ATP. 3. The apparent affinity of ATP to regulated acto-HMM (which contained tropomyosin-troponin) was lower than to unregulated acto-HMM (which was devoid of tropomyosin-troponin). In the presence of rigor complexes (indicated by an incomplete decrease of light scattering) the ATPase activity of regulated acto-HMM was higher than that of unregulated acto-HMM. At increasing ATP concentrations the ATPase activity of regulated acto-HMM stopped rising at a similar degree of saturation with ATP as the ATPase activity of unregulated acto-HMM at the same ionic strength.

Calcium-activated tension of skinned muscle fibers of the frog. Dependence on magnesium adenosine triphosphate concentration.

The influence of MgATP on the Ga(++)-activated isometric tension of skinned frog muscle fibers was examined in solutions containing: Mg(++) = 5 mM, creatine phosphate (CP) = 14.5 mM, creatinephosphokinase (CPK) = 1 mg/ml, total EGTA = 7 mM, CaCl(2), KCl, imidazole >/= 20 mM so that ionic strength = 0.15, pH = 7.00, and MgATP = 2 mM, 0.1 mM, or 20 microM. CP and CPK were necessary for these experiments as determined experimentally by their effect on the tension-Ca(++) relation, which was saturated for CP >/= 14.5 mM. This was interpreted to mean that sufficient CP was present to effectively buffer MgATP intracellularly. Decreasing MgATP shifts the tension-pCa curve to higher pCa (-log Ca(++)) so that, for half-maximal tension: pCa(1/2) = 4.5 for MgATP = 2 mM, pCa(1/2) = 5.1 for MgATP = 0.1 mM, and pCa(1/2) = 5.8 for MgATP = 20 microM; maximum isometric tension is the same in all cases, however. If MgATP was decreased to 1 microM, tension at Ga(++) > 10(-8) M was 84% of the maximum Ca(-+)-activated tension in 2 mM MgATP and increased only slightly to 90% for pCa = 4.5. Weber (1970, In The Physiology and Biochemistry of Muscle as Food, Volume 2, E. J. Briskey, R. G. Cassens, and B. B. Marsh, University of Wisconsin Press, Madison, Wis.), using similar solutions, observed similar shifts in half-maximal calcium activation of rabbit myofibril ATPase rates. In explanation, Weber and Bremel (1971, In Contractility of Muscle Cells and Related Processes, R. J. Podolsky, editor, Prentice-Hall, Inc., Englewood Cliffs, N.J.; Bremel and Weber, 1972, Nat. New Biol., 238:97) have described a mechanism whereby, at low ATP, "rigor complexes" are formed between myosin and thin filament actin and, in turn, alter the calcium affinity of one class of the two Ca(++)-binding sites on troponin, so that the thin filament is "turned on" for contraction at lower Ca(++) levels. Tension data from skinned fibers substantially supports this hypothesis. A stability constant for CaEGTA of 2.62 x 10(10) M(-1) was determined, with the help of F. N. Briggs, in solutions similar to those used for skinned fibers and was the same for 100 and 300 mM KCl.

Kinetics of steady state ATPase activity and rigor complex formation of acto-heavy meromyosin

1. Actin and heavy meromyosin, initially mixed in a Mg-ATP solution, began to form the rigor complex slowly after ATP in the solution had been completely hydrolyzed. 2. This was because the heavy meromyosin-product complex formed via ATP hydrolysis was almost completely dissociated from actin even in the absence of ATP and as soon as this heavy meromyosin-product complex was decomposed, the heavy meromyosin combined with actin forming the rigor complex. 3. Linear plots were obtained when the reciprocal of the excess rate of the actin-accelerated rigor complex formation was plotted against the reciprocal of the added actin concentration as similar with those made on the steady acto-heavy meromyosin ATPase. 4. The V of the rigor complex formation process was about 1/5 of that of the steady acto-heavy meromyosin ATPase activity, showing that the actomyosin ATPase activity could not be explained merely by the actin-accelerated decomposition of the heavy meromyosin-product complex. 5. The same analyses were carried out on myosin subfragment 1. 6. Our results could be explained by considering the two non-identical active sites of myosin, and we propose the following scheme for the actomyosin ATPase. 7. Actin accelerates the rate-limiting bond hydrolysis in the ATPase occurring at one active site of myosin, as well as the rate-limiting decomposition of the heavy meromyosin-product complex formed at another site.

Cooperation within actin filament in vertebrate skeletal muscle.

When actin molecules form “rigor complexes” with nucleotide-free myosin, tropomn binds calcium with greater affinity and in a cooperative response the remaining actin molecules not cornplexed with myosin are “turned on” even though calcium is absent.