Binding of ADP and adenosine 5′‐[β,γ‐imido]triphosphate to insect flight muscle fibrils

Abstract

We have studied the binding of ADP and adenosine 5'-[/?,y-imido]triphosphate (AdoPP[NH]P) to insect flight muscle fibrils. We find that 25% of the myosin heads, presumably those which do not interact with actin, bind AdoPP[NH]P with a binding constant greater than 3 x lo6 M-', similar to the binding constant of the same compound to the rabbit myosin heads which do not overlap with actin. The remaining heads in insect myofibrils bind AdoPP[NH]P with an association constant of 8 x lo3 M-', which is eight times stronger than the affinity of this compound for rabbit myosin heads in overlap with actin. Therefore, in contrast to the situation with rabbit myofibrils where AdoPP[NH]P binds much more weakly than ADP, with insect myofibrils these two adenosine phosphates bind with almost equal affinity. This is consistent with the numerous structural studies on insect flight muscle which were interpreted on the basis that most of the actomyosin sites were saturated with nucleotide at an AdoPP[NH]P concentration of 1 mM.The highly regular structure of insect fibrillar flight muscle has made it useful for X-ray diffraction and electron microscopy studies of myosin crossbridge conformation in the presence and absence of nucleotides [l, 21. The most extensively studied condition occurs in the absence of ATP [3 -51 ; this rigor state is widely used as a model for the end of the crossbridge power stroke (the 45" state). The beginning of the power stroke has been modeled after the 90" detached crossbridges seen in Lethocerus muscle relaxed by ATP [3, 61. Attempts to visualize intermediate crossbridge conformations have centered around the use of nucleotide analogs to trap myosin heads at some position between the 90" and 45" states. Adenosine 5'-[P,y-imido]triphosphate (AdoPP[NH]P), an ATP analog not hydrolyzed by myosin [7], is the most widely used of these analogs [8,9] because it causes a large drop in rigor tension without reducing stiffness. This suggests that a large number of crossbridges remain attached but are caught at a different part of the crossbridge stroke. In Lethocerus flight muscle the structure resulting from exposure to AdoPP[NH]P at 4 "C defies simple interpretation because, in the cold, AdoPP[NH]P induces a mixed structural state in which attached crossbridges are altered by the analogue and show both rigor and relaxed features. In addition, although muscles from other species show a drop in tension with constant stiffness in the presence of AdoPP[NH]P, structural responses are varied [lo] or even undetectable 1111.Moreover, the binding of AdoPP[NH]P shows a strong dependence on whether or not the myosin head is bound to

Cross‐bridge kinetics in the presence of MgADP investigated by photolysis of caged ATP in rabbit psoas muscle fibres.

“…These values for KD contrast with estimates at saturating levels of Ca2+: Biosca, Greene & Eisenberg, 1988). Dissociation of the nucleotide analogue 1-N6-ethenoadenosine 5'-diphosphate from regulated acto S-1 (with troponin and tropomyosin present) is 10-to 15-fold more rapid in the presence than in the absence of Ca2+ (Rosenfeld & Taylor, 1987a, b) To explain the tension decrease on adding MgADP to a fibre in rigor we should consider either cross-bridge detachment (possibly one or both heads) or else a structural change in the heads themselves.…”

Three-dimensional Structure of Nucleotide-bearing Crossbridgesin Situ: Oblique Section Reconstruction of Insect Flight Muscle in AMPPNP at 23°C

“…However, almost no further loss of tension or stiffness is observed between 23°C and 4°C. Second, insect and vertebrate myosins differ in their affinities for AMPPNP, which binds about eight times more strongly to IFM myofibrils than it does to rabbit myofibrils (Biosca et al, 1990), confirming original determinations that IFM is saturated at the 0.5 to 1.0 mM AMPPNP concentrations normally used. Third, in IFM, 20 to 25% of myosin heads are not attached to actin even in rigor, in contrast to vertebrate muscle, in which virtually all heads are thought to attach to actin in rigor (Lovell et al, 1981;Goody et al, 1985;Thomas et al, 1983).…”

“…Third, in IFM, 20 to 25% of myosin heads are not attached to actin even in rigor, in contrast to vertebrate muscle, in which virtually all heads are thought to attach to actin in rigor (Lovell et al, 1981;Goody et al, 1985;Thomas et al, 1983). Since AMPPNP has a higher binding affinity for myosin heads not bound to actin (Biosca et al, 1988(Biosca et al, , 1990, it was thought possible that the unbound heads in IFM could form a second population of heads that would rise out of their obscurity in rigor and contribute relaxed features (e.g. 14.5 nm period, 90°angle), independently of the attached crossbridge structural and mechanical responses that were responsible for the loss of tension in AMPPNP.…”

Reverse actin sliding triggers strong myosin binding that moves tropomyosin

“…The complete suppression of Ca-activated force by Vi in IFM, in contrast to its incomplete suppression in vertebrate muscle, may reflect higheraffinity binding of Vi to IFM than to vertebrate skeletal myosin, similar to the higher affinity of AMP-PNP to insect myosin (38). That the stretch and catch response persists up to 21 min after Vi washout (Fig.…”