scholarly journals ADP binding to myosin cross-bridges and its effect on the cross-bridge detachment rate constants.

1987 ◽  
Vol 89 (6) ◽  
pp. 905-920 ◽  
Author(s):  
M Schoenberg ◽  
E Eisenberg
Keyword(s):  
Dissociation Constant ◽  
Active Site ◽  
Rate Constants ◽  
Competitive Inhibition ◽  
Psoas Muscle ◽  
Nucleotide Analogues ◽  
Cross Bridge ◽  
Detachment Rate ◽  
The Cross ◽  
Cross Bridges

We have studied the binding of adenosine diphosphate (ADP) to attached cross-bridges in chemically skinned rabbit psoas muscle fibers and the effect of that binding on the cross-bridge detachment rate constants. Cross-bridges with ADP bound to the active site behave very similarly to cross-bridges without any nucleotide at the active site. First, fiber stiffness is the same as in rigor, which presumably implies that, as in rigor, all the cross-bridges are attached. Second, the cross-bridge detachment rate constants in the presence of ADP, measured from the rate of decay of the force induced by a small stretch, are, over a time scale of minutes, similar to those seen in rigor. Because ADP binding to the active site does not cause an increase in the cross-bridge detachment rate constants, whereas binding of nucleotide analogues such as adenyl-5'-yl imidodiphosphate (AMP-PNP) and pyrophosphate (PPi) do, it was possible, by using ADP as a competitive inhibitor of PPi or AMP-PNP, to measure the competitive inhibition constant and thereby the dissociation constant for ADP binding to attached cross-bridges. We found that adding 175 microM ADP to 4 mM PPi or 4 mM AMP-PNP produces as much of a decrease in the apparent cross-bridge detachment rate constants as reducing the analogue concentration from 4 to 1 mM. This suggests that ADP is binding to attached cross-bridges with a dissociation constant of approximately 60 microM. This value is quite similar to that reported for ADP binding to actomyosin subfragment-1 (acto-S1) in solution, which provides further support for the idea that nucleotides and nucleotide analogues seem to bind about as strongly to attached cross-bridges in fibers as to acto-S1 in solution (Johnson, R.E., and P. H. Adams. 1984. FEBS Letters. 174:11-14; Schoenberg, M., and E. Eisenberg. 1985. Biophysical Journal. 48:863-871; Biosca, J.A., L.E. Greene, and E. Eisenberg. 1986. Journal of Biological Chemistry. 261:9793-9800).

BDM affects nucleotide binding and force generation steps of the cross-bridge cycle in rabbit psoas muscle fibers

1994 ◽  
Vol 266 (2) ◽  
pp. C437-C447 ◽  
Author(s):  
Y. Zhao ◽  
M. Kawai
Keyword(s):  
Equilibrium Constants ◽  
Muscle Fibers ◽  
Psoas Muscle ◽  
Isometric Tension ◽  
Activation Mechanism ◽  
Phosphate Binding ◽  
Rabbit Psoas ◽  
Cross Bridge ◽  
The Cross ◽  
Cross Bridges

The effect of 2,3-butanedione monoxime (BDM) on elementary steps of the cross-bridge cycle was studied with the sinusoidal analysis technique in skinned rabbit psoas muscle fibers. Our results showed that isometric tension and stiffness decreased progressively with an increase in the BDM concentration. The MgATP and MgADP binding constants increased 27 and 6 times, respectively, when BDM was increased from 0 to 18 mM, whereas the phosphate binding constant did not change significantly. The equilibrium constants of the ATP isomerization and detachment step were not sensitive to BDM, whereas the equilibrium constant of the attachment (power stroke) step decreased with BDM. Thus, in the presence of BDM, the number of attached cross bridges decreases; more cross bridges accumulate in the detached state, causing isometric tension and stiffness to decline. However, our detailed analysis shows that the decrease in the number of attached cross bridges is approximately 40%, which is not adequate to account for the 84% decrease in the isometric tension when 18 mM BDM was present. Therefore we suggest that a thin-filament activation mechanism is also affected by BDM.

Inhibition of cycling and noncycling cross bridges in skinned smooth muscle by vanadate

1986 ◽  
Vol 250 (2) ◽  
pp. C325-C332 ◽  
Author(s):  
R. A. Nayler ◽  
M. P. Sparrow
Keyword(s):  
Smooth Muscle ◽  
Active Site ◽  
Skinned Fibers ◽  
Cross Bridge ◽  
The Cross ◽  
High Concentrations ◽  
Cross Bridges ◽  
Myosin X ◽  
A Site ◽  
Small Inhibition

Vanadate (Vi, 3-300 microM) reversibly inhibited force development elicited by micromolar Ca2+ in membrane-skinned fibers of smooth muscle from taenia coli and trachea of guinea pig. When relaxed fibers were preincubated with Vi, the contraction to Ca2+ was characterized by a peak response followed by a lower steady-state phase. The peak phase depended on the rate of contraction and the [Vi]and was absent after Vi incubation during a previous contraction. These observations were consistent with Vi binding to a site that was exposed during the cross-bridge cycle but absent in the relaxed state. The actin X myosin X ADP intermediate formed at the active site during the cross-bridge cycle is suggested as the site of action of Vi. A weak antagonism between Pi and Vi was demonstrated during contractions activated by myosin thiophosphorylation. High concentrations of Pi (6-12 mM) were needed to produce a small inhibition (10%) of maximal Ca2+-activated tension. Skinned fibers relaxed slowly after Ca2+ removal, and the absence of an active state suggested that tension was maintained by noncycling cross bridges. Both Vi and Pi increased the rate of tension loss by 10-fold, but Vi was 5-10 times more potent than Pi. It is suggested that Vi and Pi both act on the active site but that Pi has a more efficacious action on slowly cycling than rapidly cycling cross bridges.

scholarly journals FmhA and FmhC of Staphylococcus aureus incorporate serine residues into peptidoglycan cross-bridges

2020 ◽  
Vol 295 (39) ◽  
pp. 13664-13676 ◽  
Author(s):  
Stephanie Willing ◽  
Emma Dyer ◽  
Olaf Schneewind ◽  
Dominique Missiakas
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Binding Proteins ◽  
Penicillin Binding Proteins ◽  
Daughter Cells ◽  
Cross Bridge ◽  
Resistant Strains ◽  
The Cross ◽  
Cross Bridges ◽  
Adjacent Wall

Staphylococcal peptidoglycan is characterized by pentaglycine cross-bridges that are cross-linked between adjacent wall peptides by penicillin-binding proteins to confer robustness and flexibility. In Staphylococcus aureus, pentaglycine cross-bridges are synthesized by three proteins: FemX adds the first glycine, and the homodimers FemA and FemB sequentially add two Gly-Gly dipeptides. Occasionally, serine residues are also incorporated into the cross-bridges by enzymes that have heretofore not been identified. Here, we show that the FemA/FemB homologues FmhA and FmhC pair with FemA and FemB to incorporate Gly-Ser dipeptides into cross-bridges and to confer resistance to lysostaphin, a secreted bacteriocin that cleaves the pentaglycine cross-bridge. FmhA incorporates serine residues at positions 3 and 5 of the cross-bridge. In contrast, FmhC incorporates a single serine at position 5. Serine incorporation also lowers resistance toward oxacillin, an antibiotic that targets penicillin-binding proteins, in both methicillin-sensitive and methicillin-resistant strains of S. aureus. FmhC is encoded by a gene immediately adjacent to lytN, which specifies a hydrolase that cleaves the bond between the fifth glycine of cross-bridges and the alanine of the adjacent stem peptide. In this manner, LytN facilitates the separation of daughter cells. Cell wall damage induced upon lytN overexpression can be alleviated by overexpression of fmhC. Together, these observations suggest that FmhA and FmhC generate peptidoglycan cross-bridges with unique serine patterns that provide protection from endogenous murein hydrolases governing cell division and from bacteriocins produced by microbial competitors.

Cross-bridge phosphorylation and regulation of latch state in smooth muscle

1988 ◽  
Vol 254 (1) ◽  
pp. C99-C106 ◽  
Author(s):  
C. M. Hai ◽  
R. A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Experimental Observation ◽  
Rate Constants ◽  
Regulatory Mechanism ◽  
Myosin Phosphorylation ◽  
Model Simulations ◽  
Cross Bridge ◽  
Cross Bridges ◽  
Latch State

We have developed a minimum kinetic model for cross-bridge interactions with the thin filament in smooth muscle. The model hypothesizes two types of cross-bridge interactions: 1) cycling phosphorylated cross bridges and 2) noncycling dephosphorylated cross bridges ("latch bridges"). The major assumptions are that 1) Ca2+-dependent myosin phosphorylation is the only postulated regulatory mechanism, 2) each myosin head acts independently, and 3) latch bridges are formed by dephosphorylation of an attached cross bridge. Rate constants were resolved by fitting data on the time courses of myosin phosphorylation and stress development. Comparison of the rate constants indicates that latch-bridge detachment is the rate-limiting step. Model simulations predicted a hyperbolic dependence of steady-state stress on myosin phosphorylation, which corresponded with the experimental observation of high values of stress with low levels of phosphorylation in intact tissues. Model simulations also predicted the experimental observation that an initial phosphorylation transient only accelerates stress development, with no effect on the final steady-state levels of stress. Because the only Ca2+-dependent regulatory mechanism in this model was activation of myosin light chain kinase, these results are consistent with the hypothesis that myosin phosphorylation is both necessary and sufficient for the development of the latch state.

scholarly journals Mechanical transients initiated by photolysis of caged ATP within fibers of insect fibrillar flight muscle.

1991 ◽  
Vol 98 (4) ◽  
pp. 657-679 ◽  
Author(s):  
M Yamakawa ◽  
Y E Goldman
Keyword(s):  
Rate Constant ◽  
Time Course ◽  
Flight Muscle ◽  
Order Rate Constant ◽  
Stretch Activation ◽  
Pulse Photolysis ◽  
Caged Atp ◽  
Cross Bridge ◽  
The Cross ◽  
Cross Bridges

Kinetics of the cross-bridge cycle in insect fibrillar flight muscle have been measured using laser pulse photolysis of caged ATP and caged inorganic phosphate (Pi) to produce rapid step increases in the concentration of ATP and Pi within single glycerol-extracted fibers. Rapid photochemical liberation of 100 microM-1 mM ATP from caged ATP within a fiber caused relaxation in the absence of Ca2+ and initiated an active contraction in the presence of approximately 30 microM Ca2+. The apparent second order rate constant for detachment of rigor cross-bridges by ATP was between 5 x 10(4) and 2 x 10(5) M-1s-1. This rate is not appreciably sensitive to the Ca2+ or Pi concentrations or to rigor tension level. The value is within an order of magnitude of the analogous reaction rate constant measured with isolated actin and insect myosin subfragment-1 (1986. J. Muscle Res. Cell Motil. 7:179-192). In both the absence and presence of Ca2+ insect fibers showed evidence of transient cross-bridge reattachment after ATP-induced detachment from rigor, as found in corresponding experiments on rabbit psoas fibers. However, in contrast to results with rabbit fibers, tension traces of insect fibers starting at different rigor tensions did not converge to a common time course until late in the transients. This result suggests that the proportion of myosin cross-bridges that can reattach into force-generating states depends on stress or strain in the filament lattice. A steady 10-mM concentration of Pi markedly decreased the transient reattachment phase after caged ATP photolysis. Pi also decreased the amplitude of stretch activation after step stretches applied in the presence of Ca2+ and ATP. Photolysis of caged Pi during stretch activation abruptly terminated the development of tension. These results are consistent with a linkage between Pi release and the steps leading to force production in the cross-bridge cycle.

scholarly journals Different structural states of a microtubule cross-linking molecule, captured by quick-freezing motile axostyles in protozoa.

1986 ◽  
Vol 103 (6) ◽  
pp. 2209-2227 ◽  
Author(s):  
J E Heuser
Keyword(s):  
Active Role ◽  
Longitudinal Shear ◽  
The Other ◽  
Cross Bridge ◽  
Other Hand ◽  
Quick Freezing ◽  
Cross Links ◽  
Dynein Arms ◽  
The Cross ◽  
Cross Bridges

Freeze-etch preparation of the laminated bundles of microtubules in motile axostyles demonstrates that the cross-bridges populating individual layers or laminae are structurally similar to the dynein arms of cilia and flagellae. Also, like dynein, they are extracted by high salt and undergo a change in tilt upon removal of endogenous ATP (while the axostyle as a whole straightens and becomes stiff). On the other hand, the bridges running between adjacent microtubule laminae in the axostyle turn out to be much more delicate and wispy in appearance, and display no similarity to dynein arms. Thus we propose that the internal or "intra-laminar" cross-bridges are the active force-generating ATPases in this system, and that they generate overall bends or changes in the helical pitch of the axostyle by altering the longitudinal and lateral register of microtubules in each lamina individually; e.g., by "warping" each lamina and creating longitudinal shear forces within it. The cross-links between adjacent laminae, on the other hand, would then simply be force-transmitting elements that serve to translate the shearing forces generated within individual laminae into overall helical shape changes. (This hypothesis differs from the views of earlier workers who considered a more active role for the later cross-links, postulating that they cause an active sliding between adjacent layers that somehow leads to axostyle movement.) Also described here are physical connections between adjacent intra-laminar cross-bridges, structurally analogous to the overlapping components of the outer dynein arms of cilia and flagella. As with dynein, these may represent a mechanism for propagating local changes from cross-bridge to cross-bridge down the axostyle, as occurs during the passage of bends down the length of the organelle.

scholarly journals Phosphate binding induced force-reversal occurs via slow backward cycling of cross-bridges

2020 ◽  
Author(s):  
R Stehle
Keyword(s):  
Rate Constant ◽  
Force Generation ◽  
Phosphate Binding ◽  
Reversible Process ◽  
Cross Bridge ◽  
Force Relation ◽  
The Cross ◽  
Cross Bridges ◽  
Bridge Models ◽  
Kinetics Of

ABSTRACTThe release of inorganic phosphate (Pi) from the cross-bridge is a pivotal step in the cross-bridge ATPase cycle leading to force generation. It is well known that Pi release and the force-generating step are reversible, thus increase of [Pi] decreases isometric force by product inhibition and increases the rate constant kTR of mechanically-induced force redevelopment due to the reversible redistribution of cross-bridges among non-force-generating and force-generating states. The experiments on cardiac myofibrils from guinea pig presented here show that increasing [Pi] increases kTR almost reciprocally to force, i.e., kTR ≈ 1/force. To elucidate which cross-bridge models can explain the reciprocal kTR-force relation, simulations were performed for models varying in sequence and kinetics of 1) the Pi release-rebinding equilibrium, 2) the force-generating step and its reversal, and 3) the transitions limiting forward and backward cycling of cross-bridges between non-force-generating and force-generating states. Models consisting of fast reversible force generation before/after rapid Pi release-rebinding fail to describe the kTR–force relation observed in experiments. Models consistent with the experimental kTR-force relation have in common that Pi binding and/or force-reversal are/is intrinsically slow, i.e., either Pi binding or force-reversal or both limit backward cycling of cross-bridges from force-generating to non-force-generating states.STATEMENT OF SIGNIFICANCEPrevious mechanical studies on muscle fibers, myofibrils and myosin interacting with actin revealed that force production associated to phosphate release from myosin’s active site presents a reversible process in the cross-bridge cycle. The correlation of this reversible process to the process(es) limiting kinetics of backward cycling from force-generating to non-force-generating states remained unclear.Experimental data of cardiac myofibrils and model simulations show that the combined effects of [Pi] on force and the rate constant of force redevelopment (kTR) are inconsistent with fast reversible force generation before/after rapid Pi release-rebinding. The minimum requirement in sequential models for successfully describing the experimentally observed nearly reciprocal change of force and kTR is that either the Pi binding or the force-reversal step limit backward cycling.

scholarly journals Milrinone inhibits contractility in skinned skeletal muscle fibers

1998 ◽  
Vol 274 (5) ◽  
pp. C1306-C1311 ◽  
Author(s):  
C. Y. Seow ◽  
L. Morishita ◽  
B. H. Bressler
Keyword(s):  
Skeletal Muscle ◽  
Direct Action ◽  
Force Production ◽  
Isometric Tension ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Reduction In Force ◽  
Cross Bridge ◽  
The Cross ◽  
Cross Bridges

Direct action of the cardiotonic bipyridine milrinone on the cross bridges of single fibers of skinned rabbit skeletal muscle was investigated. At 10°C and pH 7.0, milrinone reduced isometric tension in a logarithmically concentration-dependent manner, with a 55% reduction in force at 0.6 mM. Milrinone also reduced Ca2+ sensitivity of skinned fibers in terms of force production; the shift in the force-pCa curve indicated a change in the pCa value at 50% maximal force from 6.10 to 5.94. The unloaded velocity of shortening was reduced by 18% in the presence of 0.6 mM milrinone. Parts of the transient tension response to step change in length were altered by milrinone, so that the test and control transients could not be superimposed. The results indicate that milrinone interferes with the cross-bridge cycle and possibly detains cross bridges in low-force states. The results also suggest that the positive inotropic effect of milrinone on cardiac muscle is probably not due to the drug’s direct action on the muscle cross bridges. The specific and reversible action of the bipyridine on muscle cross bridges makes it a potentially useful tool for probing the chemomechanical cross-bridge cycle.

The Z band in skeletal muscle in rigor exhibits the activated lattice form

1989 ◽  
Vol 47 ◽  
pp. 252-253
Author(s):  
R. J. Edwards
Keyword(s):  
Skeletal Muscle ◽  
Cross Sections ◽  
Active Tension ◽  
Thin Filaments ◽  
Cross Bridge ◽  
The Cross ◽  
Cross Bridges ◽  
Glycerinated Muscle

The Z band of skeletal muscle is a tetragonal array of interdigitating thin filaments from adjacent sarcomeres held together by cross connecting filaments. Two visually unique forms of the Z band (small square, ss, and basketweave, bw) can be observed by TEM of cross sections. The ss form is found in relaxed muscle and the bw is found in maximally activated muscle. The average Z spacing in the bw form is 20% largerthan in the ss form. There is a correlation between active tension and the form of the Z band. This correlation suggests that cross bridge binding in the A band is directly related to the form of the Z band. In rigor, the cross bridges are completely bound; therefore, we predicted that the Z band would exhibit the bw form. To test this hypothesis we compared unstimulated muscle to glycerinated muscle in rigor.

scholarly journals Effect of cross-bridge kinetics on apparent Ca2+ sensitivity.

1982 ◽  
Vol 79 (6) ◽  
pp. 997-1016 ◽  
Author(s):  
P W Brandt ◽  
R N Cox ◽  
M Kawai ◽  
T Robinson
Keyword(s):  
Ionic Strength ◽  
Phosphate Concentration ◽  
Hill Coefficient ◽  
Cross Bridge ◽  
Left Shift ◽  
Hill Coefficients ◽  
Tension Curve ◽  
The Cross ◽  
Cross Bridges ◽  
The Hill

Three different ways of shifting the pCa/tension curve on the pCa axis have been studied and related to changes in the rate constants of the cross-bridge cycle. The curve midpoint shifts to higher pCa's when the substrate (Mg-ATP) is reduced from 5 to 0.25 mM, when the phosphate concentration is reduced from 7.5 mM to 0, and when the ionic strength is reduced from 0.200 to 0.120. The Hill coefficients of the pCa/tension curve in our standard saline (5 mM substrate, 5 mM free ATP, 7.5 mM phosphate, ionic strength 0.200, 15 degree C) are between 5.1 and 5.6 and fall to 3.0 with the left shift of the curve brought about by reducing both substrate and phosphate. Left shifts of the curve produced by reduction in the ionic strength do not result ina lower Hill coefficient. Reducing eigher substrate or phosphate is associated with a reduction in the optimal frequency for oscillatory work, but reduction in ionic strength is not so associated. Maximum tension increases with the left shift of the curve brought about by reducing phosphate concentration or ionic strength, but tension decreases with the left shift of the curve accompanying substrate concentration reduction in phosphate-free saline. We argue that one mechanism for the observed shift of the curve along the pCa axis is the relationship between the time a cross-bridge takes to complete a cycle and the time Ca2+ stays bound to troponin C (TnC). If the cycle rate is decreased, a smaller fraction to TnC sites must be occupied to keep a given fraction of cross-bridges active. To illustrate this concept, we present a simplified model of the cross-bridge cycle incorporating the kinetics of Ca binding to TnC.

Sign in / Sign up

Export Citation Format

Share Document