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M Schoenberg

Publications and source records attributed to M Schoenberg.

At least 55 records · Page 3Linked to original sources
PubMed1990

Substance P contracts bovine tracheal smooth muscle via activation of myosin light chain kinase.

At near-threshold substance P concentrations, the isometric tension response of bovine tracheal strips is almost completely abolished by atropine, indicating mediation of contraction via substance P-stimulated release of acetylcholine from prejunctional nerve terminals. At near-maximal concentrations, the atropine-inhibited component of the tension response is less than 25%, indicating mainly direct activation. Under conditions in which activation by substance P is direct, peak tension is reached in approximately 11 min. Immunoblot analysis of the time course of phosphorylation of the 20-kDa myosin light chain (LC20) reveals incorporation of approximately 0.5 mol phosphate/mol light chain at 10 min. Two-dimensional tryptic phosphopeptide analysis of phosphorylated light chain reveals a single major phosphopeptide. The peptide migrates identically with that produced by myosin light chain kinase phosphorylation of purified tracheal myosin in vitro. Contraction stimulated by acetylcholine is more rapid, with attainment of peak tension in 2.5 min and a peak LC20 phosphorylation of 0.65 mol/mol. These results indicate that 1) substance P mediates contraction of bovine trachea both directly and indirectly, and 2) under conditions in which activation is direct, the tension and phosphorylation responses qualitatively resemble those observed with acetylcholine.

Animals↗
PubMed1989

Effect of adenosine triphosphate analogues on skeletal muscle fibers in rigor.

It is commonly believed, for both vertebrate striated and insect flight muscle, that when the ATP analogue adenyl-5'-yl imidodiphosphate (AMPPNP) is added to the muscle fiber in rigor, it causes the fiber to lengthen by 0.15%. This has been interpretated (Marston S.B., C.D. Roger, and R.T. Tregear. 1976. J. Mol. Biol. 104:263-267) as suggesting (a) that in rigor the crossbridge is fixed to, i.e., almost never detaches from the actin filament; (b), that the crossbridge remains fixed to the actin filament after AMPPNP addition; and (c) that the ability of AMPPNP to cause apparent lengthening of a muscle fiber is due to its ability to cause a conformational change in the myosin crossbridge that has an axial component of approximately 1.6 nm/half-sarcomere. The present study, done only on chemically-skinned rabbit psoas fibers, confirms that AMPPNP can cause muscle fibers to lengthen by 0.15% but only for a narrow set of experimental conditions. When experimental conditions are varied over a wider range, it becomes apparent that the extent of lengthening of a rigor muscle fiber upon AMPPNP addition depends almost entirely on the strain present in the rigor fiber before AMPPNP addition. Addition of AMPPNP to an unstrained rigor fiber (one supporting zero tension), induces zero length change while addition of AMPPNP to very highly strained rigor fibers induces length changes greater than 0.15%. The data thus do not support the hypotheses that the crossbridges remain fixed to the actin filament after AMPPNP addition and that the size of the apparent length change induced by AMPPNP is related to the size of the axial component of a conformational change. Instead, the data support the idea that the ability of AMPPNP to cause lengthening of a rigor muscle fiber is related to its ability to accelerate the rate at which strained crossbridges detach from actin and reattach in positions in lesser strain. The data do not rule out a conformational change upon AMPPNP binding, they simply make clear that any attempt to measure a force response conceivably due to a conformational change, would be more than obscured by the force changes due to crossbridges detaching and reattaching in positions of lesser strain.

Adenosine Triphosphate↗
PubMed1988

Characterization of the myosin adenosine triphosphate (M.ATP) crossbridge in rabbit and frog skeletal muscle fibers.

In the presence of ATP and absence of Ca2+, muscle crossbridges have either MgATP or MgADP.Pi bound at the active site (S. B. Marston and R. T. Tregear, Nature [Lond.], 235:22:1972). The behavior of these myosin adenosine triphosphate (M.ATP) crossbridges, both in relaxed skinned rabbit psoas and frog semitendinosus fibers, was analyzed. At very low ionic strength, T = 5 degrees C, mu = 20 mM, these crossbridges spend a large fraction of the time attached to actin. In rabbit, the attachment rate constants at low salt are 10(4) - 10(5) s-1, and the detachment rate constants are approximately 10(4) s-1. When ionic strength is increased up to physiological values by addition of 140 mM potassium propionate, the major effect is a weakening of the crossbridge binding constant approximately 30-40-fold. This effect occurs because of a large decrease, approximately 100-fold, in the crossbridge attachment rate constants. The detachment rate constants decrease only 2-3-fold. The effect of ionic strength on crossbridge binding in the fiber is very similar to the effect of ionic strength on the binding of myosin subfragment-1 to unregulated actin in solution. Thus, the effect of increasing ionic strength in fibers appears to be a direct effect on crossbridge binding rather than an effect on troponin-tropomyosin. The finding that crossbridges with ATP bound at the active site can and do attach to actin over a wide range of ionic strengths strongly suggests that troponin-tropomyosin keeps a muscle relaxed by blocking a step subsequent to crossbridge attachment. Thus, rather than troponin-tropomyosin serving to keep a muscle relaxed by inhibiting attachment, it seems quite possible that the main way in which troponin-tropomyosin regulates muscle activity is by preventing the weakly-binding relaxed crossbridges from going on through the crossbridge cycle into more strongly-binding states.

Adenosine Triphosphate↗
PubMed1988

A model of force production that explains the lag between crossbridge attachment and force after electrical stimulation of striated muscle fibers.

Whereas the mechanical behavior of fully activated fibers can be explained by assuming that attached force-producing crossbridges exist in at least two configurations, one exerting more force than the other (Huxley A. F., and R. M. Simmons. 1971. Nature [Lond.]. 233:533-538), and the behavior of relaxed fibers can be explained by assuming a single population of weakly binding rapid-equilibrium crossbridges (Schoenberg, M. 1988. Biophys. J. 54:135-148), it has not been possible to explain the transition between rest and activation in these terms. The difficulty in explaining why, after electrical stimulation of resting intact frog skeletal muscle fibers at 1-5 degrees C, force development lags stiffness development by more than 15 ms has led a number of investigators to postulate additional crossbridge states. However, postulation of an additional crossbridge state will not explain the following three observations: (a) Although the lag between force and stiffness is very different after stimulation, during the redevelopment of force after an extended period of high velocity shortening, and during relaxation of a tetanus, nonetheless, the plots of force versus stiffness in each of these cases are approximately the same. (b) When the lag between stiffness and force during the rising phase of a twitch is changed nearly fourfold by changing temperature, again the plot of force versus stiffness remains essentially unchanged. (c) When a muscle fiber is subjected to a small quick length change, the rate constant for the isometric force recovery is faster when the length change is applied during the rising phase of a tenanus than when it is applied on the plateau. We have been able to explain all the above findings using a model for force production that is similar to the 1971 model of Huxley and Simmons, but which makes the additional assumption that the force-producing transition envisioned by them is a cooperative one, with the back rate constant of the force-producing transition decreasing as more crossbridges attach.

Animals↗
PubMed1988

The kinetics of weakly- and strongly-binding crossbridges: implications for contraction and relaxation.

The properties of the weakly-binding crossbridge formed when MgATP binds to the active site in the absence of Ca2+ were studied and contrasted with the behavior of the strongly-binding crossbridge formed when MgADP binds to the active site. It was found that salt causes a large decrease in the attachment rate-constants of the weakly-binding state and a small decrease in the detachment ones. This results in a decrease in the number of weakly-attached crossbridges with increasing salt. The effect is about 15-fold when K Propionate is used to increase ionic strength from mu = 20 mM to mu = 160 mM. Frog skeletal fibers show fewer weakly binding crossbridges than the rabbit. Taken as a whole, the data suggest a model in which the major role of troponin-tropomyosin, at both normal and low ionic strength, is to prevent the weakly-binding crossbridges of the relaxed fiber from going on through the crossbridge cycle to more strongly-binding states. Upon activation by Ca2+, the weakly-binding crossbridges go on through the cycle to a series of more strongly-binding intermediates, leading to an increase in the number of attached crossbridges and the appearance of force.

Actins↗
PubMed1987

Possible cooperativity in crossbridge detachment in muscle fibers having magnesium pyrophosphate at the active site.

When rabbit psoas muscle fibers bathed in solutions containing the ATP analogue magnesium pyrophosphate (MgPPi) are first stretched rapidly and then held isometric, a force is generated during the stretch which decays during the subsequent isometric period (Schoenberg, M., and E. Eisenberg. 1985. Biophys. J. 48:863-871). Previously we showed that the force decay is due to crossbridge heads detaching and reattaching in positions of lesser strain, the rate of decay of force reflecting the crossbridge detachment rate constants. Since the crossbridge detachment rate constants with MgPPi bound to the active site are so much faster than without analogue bound, at subsaturating concentrations of analogue, if the heads act independently and nucleotide association and dissociation is rapid, the rate of force decay should simply be proportional to the number of heads with bound analogue. That is, the analogue concentration dependence of the rate of force decay should have the same form as the Michaelis-Menten equation. Here we report that the concentration dependence of the rate of force decay is not described by the Michaelis equation, but is instead sigmoidal. This suggests possible cooperativity in the detachment of the crossbridge heads, the amount of cooperativity being described by an interaction coefficient of approximately 2. One idea put forward to explain the data is that both of the heads of a crossbridge may need to bind analogue before the crossbridge can relax a substantial fraction of the tension it supports.

Adenosine Triphosphate↗
PubMed1987

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

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).

Adenosine Diphosphate↗
PubMed1986

Stiffness of skinned rabbit psoas fibers in MgATP and MgPPi solution.

The stiffness of single skinned rabbit psoas fibers was measured during rapid length changes applied to one end of the fibers. Apparent fiber stiffness was taken as the initial slope when force was plotted vs. change in sarcomere length. In the presence of MgATP, apparent fiber stiffness increased with increasing speed of stretch. With the fastest possible stretches, the stiffness of relaxed fibers at an ionic strength of 20 mM reached more than 50% of the stiffness measured in rigor. However, it was not clear whether apparent fiber stiffness had reached a maximum, speed independent value. The same behavior was seen at several ionic strengths, with increasing ionic strength leading to a decrease in the apparent fiber stiffness measured at any speed of stretch. A speed dependence of apparent fiber stiffness was demonstrated even more clearly when stiffness was measured in the presence of 4 mM MgPPi. In this case, stiffness varied with speed of stretch over about four decades. This speed dependence of apparent fiber stiffness is likely due to cross-bridges detaching and reattaching during the stiffness measurement (Schoenberg, 1985. Biophys. J. 48:467). This means that obtaining an estimate of the maximum number of cross-bridges attached to actin in relaxed fibers at various ionic strengths is not straightforward. However, the data we have obtained are consistent with other estimates of cross-bridge affinity for actin in fibers (Brenner et al., 1986. Biophys. J. In press.) which suggest that ~60-90% of the cross-bridges attached in rigor are attached in relaxed fibers at an ionic strength of 20 mM and ~2-10% of this number of cross-bridges are attached in a relaxed fiber at an ionic strength of 170 mM.

Adenosine Triphosphate↗
PubMed1986

The effect of cross-bridge clustering and head-head competition on the mechanical response of skeletal muscle under equilibrium conditions.

The effect of cross-bridge clustering and head-head competition on the mechanical response of skeletal muscle under equilibrium conditions is considered. For this purpose, the recent multiple site equilibrium cross-bridge model of Schoenberg (Schoenberg, M., 1985, Biophys. J., 48:467-475) is extended in accordance with the formalism of T.L. Hill (1974, Prog. Biophys, Mol. Biol., 28:267-340) to consider the case where groups of independent cross-bridge heads compete with each other for binding to multiple actin sites. Cooperative behavior between heads is not allowed. Computations indicate that for the double-headed cross-bridge with two independent equivalent heads, the time course of force decay after a stretch is similar to that for the single-headed cross-bridge; that is, the rate constant for force decay is approximately equal to the cross-bridge head detachment rate constant. The results also show that the force decay after a stretch becomes slower than the detachment rate constant of a single head when cross-bridge heads bind adjacently in clusters so that competition between heads for binding to the available actin sites increases. However, if one assumes that the detachment rate constant of an unstrained head in a fiber is comparable to that of an S1 molecule in solution, this effect is not large enough to explain why some of the rate constants for force decay after a stretch in rigor, or in the presence of ATP analogues such as adenyl-5'-yl imidodiphosphate, appear to be significantly slower than the detachment rate constant of S1 from actin in solution.

Animals↗
PubMed1986

Ca2+-sensitive cross-bridge dissociation in the presence of magnesium pyrophosphate in skinned rabbit psoas fibers.

We find that at 6 degrees C in the presence of 4 mM MgPPi, at low or moderate ionic strength, skinned rabbit psoas fibers exhibit a stiffness and an equatorial x-ray diffraction pattern similar to that of rigor fibers. As the ionic strength is increased in the absence of Ca2+, both the stiffness and the equatorial x-ray diffraction pattern approach those of the relaxed state. This suggests that, as in solution, increasing ionic strength weakens the affinity of myosin cross-bridges for actin, which results in a decrease in the number of cross-bridges attached. The effect is Ca2+-sensitive. Assuming that stiffness is a measure of the number of cross-bridge heads attached, in the absence of Ca2+, the fraction of attached cross-bridge heads varies from approximately 75% to approximately 25% over an ionic strength range where ionic strength in solution weakens the binding constant for myosin subfragment-1 binding to unregulated actin by less than a factor of 3. Therefore, this phenomenon appears similar to the cooperative Ca2+-sensitive binding of S1 to regulated actin in solution (Greene, L. E., and E. Eisenberg, 1980, Proc. Natl. Acad. Sci. USA, 77:2616). By comparing the binding constants in solution and in the fiber under similar conditions, we find that the "effective actin concentration," that is, the concentration that gives the same fraction of S1 molecules bound to actin in solution as cross-bridge heads are bound to actin in a fiber, is in the millimolar range. An effective actin concentration in the millimolar range suggests that the strength of actin binding to cross-bridges in fibers may be several orders of magnitude weaker than the strength of ATP binding. Previously, it has been assumed that these two quantities were equal, as this gives the minimum energy loss when ATP dissociates the cross-bridge from actin (Morales, 1980, J. Supramol. Struct., 3:105:1975; Eisenberg, E.,Hill, T. L. and Y. Chen, 1980, Biophys. J., 29:195).

Actins↗
PubMed1985

Equilibrium muscle cross-bridge behavior. Theoretical considerations.

We have developed a model for the equilibrium attachment and detachment of myosin cross-bridges to actin that takes into account the possibility that a given cross-bridge can bind to one of a number of actin monomers, as seems likely, rather than to a site on only a single actin monomer, as is often assumed. The behavior of this multiple site model in response to constant velocity, as well as instantaneous stretches, was studied and the influence of system parameters on the force response explored. It was found that in the multiple site model the detachment rate constant has considerably greater influence on the mechanical response than the attachment rate constant. It is shown that one can obtain information about the detachment rate constants either by examining the relationship between the apparent stiffness and duration of stretch for constant velocity stretches or by examining the force-decay rate constants following an instantaneous stretch. The main effect of the attachment rate constant is to scale the mechanical response by influencing the number of attached cross-bridges. The significance of the modeling for the interpretation of experimental results is discussed.

Actins↗
PubMed1985

Muscle cross-bridge kinetics in rigor and in the presence of ATP analogues.

Recently we reported preliminary mechanical experiments on freshly skinned rabbit psoas fibers that suggested that while almost all of the cross-bridges are attached to actin in the presence of 4 mM adenyl-5'-yl-imidodiphosphate (AMP-PNP) (ionic strength, 0.13 M), there is an equilibrium between the attached and detached states, so that, in the presence of 4 mM AMP-PNP, fibers should not be able to maintain tension (Schoenberg, et al., 1984, in Contractile Mechanisms in Muscle, Pollack and Sugi, editors., Plenum Publishing Corp., NY). Since this suggestion was at variance with published results of Clarke and Tregear (1982, FEBS [Fed. Eur. Biochem. Soc.] Lett, 143:217), we reinvestigated the ability of rabbit psoas fibers to support tension following a 2-nm stretch in rigor and in the presence of the nucleotide analogues, PPi and AMP-PNP, for analogue concentrations ranging from 0.25 to 4 mM. We find that, whereas in rigor there is very little tension decay following a stretch, in 4 mM nucleotide analogue solution, the force generated by stretch quickly decays to zero. The force decay is not exponential; rather, it can be described by rate constants that range from approximately 0.1 to 100 s-1 in 4 mM PPi, and 0.01 to 10 s-1 in 4 mM AMP-PNP. This large range of decay rate constants may be partially related to the dependence of either analogue binding or cross-bridge dissociation upon strain, since we find that the rate constants for force decay decrease with decreasing size of stretch or with decrease of analogue concentration below the maximum studied (4 mM). In general the results are consistent with an equilibrium model for cross-bridge binding to actin, where the rate constants for cross-bridge detachment determine the rate of tension decay.

Actins↗
PubMed1984

Cross-bridge attachment in relaxed muscle.

We have measured the stiffness of relaxed, skinned rabbit psoas fibers at 5 degrees C in low ionic strength relaxing solution (mu = 0.02 M) by stretching the fibers and measuring the resulting force and sarcomere length changes. This stiffness is very dependent upon the velocity of stretch. With very slow stretches (0.5% of fiber length in greater than 30 ms), it is almost negligible but with stretches as fast as 0.5% of fiber length in 150 microseconds, the stiffness approaches 1/3 that of the rigor fiber. This stiffness is also very sensitive to ionic strength, being reduced more than 20-fold at an ionic strength of 0.17 M. This ionic strength sensitive stiffness scales with the amount of overlap between the actin and myosin filaments which strongly suggests that it is due to attached cross-bridges. The speed dependence suggests that the attached cross-bridges are not statically attached but in rapid equilibrium between attached and detached states. Experiments with adenylyl-imido-diphosphate suggest that the rates of attachment and detachment depend upon nucleotide.

Adenosine Triphosphate↗
PubMed1984

Stiffness, force, and sarcomere shortening during a twitch in frog semitendinosus muscle bundles.

The time course of force and stiffness during a twitch was determined at 6 and 26 degrees C in frog semitendinosus muscle bundles using the transmission time technique of Schoenberg, M., J.B. Wells, and R.J. Podolsky, 1974, J. Gen. Physiol. 64:623-642. Sarcomere shortening due to series compliance was also measured using a laser light diffraction technique. Following stimulation, stiffness developed more rapidly than force, but had a slower time course than published Ca2+ transients determined from light signals using Ca2+ sensitive dyes (Baylor, S.M., W.K. Chandler, and M.W. Marshall, 1982, J. Physiol. (Lond.). 331:139-177). Stiffness (S) did not reach its tetanic value during a twitch at 6 or 26 degrees C, although at 6 degrees C, it approached close to this value with S-twitch/S-tetanus = 0.82 +/- 0.07 (+/- SEM). During relaxation, force fell more rapidly than stiffness both for a twitch and also a tetanus. Also in this paper, several of the assumptions inherent in using the transmission time technique for the measurement of stiffness are considered in detail.

Animals↗
PubMed1983

[Changes of the acid-base status, serum lactate concentration and purine metabolism in reconstructive arterial surgery].

Hypoxic damages are normally determined by acid-base status as well as lactate and pyruvate levels. Since several other illnesses lead also to raised levels of these parameter, they are not specific for tissue hypoxia. In animal experiments it was shown that purine metabolites, especially hypoxanthine, are a very exact indicator for hypoxia. To test its validity in clinical situations the changes in acid-base status, lactate, pyruvate and purine metabolism were measured in 17 patients undergoing reconstructive aortic surgery. Blood samples were taken just before and after declamping of the aorta systemically and from the femoral vein. After declamping especially the systemic hypoxanthine levels increased 3 fold, due to a 20 times higher hypoxanthine concentration in the femoralis vein. The concentration changes of hypoxanthine in percent correlated very good with the aorta clamping time (R = 0,85). The changes in acid-base status and lactate levels were erratic and showed no correlation with the duration of ischemic period. Systemic hypoxanthine concentrations are a more specific and reliable parameter for hypoxic damages.

Acid-Base Equilibrium↗
PubMed1982

Evidence for cross-bridge attachment in relaxed muscle at low ionic strength.

In vitro at low ionic strength (mu = 0.02 M) and 5 degrees C, myosin subfragment-1 shows significant binding to regulated actin in the presence of ATP, independent of the concentration of free Ca2+. Under the same conditions, single skinned rabbit psoas muscle fibers develop force only in the presence of Ca2+ and are relaxed in its absence. However, the stiffness, measured with very rapid stretches (0.5% of muscle length in 0.1 ms), is high even when the fibers are relaxed. This "rapid stiffness" of the resting muscle is sensitive to ionic strength, becoming small at normal ionic strength (mu = 0.17 M). At low ionic strength, the rapid stiffness is approximately proportional to the overlap between the actin and myosin filaments. At zero overlap (sarcomere length = 3.8 microns), the stiffness is less than 20% of the value measured at full overlap. This remaining 20% is relatively insensitive to ionic strength, like the passive resting tension, and it may in fact be due to the structures responsible for the resting tension. Thus, both in vitro binding and the effect of overlap on rapid stiffness measurements in fibers suggest that cross-bridges are attached to actin in relaxed muscle at low ionic strength.

Actins↗
PubMed1982

Oxygen-transporting fluids and oxygen delivery with hemodilution.

The hemodynamic effectiveness and the oxygen characteristics of stroma-free hemoglobin (SFH) solutions were studied in 11 dogs. the animals were bled two-thirds of the estimated blood volume or until cardiac arrest. The shed blood was immediately replaced by equivalent amounts of either SFH or polyhemoglobin (SFH-PLP). Sixty min later, Ringer's lactate was given iv for 180 min to maintain right atrial pressure at the initial value. In a second set of experiments, the intravascular persistence of SFH-PLP was investigated in 5 dogs by withdrawal of blood (7 ml/kg bw) and immediate replacement with 131I-labeled SFH-PLP. Hemodynamic disorders after severe blood loss could be reversed by infusion of hemoglobin solutions. Due to the short intravascular persistence, cardiac output did not increase as one would expect from the degree of hemodilution. Pyridoxalation of the hemoglobin molecule reduced oxygen affinity and improved oxygen unloading at the tissue level. The short intravascular half-time of this compound could be overcome by crosslinking of the pyridoxalated hemoglobin molecules. Further studies must prove whether this polyhemoglobin can be used as a long-term oxygen-carrying blood substitute.

Animals↗