Polarization of tryptophan fluorescence in muscle

The presteady state and steady state release of protons during hydrolysis of ATP by myosin has been studied by using a proteolytic subfragment of myosin, heavy meromyosin, isolated from rabbit skeletal muscle. Under the conditions of 0.5 M KCI, 9 mM MgCI2, pH 8, and 250, the rate of the H+ initial burst is proportional to the concentration of ATP added, and shows no plateau even when the concentration of ATP added is increased up to 750 ,AM. The apparent second order rate constant is approximately 7 X 105 M-1 sec1. When the concentration of ATP added is equal to or less than the concentration of heavy meromyosin catalytic sites, the H+ initial burst is followed by a slow exponential release of H+ with a rate constant of 0.017 sec'. This rate constant is equal to the steady state rate constant calculated for each heavy meromyosin catalytic site, suggesting that both sites split ATP independently. The magnitude of the H+ released per ATP bound is always 0.4 in the initial burst, and 0.6 in the slow exponential phase, which equals to a total of 1 H+ released per ATP hydrolyzed. On the basis of these observations, it is concluded that the H+ initial burst may well represent a fast ionization of H+ due to a conformational change as a result of nucleotide binding.It is now generally accepted that when ATP is added to rabbit skeletal myosin, heavy meromyosin (HMM), or subfragment-1 S-1, in the presence of excess Mg++, an initial rapid release of phosphate is observed prior to the onset of the steady state rate of phosphate release (1-3). Several workers have suggested that this initial phosphate burst might be correlated with the formation of an intermediate involved in the mechanism of muscle contraction in vivo (4-7). The rate of the initial phosphate burst has generally been reported to plateau at a rate of approximately 100 sec , but there is disagreement on the magnitude of the burst, with data varying from I to 2 mole of Pi released per mole of myosin or HMM (3,8,9 Eisenberg and Moos (17). The HMM was dialyzed against 0.5 M KCl before use. The stopflow experiments were performed using a two-syringe Aminco-Morrow stopflow mixer equipped with a 1-cm light path flow cell thermostated at 250C. The exhaust of the mixing chamber was slightly enlarged to reduce the flow resistance, which reduced the dead time of the instrument to approxiimately 2.1 msec for a reacting system containing 0.6-0.7 mg/ml of HMIM solution operating at 60 psi sampling pressure. The photodetection system was designed and built by Dr. P. 13. Chock and Mr. Ml.Greifner here at the National Institutes of Health. A detailed description of it will be published elsewhere. The time course

Section: Rates Of the H+ Releasementioning

confidence: 62%

Heavy Meromyosin Mg-ATPase: Presteady-State and Steady-State H ⁺ Release

Chock¹,

Eisenberg²

1974

“…5A). [10][11][12][13][14][15][16][17][18][19][20] Hz). Third is that motion may be slower under isometric tension than in isotonic shortening (3).…”

Section: Methodsmentioning

confidence: 99%

Fluctuations in polarized fluorescence: evidence that muscle cross bridges rotate repetitively during contraction.

Borejdo¹,

Putnam²,

Morales³

1979

Particular thiols of the myosin subfragment 1 moieties of single glycerinated muscle fibers are cova ently labeled with rhodamine. By using appropriate solutions such fibers can be relaxed, be in rigor, or develop active isometric tension. The rhodamine is excited by polarized 514.5-nm laser light; the >580-nm fluorescence is resolved into orthogonal components and the intensity of each is measured by a computer-interfaced photon counting system. Fluctuations overand-above noise appear in steady-state activity but not in relaxation or rigor and not when the fluorophore is actin-attached instead of myosin-attached. Fluctuations also appear in ratios of polarized intensities-quantities sensitive to fluorophore attitude but not to fluorophore number. The fluctuations are dominated by low (t2 Hz) frequencies similar to separately measured ATPase frequencies. The fluctuations are ascribed to repetitive motion of the cross bridges to which the rhodamine is attached.The "cycling cross bridge" theory of muscle contraction postulates that myosin projections ("S-1" moieties) execute repetitive rotational motion coupled (1) to repetitive (enzymatic) chemistry occurring at myosin S-1 ATPase sites. According to this theory, a cross bridge attaches to actin at a specific attitude (2, 3)-e.g., 900 ("relaxation") to the fiber axis. Attachment is followed by a "power stroke" in which the S-1 "rolls" on actin (2), passing through many states-down to, say, 450 ("rigor"). After the roll and the binding of a new ATP, the moiety detaches from actin. Detachment allows reorientation to 900. A new cycle begins on the next attachment; if shortening occurs, attachment is to a "downstream" actin site. This theory is supported by much circumstantial evidence but not yet by direct test. One aspect-on stimulation is there a transient increase in myosin-actin attachments?-is being investigated elsewhere by time-resolved x-ray diffraction (4, 5). Here we attempt to test directly whether in steady-state (isometric) tension development the cross bridges execute repetitive rotational motion.The ideas underlying our examination of the theory follow. The steady-state tension (or the time-averaged value of any other cross bridge-related parameter) may, as the theory assumes, be the summation of the individual activities of a large population of molecular impellers behaving stochastically. Therefore, if the parameter is measured with sufficient sensitivity there will be revealed fluctuations around the "average" or steady-state value of the parameter. A Fourier analysis of these fluctuations gives an account of the frequencies that compose them. Of course, we must be sure that the fluctuations are "over-and-above" those generated by measuring instruments or other artifactual sources. What we will report (interchangeably) about such analyzed fluctuations is their "autocorrelation function", G(T), and their "power spectrum", S(v). G(T) expresses the similarity between two measurements of the parameter separated in time by r; in particular, th...

“…The unitary impulses are assumed to result from the interaction of nucleotides with enzymatic sites on the cross-bridges (1). One of the potentially powerful methods of studying these events consists of recording spectroscopic data from dipolar (fluorescent or paramagnetic) probes residing in, or artificially attached to, the cross-bridges (2). Recently, investigators using directional EPR to monitor the orientation of spin labels attached to cross-bridges have concluded that, during the generation of isometric tension, only a small fraction (20%) of the cross-bridges are attached to actin while the rest are unattached (3).…”

mentioning

confidence: 99%

“…§1734 solely to indicate this fact. servable absorbance of the system: [2] where flo is the unit sphere. Instead of assuming in advance that the probes have a particular distribution in the fiber, we acknowledge only that N will depend on ( and 4 and that it will reflect something of the fiber shape in relation to the arrangement of the experiment.…”

mentioning

confidence: 99%

Evidence for cross-bridge order in contraction of glycerinated skeletal muscle.

Burghardt¹,

Ando²,

Borejdo³

1983

The linear dichroism of iodoacetylrhodamine labels attached to the single reactive thiol groups of myosin heads was measured to determine the spatial orientation of myosin crossbridges in single glycerinated skeletal muscle fibers. We have shown previously that in rigor the chromophoric labels are well ordered and assume an-orientation nearly perpendicular to the fiber axis; in the presence of MgADP, a large fraction of probe remains well ordered but the probe attitude assumes a more slanted orientation; in relaxed muscle, the probe order is largely lost, implying a high degree of cross-bridge disorder. In this paper, we report that during isometric contraction a large fraction of the probe shows a high degree of order, suggesting the attachment-of 465% of the cross-bridges to actin. These ordered cross-bridges have a probe attitude similar to that of the MgADP-induced static state and hence are in a mechanical state quite distinct from rigor.In seeking a molecular explanation of how force is generated in muscle at the expense of ATP hydrolysis, a particular hypothesis has seemed to rationalize many of the biochemical and physiological data. In this hypothesis, myosin cross-bridges (S-1 moieties of the myosin molecules) are assumed to deliver mechanical impulses to adjacent actin filaments in a cyclical manner. The unitary impulses are assumed to result from the interaction of nucleotides with enzymatic sites on the cross-bridges (1). One of the potentially powerful methods of studying these events consists of recording spectroscopic data from dipolar (fluorescent or paramagnetic) probes residing in, or artificially attached to, the cross-bridges (2). Recently, investigators using directional EPR to monitor the orientation of spin labels attached to cross-bridges have concluded that, during the generation of isometric tension, only a small fraction (20%) of the cross-bridges are attached to actin while the rest are unattached (3). Moreover, they concluded that the predominant probe orientation is identical to that of rigor, suggesting that those crossbridges that are attached to actin have the rigor-state orientation. These findings imply that the cross-bridge, or a domain of the cross-bridge where the probe is attached, maintains a constant orientation during force generation. In this work, we have observed the active state by using a method based on the absorption dichroism of fluorescent probes attached to the crossbridges in single fibers (4). Our data suggest that 65% of the cross-bridges are attached to actin and that those that are attached to actin do not have the rigor-state orientation but have an orientation characteristic of a nucleotide-ligated state that can be simulated statically by addition of MgADP. THEORYThe linear dichroism technique for measuring the spatial orientation of cross-bridges in a muscle fiber has been described (4). In this section, we outline the central points of the optical technique wherein the total emitted fluorescence is used to measure the absorption of the fluor...