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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...

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Time-dependent fluorescence depolarization and lifetime studies have been made on myosin subfragment 1 to obtain information about mobility changes and dye environment changes when different nucleotides are added. Data are reported for static and actively hydrolyzing systems containing G- and F-actin. Preliminary data indicate that myosin labeled with the fluorophore 1, 5 IAEDANS and treated with DTT preserves its actin-activated Vmax. S1 prepared in this manner gives lifetime changes which are nearly identical for all systems studied. S1 labeling without DTT addition gives a pattern of lifetimes similar, though not identical to ESR work. Either type of labeling produces no observable change in the polarization decay, and we set an upper limit of 15% length change for the elongate S1. An unusually long fluorescence decay lifetime for the S1-Mg++ ATP-G-actin system is found which may indicate a new acto-S1 state stabilized by G-actin. The method for obtaining the bound fraction of S1's in the presence of actin is presented and applied to the S1-F-actin-Mg++ ATP system. Qualitative agreement is obtained with other methods.