Absorbance signals from resting frog skeletal muscle fibers injected with the pH indicator dye, phenol red

Singly dissected twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with the pH indicator dye, phenol red. Dye-related absorbances in myoplasm, denoted by A0(lambda) and A90(lambda), were estimated as a function of wavelength lambda (450 nm less than or equal to lambda less than or equal to 640 nm) with light polarized parallel (0 degrees) and perpendicular (90 degrees) to the fiber axis respectively. At all lambda, A0(lambda) was slightly greater than A90(lambda), indicating that some of the phenol red molecules were bound to oriented structures accessible to myoplasm. The phenol red "isotropic" signal, [A0(lambda) + 2A90(lambda)]/3, a quantity equal to the average absorbance of all the dye molecules independent of their orientation, had a spectral shape that was red-shifted by approximately 10 nm in comparison with in vitro dye calibration curves measured in 140 mM KCl. The red-shifted spectrum also indicates that some phenol red molecules were bound in myoplasm. A quantitative estimate of indicator binding was obtained from measurements of the dye's apparent diffusion constant in myoplasm, denoted by Dapp. The small value of Dapp, 0.37 x 10(-6) cm2 s-1 (at 16 degrees C), can be explained if approximately 80% of the dye was bound to myoplasmic sites of low mobility. To estimate the apparent myoplasmic pH, denoted by pHapp, the isotropic absorbance of phenol red was fitted by in vitro calibration spectra. pHapp was found to be independent of dye concentration (0.2-2 mM), but varied widely (range, 6.8-7.5; mean value, 7.17) among fibers judged from functional characteristics to be normal. When fibers were subjected to acid or alkaline loads by exposure to Ringer's solution containing, respectively, dissolved CO2 or NH3, the changes in pHapp were in agreement with those expected from pH micro-electrode studies. It is concluded that in spite of the several indications for the presence of bound phenol red inside muscle cells, the pHapp signal from the indicator is useful for monitoring changes in myoplasmic pH in response to physiological and pharmacological manipulations.     

Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra

Furaptra (Raju, B., E. Murphy, L. A. Levy, R. D. Hall, and R. E. London. 1989. Am. J. Physiol. 256:C540-C548) is a "tri-carboxylate" fluorescent indicator with a chromophore group similar to that of fura-2 (Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. J. Biol. Chem. 260:3440-3450). In vitro calibrations indicate that furaptra reacts with Ca2+ and Mg2+ with 1:1 stoichiometry, with dissociation constants of 44 microM and 5.3 mM, respectively (16-17 degrees C; ionic strength, 0.15 M; pH, 7.0). Thus, in a frog skeletal muscle fiber stimulated electrically, the indicator is expected to respond to the change in myoplasmic free [Ca2+] (delta[Ca2+]) with little interference from changes in myoplasmic free [Mg2+]. The apparent longitudinal diffusion constant of furaptra in myoplasm was found to be 0.68 (+/- 0.02, SEM) x 10(-6) cm2 s-1 (16-16.5 degrees C), a value which suggests that about half of the indicator was bound to myoplasmic constituents of large molecular weight. Muscle membranes (surface and/or transverse-tubular) appear to have some permeability to furaptra, as the total quantity of indicator contained within a fiber decreased after injection; the average time constant of the loss was 302 (+/- 145, SEM) min. In fibers containing less than 0.5 mM furaptra and stimulated by a single action potential, the calibrated peak value of delta[Ca2+] averaged 5.1 (+/- 0.3, SEM) microM. This value is about half that reported in the preceding paper (9.4 microM; Konishi, M., and S. M. Baylor. 1991. J. Gen. Physiol. 97:245-270) for fibers injected with purpurate-diacetic acid (PDAA). The latter difference may be explained, at least in part, by the likelihood that the effective dissociation constant of furaptra for Ca2+ is larger in vivo than in vitro, owing to the binding of the indicator to myoplasmic constituents. The time course of furaptra's delta[Ca2+], with average values (+/- SEM) for time to peak and half-width of 6.3 (+/- 0.1) and 9.5 (+/- 0.4) ms, respectively, is very similar to that of delta[Ca2+] recorded with PDAA. Since furaptra's delta[Ca2+] can be recorded at a single excitation wavelength (e.g., 420 nm) with little interference from fiber intrinsic changes, movement artifacts, or delta[Mg2+], furaptra represents a useful myoplasmic Ca2+ indicator, with properties complementary to those of other available indicators.     

Simultaneous monitoring of changes in magnesium and calcium concentrations in frog cut twitch fibers containing antipyrylazo III

Antipyrylazo III was introduced into frog cut twitch fibers (17-19 degrees C) by diffusion. After action potential stimulation, the change in indicator absorbance could be resolved into two components that had different time courses and wavelength dependences. The first component was early and transient and due to an increase in myoplasmic free [Ca] (Maylie, J., M. Irving, N.L. Sizto, and W.K. Chandler, 1987, Journal of General Physiology, 89:83-143). The second component, usually measured at 590 nm (near the isosbestic wavelength for Ca), developed later than the Ca transient and returned towards baseline about 100 times more slowly. Although the wavelength dependence of this component is consistent with an increase in either free [Mg] or pH, its time course is clearly different from that of the signals obtained with the pH indicators phenol red and 4',5'-dimethyl-5-(and -6-) carboxyfluorescein, suggesting that it is mainly due to an increase in free [Mg]. After a single action potential in freshly prepared cut fibers that contained 0.3 mM antipyrylazo III, the mean peak amplitude of delta A (590) would correspond to an increase in free [Mg] of 47 microM if all the signal were due to a change in [Mg] and all the intracellular indicator reacted with Mg as in cuvette calibrations. With either repetitive action potential stimulation or voltage-clamp depolarization, the delta A (590) signal continued to develop throughout the period when free [Ca] was elevated and then recovered to within 40-90% of the prestimulus baseline with an average rate constant between 0.5 and 1.0 s-1. With prolonged voltage-clamp depolarization, both the amplitude and rate of development of the delta A(590) signal increased with the amplitude of the depolarization and appeared to saturate at levels corresponding to an increase in free [Mg] of 0.8-1.4 mM and a maximum rate constant of 3-4 s-1, respectively. These results are consistent with the idea that the delta A(590) signal is primarily due to changes in myoplasmic free [Mg] produced by a change in the Mg occupancy of the Ca,Mg sites on parvalbumin that results from the Ca transient.     

The amplitude and time course of the myoplasmic free [Ca2+] transient in fast-twitch fibers of mouse muscle

Bundles of 10-100 fibers were dissected from the extensor digitorum longus muscle of mouse, mounted in an apparatus for optical recording, and stretched to long sarcomere length (> or = 3.6 microns). One fiber within the bundle was microinjected with furaptra, a fluorescent indicator that responds rapidly to changes in myoplasmic free [Ca2+] (delta [Ca2+]). Twitches and brief tetani were initiated by external stimulation. At myoplasmic furaptra concentrations of approximately 0.1 mM, the indicator's fluorescence signal during fiber activity (delta F/F) was well resolved. delta F/F was converted to delta [Ca2+] under the assumption that furaptra's myoplasmic dissociation constant for Ca2+ is 98 microM at 16 degrees C and 109 microM at 28 degrees C. At 16 degrees C, the peak amplitude of delta [Ca2+] during a twitch was 17.8 +/- 0.4 microM (+/-SEM; n = 8) and the half-width of delta [Ca2+] was 4.6 +/- 0.3 ms. At 28 degrees C, the peak and half-width values were 22.1 +/- 1.8 microM and 2.0 +/- 0.1 ms, respectively (n = 4). During a brief high-frequency tetanus, individual peaks of delta [Ca2+] were also well resolved and reached approximately the same amplitude that resulted from a single shock; the initial decays of delta [Ca2+] from peak slowed substantially during the tetanus. For a single twitch at 16 degrees C, the amplitude of delta [Ca2+] in fast-twitch fibers of mouse is not significantly different from that recently measured in fast- twitch fibers of frog (16.5 +/- 0.9 microM; Zhao, M., S. Hollingworth, and S.M. Baylor. 1996. Biophys. J. 70:896-916); in contrast, the half- width of delta [Ca2+] is surprisingly brief in mouse fibers, only about half that measured in frog (9.6 +/- 0.6 ms). The estimated peak rate at which Ca2+ is released from the sarcoplasmic reticulum in response to an action potential is also similar in mouse and frog, 140-150 microM/ms (16 degrees C).     

Calcium release and its voltage dependence in frog cut muscle fibers equilibrated with 20 mM EGTA

Sarcoplasmic reticulum (SR) Ca release was studied at 13-16 degrees C in cut fibers (sarcomere length, 3.4-3.9 microns) mounted in a double Vaseline-gap chamber. The amplitude and duration of the action- potential stimulated free [Ca] transient were reduced by equilibration with end-pool solutions that contained 20 mM EGTA with 1.76 mM Ca and 0.63 mM phenol red, a maneuver that appeared to markedly reduce the amount of Ca complexed by troponin. A theoretical analysis shows that, under these conditions, the increase in myoplasmic free [Ca] is expected to be restricted to within a few hundred nanometers of the SR Ca release sites and to have a time course that essentially matches that of release. Furthermore, almost all of the Ca that is released from the SR is expected to be rapidly bound by EGTA and exchanged for protons with a 1:2 stoichiometry. Consequently, the time course of SR Ca release can be estimated by scaling the delta pH signal measured with phenol red by -beta/2. The value of beta, the buffering power of myoplasm, was determined in fibers equilibrated with a combination of EGTA, phenol red, and fura-2; its mean value was 22 mM/pH unit. The Ca content of the SR (expressed as myoplasmic concentration) was estimated from the total amount of Ca released by either a train of action potentials or a depleting voltage step; its mean value was 2,685 microM in the action-potential experiments and 2,544 microM in the voltage- clamp experiments. An action potential released, on average, 0.14 of the SR Ca content with a peak rate of release of approximately 5%/ms. A second action potential, elicited 20 ms later, released only 0.6 times as much Ca (expressed as a fraction of the SR content), probably because Ca inactivation of Ca release was produced by the first action potential. During a depolarizing voltage step to 60 mV, the rate of Ca release rapidly increased to a peak value of approximately 3%/ms and then decreased to a quasi-steady level that was only 0.6 times as large; this decrease was also probably due to Ca inactivation of Ca release. SR Ca release was studied with small step depolarizations that open no more than one SR Ca channel in 7,000 and increase the value of spatially averaged myoplasmic free [Ca] by only 0.2 nM.     

Myoplasmic calcium transients monitored with purpurate indicator dyes injected into intact frog skeletal muscle fibers

Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after microinjection with tetramethylmurexide (TMX) or purpurate-3,3' diacetic acid (PDAA), two compounds from the purpurate family of absorbance Ca2+ indicators previously used in cut muscle fibers (Maylie, J., M. Irving, N. L. Sizto, G. Boyarsky, and W. K. Chandler. 1987. J. Gen. Physiol. 89:145-176; Hirota, A., W. K. Chandler, P. L. Southwick, and A. S. Waggoner. 1989. J. Gen. Physiol. 94:597-631.) The apparent longitudinal diffusion constant of PDAA (mol wt 380) in myoplasm was 0.99 (+/- 0.04, SEM) x 10(-6) cm2 s-1 (16-17 degrees C), a value which suggests that 24-43% of the PDAA molecules were bound to myoplasmic constituents of large molecular weight. The corresponding values for TMX (mol wt 322) were 0.98 (+/- 0.05) x 10(-6) cm2 s-1 and 44-50%, respectively. Muscle membranes (surface and/or transverse-tubular) appear to be permeable to TMX and, to a lesser extent, to PDAA, since the total amount of indicator contained within a fiber decreased with time after injection. The average time constants for disappearance of indicator were 46 (+/- 7, SEM) min for TMX and 338 (+/- 82) min for PDAA. The fraction of indicator in the Ca2(+)-bound state in resting fibers was significantly different from zero for TMX (0.070 +/- 0.008) but not for PDAA (0.026 +/- 0.009). In in vitro calibrations PDAA but not TMX appeared to react with Ca2+ with 1:1 stoichiometry. In agreement with Hirota et al. (Hirota, A., W. K. Chandler, P. L. Southwick, and A. S. Waggoner. 1989. J. Gen. Physiol. 94:597-631), we conclude that PDAA is probably a more reliable myoplasmic Ca2+ indicator than TMX. In fibers that contained PDAA and were stimulated by a single action potential, the calibrated peak value of the myoplasmic free [Ca2+] transient (delta[Ca2+]) averaged 9.4 (+/- 0.6) microM, a value about fivefold larger than that calibrated with antipyrylazo III under otherwise identical conditions (Baylor, S. M., and S. Hollingworth. 1988. J. Physiol. 403:151-192). The fivefold difference is similar to that previously reported in cut fibers with antipyrylazo III and PDAA. Since in both intact and cut fibers the percentage of PDAA bound to myoplasmic constituents is considerably smaller than that found for antipyrylazo III, the PDAA calibration of delta[Ca2+] is likely to be more accurate. Interestingly, in intact fibers the peak value of delta[Ca2+] calibrated with either PDAA or antipyrylazo III is about half that calibrated in cut fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Perturbation of sarcoplasmic reticulum calcium release and phenol red absorbance transients by large concentrations of fura-2 injected into frog skeletal muscle fibers

Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with two indicator dyes: phenol red, to monitor a previously described signal (denoted delta pHapp; Hollingworth and Baylor. 1990. J. Gen. Physiol. 96:473-491) possibly reflective of a myoplasmic pH change following action potential stimulation; and fura-2, to monitor the associated change in the myoplasmic free calcium concentration (delta[Ca2+]). Additionally, it was expected that large myoplasmic concentrations of fura-2 (0.5-1.5 mM) might alter delta pHapp, since it was previously found (Baylor and Hollingworth. 1988. J. Physiol. 403:151-192) that the Ca2(+)-buffering effects of large fura-2 concentrations: (a) increase the estimated total concentration of Ca2+ (denoted by delta[CaT]) released from the sarcoplasmic reticulum (SR), but (b) reduce and abbreviate delta[Ca2+]. The experiments show that delta pHapp was increased at the larger fura-2 concentrations; moreover, the increase in delta pHapp was approximately in proportion to the increase in delta[CaT]. At all fura-2 concentrations, the time course of delta pHapp, through time to peak, was closely similar to, although probably slightly slower than, that of delta[CaT]. These properties of delta pHapp are consistent with an hypothesis proposed by Meissner and Young (1980. J. Biol. Chem. 255:6814-6819) and Somlyo et al. (1981. J. Cell Biol. 90:577-594) that a proton flux from the myoplasm into the SR supplies a portion of the electrical charge balance required as Ca2+ is released from the SR into the myoplasm. A comparison of the amplitude of delta pHapp with that of delta[CaT] indicates that, in response to a single action potential, 10-15% of the charge balance required for Ca2+ release may be carried by protons.     

Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers.

Fura red, a fluorescent Ca2+ indicator with absorbance bands at visible wavelengths, was injected into intact single muscle fibers that had been stretched to a long sarcomere length (approximately 3.8 microns) and bathed in a 'high-Ca2+' Ringer ([Ca2+] = 11.8 mM). From fura red's slow diffusion coefficient in myoplasm, 0.16 (+/- 0.01, SEM) x 10(-6) cm2 s-1 (N = 5; 16 degrees C), it is estimated that approximately 85% of the indicator molecules are bound to muscle constituents of large molecular weight. Binding appears to elevate, by 3- to 4-fold, the indicator's apparent dissociation constant for Ca2+ (KD), which is estimated to be 1.1-1.6 microM in myoplasm. Fura red's myoplasmic absorbance spectrum was used to estimate fr, the fraction of fura red molecules in the Ca2+-bound form at rest. In 3 fibers thought to be minimally damaged by the micro-injection, fr was estimated to be 0.15 (+/- 0.01). Thus, resting myoplasmic free [Ca2+] ([Ca2+]r) is estimated to be 0.19-0.28 microM. For fibers in normal Ringer solution ([Ca2+] = 1.8 mM), at shorter sarcomere length (approximately 2.7 microns), and containing a nonperturbing concentration of indicator (< or = 0.2 mM), [Ca2+]r is estimated to be 0.18-0.27 microM. This range is higher than estimated previously in frog fibers with other techniques. In 6 fibers, R, the indicator's fluorescence ratio signal (equal to the emission intensity measured with 420 nm excitation divided by that measured with 480 nm excitation), was measured at rest and following electrical stimulation and compared with absorbance measurements made from the same fiber region. The analysis implies that RMIN and RMAX (the values of R that would be measured if all indicator molecules were in the Ca(2+)-free and Ca(2+)-bound states, respectively) were substantially smaller in myoplasm than in calibration solutions lacking muscle proteins. Several methods for estimation of [Ca2+]r from R are analyzed and discussed.     

Quantitative mapping of the orientation of fibroin beta-sheets in B. mori cocoon fibers by scanning transmission X-ray microscopy

The spatial distribution of the linear dichroic signal associated with aligned beta-sheets in a microtomed section of a Bombyx mori cocoon silk fiber was derived from scanning transmission X-ray microscopy (STXM). The intense C 1s --> pi(amide) peak at 288.25 eV was found to have negligible dichroic signal in transverse sections but a large dichroic signal in longitudinal sections. This is consistent with other measurements of the orientation of the aligned beta-sheets in silk fibers, in particular with those obtained by polarized Raman microspectroscopy to which our results are compared. When the dichroic signal strength is mapped at better than 100 nm spatial resolution, microscopic variations are found. Although the magnitude of the dichroic signal changes over a fine spatial scale, the direction of the maximum signal at any position does not change. We interpret the spatial variation of the intensity of the dichroic signal as a map of the quality of local orientation of the beta-sheets in the fiber. At sufficiently high magnification and resolution, this technique should image individual beta-sheet crystallites, although the present implementation does not achieve that. A map of the orientation parameter P(2) is derived. The average value of P(2) (-0.20 +/- 0.04) from STXM is smaller than that derived from the analysis of the amide I band in polarized Raman spectra (-0.41 +/- 0.03). This deviation is attributed to the fact that the STXM results also include the signal from unaligned regions of the protein.     

Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3.

Fluo-3 is an unusual tetracarboxylate Ca2+ indicator. For recent lots supplied by Molecular Probes Inc. (Eugene, OR), FMAX, the fluorescence intensity of the indicator in its Ca(2+)-bound form, is approximately 200 times that of FMIN, the fluorescence intensity of the indicator in its Ca(2+)-free form. (For earlier lots, impurities may account for the smaller reported values of FMAX/FMIN, 36-40). We have injected fluo-3 from a high-purity lot into intact single fibers from frog muscle and measured the indicator's absorbance and fluorescence signals at rest (A and F, respectively) and changes in absorbance and fluorescence following action potential stimulation (delta A and delta F signals substantially lagged behind that of the myoplasmic free Ca2+ transient. Our analysis of fluo-3's signals from myoplasm therefore focused on information about the level of resting myoplasmic free [Ca2+] ([Ca2+]r). From A, delta A, and in vitro estimates of fluo-3's molar extinction coefficients, the change in the fraction of fluo-3 in the Ca(2+)-bound form during activity (delta f) was estimated. From delta f, delta F, and F, the fraction of the indicator in the Ca(2+)-bound form in the resting fiber (fr) was estimated by fr = (delta f x F/delta F) + (1-FMAX/FMIN)-1. Since FMAX/FMIN is large, the contribution of the second term to the estimate of fr is small. At 16 degrees C, the mean value (mean +/- S.E.) of fr was 0.086 +/- 0.004 (N = 15). From two estimates of the apparent dissociation constant of fluo-3 for Ca2+ in the myoplasm, 1.09 and 2.57 microM, the average value of [Ca2+]r is calculated to be 0.10 and 0.24 microM, respectively. The smaller of these estimates lies near the upper end of the range of values for [Ca2+]r in frog fibers (0.02-0.12 microM) estimated by others with aequorin and Ca(2+)-selective electrodes. The larger of the estimates lies within the range of values (0.2-0.3 microM) previously estimated in this laboratory with fura red. We conclude that [Ca2+]r in frog fibers is at least 0.1 microM and possibly as large as 0.3 microM.     

Kinetics and mechanism for the conformational transition in p-guanidinobenzoate bovine trypsinogen induced by the isoleucine-valine dipeptide

The interaction between p-guanidinobenzoate-trypsinogen and the isoleucine-valine dipeptide has been investigated by temperature-jump relaxation spectrometry. Using the absorbance at 281 nm the concentration dependence of the relaxation parameters is consistent with the conventional induced-fit model: rapid ligand binding coupled to a slower intramolecular change; some alternative mechanisms can be excluded. At 296 K, 0.1 M Tris HCl, pH = 7.4, the dissociation equilibrium constant for the overall process is K = 5.1(+/- 0.2) X 10(-5) M; for the binding step K1 = 2.3(+/- 0.3) X 10(-3) M and the rate constants for the structural change are k2 = 26(+/-6)s-1 and k-2 = 0.61(+/- 0.04)s-1; the overall dissociation reaction enthalpy is delta H0 = 26(+/-6)KJmol-1 and the reactiom entropy is delta S0 = 4(+/- 20) kJ-1 mol-1. In combination with CD and X-ray crystallographic data, the results of this study suggest that the binding of the dipeptide to a trypsinogen-like, partially disordered conformation induces a transition to a trypsin-like highly ordered structure.     

CD of gels and suspensions: apparent CD in the soret region of sickle hemoglobin gels

CD spectra in the soret region of sickle-cell deoxyhemoglobin (deoxy-HbS) fiber gels are radically different from the CD of deoxy-HbS in solution. An explanation is found using the Stokes–Mueller representation of the interaction of a polarized beam with the instrument optical train and sample to derive expressions for the apparent CD of gels and suspensions of optically active molecules that consist of randomly oriented domains or particles that are linearly dichroic and linearly birefringent. These theoretical considerations show that the apparent CD spectra from such systems have contributions from the LD and birefringence of each domain even if no net linear birefringence and dichroism is apparent in the sample. Thus, the interpretation of the CD from gels and suspensions is problematic, unless it can be demonstrated that each domain or particle has extremely small absorbance or that the LD and birefringence of each is a very small fraction of the total absorbance. As a result, we conclude that the spectra of HbS gels are not due to the CD of the heme per se; rather, they also reflect the randomly oriented domain structure of the gels and the LD and linear birefringence associated with each domain.     

Microinjection of strong calcium buffers suppresses the peak of calcium release during depolarization in frog skeletal muscle fibers

The effects of high intracellular concentrations of various calcium buffers on the myoplasmic calcium transient and on the rate of release of calcium (Rrel) from the sarcoplasmic reticulum (SR) were studied in voltage-clamped frog skeletal muscle fibers. The changes in intracellular calcium concentration (delta[Ca2+]) for 200-ms pulses to 0-20 mV were recorded before and after the injection of the calcium buffer and the underlying Rrel was calculated. If the buffer concentration after the injection was high, the initial rate of rise of the calcium transient was slower after injection than before and was followed by a slow increase of [Ca2+] that resembled a ramp. The increase in myoplasmic [Mg2+] that accompanies the calcium transient in control was suppressed after the injection and a slight decrease was observed instead. After the injection the buffer concentration in the voltage-clamped segment of the fiber decreased as the buffer diffused away toward the open ends. The calculated apparent diffusion coefficient for fura-2 (Dapp = 0.40 +/- 0.03 x 10(-6) cm2/s, mean +/- SEM, n = 6) suggests that approximately 65-70% of the indicator was bound to relatively immobile intracellular constituents. As the concentration of the injected buffer decreased, the above effects were reversed. The changes in delta[Ca2+] were underlined by characteristic modification of Rrel. The early peak component was suppressed or completely eliminated; thus, Rrel rose monotonically to a maintained steady level if corrected for depletion. If Rrel was expressed as percentage of SR calcium content, the steady level after injection did not differ significantly from that before. Control injections of anisidine, to the concentration that eliminated the peak of Rrel when high affinity buffers were used, had only a minor effect on Rrel, the peak was suppressed by 26 +/- 5% (mean +/- SE, n = 6), and the steady level remained unchanged. Thus, the peak component of Rrel is dependent on a rise in myoplasmic [Ca2+], consistent with calcium-induced calcium release, whereas the steady component of Rrel is independent of myoplasmic [Ca2+].     

Dichroic behavior of the absorbance signals from dyes NK2367 and WW375 in skeletal muscle fibers

Absorbance signals were recorded from voltage-clamped single muscle fibers stained with the nonpenetrating potentiometric dyes NK2367 and WW375 and illuminated with quasimonochromatic light from 560 to 800 nm, linearly polarized either parallel (0 degree) or perpendicular (90 degrees) to the fiber long axis. The signals from both dyes depend strongly on the incident polarization. At any wavelength and/or polarization condition, the total absorbance signal is a superposition of the same two signal components previously identified with unpolarized light (Heiny, J. A., and J. Vergara, 1982, J. Gen. Physiol., 80:203)--namely, a fast step signal from the voltage-clamped surface membrane and a signal reflecting the slower T-system potential changes. The 0 degree and 90 degrees spectra of both membranes have similar positive and negative absorbance peaks (720 and 670 nm, respectively, for dye NK2367; 740 and 700 nm for dye WW375); in addition, they have the same dichroic maxima (670 for NK2367; 700 for WW375). However, for the surface membrane, the 0 degrees spectra are everywhere more positive than the 90 degrees spectra, whereas the reverse is true for the T-system, which results in a dichroism of opposite sign for the two membranes. These spectral characteristics were analyzed using a general model for the potential-dependent response of an absorbing dye (Tasaki, I., and A. Warashina, 1976, Photochem. Photobiol., 24:191), which takes into account both the dye response and the membrane geometries. They are consistent with the proposal that the dye responds via a common mechanism in both membranes that consists of a dye reorientation and a change in the absorption maxima.     

Dichroic Orientation of Phytochrome Intermediates in the Pathway from PR to PFR as Analyzed by Double Laser Flash Irradiations in Polarotropism of Adiantum Protonemata

The polarotropic response in protonemata of the fern Adiantumis regulated by phytochrome (Kadota et al. 1984); P_R and P_FRhave been shown to be dichroically oriented parallel and normalto the cell surface, respectively (Kadota et al. 1982). Thischange in the dichroic orientation of phytochrome during photoconversionwas analyzed by a newly-built, polarization plane-rotatabledouble laser flash irradiator. A polarotropic response was effectivelyinduced with a flash of polarized red (640 nm) light (6xl0^–7s) having the vibration plane of the electrical vector parallelto the protonemal cell axis. When a flash of polarized far-red(710 nm) light (6xl0^–7s) was given 30 sec after the redflash, the red flash-induced response was reversed by a far-redflash vibrating normal to the cell axis but not by one vibratingparallel. However, when given 2 µs or 2 ms after the redflash, the polarotropic response was not reversed by a polarizedfar-red flash vibrating normal to the cell axis but was reversedby a parallel-vibrating flash. These results suggest that theorientation of phototransformation intermediates existing 2µs or 2 ms after a red flash is still parallel to thecell surface, and that the change in the orientation of phytochromemolecules occurs between 2 ms and 30 s after the red flash. (Received February 3, 1986; Accepted April 23, 1986)     

Birefringence signals in mammalian and frog myocardium. E-C coupling implications

Birefringence signals from mammalian and frog hearts were studied. The period between excitation and the onset of contraction in which optical signals were free of movement artifact was determined by changes in scattered incandescent light and changes in laser diffraction patterns. The birefringence signal preceding contraction was found to behave as a change in retardation and was not contaminated measurably by linear dichroic or isotropic absorption changes. There were two components of the birefringence signal in mammalian heart muscles but only one component in the frog heart. The first component of the birefringence signals in both mammalian and frog hearts had a time course coincident with the action potential upstroke. The second component in mammalian preparations was sensitive to inotropic interventions, such as variation of extracellular Ca2+, stimulation frequency, temperature, and epinephrine, in a manner that correlated with the maximum rate of rise of tension. Caffeine (2-10 mM) not only failed to generate a second component in the frog heart, but also suppressed the second component in the mammalian heart while potentiating twitch tension. The results suggest that the second component of the birefringence signal in the mammalian myocardium is related to Ca2+ release from the sarcoplasmic reticulum.     

Intracellular pH changes induced by calcium influx during electrical activity in molluscan neurons

Simultaneous measurements of electrical activity and light absorbance have been made on nerve cell bodies from Archidoris monteryensis injected with indicator dyes. pH indicators, phenol red and bromocresol purple, and arsenazo III, which under normal conditions is primarily a calcium indicator have been employed. Voltage clamp pulses which induced calcium influx caused an absorbance decrease of the pH dyes indicating an internal acidification. The onset of the pH drop lagged the onset of Ca2+ influx by 200-400 ms, and pH continued to decrease for several seconds after pulse termination which shut off Ca2+ influx. Trains of action potentials also produced an internal pH decrease. Recovery of the pH change required periods greater than 10 min. The magnitude of the pH change was largely unaffected by external pH in the range 6.8-8.4. The voltage dependence of the internal p/ change was similar to the voltage dependence of calcium influx determined by arsenazo III, and removal of calcium from the bathing saline eliminated the pH signal. In neurons injected with EGTA (1-5 mM), the activity- induced internal Ca2+ changes were reduced or eliminated, but the internal pH drop was increased severalfold in magnitude. After the injection of EGTA, voltage clamp pulses produced a decrease in arsenazo III absorbance instead of the normal increase. Under these conditions the dye was responding primarily to changes in internal pH. Injection of H+ caused a rise in internal free calcium. The pH buffering capacity of the neurons was measured using three different techniques: H+ injection, depressing intrinsic pH changes with a pH buffer, and a method employing the EGTA-calcium reaction. The first two methods gave similar measurements: 4-9 meq/unit pH per liter for pleural ganglion cells and 13-26 meq/unit pH per liter for pedal ganglion cells. The EGTA method gave significantly higher values (20-60 meq/unit pH per liter) and showed no difference between pleural and pedal neurons.     

Proton and electron transfer during the reduction of molecular oxygen by fully reduced cytochrome c oxidase: a flow-flash investigation using optical multichannel detection

Proton and electron transfer events during the reaction of solubilized fully reduced bovine heart cytochrome c oxidase with molecular oxygen were investigated using the flow-flash technique. Time-resolved spectral changes resulting from ligand binding and electron transfer events were detected simultaneously with pH changes in the bulk. The kinetics and spectral changes in the visible region (450-750 nm) were probed by optical multichannel detection, allowing high spectral resolution on time scales from 50 ns to 50 ms. Experiments were carried out in the presence and absence of pH-sensitive dyes (carboxyfluorescein at pH 6.5, phenol red at pH 7.5, and m-cresol purple at pH 8.5) which permitted separation of spectral changes due to proton transfer from those caused by ligand binding and electron transfer. The transient spectra recorded in the absence of dye were analyzed by singular-value decomposition and multiexponential fitting. Five apparent lifetimes (0.93 microseconds, 10 microseconds, 36 microseconds, 90 microseconds, and 1.3 ms at pH 7.5) could consistently be distinguished and provided a basis for a reaction mechanism consistent with our most recent kinetic model [Sucheta, A., Szundi, I., and Einarsdóttir, O. (1999) Biochemistry 37, 17905-17914]. The dye response indicated that proton uptake occurred concurrently with the two slowest electron transfer steps, in agreement with previous results based on single-wavelength detection [Hallén, S., and Nilsson, T. (1992) Biochemistry 31, 11853-11859]. The stoichiometry of the proton uptake reactions was approximately 1.3 +/- 0.3, 1.4 +/- 0.3, and 1.6 +/- 0.5 protons per enzyme at pH 6.5, 7.5, and 8.5, respectively. The electron transfer between heme a and CuA was limited by proton uptake on a 90 microseconds time scale. We have established the lower limit of the true rate constant for the electron transfer between CuA and heme a to be approximately 2 x 10(5) s-1.     

Dichroism of transient absorbance changes in the red spectral region using oriented chloroplasts. II. P-700 absorbance changes

The light induced transient absorbance changes associated with the trap of photosystem I have been studied using magnetically oriented spinach chloroplasts and a polarized measuring beam. The ΔA spectra for the two polarizations parallel and perpendicular to the plane of the photosynthetic membranes have been recorded in the spectral range 630–850 nm.A dichroic ratio greater than two is observed both in the main band around 700 nm and in the radical cation band around 810 nm, leading to the conclusion that the far-red transition moment of the P-700 dimeric species is lying almost parallel to the membrane plane.Dichroic ratios smaller than one are reported in the 650–670 nm band of the ΔA spectrum. The possible attribution of this band to excitonic interactions in the dimer favors the hypothesis of a tilting out of the membrane plane of this transition. This finding ruled out an orientation parallel to the membrane plane of the two chlorophyll molecules constituting the P-700 phototrap.A small residual transient absorbance change is observed in the absence of artificial electron acceptor. Its spectrum shows significant differences as compared to the normal P-700 spectrum: the magnitude of the signal at 700 nm is only 15–25% of the normal signal, the half-band width of the band around 700 nm is nearly twice as large and the dichroic ratio in the band is only 1.5±0.1. In the presence of ferricyanide, this signal is still observed both for intact and osmotically broken chloroplasts, suggesting a heterogeneity in the population of traps in Photosystem I.     

On the flexibility of myosin in solution.

Rabbit skeletal muscle myosin from the same rabbit was prepared by two different methods, and then purified by either Sephadex or hydroxylapatite chromatography. The resulting myosin samples were analyzed in 2-10 mM sodium pyrophosphate solutions at pH 9 using transient electric birefringence. The birefringence decay signals were fitted using a Fortran program called DISCRETE and two relaxation times, 49.7 +/- 5.6 and 11.2 +/- 2.5 microseconds, were determined. These relaxation times were independent of the method of myosin preparation, the method of myosin purification, the concentration of sodium pyrophosphate between 2 and 10 mM, the concentration of myosin between 0.08 and 1.59 mg/mL, and the temperature between 4.0 and 20.0 degrees C, after correction to 20.0 degrees C. The longer relaxation time is consistent with a rigid, linear myosin molecule. The shorter relaxation time is consistent with myosin that has a completely flexible hinge region in the myosin tail. Both relaxation times are inconsistent with the previously reported single relaxation time of myosin obtained by fitting the birefringence decay data to only 90% of the decay signal. By forcing some of the birefringence decay data in the presence work to fit 90% of the decay signal with a single relaxation time, approximately the same relaxation time as previously reported was obtained.