Characterizations of Cross-Bridges in the Presence of Saturating Concentrations of MgAMP-PNP in Rabbit Permeabilized Psoas Muscle  S.M. Frisbie, S. Xu, J.M. Chalovich, L.C. Yu  Biophysical Journal  Volume 74, Issue 6, Pages 3072-3082 (June 1998) DOI: 10.1016/S0006-3495(98)78014-2 Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 1 The equatorial intensity ratio I(1,1)/I(1,0) used as a measure of the fraction of cross-bridges attached to actin as a function of increasing MgAMP-PNP concentration. Single rabbit psoas muscle fibers were studied in CaEGTA (●), in EGTA solution (○), and in 2mM MgATP with no AMP-PNP (▿). Temperature, 1°C. Ionic strength, 170mM. Sarcomere length, 2.3–2.4μm. The rigor data point in EGTA solution was set to 1.0 (the actual ratio was ∼3.0). Other data points were normalized to this value. Biophysical Journal 1998 74, 3072-3082DOI: (10.1016/S0006-3495(98)78014-2) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 2 (A) The fraction of S1-MgAMP-PNP bound as a function of actin concentration used to determine the association constant at 75mM ionic strength. (B) Association constants of S1-MgAMP-PNP and S1-MgATP for the actin-troponin-tropomyosin complex as a function of ionic strength. Constants were determined at 25°C. The association constants for MgATP are based on a variety of sources: Chalovich et al., 1983; Highsmith and Murphy, 1992; Chalovich, Sen, and Resetar, unpublished results. Biophysical Journal 1998 74, 3072-3082DOI: (10.1016/S0006-3495(98)78014-2) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 3 Two-dimensional x-ray diffraction patterns of one muscle bundle in rigor (A), in 10mM MgATP (B), and in 25mM MgAMP-PNP solution (C). The fiber bundle used was assembled from approximated 30 single skinned fibers. The ionic strength was 170mM, temperature=1°C. For the MgATP-relaxed pattern, two exposures were averaged together, one taken before treatment with 25mM MgAMP-PNP and one taken after. The patterns were recorded using the X13 beamline at EMBL Outstation (DESY, Hamburg, Germany) with image plates and scanned by BSA2000 (Fuji, Japan). Biophysical Journal 1998 74, 3072-3082DOI: (10.1016/S0006-3495(98)78014-2) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 4 Profiles of off-meridional vertical slices through the two-dimensional x-ray diffraction patterns shown in Fig. 3. (a) 10mM MgATP. (b) 25mM MgAMP-PNP. Myosin layer line at 430Å and actin layer line at 59Å are indicated. Biophysical Journal 1998 74, 3072-3082DOI: (10.1016/S0006-3495(98)78014-2) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 5 Meridional profiles of a MgATP-relaxed bundle (a) and the same bundle relaxed in MgAMP-PNP (b). Thick filament related meridional reflections at 220, 145, and 73Å are indicated. Biophysical Journal 1998 74, 3072-3082DOI: (10.1016/S0006-3495(98)78014-2) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 6 (A) Ionic strength dependence of the I11/I10 intensity ratio for fibers in saturating MgATP (10mM) (○) and MgAMP-PNP (20mM) (●) showing the similarity in behavior in the two nucleotides. (B) Ionic strength dependence of the I11/I10 intensity ratio of a fiber at 5mM MgAMP-PNP. At high ionic strengths, even at 5mM of AMP-PNP, the fiber is close to be relaxed, suggesting that at lower ionic strengths more nucleotide is required to reach saturation. The temperature was maintained at 1°C for all experiments, and the patterns were normalized to rigor condition, which is equal to 1.0. Biophysical Journal 1998 74, 3072-3082DOI: (10.1016/S0006-3495(98)78014-2) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 7 Temperature dependence of the x-ray equatorial intensity ratio I11/I10. (A) At 1°C, the pattern at 10mM MgAMP-PNP is relaxed. (B) At 30°C, the same muscle fiber is in rigor. When the concentration of MgAMP-PNP was increased to 30mM, I11 decreased greatly, rendering the diffraction pattern relaxed-like. However, the quality of the pattern was too poor for quantitative analysis. Biophysical Journal 1998 74, 3072-3082DOI: (10.1016/S0006-3495(98)78014-2) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 8 (A) HPLC of AMP-PNP and ATP showing the limit of possible ATP contamination in the AMP-PNP used. No ATP was detected in the AMP-PNP used in the x ray and solution experiments. (B) Expanded region where ATP is expected. Biophysical Journal 1998 74, 3072-3082DOI: (10.1016/S0006-3495(98)78014-2) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 9 HPLC traces of 0.5μM (A) and 1μM (B) ATP used to determine the limit of detectability of this nucleotide. All samples were run under the same conditions as in Fig. 8. Biophysical Journal 1998 74, 3072-3082DOI: (10.1016/S0006-3495(98)78014-2) Copyright © 1998 The Biophysical Society Terms and Conditions