The Malachite Green method for determination of inorganic phosphate (Pi) (Itaya K. & Ui, M. (1966) Clin. Chim. Acta 14, 361-366) was modified to measure Pi in the range of 0.2-15 nmol per ml of ATPase reaction mixture. An ATPase reaction mixture is quenched with an equal volume of 0.6 M PCA; the supernatant after centrifugation is mixed with an equal volume of the Malachite Green/molybdate reagent containing 2 g of sodium molybdate, 0.3 g of Malachite Green and 0.5 g of Triton X-100 or Sterox SE in 1 liter of 0.7 M HCl, and the absorbance at 650 nm is then measured after a 35-40 min incubation at 25 degrees C. Owing to the high sensitivity and simplicity of the modified method, the slow time course of myosin ATP hydrolysis in the presence of Mg2+ and the size of initial phosphate burst can be determined accurately using relatively low concentrations of native myosin and its subfragment-1. The phosphate burst size varied with changes in pH, ionic strength, and temperature. A typical value was 0.8-0.9 mol per site in 0.1 M KCl, 10 mM MgCl2, pH 8.0 at 25 degrees C for fresh enzyme preparations.

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We have used the method of enthalpy titration to analyze the structural changes of troponin C caused by calcium binding. Successive additions of calcium to metal-free troponin C in a microcalorimeter cell result in at least three distinguishable transitions. Analysis of the results has shown that troponin C has at least three classes of calcium binding sites; one site of highest affinity (binding constant, 10(8)-10(10) M-1), one site of next highest affinity (binding constant, 10(6)-10(7) M-1) and two low affinity sites (binding constant, 10(5)-10(6) M-1). Titrations of troponin C with calcium at various temperatures have shown that calcium binding causes large changes in the heat capacity of troponin C. Following the method of Sturtevant (1977), the magnitudes of the hydrophobic and intramolecular vibrational contributions to the heat capacity and entropy changes of troponin C on calcium binding have been estimated. In Mg-free solutions, calcium binding to the 1st site of highest affinity gives rise to a strong hydrophobic effect and to a tightening of the molecular structure. In contrast, calcium binding to the 2nd site of next highest affinity gives rise to a strong hydrophobic effect in the reverse direction and to a "softening" of the structure. Calcium binding to the two low affinity sites has a moderately strong hydrophobic effect and also causes a moderate tightening of the structure. These results are in many respects similar to those obtained with proton magnetic resonance spectroscopy by Levine et al. (1977). These studies are mutually complementary. When 1 mM magnesium is present the changes caused by calcium binding to the two high affinity sites are greatly altered, whereas those involving the two low affinity sites are not much affected. The moderate tightening of the structure which is caused by calcium binding to the two low affinity sites, and which is seen both in the absence and presence of magnesium, is most likely to be involved in the regulation of contraction.

Propagation of chemical waves in the Belousov-Zhabotinsky reaction in a thin layer of ferroin-loaded cationexchange resin beads is investigated. Dispersion relations are obtained for waves propagating in layers of resin beads. The relations in layers of resin beads are different both in the trigger wave velocity and the refractory period, when mesh size and/or percentages of cross-linkage of resin beads are different. A system with a sharp boundary between two layers with different wave velocities is made using two different resin beads exhibiting different dispersion relations. Incident waves at any angle from the high-velocity layer are refracted at the boundary. Waves from the low-velocity layer also exhibit refraction when the angle of incidence is smaller than a critical value. Refraction obeys Snell's law. Incident waves from the low-velocity layer at angles larger than the critical value are reflected. The angle of reflection is equal to the critical angle and does not depend on the incident angle. When the period of incident waves from the high-velocity layer is shorter than the refractory period in the low-velocity layer, wave propagation across the boundary causes the change of frequency.

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