hide
Free keywords:
Sarcoplasmic Reticulum. Ryanodine, Caffeine, Calcium Release
Abstract:
Heavy sarcoplasmic reticulum vesicles were reacted with ryanodine in 0.6 M KCl 0.3 M sucrose at pH 6.3 and pH 7.0 at 20 degrees C. The inhibition of caffeine induced calcium release from actively loaded vesicles by ryanodine was applied to monitor time course and attainment of equilibrium of the interaction of ryanodine with its receptors in the vesicular membranes. At ryanodine concentrations rising from 0.1-100 microM, the logarithms of the release amplitudes linearly decline with time. The dependence of the inactivation reaction on the concentration of ryanodine did not saturate in the applicable concentration range. The reaction halflife times are concentration dependent. At pH 7.0, the half times decline from 100 to 10 s when the ryanodine concentration is raised from 0.1 to 1 microM. At pH 6.3 a corresponding decline occurs between 3 microM and 100 microM. The marked dependence of the inactivation reaction on medium pH requires reaction times of one and five hours at pH 7.0 and 6.3, respectively for the attainment of reaction equilibrium at low ryanodine concentrations. The dependence of the amplitude of calcium release on the concentration of added ryanodine has been evaluated as proposed by Gutfreund (Enzymes: Physical Principles, p. 71, Wiley-Interscience, London 1972) for the preparation's affinity for ryanodine and its number of binding sites. At pH 7.0, preparations appear to contain only 0.7 pmol sites per mg protein having an affinity for ryanodine of 0.33 nM-1. The titration curves for caffeine induced calcium release, initial calcium uptake and final calcium level are identical, indicating that the three functions are controlled by the same receptor. Calcium induced calcium release, however, is only partially and differently affected by the occupancy of the high affinity ryanodine binding sites.
