No abstract

The properties of the sarcoplasmic reticulum membranes isolated from slow-twitch type I soleus and fast-twitch type II psoas muscles of control and thyroxine treated rabbits were comparatively studied. Membrane yield, maximal calcium storing capacity, ATP-supported calcium uptake, calcium-dependent ATPase activity and calcium-dependent phosphoprotein formation were found to be 3-10 fold higher in psoas than in soleus preparations. Membrane yield, calcium-dependent ATPase activity, ATP-supported calcium transport and calcium-dependent phospho­ protein are at least twice enhanced in the membranes from soleus muscles of animals treated for 14-21 days with thyroxine. The corresponding capacities of the membranes from psoas muscles are not further augmented by the same thyroxine treatment. The maximal calcium storing capaci­ ty of the psoas membranes is their sole specific property which is significantly increased. The changes in the properties of the soleus muscles’ sarcoplasmic reticulum membranes are engen­dered by an increase from 5 to 30-50% in the number of type II fibres. Since the calcium transporting properties of the sarcoplasmic reticulum membranes from type II fibres qualitatively differ from those of type I fibres, thyroxine does not only affect quantitative but also qualitative parameters of the muscles’ sarcoplasmic reticulum membrane system.

Two protein fragments with a molecular weight of 50-60 000 daltons are formed when the calcium transport ATPase of the SR is mildly digested with trypsin. The initial fragmentation of the ATPase does not interfere with calcium transport, calcium dependent ATPase activity and phosphoprotein formation. The decay of the initially formed protein fragments after prolonged tryptic digestion is accompanied by the decline of the rate of calcium uptake and the calcium concentrating ability while the activity of the calcium activated ATPase is reduced only moderately. The initial tryptic fragmentation does not give rise to any change in the morphological appearance in the SR membranes. After prolonged digestion brush border or smooth surface structures are observed depending on the agent used for negative staining

Heavy sarcoplasmic reticulum vesicles were reacted with ryanodine in 0.6 ᴍKCl 0.3 ᴍ sucrose at pH 6.3 and pH 7.0 at 20 °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 μᴍ, 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 μᴍ. At pH 6.3 a corresponding decline occurs between 3 μᴍ and 100 μᴍ. 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 nᴍ-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. The kinetic and equilibrium data for the effects of ryanodine were combined and analyzed on account of a two step reaction sequence. The corresponding dissociation and rate constants were evaluated and combined with reported data of [3H]ryanodine binding (Pessah et al., J. Biol. Chem. 261, 8643-8648 (1986))