Andrea Migala

Thyroxine induced transformation in sarcoplasmic reticulum of rabbit soleus and psoas muscles.

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.

The arsenate induced calcium release from sarcoplasmic vesicles

The efflux of calcium from calcium loaded sarcoplasmic vesicles into solutions containing low free calcium concentrations gives rise to the formation of a phosphorylated intermediate in the membranes, the phosphoryl group of which can be transferred readily to ADP [ 1,3 ] . Hence, under steady state conditions when the slow calcium efflux is balanced by an ATP driven calcium influx a slow exchange between inorganic phosphate and the y-P of ATP takes place [2] . When the calcium influx ceases because the energy donators are exhausted or removed, the rate of net calcium efflux i.e. the rate of calcium release remains low as long as there is no ADP present to serve as phosphate acceptor. The transfer of the phosphoryl group to ADP, however, leads to a tremendous acceleration of calcium release which is stoichiometrically related to a net formation of ATP [3]. The calcium efflux dependent ATP formation is neither uncoupled by dinitrophenol nor by azide. On the other hand, all treatments which increase the calcium permeability of the sarcoplasmic membranes by leak formation abolish the osmochemical energy transduction irreversibly. This paper reports the reversible uncoupling of the calcium gradient dependent phosphoryl transfer by arsenate, the classical uncoupler of substrate linked and oxidative phosphorylation.

Tryptic Fragmentation of the Calcium Transport System in the Sarcoplasmic Reticulum

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.

Activation and inhibition of the calcium gate of sarcoplasmic reticulum by high-affinity ryanodine binding

The occupancy of high‐affinity ryanodine‐binding sites of isolated heavy sarcoplasmic reticulum vesicles occurring in concentrated salt solutions affects ATP‐dependent calcium accumulation and caffeine‐induced calcium release. The initial suppression of calcium uptake is followed by a marked uptake activation resulting in a reduction of the final calcium level in the medium. Simultaneously, caffeine‐induced calcium release is blocked. The dependence of inhibition of calcium uptake and caffeine‐induced calcium release observed in assay media containing physiological concentrations of magnesium and ATP on the concentration of ryanodine corresponds to the drugs effectiveness in living muscles.

The separation of the solubilized proteins of the sarcoplasmic reticulum on deae-cellulose and its modification

In the native membranes of the sarcoplasmic reticulum (SR) two different ATP hydrolyzing enzymes have been described, the basic and the extra-ATPase. Among other characteristics, the basic ATPase splits ATP in. the absence of calcium ions [ l-41 while the extra-ATPase needs for its activity a low concentration of ionized calcium. Therefore, the latter has also been named calcium dependent ATPase (c.f. [5]). It is presumably identical with the structure which translocates calcium from outside to inside of the SR vesicles consuming ATP and which incorporates inorganic phosphate into ADP when calcium moves from inside to outside following a concentration gradient [5, 61. A most interesting property of the protein component of the energy converting structure is its high degree of functional flexibility. Its activity can be manipulated by removal and readdition of natural and artificial lipid compounds [4,7,8]. As long as natural lipids are used, none or only slight change of the activity pattern occurs but as recently shown by Walter [9] a treatment of the SR membranes with the artificial lipid compound, Triton X-100, produces a complete change of their activity pattern. In this report the interchange of the calcium dependent ATPase activity as it results when the SR membranes, dissolved in Triton X-100, are fraction-

Cations and anions as modifiers of ryanodine binding to the skeletal muscle calcium release channel

Abstract. Rate and equilibrium measurements of ryanodine binding to terminal cysternae fractions of heavy sarcoplasmic reticulum vesicles demonstrate that its activation by high concentrations of monovalent salts is based on neither elevated osmolarity nor ionic strength. The effect of the ions specifically depends on their chemical nature following the Hofmeister ion series for cations (Li+ < NH+4 < K−∼ Cs+≤ Na+) and anions (gluconate− < Cl− < NO3−∼ ClO4−∼ SCN−) respectively, indicating that both are involved in the formation of the salt-protein complex that can react with ryanodine. Activation by rising salt concentrations exhibits saturation kinetics with different dissociation constants (25–11 m) and different degrees of cooperativity (n= 1.5–4.0) for the respective salts. Maximal second order binding rates between 40,000 and 80,000 (m−1· sec−1) were obtained for chlorides and nitrates of 1a group alkali ions with the exception of lithium supporting only rates of maximally 10,000 (M−1· sec−1). The nitrogen bases, NH+4 and Tris+, in combination with chloride or nitrate, behave divergently. High maximal binding rates were achieved only with NH4NO3. The dissociation constants for the ryanodine–protein complexes obtained by measurements at equilibrium proved to depend differently on salt concentration, yet, converging to 1–3 nm for the applied salts at saturating concentrations. The salts do not affect dissociation of the ryanodine protein complex proving that the effect of salts on the proteins affinity for ryanodine is determined by their effect on the on-rate of ryanodine binding. ATP and its analogues modify salt action resulting in elevated maximal binding rates and reduction or abolition of binding cooperativity. Linear relations have been obtained by comparing the rates of ryanodine binding at different salt concentrations with the rates or the initial amplitudes (15 sec) of salt induced calcium release from actively loaded heavy vesicles indicating that the various salts promote specifically and concentration dependently channel opening and its reaction with ryanodine.

Modulation by Monovalent Anions of Calcium and Caffeine Induced Calcium Release from Heavy Sarcoplasmic Reticulum Vesicles

Both calcium and caffeine induced calcium release from actively loaded heavy sarcoplasmic reticulum vesicles were studied to analyze the dependence of both activities on the composition of the release medium with respect to monovalent anions. Calcium is unable to induce net calcium release while caffeine remains effective as releasing agent when the experimental media contain neither chloride nor nitrate ions. Caffeine induced calcium release is not suppressed by chelating residual medium calcium (approximately 0 .5 -1 μᴍ) with 2 mᴍ EGTA added 15 s prior to 10 mᴍ caffeine. Calcium release from vesicles loaded in media containing 0.2 ᴍ gluconate as monovalent anion is induced when the medium is supplemented with chloride or nitrate. The release amplitude increases linearly when K-gluconate is replaced by KCl. At constant ionic strength the release amplitude becomes maximal at a chloride concentration of 0.2 ᴍ. The chloride effect completely disappears when 2 mᴍ EGTA are added simultaneously. When chloride is replaced by nitrate, as releasing agent, maximal release is achieved already by addition of 0.1 ᴍ K-nitrate. The releasing effect of nitrate can only partially be suppressed by EGTA. The different effectiveness of gluconate, chloride and nitrate as calcium release supporting ions corresponds to their activating effect on the binding of ryanodine to the calcium release channel in the vesicular membranes.

How many ryanodine binding sites are involved in caffeine induced calcium release from sarcoplasmic reticulum terminal cysternae vesicles

Abstract The inhibition by ryanodine of caffeine induced calcium release from actively loaded heavy sarcoplasmic vesicles has been studied in order to analyse the relation between the occupancy of the vesicular calcium release channels by ryanodine and channel function. Ryanodine binding was monitored with [3H]ryanodine under ionic conditions favouring the establishment of binding equilibrium. Binding follows 1 : 1 stoichiometry yielding dissociations constants between 7 - 12 nᴍ and 12-15 pmol ryanodine/mg vesicular protein as maximum number of ryanodine binding sites. When ryanodine labeling was monitored by measuring the decline of the amplitude of caffeine induced calcium release 50% inhibition occurred at a free ryanodine concentration of 1 nM. At this concentration less than 10% of the available ryanodine binding sites are occupied. Caffeine induced calcium release is completely abolished when 3 pmol ryanodine/mg have reacted. A corresponding divergence between ryanodine binding and its effect on caffeine induced calcium release was observed when the initial rate of ryanodine binding was measured either by labeling the vesicles with [3H]ryanodine or by following the decline with time of caffeine induced calcium release. Caffein induced calcium release declines four times faster than the fraction of unoccupied ryanodine binding sites, k = 4.3 x 104 ᴍ-1 s-1 versus 1.2 x 104 ᴍ-1 s-1. The observed interrelation between the occupation of ryanodine binding sites and its effect on caffeine induced calcium release indicates that the caffeine sensitive calcium channel functions as an assembly of at least 4 ryanodine binding sites whereby the occupation of one site suffices to abolish calcium release. The stoichiometric composition appears to be not fixed but might change according to the size of the fraction of ryanodine receptors exhibiting caffeine sensitivity. The reported data were evaluated according to the algorithm derived by H. Asai and M. F. Morales, J. Biol. Chem. 4, 830-838 (1965) for the activity of a macromolecule and the extent of an inhibiting reaction.

Interaction of ryanodine with the calcium releasing system of sarcoplasmic reticulum vesicles

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))

Arrangement of proteins and lipids in the sarcoplasmic membrane.

The number of amino residues present in the proteins of the sarcoplasmic reticulum which can react with Fluram has been determined in native and sonicated SR vesicles. Sonication increases the number of amino groups accessible to Fluram from 0.57 to 0 .8 7 μmol·mg prot.-1. This increase indicates that 66% of the amino residues are present in the external and 34% in the internal membrane leaflet. The distribution of the amino phospholipids is computed from the distribution of Fluram in the membrane proteins in conjunction with the relative distribution of Fluram between protein and lipid in native and sonicated vesicles. The distribution of the calcium transport protein has been approximated under different assumptions concerning the distribution of the residual protein and taking into account that 15% of the membranes of the SR vesicles might have changed their sideness during preparation.