| | 2 | Author
| Wilhelm Hasselbach, Andrea Migala | Requires cookie* | | | Title
| Interaction of Ryanodine with the Calcium Releasing System of Sarcoplasmic Reticulum Vesicles  | | | | 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 °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 | XM, 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 [J, M. At pH 6.3 a corresponding decline occurs between 3 (XM and 100 | AM. 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 equilib-rium 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"'. 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 [ 3 H]ryanodine binding (Pessah et al., J. Biol. Chem. 261, 8643-8648 (1986)). | | | |
Reference
| Z. Naturforsch. 43c, 140—148 (1988); received November 4 1987 | | | |
Published
| 1988 | | | |
Keywords
| Sarcoplasmic Reticulum Ryanodine, Caffeine, Calcium Release | | | |
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| default:Reihe_C/43/ZNC-1988-43c-0140.pdf | | | | Identifier
| ZNC-1988-43c-0140 | | | | Volume
| 43 | |
| 3 | Author
| Wilhelm Hasselbach, Andrea Migala | Requires cookie* | | | Title
| 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 bind ing was monitored with [3H]ryanodine under ionic conditions favouring the establishment of binding equilibrium. Binding follows 1 : 1 stoichiometry yielding dissociations constants be tween 7 — 12 nM 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 m -1 s_1 versus 1.2 x 104 M" 1 s-1. The observed interrelation between the occupation of ryanodine bind ing 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 ap pears to be not fixed but might change according to the size of the fraction of ryanodine recep tors exhibiting caffeine sensitivity. The reported data were evaluated according to the algo rithm 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. | | | |
Reference
| Z. Naturforsch. 47c, 136—147 (1992); received August 7 1991 | | | |
Published
| 1992 | | | |
Keywords
| Sarcoplasmic Reticulum, Caffeine, Calcium Release, Ryanodine Binding | | | |
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| default:Reihe_C/47/ZNC-1992-47c-0136.pdf | | | | Identifier
| ZNC-1992-47c-0136 | | | | Volume
| 47 | |
| 4 | Author
| J. Kukulies, W. Stockem, K.E W Oh Lfarth-B, Tterm An | Requires cookie* | | | Title
| Caffeine-Induced Surface Blebbing and Budding in the Acellular Slime Mold Physarum polycephalum | | | | Abstract
| The mechanism of plasma m em brane proliferation was studied in the acellular slime mold Physarum polycephalum with the aid o f light and electron microscopical techniques. Treatm ent of protoplasmic drops with a Tris-buffered 15 m M caffeine solution causes surface blebbing and budding over periods of 5 -9 0 min. The process o f surface blebbing is coupled to a 5 -10-fold increase of the surface area in conjunction with characteristic changes in cytoplasmic m orphol ogy. Successive constriction of blebs exhibiting different sizes and degree o f hyalo-granulo-plasmic separation leads to the form ation of num erous spherical caffeine droplets. D uring the process of surface budding and droplet formation the total surface area o f the original (genuine) protoplasmic drop is not reduced, but continues to grow. Freeze-etch studies show that caffeine concomitantly causes characteristic changes in the fine structure of the plasma membrane. D uring the initial phase of surface blebbing the original density of intram embranous particles (IMP) is reduced from 3676/|am2 to 1669/um2 and the PF; EF ratio (IM P/|im 2 protoplasmic face: exoplasmic face) shifts from 2.4:1 to 2.8:1. W hen surface budding is completed the IMP-density in the plasm a m em brane of single caffeine droplets increases again to 2289/nm 2 and the P F :E F ratio changes to 1.5:1. Simultaneously, the isolated caffeine droplets produce numerous small hyaline m em brane protrusions, which are pinched off and contain no IMP. Control experiments dem onstrate that T ris-buffer w ithout caffeine also shows a weak capacity to induce surface blebbing, to change the IM P-density and the P F : EF ratio (2443/nm2; 1.5:1); but Tris-buffer fails to cause surface budding. On the other hand, different concentrations of sucrose (2 5 -2 0 0 m M) can supress to a certain degree both caffeine-and Tris-buffer-induced surface blebbing, but not caffeine-dependent surface budding. The caffeine-effect is reversible insofar as protoplasm ic drops with blebbing and budding activity recover to normal morphology, fine structure and locom otion when transferred to physiological conditions. The mechanisms of successive changes in plasm a m em brane morphology as well as the mode of a participation of the actomyosin system in cell surface dynamics are discussed. | | | |
Reference
| Z. Naturforsch. 38c, 589—5 (1983); received F ebruary 7 1983 | | | |
Published
| 1983 | | | |
Keywords
| Physarum polycephalum, Surface Blebbing and Budding, Caffeine, Plasmalemm a, Cytoplasmic Äctomyosin | | | |
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| default:Reihe_C/38/ZNC-1983-38c-0589.pdf | | | | Identifier
| ZNC-1983-38c-0589 | | | | Volume
| 38 | |
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