Trypsin digestion of junctional sarcoplasmic reticulum vesicles
- 19 April 1988
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 27 (8) , 2827-2833
- https://doi.org/10.1021/bi00408a025
Abstract
A putative constituent of the junctional processes, connecting the terminal cisternae of sarcoplasmic reticulum and the transverse tubules of skeletal muscle fibers, is a .gtoreq. 350,000-dalton (Da) protein that displays ryanodine binding and Ca2+ channel properties. Ryanodine modulation of Ca2+ fluxes suggests that the ryanodine receptor and calcium channel are integral parts of one functional unit corresponding to the .gtoreq. 350,000-Da protein [Inui, M., Saito, E., and Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747; Campbell, K. P., Knudson, C. M., Imagawa, T., Leung, A. L., Sutko, J. L., Kahl, S. D., Raab, C. R., and Madson, L. (1987) J. Biol. Chem. 262, 6460-6463]. We subjected vesicular fragments of junctional-cisternal membrane to stepwise trypsin digestion. The .gtoreq. 350,000-Da protein is selectively cleaved in the early stage of digestion, with consequent disappearance of the corresponding band in electrophoretic gels. The Ca2+-ATPase is cleaved at a later stage, while calsequestrin is not digested under the same experimental conditions. While the Ca2+-ATPase yields two complementary fragments that are relatively resistant to further digestion, the .gtoreq. 350,000-Da protein yields fragments that are rapidly broken down to small peptides. Under conditions producing extensive digestion of the .gtoreq. 350,000-Da protein, the junctional processes are still visualized by electron microscopy, with no discernible alterations of their ultrastructure. The functional properties of the Ca2+ release channel are also maintained following trypsin digestion, including blockage by Mg2+ and ruthenium red and activation by Ca2+ and nucleotides. Prolonged incubations with and after trypsin produce functional alterations. Ryanodine, at relatively high (micromolar) concentrations, partitions into a nonsaturable compartment of the membrane and inhibits Ca2+ efflux through the release channel. On the other hand, high-affinity ryanodine binding (Kd = 3 .times. 10-7 M) occurs with a stoichiometry approximating that of the .gtoreq. 350,000-DA protein and produces a reduction of net Ca2+ uptake by the vesicles, due to increased Ca2+ efflux through the release channel. The bound ryanodine does not interfere with digestion of the .gtoreq. 250,000-Da protein and is still bound following digestion. We suggest that the .gtoreq. 350,000-Da protein permits entrance of trypsin into a large crevice (likely at the opening of the channel) where multiple cleavage sites are readily available. The resulting proteolytic fragments remain stabilized by multiple noncovalent interactions and are only dissociated by strong detergents. Additional protein components may contribute to structural stabilization of the junctional processes. Ryanodine binds to a protein domain where it does not interfere with trypsin binding but can regulate the channel through allosteric mechanisms.This publication has 2 references indexed in Scilit:
- Single channel and 45Ca2+ flux measurements of the cardiac sarcoplasmic reticulum calcium channelBiophysical Journal, 1986
- High molecular weight proteins in cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles bind calmodulin, are phosphorylated, and are degraded by Ca2+-activated protease.Journal of Biological Chemistry, 1984