Trypsin digestion of junctional sarcoplasmic reticulum vesicles.
A putative constituent of the junctional processes, connecting the terminal cisternae of sarcoplasmic reticulum and the transverse tubules of skeletal muscle fibers, is a greater than or equal to 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 greater than or equal to 350,000-Da protein [Inui, M., Saito, E., & 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., & Madson, L. (1987) J. Biol. Chem. 262, 6460-6463]. We subjected vesicular fragments of junctional-cisternal membrane to stepwise trypsin digestion. The greater than or equal to 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 greater than or equal to 350,000-Da protein yields fragments that are rapidly broken down to small peptides. Under conditions producing extensive digestion of the greater than or equal to 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Chu
A
A
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Sumbilla
C
C
Scales
D
D
Piazza
A
A
Inesi
G
G
D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism / Q000648:ultrastructure; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000648:ultrastructure*; D014357:Trypsin
D009124:Muscle Proteins; D012433:Ryanodine; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D002118:Calcium