Trypsin destruction of the high affinity ryanodine binding sites of the junctional sarcoplasmic reticulum.
Tryptic digestion of the junctional sarcoplasmic reticulum membranes in sucrose but not NaCl buffer leads to complete loss of ryanodine binding capacity. The presence of MgCl2 in the sucrose buffer prevents the loss of ryanodine binding by the trypsin treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the treated membranes reveal that the 400-kDa protein band disappeared under all the different digestion conditions. However, the presence of 135-kDa tryptic fragment is observed only when ryanodine binding is retained. Quantitative analysis of the gels shows that the loss of ryanodine binding is well correlated with the cleavage of the 135-kDa tryptic fragment. This correlation is obtained when the cleavage was controlled either by the digestion time or by NaCl or MgCl2 concentrations. The same concentrations of MgCl2 and NaCl affect the ryanodine binding activity, the cleavage of the 135-kDa tryptic fragment, and the solubility and stability of the [3H]ryanodine-receptor complex in a detergent-containing medium. Tryptic digestion of the ryanodine receptor/junctional Ca2+ release channel, which leads to complete loss of ryanodine binding capacity, has no effect or slightly stimulates the Ca2+ accumulation activity of these membranes.
Shoshan-Barmatz
V
V
Department of Biology, Ben Gurion University of the Negev, Beer Sheva, Israel.
Zarka
A
A
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000470:Alkaloids / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism*
D000470:Alkaloids; D010446:Peptide Fragments; D012433:Ryanodine; D000266:Adenylyl Imidodiphosphate; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D014357:Trypsin; D008274:Magnesium; D002118:Calcium