POSITIONING OF MAJOR TRYPTIC FRAGMENTS IN THE CA2-MUSCLE SARCOPLASMIC-RETICULUM( RELEASE CHANNEL (RYANODINE RECEPTOR) RESULTING FROM PARTIAL DIGESTION OF RABBIT SKELETAL)

Site-specific antibodies against different regions of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor ) were developed and used as probes for immunoblotting of the major tr yptic fragments resulting from partial digestion of the ryanodine rece ptor in sarcoplasmic reticulum membranes. Five major tryptic fragments , some of which migrated as doublets, with apparent masses of 150/140, 110/100, 55, 170/160, and 76 kDa were ordered so that they covered th e bulk of the protein from the NH2 to the COOH terminus. Tryptic subfr agments of 53, 63, and 115/95 kDa were also derived from the 150/140-, 110/100-, and 170/160-kDa fragments, respectively. All of these fragm ents and subfragments were detected only in the insoluble membrane fra ction of the trypsinized sarcoplasmic reticulum. Upon Na2CO3 extractio n, the 150/140-, 110/100-, and 55-kDa fragments could be solubilized, suggesting their origin in the cytoplasmic domain of the ryanodine rec eptor. The 170/160- and 76-kDa fragments and the 115/195-kDa subfragme nt remained insoluble, suggesting their origin in the transmembrane re gion of the ryanodine receptor. The 150/140-,110/100-,170/160-, and 76 -kDa fragments and the 115/95 subfragment co-migrated near the bottom of a sucrose density gradient after CHAPS solubilization, suggesting t hat they were associated in an oligomeric complex. By contrast, the 53 - and 63-kDa subfragments and the 55-kDa fragment were detected near t he top of the sucrose gradient after CHAPS solubilization, suggesting that they were not involved in the formation of the core of the oligom eric complex. These studies identify 7 sites that are exposed to tryps in in the ryanodine receptor in sarcoplasmic reticulum, 3 of which are novel and 4 of which are in the same location as proteolytic cleavage sites identified previously (Marks, A. R., Fleischer, S., and Tempst, P. (1990) J. Biol. Chem. 265, 13143-13149).