Identification of heterotrimeric and low molecular weight GTP-binding proteins in rabbit skeletal muscle longitudinal sarcoplasmic reticulum

Abstract

Direct photoaffinity labeling of proteins of longitudinal sarcoplasmic reticulum (LSR) of rabbit skeletal muscle with [ 32P]GTP revealed GTP-binding proteins of about 52, 45 and 30 kDa. ADP-ribosylation with [ 32P]NAD in the presence of cholera toxin (CTX) or pertussis toxin (PTX) indicates the existence of a CTX substrate (about 45 kDa); no PTX substrates were observed. Western blots of LSR probed with RM/1, an antiserum against a decapeptide from the C-terminus of G sα, showed an immunoreactive band at about 45 kDa. [ 32P]GTP overlays of Western blots of LSR showed a heavily-labeled protein of about 29 kDa and one or more additional slightly smaller proteins that were more weakly labeled. When LSR was subjected to mild trypsin hydrolysis, the Western blot overlay revealed three bands at about 23, 25 and 29 kDa. Western blots of LSR proteins showed no significant immunoreactivity with the anti-(pan)- ras monoclonal antibodies 142-24E05 and Ras 11. ADP-ribosylation of LSR with [ 32P]NAD in the presence of C3 exoenzyme of Clostridium botulinum yielded a labeled band at about 23 kDa. Our results indicate the presence in rabbit LSR of a G sα, the absence of G i and G o, and the presence of several low molecular weight GTP-binding proteins, distinct from p21 ras, one of which belongs to the rho or rac family.