DETECTION OF G-PROTEINS IN PURIFIED BOVINE BRAIN MYELIN

Following a previous report on detection of muscarinic receptors in myelin with the implied presence of G proteins, we now demonstrate by more direct means the presence of such proteins and their quantification. Using [S-35]guanosine 5'-O-(3-thiotriphosphate) ([S-35]GTP-gamma-S) as the binding ligand, purified myelin from bovine brain was found to contain approximately half the binding activity of whole white matter (138 +/- 9 vs. 271 +/- 18 pmol/mg of protein). Scatchard analysis of saturation binding data revealed two slopes, a result suggesting at least two binding populations. This binding was inhibited by GTP and its analog but not by 5'-adenylylimidodiphosphate [App(NH)p], GMP, or UTP. Following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of myelin proteins and blotting on nitrocellulose, [alpha-P-32]GTP bound to three bands in the 21-27-kDa range in a manner inhibited by GTP and GTP-gamma-S but not App(NH)p. ADP-ribosylation of myelin with [P-32]NAD+ and cholera toxin labeled a protein of 43 kDa, whereas reaction with pertussis toxin labeled two components of 40 kDa. Cholate extract of myelin subjected to chromatography on a column of phenyl-Sepharose gave at least three major peaks of [S-35]GTP-gamma-S binding activity. SDS-PAGE and immunoblot analyses of peak I indicated the presence of Go-alpha, Gi-alpha, and Gs-alpha. Further fractionation of peak II by diethylaminoethyl-Sephacel chromatography gave one [S-35]GTP-gamma-S binding peak with the low-molecular-mass (21-27 kDa) proteins and a second showing two major protein bands of 36 and 40 kDa on SDS-PAGE. Immunoblot analysis of this material identified the 36-kDa protein as the beta-subunit, whereas the fraction containing 40-kDa polypeptides reacted with specific antibodies to Go-alpha and Gi-alpha. Thus, purified myelin from bovine brain has been shown to contain several GTP-binding proteins resembling in broad outline the G proteins of whole brain and potentially able to transduce signals received by muscarinic (and perhaps other) receptors in this membrane.