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Detection of G Proteins in Purified Bovine Brain Myelin
Author(s) -
Larocca J. N.,
Golly F.,
Ledeen R. W.
Publication year - 1991
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1991.tb02095.x
Subject(s) - g protein , myelin , gtp' , pertussis toxin , biochemistry , gel electrophoresis , chemistry , polyacrylamide gel electrophoresis , adp ribosylation , cholera toxin , gtp binding protein regulators , microbiology and biotechnology , receptor , biology , nad+ kinase , enzyme , neuroscience , central nervous system
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 [ 35 S]guanosine 5′‐ O ‐(3‐thiotriphosphate) ([ 35 S]GTPγS) 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, [α‐ 32 P]GTP bound to three bands in the 21–27‐kDa range in a manner inhibited by GTP and GTPγS but not App(NH)p. ADP‐ribosylation of myelin with [ 32 P]NAD + and cholera toxin labeled a protein of 43 kDa, whereas reaction with pertussis toxin labeled two components of 40 kDa. Cholatc extract of myelin subjected to chromatography on a column of phenyl‐Sepharose gave at least three major peaks of [ 35 S]GTPγS binding activity. SDS‐PAGE and immunoblot analyses of peak I indicated the presence of Goα, Giα, and Gsα. Further fractionation of peak II by diethyl‐aminoethyl‐Sephacel chromatography gave one [ 35 S]GTPγS 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 β subunit, whereas the fraction containing 40‐kDa polypeptides reacted with specific antibodies to Goα and Giα. 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.