ISSN:
1471-4159
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Medicine
Notes:
Abstract: 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 [35S]guanosine 5′-O-(3-thiotriphosphate) ([35S]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-32P]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 [32P]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 [35S]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 [35S]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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1111/j.1471-4159.1991.tb02095.x
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