Identification and characterization of the 35-kDa β subunit of guanine-nucleotide-binding proteins by an antiserum raised against transducin

Authors


Correspondence to W. Rosenthal, Pharmakologisches Institut der Freien Universität Berlin, Thielallee 69–73, D-1000 Berlin 33, Federal Republic of Germany

Abstract

Antisera were raised against the retinal guanine-nucleotide-binding protein (N-protein), transducin, purified from bovine rod outer segments. Sera obtained after repeated injections of antigen recognized all transducin subunits (α,β and γ). One antiserum, tested for cross-reactivity with non-retinal N-proteins, was found to cross-react with the β subunits of the ubiquitously occurring N-proteins, Ns and Ni, but not with their respective α and γ subunits. The antiserum also cross-reacted with the β subunit of the recently identified N-protein, No, which has been found in high abundance in the central nervous system. These data support the similarity of the β subunits of the N-proteins identified so far.

Purification of N-proteins from porcine cerebral cortex without the use of activating ligands yielded fractions containing the isolated α subunit of No, free βγ complex, Ni, No and fractions containing both N-proteins in various proportions. The purity of the preparations was at least 80% as judged by Coomassie-blue-stained SDS gels. No pure Ns was obtained. Use of the transducin antibody during the course of the purification revealed that the β subunits coeluted from a gel filtration column largely with the α subunits of Ni and No but were hardly detectable in fractions that were able to reconstitute Ns activity into membranes of an Ns-deficient cell line (S49 cyc lymphoma cells). This indicates that in the central nervous system the concentrations of Ni and No are of magnitudes higher than that of Ns.

Two-dimensional gel electrophoresis of N-proteins, purified from porcine cerebral cortex, resulted in the resolution of two major peptides in the 35-kDa region, which differed in their pI values and were identified as β subunits by the use of the antiserum. Identical results were achieved using crude cholate extracts from membranes of the same tissue instead of purified proteins. The occurrence of different β subunits may be explained by posttranslational N-protein modification.

Abbreviations
N-protein

guanine-nucleotide-binding protein

Chaps

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

ABTS

2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)

SDS-PAGE

sodium dodecyl sulfate/polyacrylamide gel electrophoresis

NaCl/Tris/HCl buffer

150 mM NaCl plus 50 mM Tris/HCl (pH 8.0)

ELISA

enzyme-linked immunosorbent assay

NaCl/Pi

120 mM NaCl plus 25 mM phosphate (pH 7.8)

BSA

bovine serum albumin

Enzyme
 

Adenylate cyclase or ATP pyrophosphate-lyase (cyclizing) (EC 4.6.1.1)

Ancillary