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.
sodium dodecyl sulfate/polyacrylamide gel electrophoresis
- NaCl/Tris/HCl buffer
150 mM NaCl plus 50 mM Tris/HCl (pH 8.0)
enzyme-linked immunosorbent assay
120 mM NaCl plus 25 mM phosphate (pH 7.8)
bovine serum albumin
Adenylate cyclase or ATP pyrophosphate-lyase (cyclizing) (EC 126.96.36.199)