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.
|Number of pages
|European Journal of Biochemistry
|Published - Jul 1986
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