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The Biological Bulletin, Vol 185, Issue 3 346-354, Copyright © 1993 by Marine Biological Laboratory

CELL BIOLOGY

Differential Susceptibility of Guanine Nucleotide-binding Proteins to Pertussis Toxin-catalyzed ADP-ribosylation in Brain Membranes of Two Congeneric Marine Fishes

T. F. Murray and J. F. Siebenaller
College of Pharmacy, Oregon State University, Corvallis, Oregon 97331

Pertussis toxin-catalyzed [32P]ADP-ribosylation was used to probe the guanine nucleotide binding regulatory proteins Gi and Go in brain membranes from two scorpaenid fishes, Sebastolobus alascanus and S. altivelis. The membranes of the two species exhibit a differential sensitivity to [32P]ADP-ribosylation produced by a fixed concentration of pertussis toxin. The membranes from the deeper-living S. altivelis consistently incorporated more [32P]ADP than the membranes from S. alascanus. Proteins of 39 and 41 kDa are specifically labeled in both species, corresponding to the apparent molecular masses of the {alpha} subunits of Gi and Go. At 5{deg}C the ribosylation reaction is linear for at least 7 h. The pertussis toxin concentration-response relationship was evaluated with concentrations of pertussis toxin from 0 to 100 ng/ {mu}l. The extent of [32P]ADP-ribosylation was quantified by autoradiography and computer-assisted image analysis. The EC50 values for pertussis toxin were similar for the two species, but the maximum level of [32P]ADP-ribosylation was significantly greater in S. altivelis brain membranes. Because the heterotrimeric holoprotein is the substrate for ribosylation, the modulatory effects of the guanyl nucleotides GDP and GTP{gamma}S on the ribosylation were assessed. GDP increased [32P]ADP-ribosylation of the {alpha} subunits in S. altivelis. Only the highest concentration tested (1000 {mu}M) increased [32P]ADP-ribosylation in S. alascanus brain membranes and only to a modest extent. Increasing concentrations of GTP{gamma}S suppressed [32P]ADP-ribosylation in S. alascanus brain membranes, presumably by promoting dissociation of the holotrimer. GTP{gamma}S had much less of an effect on the S. altivelis brain membranes. These differences in the extent of ADP-ribosylation and the modulatory effects of guanyl nucleotides may reflect different coupling efficiencies of G proteins and receptors. The expression of the {alpha} and {beta} subunits of Gi and Go in the two Sebastolobus species, the deep-sea morid teleost fish Antimora rostrata, and the rat were compared by Western immunoblotting of brain membranes with antipeptide antisera. Levels of Gi{alpha}3 were 63% higher in brain membranes of S. altivelis than those in S. alascanus. The levels of Gi{alpha}1, Gi{alpha}2, Go and {beta}36 were similar in the two species. Although the complement of G proteins identified by the array of antisera used was similar in all the species, there appears to be additional diversity of {alpha} subunits in the teleost brain membranes. In fish, antiserum to Go{alpha} reacted with an additional 41 to 42 kDa protein that was not expressed in rat brain.




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Copyright © 1993 by the Marine Biological Laboratory.