Endogenous ADP‐Ribosylation in Brain: Initial Characterization of Substrate Proteins

Abstract

Cholera and pertussis toxin-mediated ADP-ribo-sylation has been used extensively to study regulation of guanine nucleotide binding proteins (G proteins) in the nervous system, but much less is known about possible endogenous ADP-ribosylation of G proteins in brain. The present study demonstrates endogenous ADP-ribosylation, in the absence of cholera and pertussis toxins, of four predominate proteins in homogenates of rat cerebral cortex. These proteins showed apparent molecular masses of 20, 42, 45, and SO kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 42- and 45-kDa proteins comigrated precisely with the major cholera toxin-labeled bands. Furthermore, the endogenous ADP-ribosylated and cholera toxin-ADP-ribosylated bands yielded identical ³²p-labeled peptide fragments by one-dimensional peptide mapping, indicating that they are probably the same proteins, presumably the α-subunits of G_s. In contrast, peptide maps of the 50-kDa protein, which migrated close to a 48-kDa cholera toxin-labeled band, demonstrated that this protein is distinct from the toxin-labeled band and from G_sα. Levels of endogenous ADP-ribosylation activity showed regional heterogeneity in brain, with a nearly threefold variation observed among the brain regions examined. Chronic administration (7 days) of corticosterone significantly increased overall levels of endogenous ADP-ribosylation, indicating that components of this system may be under hormonal control in vivo. Attempts to identify neurotransmitters or second messenger systems that regulate endogenous ADP-ribosylation activity in brain have so far been unsuccessful with one exception. Sodium nitroprusside, which increases the formation of nitric oxide, dramatically stimulated the endogenous ADP-ribosylation of a 36-kDa protein, as reported previously by others, and, under certain conditions, also stimulated ADP-ribosylation of the 42-, 45-, and 50-kDa proteins, although to a lesser extent than the 36-kDa protein. These findings demonstrate that G_Sα and additional, as yet unidentified, proteins are ADP-ribosylated in brain in the absence of bacterial toxins and suggest that such endogenous covalent modification may regulate the functional activity of these proteins in vivo.