Characterization of α s ‐immunoreactive ADP‐ribosylated proteins in postmortem human brain

ADP‐ribosylation of the stimulatory G protein α subunit, α s , has been demonstrated in a number of different mammalian tissues. However, little is known about the occurrence and role of this process in modifying α s levels/function in human brain. In the present study, endogenous and cholera toxin (CTX)‐catalyzed [ 32 P]ADP‐ribosylated products were characterized in postmortem human temporal cortex by (1) immunoprecipitation with α s antisera (RM/1), (2) comparisons of immunoblots and autoradiograms of the [ 32 P]ADP‐ribosylated products, and (3) limited protease digestion. Of the three major endogenous [ 32 P]ADP‐ribosylated products (48, 45, and 39 kDa) in postmortem brain, the 48‐kDa and 45‐kDa bands were clearly identified as α s‐L (long isoform) and α s‐S (short isoform), respectively. RM/1 immunoprecipitated the 39‐kDa [ 32 P]ADP‐ribosylated protein, and overlays of immunoblots and autoradiograms showed that this product corresponded to an α s ‐like‐immunoreactive protein. Furthermore, limited protease digestion of the 39‐kDa endogenous [ 32 P]ADP‐ribosylated band generated peptide fragments similar to both endogenous and CTX‐catalyzed [ 32 P]ADP‐ribosylated α s‐S . Two major CTX‐catalyzed [ 32 P]ADP‐ribosylated products were also identified as α s‐L (52 kDa) and α s‐S (45 kDa). These findings clearly demonstrate that α s is a substrate for endogenous and CTX‐catalyzed [ 32 P]ADP‐ribosylation in postmortem human brain. Furthermore, a lower molecular weight α s ‐like immunoreactive protein is also expressed in human brain and is a substrate for endogenous but not CTX‐catalyzed [ 32 P]ADP‐ribosylation. J. Neurosci. Res. 56:632–643, 1999. © 1999 Wiley‐Liss, Inc.