Characterization of alpha(s)-immunoreactive ADP-ribosylated proteins in postmortem human brain

ADP-ribosylation of the stimulatory G protein alpha subunit, alpha(s), has been demonstrated in a number of different mammalian tissues. However, litt le is known about the occurrence and role of this process in modifying alph a(s) levels/function in human brain. In the present study, endogenous and c holera toxin (CTX)-catalyzed [P-32]ADP-ribosylated products were characteri zed in postmortem human temporal cortex by (1) immunoprecipitation with alp ha(s) antisera (RM/1), (2) comparisons of immunoblots and autoradiograms of the [P-32]ADP-ribosylated products, and (3) limited protease digestion. Of the three major endogenous [P-32]ADP-ribosylated products (48, 45, and 39 kDa) in postmortem brain, the 48-kDa and 45-kDa bands were clearly identifi ed as alpha(s-L) (long isoform) and alpha(s-)S (short isoform), respectivel y. RM/1 immunoprecipitated the 39-kDa [P-32]ADP-ribosylated protein, and ov erlays of immunoblots and autoradiograms showed that this product correspon ded to an alpha(s)-like-immunoreactive protein. Furthermore, limited protea se digestion of the 39-kDa endogenous [P-32]ADP-ribosylated band generated peptide fragments similar to both endogenous and CTX-catalyzed [P-32]ADP-ri bosylated alpha(s-S-). Two major CTX-catalyzed [P-32]ADP-ribosylated produc ts were also identified as alpha(s-L) (52 kDa) and alpha(s-S) (45 kDa), The se findings clearly demonstrate that (alpha(s), is a substrate for endogeno us and CTX-catalyzed [P-32]ADP-ribosylation in postmortem human brain. Furt hermore, a lower molecular weight alpha(s)-like immunoreactive protein is a lso expressed in human brain and is a substrate for endogenous but not CTX- catalyzed [P-32]ADP-ribosylation. (C) 1999 Wiley-Liss, Inc.