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, little is known about the occurrence and role of this process in modifying alpha(s) levels/function in human brain. In the present study, endogenous and cholera toxin (CTX)-catalyzed [32P]ADP-ribosylated products were characterized in postmortem human temporal cortex by (1) immunoprecipitation with alpha(s) antisera (RM/1), (2) comparisons of immunoblots and autoradiograms of the [32P]ADP-ribosylated products, and (3) limited protease digestion. Of the three major endogenous [32P]ADP-ribosylated products (48, 45, and 39 kDa) in postmortem brain, the 48-kDa and 45-kDa bands were clearly identified as alpha(s-L) (long isoform) and alpha(s-S) (short isoform), respectively. RM/1 immunoprecipitated the 39-kDa [32P]ADP-ribosylated protein, and overlays of immunoblots and autoradiograms showed that this product corresponded to an alpha(s)-like-immunoreactive protein. Furthermore, limited protease digestion of the 39-kDa endogenous [32P]ADP-ribosylated band generated peptide fragments similar to both endogenous and CTX-catalyzed [32P]ADP-ribosylated alpha(s-S). Two major CTX-catalyzed [32P]ADP-ribosylated products were also identified as alpha(s-L) (52 kDa) and alpha(s-S) (45 kDa). These findings clearly demonstrate that alpha(s) is a substrate for endogenous and CTX-catalyzed [32P]ADP-ribosylation in postmortem human brain. Furthermore, a lower molecular weight alpha(s)-like immunoreactive protein is also expressed in human brain and is a substrate for endogenous but not CTX-catalyzed [32P]ADP-ribosylation.
Andreopoulos
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S
Section of Biochemical Psychiatry, Clarke Institute of Psychiatry, Toronto, Ontario, Canada.
Li
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PP
Siu
K P
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Warsh
J J
JJ
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001344:Autopsy; D001345:Autoradiography; D001921:Brain / Q000378:metabolism*; D002772:Cholera Toxin / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D009243:NAD / Q000378:metabolism*; D010449:Peptide Mapping; D010761:Phosphorus Radioisotopes; D013702:Temporal Lobe / Q000378:metabolism
D010761:Phosphorus Radioisotopes; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; D019205:GTP-Binding Protein alpha Subunits, Gs; D004229:Dithiothreitol