Post-mortem degradation of brain glutamate decarboxylase.
The post-mortem stability of the GABA synthesizing enzyme glutamate decarboxylase (GAD) was studied by using SDS-PAGE and quantitative immunoblotting to measure the rates of degradation of GAD in the cerebral cortex, hippocampus, and cerebellum of rats and mice as a function of time after death. The intact 65- and 67-kDa isoforms of GAD (GAD(65) and GAD(67)) disappeared gradually over a 24-h period. In both rats and mice, the degraded GAD appeared as a band with an apparent molecular mass of 55-57 kDa; no significant amounts of smaller forms were observed. The 55-57 kDa band reacted with antiserum W887, which recognizes a shared epitope at the carboxyl-terminal end of both GADs, indicating that GAD was cleaved near the amino-terminal end of the molecule. GAD(67) was cleaved at a site between the amino-terminus and the epitope for antiserum W883 (located within residues 79-93 of GAD(67)), as antiserum W883 stained a 56-kDa band on the blots. The appearance of degraded GAD paralleled the loss of total GAD (GAD(65)+GAD(67)), and after 24h the 55-57 kDa band accounted for 97, 88, and 59% of the intact GAD lost from rat cerebellum, cerebral cortex and hippocampus. On a percentage basis, GAD(67) was degraded more rapidly than was GAD(65) in all brain regions studied. The loss of GAD activity was greater in rat than mouse brain, even though the percent loss of intact GAD protein was similar.
Martin
Sandra B
SB
Wadsworth Center, New York State Department of Health, P.O. Box 509, Albany, NY 12201-0509, USA.
Waniewski
Robert A
RA
Battaglioli
Gino
G
Martin
David L
DL
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002531:Cerebellum / Q000201:enzymology; D002540:Cerebral Cortex / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005968:Glutamate Decarboxylase / Q000378:metabolism*; D006624:Hippocampus / Q000201:enzymology; D015151:Immunoblotting; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D011180:Postmortem Changes; D051381:Rats; D017208:Rats, Wistar; D013045:Species Specificity; D013997:Time Factors; D014018:Tissue Distribution
D007527:Isoenzymes; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; C401141:glutamate decarboxylase 2