Characterization of the eIF2-associated protein p67 during brain ischemia and reperfusion.
OBJECTIVE
Within the first few minutes of reperfusion after global brain ischemia, there is a severe depression of protein translation owing to phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2). There is a 67 kDa peptide (p67) that, in its glycosylated form, binds to eIF2 and protects eIF2alpha from phosphorylation. Moreover, cells with high p67 content exhibit enhanced resistance to eIF2alpha phosphorylation. To examine the possibilities that deglycosylation of brain p67 occurs during ischemia and/or early reperfusion or that p67 deglycosylation may be more extensive in the vulnerable neurons, these experiments were undertaken to characterize the localization and activation state of p67 during early brain reperfusion


METHODS
Western blots using antibodies that recognize total p67, glycosylated p67 and phosphorylated eIF2alpha were used to characterize total p67 and glycosylated p67 during reperfusion-induced phosphorylation of eIF2alpha. We also characterized the immunohistochemical distribution of glycosylated p67 before and after brain ischemia and reperfusion.


RESULTS
There was a large increase in phosphorylated eIF2alpha, but there was no decrease in the levels of total or glycosylated p67 from those observed in controls following 10 minutes complete brain ischemia and 10 or 60 minutes subsequent reperfusion. Furthermore, there was no reduction in localized immunostaining for glycosylated p67 in vulnerable neurons during ischemia and reperfusion.


CONCLUSIONS
It does not appear that p67 plays a significant role in regulating the phosphorylation of eIF2alpha following transient brain ischemia.
Owen
Cheri
C
Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Lipinski
Christopher
C
Page
Andrea
A
White
Blaine
B
Sullivan
Jonathon
J
Rafols
José
J
Krause
Gary
G
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002545:Brain Ischemia / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D006023:Glycoproteins / Q000378:metabolism*; D006624:Hippocampus / Q000378:metabolism / Q000473:pathology; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D010766:Phosphorylation; D051381:Rats; D020318:Rats, Long-Evans; D015424:Reperfusion; D013997:Time Factors
D006023:Glycoproteins