Distribution of ankyrin isoforms and their proteolysis after ischemia and reperfusion in rat brain.
The distribution of brain-type ankyrin (ankyrinB, 212 kDa) and erythrocyte-type ankyrin (ankyrinR, 239 kDa) was investigated in the subcellular fractions of rat forebrain (P1, 1,000 g pellet; P2, 15,000 g pellet; P3, 100,000 g pellet; S, 100,000 g supernatant) by immunoblotting using specific antibodies. The P2 fraction contained approximately 40% of the 212- and 163-kDa isoforms of ankyrinB and the 239-kDa isoform of ankyrinR. Further subfractionation of the P2 by Percoll gradient centrifugation followed by separation of myelin showed association of the three ankyrin isoforms with the synaptosome-rich fraction but not with the myelin-rich fraction. The plasma membrane-rich P3 fraction contained a concentration of ankyrin isoforms similar to that in the P2 fraction. In vitro proteolysis of ankyrin in the P2 fraction with calpain showed that the 212-kDa ankyrinB was more susceptible to calpain than was ankyrinR. In the two-vessel occlusion model, ischemia for 30 min generated the 160-kDa fragment of ankyrinR, and reperfusion for 60 min after 30 min of ischemia remarkably increased the 160-kDa fragment. The reperfusion also significantly decreased the 212-kDa isoform of ankyrinB. Both ischemia-reperfusion and in vitro proteolysis with calpain generated the 160-kDa fragment of ankyrinR, suggesting the involvement of calpain.
Harada
K
K
Department of Legal Medicine, Yamaguchi University School of Medicine, Ube, Japan.
Fukuda
S
S
Kunimoto
M
M
Yoshida
K
K
D000818:Animals; D017487:Ankyrins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D001923:Brain Chemistry / Q000502:physiology; D002545:Brain Ischemia / Q000378:metabolism*; D002154:Calpain / Q000494:pharmacology; D007536:Isomerism; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000378:metabolism*; D013347:Subcellular Fractions / Q000737:chemistry
D017487:Ankyrins; D002154:Calpain