Distribution of ankyrin isoforms and their proteolysis after ischemia and reperfusion in rat brain

The distribution of brain-type ankyrin (ankyrin(B), 212 kDa) and erythrocyte-type ankyrin (ankyrin(R), 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 similar to 40% of the 212- and 163-kDa isoforms of ankyrin(B) and the 239-kDa isoform of ankyrin(R). 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 ankyrin(B) was more susceptible to calpain than was ankyrin(R). In the two-vessel occlusion model, ischemia for 30 min generated the 160-kDa fragment of ankyrin(R), 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 ankyrin(B). Both ischemia-reperfusion and in vitro proteolysis with calpain generated the 160-kDa fragment of ankyrin(R), suggesting the involvement of calpain.