Distribution of Ankyrin Isoforms and Their Proteolysis After Ischemia and Reperfusion in Rat Brain

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Abstract
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 ∼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.

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