Proteolysis of erythrocyte-type and brain-type ankyrins in rat heart after postischemic reperfusion.
Ankyrin links cytoskeleton and integral membrane proteins and is proteolyzed in vitro by calpain, a Ca2+-dependent protease. In the present study, we examined the localization of two ankyrin isoforms, erythrocyte (red blood cell)-type (ankyrin(R)) and brain-type (ankyrin(B)), and their proteolysis after ischemia-reperfusion in the subcellular fractions of perfused rat heart by immunoblotting and by immunohistochemistry using specific antibodies. Both isoforms were observed to be distributed chiefly in the myofibril-nucleus (1,OOOx g pellet: P1) fraction, while ankyrin(R) was located substantially in the membrane (100,000x g pellet: P2) fraction. Reperfusion after 10 min or more of global ischemia induced preferential proteolysis of ankyrin(R) in the P2 fraction and ankyrin(B) in the P1 fraction. The proteolysis of ankyrin(R), but not ankyrin(B), was effectively inhibited by the synthetic calpain inhibitor acethyl-leucyl-leucyl-norleucinal. The immunohistochemical examination showed that anti-ankyrin(R) delineated striations, sarcolemma and nuclei, and the staining was decreased after ischemia-reperfusion, while anti-ankyrin(B) showed diffuse staining. The proteolysis of ankyrin(R) may interfere with force conduction through disruption of the linkage between integral membrane proteins and the myofibril-cytoskeleton.
Yoshida
K
K
Department of Legal Medicine, Yamaguchi University School of Medicine, Ube.
Harada
K
K
D000818:Animals; D017487:Ankyrins / Q000737:chemistry / Q000378:metabolism*; D001921:Brain; D002154:Calpain / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002458:Cell Fractionation; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004912:Erythrocytes; D066298:In Vitro Techniques; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D017202:Myocardial Ischemia / Q000378:metabolism*; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments; D051381:Rats; D017208:Rats, Wistar
D017487:Ankyrins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D010446:Peptide Fragments; C063261:acetylleucyl-leucyl-norleucinal; D002154:Calpain; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde