Purification of cardiac annexin V from the beagle dog heart and changes in its localization in the ischemic rat heart

Abstract

We isolated and purified 35 kDa protein from the myocardium of the beagle dog and identified it to be annexin V from partial amino acid sequence determination. It was confirmed that anticanine cardiac annexin V rabbit polyclonal antibody, which was produced using the 35 kDa protein, cross-reacts with annexin V of the myocardium, lung, liver, kidney, and brain of the rat. The localization of cardiac annexin V and the effect of ischemia for 30-180 min in the rat were immunohistochemically studied with the use of the Langendorff perfusion heart. In the normal myocardium, annexin V, accompanied by cross-striation, was observed throughout the cell. In ischemia of 30 min, extracellular leakage of annexin V was observed with uneven staining in the cytoplasm. When the ischemic time exceeded 60 min, annexin V was observed in the cell membrane with a decrease of annexin V in the cytoplasm. Also, extracellular leakage of annexin V was observed prominently. In ischemia for 180 min, almost all the annexin V in the cytoplasm disappeared. These results suggest that the level of ischemia can be estimated from the changes in localization of annexin V.