Mammalian-heart adenylate deaminase: Cross-species immunoanalysis of tissue distribution with a cardiac-directed antibody

Abstract

A sheep antiserum against purified rabbit-heart adenylate deaminase (EC 3.5.4.6) (AMPD) was developed and validated as an immunologic probe to assess the cross-species tissue distribution of the mammalian cardiac AMPD isoform. The antiserum and the antibodies purified therefrom recognized both native and denatured rabbit-heart AMPD in immunoprecipitation and immunoblot experiments, respectively, and antibody binding did not affect native enzyme activity. The immunoprecipitation experiments further demonstrated a high antiserum titer. Immunoblot analysis of either crude rabbit-heart extracts or purified rabbit-heart AMPD revealed a major immunoreactive band with the molecular mass (approximately 81 kDa) of the soluble rabbit-heart AMPD subunit. AMPD in heart extracts from mammalian species other than rabbit (including human) was equally immunoreactive with this antiserum by quantitative immunoblot criteria. Although generally held to be in the same isoform class as heart AMPD, erythrocyte AMPD was not immunoreactive either within or across species. Nor was AMPD from most other tissues [e.g., white (gastrocnemius) muscle, lung, kidney] immunoreactive with the cardiac-directed antibody. Limited immunoreactivity was evidenced by mammalian liver, red (soleus) muscle, and brain extracts across species, indicating the presence of a minor cardiac(-like) AMPD isoform in these tissues. The results of this study characterize the tissue distribution of the cardiac AMPD isoform using a molecular approach with the first polyclonal antibodies prepared against homogeneous cardiac AMPD. This immunologic probe should prove useful at the tissue level for AMPD immunohistochemistry.

AMP deaminase in rat brain: Localization in neurons and ependymal cells

“…Therefore, it is difficult to predict which isoform is most engaged in a given aspect of the cellular physiology of ependymal cells and neurons. In this context it appears noteworthy that the heart isoform has been detected in brain extracts by immunological techniques (Thakkar et al, 1995). To establish the correlation between the expression of a certain AMPD isoenzyme and a cellular function it would be necessary to elucidate the regional location of such a neuronal isoenzyme and its subcellular location.…”