MAMMALIAN-HEART ADENYLATE DEAMINASE - CROSS-SPECIES IMMUNOANALYSIS OFTISSUE DISTRIBUTION WITH A CARDIAC-DIRECTED ANTIBODY

A sheep antiserum against purified rabbit-heart adenylate deaminase (E C 3.5.4.6) (AMPD) was developed and validated as an immunologic probe to assess the cross-species tissue distribution of the mammalian cardi ac AMPD isoform. The antiserum and the antibodies purified therefrom r ecognized both native and denatured rabbit-heart AMPD in immunoprecipi tation and immunoblot experiments, respectively, and antibody binding did not affect native enzyme activity. The immunoprecipitation experim ents further demonstrated a high antiserum titer. Immunoblot analysis of either crude rabbit-heart extracts or purified rabbit-heartAMPD rev ealed a major immunoreactive band with the molecular mass (approximate to 81 kDa) of the soluble rabbit-heartAMPD subunit. AMPD in heart ext racts from mammalian species other than rabbit (including human) was e qually immunoreactive with this antiserum by quantitative immunoblot c riteria. Although generally held to be in the same isoform class as he artAMPD, erythrocyte AMPD was not immunoreactive either within or acro ss species. Nor was AMPD from most other tissues [e.g., white (gastroc nemius) 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 car diacAMPD isoform using a molecular approach with the first polyclonal antibodies prepared against homogeneous cardiacAMPD. This immunologic probe should prove useful at the tissue level for AMPD immunohistochem istry.