PURIFICATION, CHARACTERIZATION, AND LOCALIZATION OF 2 ATP-DIPHOSPHOHYDROLASE ISOFORMS IN BOVINE HEART

Two ATP diphosphohydrolase (ATPDase) isoforms have been purified from the bovine heart ventricle. The purification procedure includes the fo llowing steps: differential centrifugation, sucrose cushion centrifuga tion, solubilization with Triton X-100, DEAE agarose ion exchange, and Affi-Gel blue-Sepharose and concanavalin A (con A)-Sepharose chromato graphies. The purified enzyme has an optimum pH of catalysis of 7.5 an d requires Ca2+ or Mg2+. The apparent Michaelis constant of the enzyme , with ADP as the substrate, is 29 mu M, and the apparent maximal velo city is 1.6 mu mol.min(-1).mg protein(-1). Substrate specificity, heat -inactivation curves, and copurification of adenosinetriphosphatase (A TPase) and adenosinediphosphatase (ADPase) activities confirmed the id entity of the purified enzyme as an ATPDase. In addition, polyacrylami de gel electrophoresis, under nondenaturing conditions, showed identic al migration patterns for the protein involved in ATPase and ADPase ac tivities. Western blot analysis, with an antibody that specifically re cognizes the NH2-terminal sequence of pig pancreas ATPDase and specifi cally reacts with bovine and human ATPDases, showed cross-reactivity w ith the purified ATPDase isoforms from the bovine heart. Immunocytoche mical localization in the ventricle produced strong reactions with the plasma membrane of Purkinje fiber cells and the majority of myocardia l cells. Immunoreactivity was variable, producing a mosaic-like aspect . As expected, smooth muscle cells and endothelial cells of coronary v essels were highly reactive. This ectoenzyme could play a protective r ole against the potentially deleterious effects of extracellular ATP. In tandem with 5'-nucleotidase, it produces adenosine, a powerful vaso dilator, especially in hypoxic or ischemic conditions that favor the r elease of ATP.