Combined biochemistry and histocytochemistry as a tool to investigate Ecto-ATPase in the cardiac muscle

Ecto-ATPase (ecto-adenosine triphosphatase), a key enzyme of cardiac metabolism, is responsible for modulation of the concentration of extracellular nucleotides in the heart. We present methodology consisting of the combined use of biochemical and histocytochemical techniques to study its properties. Using samples from essentially the same preparation, we applied biochemistry and histocytochemistry to determine biochemical characteristics of ecto-ATPase and an in situ localization of its reactivity. Our results indicated that detected enzyme resists fixation, depends on divalent ions, and hydrolyzes ATP, but not AMP or ADP-beta-S. Reaction product of the enzyme activity was found confined to the extracellular surface of the plasma membrane of cardiac myocytes and endothelial cells due to the corresponding orientation of the enzyme active sites. Experiments using an inhibitor justified specificity of the reaction. When used together with molecular biological and immunocytochemical techniques, the present methodological approach should be capable of yielding important information about the actual ability of ecto-ATPase to operate.