Ultrastructural localization of adenosine triphosphatase activity in lymphocytes activated in vitro by phytohaemagglutinin

Summary The ultrastructural localization of Ca²⁺, Mg²⁺-activated ATPase was studied in phytohaemagglutinin activated lymphocytes and in normal unstimulated lymphocytes. Cells, fixed in paraformaldehyde-glutaraldehyde, were incubated in a medium containing 3mm ATP, 5mm CaCl₂ and 2.4mm Pb(NO₃)₂ in 0.1m tris buffer at pH 8.5, the optimum pH for histochemical demonstration of this enzyme. Reaction product was localized i the endoplasmic reticulum, nuclear membrane, Golgi apparatus and mitochondria and on the membrane surrounding large electron-dense bodies. Cytoplasmic vesicles and the plasma membrane were negative. Activity in unstimulated lymphocytes showed a similar localization but the amount of endoplasmic reticulum was much less than in activated lymphocytes.The pH of the medium was critical for the localization of the enzyme. At pH 7.5, the cytoplasmic reaction was almost completely inhibited but a dense precipitate was present on the outer surface of the plasma membrane. The reaction was stimulated by either Ca²⁺ or Mg²⁺ and was greatly decreased in the absence of these cations or in the presence ofp-chloromercuribenzoate orN-ethylmaleimide. Oligomycin inhibited selectively the reaction in mitochondria but not the reaction at other sites. While the reaction in mitochondria showed complete substrate specificity, a mild reaction was obtained at the other sites with uridine diphosphate or sodium -glycophosphate as substrate. ATP was, however, the preferential substate.