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

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 3 mM ATP, 5 mM CaCl2 and 2.4 mM Pb(NO3)2 in 0.1 M tris buffer at pH 8.5, the optimum pH for histochemical demonstration of this enzyme. Reaction product was localized in 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 of p-chloromercuribenzoate or N-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 beta-glycophosphate as substrate. ATP was, however, the preferential substrate.