1981 年 14 巻 6 号 p. 705-726
A new one-step lead citrate method is introduced for the histochemical and cytochemical demonstration of Ca2+-ATPase activity. The standard incubation medium contains 3mM ATP as the substrate, 10mM CaCl2 as the activator, 2mM lead citrate as the capture reagent, and 250mM glycine-NaOH or-KOH buffer, pH 9.0. The incubation medium was clear and stable.
Cytochemical reactions in this incubation medium were examined in various tissues; liver, kidney, heart, small intestine, trachea, bone marrow, and femoral muscle. The reaction products were found in plasma membrane, mitochondria, myofilaments, axoneme of cilia, sarcoplasmic reticulum, transverse tubules, gap junctions, lysosomes, and others. The cytochemical reaction showed Ca2+ and substrate dependency. Preheating sections in boiling water resulted in complete loss of the reaction, but pretreatment with 10mMEDTA or EGTA did not affect it. Inhibitors such as 10mM PCMB, 0.1mM quercetin, and 0.1mM oligomycin, partly reduced the cytochemical reaction in some tissues, but 0.5mM bromotetramisole left it unchanged. Substrate specificity was proved by the substitution of various substrates. Reaction products were found to be composed of lead phosphate with X-ray microanalysis.
From these results, it was concluded that the cytochemical demonstration of Ca2+-ATPase activity was successful by this method at both light and electron microscopic levels.