A NEW ONE-STEP METHOD FOR THE HISTOCHEMISTRY AND CYTOCHEMISTRY OF CA2+-ATPASE ACTIVITY

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Abstract
A new 1-step lead citrate method is introduced for the histochemical and cytochemical demonstration of Ca2+-ATPase activity. The standard incubation medium contains 3 mM ATP as the substrate, 10 mM CaCl2 as the activator, 2 mM lead citrate as the capture reagent and 250 mM 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 [rat] 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 10 mM EDTA or EGTA [ethyleneglycol-bis(.beta.-aminoethyl ether)N,N,N''N''-tetraacetic acid] did not effect it. Inhibitors such as 10 mM p-chloromercuribenzoic acid, 0.1 mM quercetin and 0.1 mM oligomycine partly reduced the cytochemical reaction in some tissues, but 0.5 mM bromotetramisole left it unchanged. Substrate specificity was proved by the substitution of various substrates. Reaction products were found to be composed of lead phosphate by X-ray microanalysis. The cytochemical demonstration of Ca2+-ATPase activity was successful by this method at both light microscopic and EM levels.

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