A new one-step method for the cytochemical localization of ouabain-sensitive,potassium-dependent p-nitrophenylphosphatase activity

Summary A new one-step method for the light and electron microscopic localization of the ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na-K-ATPase complex is introduced. The incubation medium contains p-nitrophenylphosphate (NPP) as substrate, lead citrate as the capture reagent, and dimethylsulfoxide (DMSO) as an activator. It is usable at the optimal pH of the K-NPPase, which is about pH 9.0 in the presence of 25% DMSO. The effects of fixation, lead concentration, and DMSO on the enzyme activity were studied using rat kidney as a test tissue. The fixation of tissues in a mixture of 2% paraformaldehyde and 0.5% glutaraldehyde for 60 min at 0°–4° C preserved 45% of the enzyme activity. In the absence of DMSO, lead citrate (4.0 mM) caused 82% inhibition of the enzyme activity in fixed tissue. However, the addition of DMSO (25%) caused about 3-fold activation of the remaining activity. Cytochemical demonstration of the ouabain-sensitive K-NPPase activity was successfully made by this method at both light and electron microscopic levels.This study was supported partly by grants-in-aid for scientific reasearch from the Ministry of Education, the Japanese Government (Nos. 244016 and 337001)Part of this paper was presented at the 19th Annual Meeting of the Japan Society of Histochemistry and Cytochemistry held in Gifu, November 1–2, 1978 (Mayahara et al. 1979a), and the 35th Annual Meeting of the Japanese Society of Electron Microscopy, held at Takarazuka, May 23–25, 1979 (Mayahara et al. 1979b)