title: A new one-step method for the cytochemical localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity.
abstract: 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% of 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 degrees--4 degrees 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.
mesh_terms: D009597:4-Nitrophenylphosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002951:Citrates / Q000494:pharmacology; D019343:Citric Acid; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005404:Fixatives / Q000494:pharmacology; D006651:Histocytochemistry; D007668:Kidney / Q000201:enzymology*; D007854:Lead / Q000494:pharmacology; D008297:Male; D008854:Microscopy, Electron; D010042:Ouabain / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D051381:Rats
chemical_list: D002951:Citrates; D005404:Fixatives; D019343:Citric Acid; D007854:Lead; D010042:Ouabain; D010744:Phosphoric Monoester Hydrolases; D009597:4-Nitrophenylphosphatase; D011188:Potassium; D004121:Dimethyl Sulfoxide