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.

ULTRACYTOCHEMICAL LOCALIZATION OF OUABAIN-SENSITIVE, POTASSIUM-DEPENDENT <i>P</i>-NITROPHENYLPHOSPHATASE ACTIVITY IN THE GUINEA PIG RETINA

“…The sections were immersed in the following incubation medium at 37°C for 5 to 25 min : A) For K-NPPase (Mayahara et al) (43)(44)(45) : 250 mM glycine-KOH buffer (pH 9.0), 10 mM p-nitrophenylphosphate (Mg-salt, Sigma Chem. Co., St. Louis) ; 25% v/v DMSO ; 4 mM lead citrate (in 50 mM KOH) ; 2.5 mM Levamisole (Sigma Chem.…”

“…As a method to localize the K-NPPase activity, Ernst proposed a two step (Sr-Pb) cytochemical method (14)(15)(16). However, as his method has two fundamental drawbacks (45), Mayahara et al developed an alternative one step lead citrate method (42)(43)(44)(45). This K-NPPase activity has been studied in several tissues and, undoubtedly, seems to be the most widely accepted approach for localizing Na-K-ATPase activity (18).…”

An Immuno- and Enzyme Cytochemical Study of the H+-K+ ATPase in Human Parietal Cells after Administration of Tetragastrin and Omeprazole.

“…All of these are K+-dependent, and gastric K+-stimulated p-nitrophenylphosphatase (K+-pNPPase) is correlative to H+-K+ ATPase activity (LJUNGSTROM et al, 1984;KOENIG, 1984;RAY and NANDI,1986). To determine the activity of H+-K+ ATPase, FUJIMOTO et al (1986) devised a cytochemical method for demonstration of H+-K+ ATPase in ouabain-insensitive, K+-pNPPase with a lead based method, derived from the method for measuring Na+-K+ ATPase activity (MAYAHARA et al, 1980). However, their lead-based method was performed under non-physiological pH conditions.…”

Cytochemical localization of Ca++-ATPase, H+,K+-ATPase and Na+,K+-ATPase in acid-secreting parietal cell and non-secreting parietal cell.

“…Ca++-activated adenosine triphosphatase (Ca++-ATPase), ouabaininsensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase), a component of H+, K+-ATPase system, and ouabain-sensitive K+-NPPase, a component of Na+, K+-ATPase system, were studied in the gastric parietal cells of guinea pigs fed ad libitum and/or starved using the recently improved cytochemical methods introduced by Ando et al (1981), Fujimoto et al (1986) and Mayahara et al (1980), respectively. Ca++-ATPase activity was localized: a) on the exterior side of basal and lateral membranes, b) on the interior side of the membrane covering some tubulovesicles, c) just on the membrane covering Golgi apparatus and d) on the matrix of mitochondria.…”