K-dependent P-nitrophenylphosphatase Activity Research Articles

Ultrastructural localization of ouabain-sensitive, K-dependent p-nitrophenyl phosphatase activity in monkey eccrine sweat gland.

We studied the electron microscopic localization of ouabain-sensitive, potassium-dependent p-nitrophenyl phosphatase (K-pNPPase) activity of the Na K-ATPase complex in Rhesus monkey eccrine sweat gland by use of the one-step lead citrate method of Mayahara et al. (Histochemistry 1980; 67:125). Reaction product was observed predominantly in the cytoplasmic side of the basolateral membranes of clear (secretory) cells, especially in the interdigitating membrane folds in the basal labryinth, and were completely abolished by 10 mM ouabain or by removal of K+. Little or no enzyme activity was noted in membrane processes in the intercellular canaliculi and in the secretory coil lumen. Basolateral membranes of the dark cells also showed moderate enzyme activity. The myoepithelial cell membrane was devoid of reaction product, except in a few membrane processes arising from the inner aspect of myoepithelial cells. In the coiled duct, K-pNPPase activity was present predominantly in the entire cell membrane of the peripheral ductal cells. The predominantly basolateral distribution of Na-K-ATPase in the eccrine sweat secretory cells is consistent with the concept that a Na-K-Cl co-transport model may be involved in the mechanism of eccrine sweat secretion.

Cytochemical localization of ouabain-sensitive, K-dependent p-nitrophenylphosphatase in the rat hepatocyte.

To determine the localization of Na-K-ATPase activity in the rat hepatocyte, ouabain-sensitive, K-dependent p-nitrophenylphosphatase activity was studied cytochemically with a newly developed one-step lead citrate method devised by Mayahara et al. (1980). The liver was fixed with a mixture of 0.125% glutaraldehyde and 2% paraformaldehyde buffered with 30mM NaOH-PIPES instead of conventionally used 0.1M sodium cacodylate. Reaction products were localized predominantly on the sinusoidal and lateral membranes of the rat hepatocyte, which were remarkably abolished by addition of 10mM ouabain or by the deletion of K. They were localized on the cytoplasmic side of the membranes. Occasionally, reaction products were seen on the bile canalicular membrane, which were insensitive to ouabain and independent of K. These findings indicate that Na-K-ATPase is localized to the sinusoidal and lateral (basolateral) membranes of rat hepatocytes.

Some properties of thyroidal membrane adenosinetriphosphatase and iodide uptake: effects of basic polyamino acids.

Poly L-lysine, poly L-ornithine, and histone significantly inhibited the iodide uptake by the thyroid slices, as previously reported. These basic polymers diminshed Na, K-ATPase and concomitantly markedly elevated Mg-ATPase activity in the NaI-treated microsomal preparation and the plasma membrane fraction obtained from thyroid. Poly L-glutamic acid, which was noneffetive to the iodide uptake in vitro, did not show such phenomenon. K-dependent p-nitrophenylphosphatase activity which is considered to reflect the terminal step of the reaction sequence of Na, K-ATPase was also inhibited by poly L-lysine. The effects mentioned above of poly L-lysine and other basic polyamino acids on membrane ATPase system were only found in the preparations from thyroid. The inhibitory effect of these reagents on thyroidal iodide uptake was discussed in terms of the change in membrane ATPase activities.