Demonstration of lysosomal and extralysosomal sites for acid phosphatase in mouse kidney tubule cells with p-nitrophenylphosphate lead-salt technique.
Dual localization of acid phosphatase in lysosomal and extralysosomal sites of the tubule epithelial cells of normal mouse kidney was observed at the light and electron microscope level using a modified Gomori lead-salt method with p-nitrophenylphosphate (pNPP) as substrate. Based on previous biochemical and cytochemical findings, we developed optimal conditions for the enzyme activity in extralysosomal sites. The conditions used for the light microscopic level consisted of 1.5 mM PNPP, 2.0 MM Pb(NO3)2 and 0.05 M acetate buffer (pH 5.8). Those for the electron microscopic study required 3.0 mM PNPP, 3.6 MM Pb(NO3)2 and 0.1 M acetate buffer (pH 5.8). This modified lead-salt technique was highly specific and provided a suitable method for the demonstration of nonlysosomal as well as lysosomal sites of acid phosphatase activity in the tubule epithelial cells of normal mouse kidney. As expected, the enzyme activity appeared in the lysosomes, but the prominent reaction in the brush border, the rough endoplasmic reticulum and basal infolding plasma membranes was not anticipated. We were able to demonstrate in situ organelle precursors of microsomal acid phosphatase such as endoplasmic reticulum, plasma membrane and basal infolding membranes showing the same substrate preference, which had been observed previously in biochemical studies in our laboratory. Since the possible participation of alkaline phosphatases, K+-pNPPase or Na+-K+-adenosine triphosphatase was ruled out by use of appropriate inhibitors, the enzyme-reactive sites can be interpreted as reflecting nonspecific acid phosphatase.
D000085:Acetates; D000135:Acid Phosphatase / Q000032:analysis; D000818:Animals; D002021:Buffers; D002462:Cell Membrane / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology; D006651:Histocytochemistry / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007684:Kidney Tubules / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D007854:Lead; D008247:Lysosomes / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D009566:Nitrates; D009596:Nitrophenols; D009943:Organophosphorus Compounds
D000085:Acetates; D002021:Buffers; D009566:Nitrates; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D007854:Lead; C008644:nitrophenylphosphate; D000135:Acid Phosphatase