ArticlePDF Available

A quantitative biochemical and histochemical study of the lead method for localization of adenosine triphosphate-by-drolyzing enzymes

Authors:

Abstract and Figures

The purpose was to study the nature of the adenosine triphosphatase (ATPase) localized to plasma membranes by the lead method and the significance of the lead-catalyzed hydrolysis of adenosine triphosphate (ATP) for the staining of fixed kidney. A modified Wachstein and Meisel's medium was used. The staining of sections of microsomal sediments from rat kidney was compared with the in vitro activity of (Na+ + K+)–, Mg++– and Ca++– ATPase in the presence of lead. The histochemical relevance of these observations was tested on sections of rat kidney. Localization of (Na+ + K+)–ATPase was not possible at concentrations of lead below the inactivating level (0.5 mM). Mg++–ATPase and Ca++– ATPase were only partially inhibited by 3.6 mM lead and were localized to plasma membranes in glutaraldehyde-fixed kidney. The staining followed a course of activation by Ca++ and Mg++ characteristic for enzymatic hydrolysis and different from the activation of nonenzymatic hydrolysis of ATP. Staining of fixed tissue was maximal at 2-3 mM lead and decreased at higher concentrations. In Mg++–deficient media membrane staining was weak or absent at 5.2-6.8 mM lead, where the rate of nonenzymatic hydrolysis of ATP was high. Thus, in the medium used the lead-catalyzed hydrolysis of ATP contributed little to the staining of fixed kidney.
No caption available
… 
No caption available
… 
No caption available
… 
No caption available
… 
Content may be subject to copyright.
A preview of the PDF is not available
Article
The histochemical distribution of selected enzymes were examined in the small intestine of 5 about 3-week-old normal calves fed on whole cow’s milk. Alkaline phosphatase and β-D-galactosidase (= lactase) in the epithelial brush border, and non-specific esterase in the cytoplasm showed a strong reaction in the villi of the anterior small intestine and a marked decrease in the posterior regions. Aminopeptidase in the brush border of the villi showed a reverse distribution, with the strongest reaction in the posterior small intestine. Adenosine-triphosphate-splitting enzyme in the epithelial brush border, acid phosphatase and succinate dehydrogenase in the cytoplasm of the epithelial cells gave a relatively uniform reaction in the villi throughout the small intestine. A fluoride-resistant acid phosphatase was demonstrated in the brush border of the villi in the anterior small intestine. The distribution of enzymes demonstrated in this study was generally compatible with the known absorptive functions of the various parts of the small intestine.
Article
During the first four mitotic division cycles of Lymnaea stagnalis embryos, we have detected cell cycle-dependent changes in the pattern of transcellular ionic currents and membrane-bound Ca2+-stimulated ATPase activity. Ionic currents ranging from 0.05 to 2.50 µA/cm2 have been measured using the vibrating probe technique. Enzyme activity was detected using Ando's cytochemical method (Ando et al. 1981) which reveals Ca2+/Mg2+ ATPase localization at the ultrastructural level, and under high-stringency conditions with respect to calcium availability, it reveals Ca2+-stimulated ATPase. The ionic currents and Ca2+-stimulated ATPase localization have in common that important changes occur during the M-phase of the cell cycles. Minimal outward current at the vegetal pole coincides with metaphase/anaphase. Maximal inward current at the animal pole coincides with the onset of cytokinesis at that pole. Ca2+-stimulated ATPase is absent from one half of the embryo at metaphase/anaphase of the two- and four-cell stage, whereas it is present in all cells during the remaining part of the cell cycle. Since fluctuations of cytosolic free calcium concentrations appear to correlate with both karyokinesis and cytokinesis, we speculate that part of the cyclic pattern of Ca2+-stimulated ATPase localization and of the transcellular ionic currents reflects the elevation of cytosolic free calcium concentration during the M-phase.
Article
Summary During extrusion of the first polar body in eggs ofLymnaea stagnalis andBithynia tentaculata a localized Ca2+ /Mg2+ ATPase activity was detected, using Ando's enzyme-cytochemical method for electron microscopy [Ando et al. (1981) Acta Histochem Cytochem 14:705–726]. The enzyme activity was distributed in a polar fashion, along the cytoplasmic face of the plasma membrane. In the eggs ofLymnaea it was found only in the vegetal hemisphere, whereas inBithynia eggs it was localized both in the vegetal hemisphere and at the animal pole. This pattern of enzyme activity corresponds to the polar pattern of transcellular ionic currents measured with the vibrating probe, which we showed to be partially carried or regulated by calcium [Zivkovic and Dohmen (1989) Biol Bull (Woods Hole) 176 (Suppl):103–109]. The characteristics of the ATPase were studied using a variety of approaches such as ion and substrate depletions and substitutions, addition of specific inhibitors of ATPase activity, treatment with EDTA/EGTA and electron energy-loss spectrometry. The results indicate that, inLymnaea, there are at least two enzymatic entities. The first one is a Ca2+ /Mg2+ ATPase localized along the membrane and in the cortex of the vegetal hemisphere. The second one is a Ca2+-stimulated ATPase (calcium pump of the plasma membrane) localized in a small region of the membrane at the vegetal pole. We speculate that in the eggs ofLymnaea andBithynia a functional relationship exists between the plasma-membrane-associated ATPase activity and the transcellular ionic currents measured in the same region.
Article
The cytochemical localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase in the guinea pig inner ear was studied using both the lead-based method and the cerium-based method. the influence of primary fixation and incubation time was investigated under both incubation conditions. a high p-NPPase activity was observed in the basolateral infoldings of the stria vascularis in the cochlea and the dark cells in the vestibular labyrinth. the stromal cells of the spiral prominence only showed a weak reaction with the lead-based method. No specific enzyme activity was found in the endolymphatic sac. the results of the present study are discussed with emphasis on the cation transport mechanisms of the inner ear.
Article
Adenylate cyclase activity has been demonstrated in tissue of the blowfly, Phormia regina, chemoreceptor sensillium and in labellar epidermal cells. Sensillar tissue included sensory neurons innervating the sensillum and associated supportive cells. Epidermal cells which surround the internal labellar apophysis and those below the external cuticles both displayed enzyme activity. Possible explanations for these data are discussed.
Article
1.1. Factors which influence the validity of cytochemical staining methods for enzymes are briefly discussed.2.2. Objective assessment of the validity of staining methods for labile enzyme systems requires fairly extensive study to determine conditions under which they are best preserved and visualized.3.3. Application of the Gomori acid phosphatase procedure to rat kidney and liver has been studied by a correlated enzymic, analytical and morphological approach, and by observing the effect of deliberate alterations to the integrity of the tissues and to the composition of the staining medium.4.4. Formol-calcium fixation followed by treatment with hypertonic sucrose containing gum acacia (“gum-sucrose”) gives the best preservation of the acid phosphatase integrity of rat kidney and liver cells.5.5. When stained by the Gomori procedure, the tissues prepared in this way give the most accurate visualization of the cytological distribution of the enzyme.6.6. Adequate additional investigations should be made before the conclusions drawn here with respect to rat kidney and liver are applied to other tissues.
Article
The lead method of Wachstein and Meisel for the histochemical localization of adenosine triphosphatase (ATPase) involves the incubation of sections of fixed tissue in reaction mixtures containing ATP, lead nitrate, magnesium sulfate and a Tris-maleate buffer, pH 7.2. Both fixation and the presence of lead ion were shown to inhibit tissue ATPase activity markedly and to inactivate the sodium- plus potassium-dependent membrane ATPase. In addition, recent studies have demonstrated that lead ion, in the concentration used in the Wachstein-Meisel system, will catalyze the hydrolysis of ATP. Studies on the effect of this nonenzymatic reaction on the histochemical localization of ATPases demonstrated that plasma membrane localization occurred only with lead and ATP concentrations which gave significant nonenzymatic hydrolysis of ATP by lead. In addition, nuclear and mitochondrial localization without accompanying plasma membrane localization could be obtained in formalin-fixed tissue with decreased concentrations of lead or with increased concentrations of ATP in the reaction mixture. The amount of lead-catalyzed hydrolysis was in the same order of magnitude as fixed tissue ATPase activity and could quantitatively account for the amount of phosphate needed to give recognizable localization of lead salt deposits in sections of fixed tissue.
Article
The localization of ATPase(1) activity has been studied by light and electron microscopy in the epidermis of Rana pipiens, Rana catesbiana, and Bufo marinus. The reaction was carried out on skin (glutaraldehyde-fixed or fresh) sectioned with or without freezing. Best results were obtained with nonfrozen sections of fixed tissue. The incubation mixture was either a Wachstein-Meisel medium, or a modification which approximates assay systems used in biochemical studies of transport ATPases. The reaction product was found localized in contact with the outer leaflet of all cell membranes facing the labyrinth of intercellular spaces of the epidermis. It was absent from: (a) membrane areas involved in cell junctions (desmosomes, zonulae and maculae occludentes); (b) cell membranes facing the external medium (i.e., those on the distal aspect of the ultimate cell layer in s. corneum); (c) cell membranes facing the dermis (those on the proximal aspect of cells in s. germinativum). In the presence of (Na(+) + K(+)) the localization did not change, but the reaction was not appreciably activated. A similar though less intense reaction was obtained with ITP, but not with ADP, AMP, and GP as substrates. The results are discussed in relation to available data on transport ATPases in general, and on the morphology and physiology of amphibian skin in particular. Assuming that the ATPase studied is related to transport ATPase, the findings suggest a series of modifications to the frog skin model proposed by Koefoed-Johnsen and Ussing. The salient feature of this modified model is the localization of the Na(+) pump along all cell membranes facing the intercellular spaces of the epidermis.
Pitfalls in the use of lead ion for histochemnicallocalizations of nucleoside phosphatases The localization of adenosine triphos-phatase in liver: in situ staining and cell fractionation studies Gewinnung voms (32P) Adenosims-triphosphorsaure mit hoher spezifischer Radioaktivitat
  • H L Moses
  • A S J Rosemsthal
  • Histo-Chemn
  • Cytochemmn
  • A B Novikoff
  • D H Hausman
  • E Podber
Moses, H. L. and Rosemsthal, A. S.: Pitfalls in the use of lead ion for histochemnicallocalizations of nucleoside phosphatases. J. Histo-chemn. Cytochemmn. 16: 530, 1968. 11. Novikoff, A. B. Hausman, D. H. anud Podber, E.: The localization of adenosine triphos-phatase in liver: in situ staining and cell fractionation studies. J. Histochem.Cytochemmi. 6: 61, 1958. 12. Pfleiderer, G.: Gewinnung voms (32P) Adenosims-triphosphorsaure mit hoher spezifischer Radioaktivitat. Biochemn. Biophys. Acta 47: 389, 1961.
Zur Bestimniung des Orthophosphats nebeni S#{228}ure-molybdat-labilenPhosphsors#{228}urever- bindungen
  • B E Wahier
  • A Wollemsberger
Wahier, B. E. amid Wollemsberger, A.: Zur Bestimniung des Orthophosphats nebeni S#{228}ure-molybdat-labilenPhosphsors#{228}urever- bindungen. Biochem. Z. 329: 508. 1958. by guest on July 11, 2011 jhc.sagepub.com Downloaded from