A Quantitative Histochemical Study of Alkaline Phosphatase Activity in Isolated Rat Duodenal Epithelial Cells

A simple separation method enabling the quantification of alkaline phosphatase activity in unfixed, isolated, individual, duodenal epithelial cells has been presented. The activity of intestinal brush border-bound alkaline phosphatase has been demonstrated using naphthol AS-BI phosphate as a substrate and hexazotized New Fuchsin as a simultaneous coupling agent. The amount of final reaction product, as measured cytophotometrically, increases linearly with incubation time (up to 10 min) and with substrate concentration (up to 0.4 mM). Maximum enzyme activity was obtained at pH 8.9. Variation of the substrate concentration revealed the kinetic parameters for naphthol AS-BI phosphate as K_m = 0.17 ± 0.015 and V_max = 13.9 ± 1.38. The specificity of the enzyme reaction was confirmed by the complete inhibition of the enzyme activity in the presence of l-cysteine (10 mm) and 80% inhibition with L - phenylalanine (30 mM). Comparison of alkaline phosphatase activity in 8-m cryostat sections (beginning at the tip and proceeding to the cryptal part) along the villus axis, with the activity of individual cells obtained by successive separation, revealed similar values of the percentage quotient derived from the entire activities in these two different methods. This suggests that the presented separation procedure gives rise to isolation of the respective cells from the corresponding areas of the villus. Finally, the isolated cells can be used as a valuable tool for the quantitative analysis of alkaline phosphatase activity along the length of the villus.     

A quantitative cytochemical study of acid phosphatases in rat liver parenchymal cells of different ploidy values

Synopsis The Vickers M86 integrating microdensitometer has been used to quantify the cytochemical reaction for acid naphthol AS-BI phosphatase activity in isolated, rat liver parenchymal cells. Data are presented which valudate the method. The use of this method together with that of the Feulgen reaction to estimate nuclear ploidy value in the same cell, has shown that there is an increase in acid phosphatase activity of up to 100% when the euploidy value of the liver cell doubles. It has been further shown that 70–80% of this enzyme activity is ouabain-sensitive, regardless of the euploidy value. The data may be interpreted to indicate that the extra gene copies of the polyploid cells are operative.     

A multiple-staining procedure for the detection of different DNA fragments on a single blot.

We have developed a method that implies the use of a particular type of substrate which can be used in combination with alkaline phosphatase in detecting nucleic acid on filters. The method allows the detection of several different nucleic acid sequences on a single filter. In consecutive steps, the target DNA molecules are hybridized with different digoxigenin-labeled DNA probes. After each hybridization step, digoxigenin is detected with an antibody-alkaline phosphatase conjugate. This enzyme is subsequently visualized by a color reaction using different 2-hydroxy-3-naphthoic acid anilide (naphthol AS) phosphates as substrates in combination with varying diazonium salts. The multiple-staining procedure is based on the fact that the probe DNA-antibody complex can be removed while the color precipitate remains stably bound at its place on the filter. This allows several repeated hybridizations with other digoxigenin-labeled probes followed by antibody detection and color reaction with other naphthol AS phosphate-diazonium salt combinations. Aside from the ability to simultaneously visualize different target DNAs on a single filter, this new method provides several important features that are more powerful than the conventional 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (BCIP-NBT) color reaction for alkaline phosphatase. The colors are more stable and brilliant than BCIP-NBT; their development is faster, the resolution of closely spaced bands is greater, and the background is much lower. The detection limit for alkaline phosphates is as good as with BCIP-NBT (0.1 pg of DNA). One major advantage is the simplicity of removing the colors by ethanol incubation. In this paper, the method is described using the example of Southern blotted DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photographic microdensitometry for evaluation of acid phosphatase activity at the electron microscope level

Summary The dry mass of reaction products in ultrathin sections was determined using electron micrographs of polystyrene spheres of known weight deposited on Formvar membranes and evaluating the negatives photometrically. This method was applied to the quantification of the final reaction product of the acid phosphatase reaction in a model system in which enzyme was incorporated in gelatin. The enzyme activity was demonstrated by the lead precipitation method and quantified by direct microphotometry at the light microscope level. Models were then embedded and sectioned for electron microscopy. Microphotometric values afforded by the electron negatives were in linear correlation with incubation times and enzyme concentration. Section thickness and its possible variations due to deformation or contamination under the electron beam were also evaluated. Measurements of lysosomal acid phosphatase activity in rat kidney sections served to illustrate the application of the technique.     

Nature of human spleen red pulp cells with special reference to sinus lining cells

Summary A histological and cytological as well as enzyme histo- and cytochemical analysis (alpha-naphthyl acetate esterases, naphthol AS acetate esterases, naphthol AS-D chloroacetate esterases, acid and alkaline phosphatases) of human spleen cells in sections and imprints was carried out with special reference to sinus lining cells. These cells show strong naphthol AS esterase activity and no or only little alpha-naphthyl acetate esterase and acid phosphatase activity. Thus they can be distinguished from reticular cells in pulp cords and from other macrophages in cords and sinuses. From the morphological and enzyme histochemical aspect it can be deduced that the sinus lining cells are a distinct cell type of the human spleen. The comparison of these enzyme cytochemical findings with the results of biochemical and electron microscopical investigations suggests that reticular cells of pulp cords and littoral cells of sinuses also have different functions: reticular cells seem to have a high phagocytotic activity while littoral cells seem to be only facultatively phagocytic.This work was supported by the Deutsche Forschungsgemeinschaft.     

The Stability Of So-Called Axonal Acid Phosphatase As Determined By Experiments In Its “Stainability”

Since the histochemical method for exhibiting acid phosphatase in bodily tissues is said to depend upon the enzyme acting on suitable substrates, it is possible to test its stability by various tests. It has been found that the background element or elements, whatever they may b e, concerned with the “staining” properties of the reaction are very stable and somewhat resistant to destruction. So-called acid phosphatase in spinal cord axons has not been inactivated by subjecting it to various fixing solutions, changes in temperature and pH, relatively prolonged post-mortem autolysis nor by well known enzyme inactivators. It is believed that the “staining” reaction may be dependent fundamentally on other factors than enzymatic activity.     

Quantitative histochemical study of acid phosphatase activity in rat liver using a semipermeable membrane technique

Acid phosphatase activity has been demonstrated in rat liver with the semipermeable membrane technique using naphthol AS-BI phosphate as substrate and hexazotized pararosaniline (HPRA) as simultaneous coupling agent. With this method the final reaction product (FRP) appeared in rat liver as intensely colored red granules in liver parenchymal cells and in Küpffer cells. The absorbance spectrum of the FRP peaks between 510 and 550 nm. A nonspecific reaction product, as has been found in skeletal muscle, did not occur in rat liver. A substrate concentration of 5 mM and a HPRA concentration of 10 mM result in optimum localization and activity. We concluded from the results with different enzyme inhibitors that lysosomal acid phosphatase was demonstrated. The mean absorbance of the FRP increased linearly with incubation time (15-60 min). Furthermore, we found a linear increase of the FRP with increasing section thickness (4-10 micron). When the simultaneous coupling method was replaced by a post-coupling technique, the colored reaction product was diffusely located throughout the cytoplasm. In conclusion, the simultaneous coupling technique in combination with the semipermeable membrane method is a valuable tool for detecting and quantifying lysosomal acid phosphatase activity in rat liver. We demonstrated that acid phosphatase activity is 1.2 times higher periportally than pericentrally in rat liver, and that 24 hr fasting before the experiments did not change the acid phosphatase activity.     

Acid phosphatase and esterase activity in orchid mycorrhiza

Summary A cytochemical study was made to examine the possibility that acid phosphatase may be specifically involved in the digestion of endophytic hyphae in orchid mycorrhiza. Esterase activity was studied for comparison. Frozen sections of unfixed or glutaraldehyde-fixed protocorms of Dactylorhiza purpurella infected by Thanatephorus cucumeris (Rhizoctonia solani) were reacted for acid naphthol AS BI phosphatase, acid -glycerophosphatase or naphthol AS D esterase.A marked increase in particulate acid naphthol AS BI phosphatase activity was observed during infection of host, central, parenchyma cells shortly before hyphae lysed; a diffuse reaction of high activity was localised on lysed fungus. Acid -glycerophosphatase was present at particulate sites only in fungal cytoplasm and as a diffuse reaction on lysed fungus.Naphthol AS D esterase showed highest activity at hyphal apices. Esterase seems to be associated with growth and differentiation of hyphae in orchid cells, rather than lysis of the fungus.     

A study of acid phosphatase in mouse kidney using naphthol AS/BI phosphate as a substrate

Summary A kinetic study of mouse kidney acid phosphatase has been performed using an application of the histochemical method ofBurstone (1958a, b). The suitability of the use of naphthol AS/BI phosphate as a substrate for biochemical assays of acid phosphatase has been ascertained. However, the rate of inhibition of the enzyme by sodium molybdate and sodium fluoride suggests that naphthol AS/BI phosphate may represent a substrate for an acid phosphatase different from-glycerophosphatase.     

Fluorescent cytochemistry of acid phosphatase and demonstration of fluid-phase endocytosis using an azo dye method

 The aim of this work was the development of a fluorescent microscopy technique to visualize acid phosphatase activity in living and pre-fixed cells. We have shown that a coupling azo dye method, using naphthol AS-MX phosphate (NP) as substrate and fast red TR (FR) as a diazonium salt coupling agent, gives rise to a fluorescent azo dye reaction product which permits a highly sensitive demonstration of lysosomal acid phosphatase in both living and pre-fixed monolayer cell cultures. The granular staining is prevented by inhibition of acid phosphatase activity using fluoride and/or orthovanadate in both living and pre-fixed preparations. Lysosomal staining in living cells is also abolished by inhibition of fluid-phase endocytosis using low temperatures or inhibition of oxidative phosphorylation. It was shown that whilst NP entered living cells by passive diffusion, occurrence of FR in lysosomes resulted from fluid-phase endocytosis. Spectroscopic analysis of the emission and absorption features of FR, NP, naphthol AS-MX (N), and the N–FR azo dye reaction product in solution corroborated our microscopic results. The differing uptake mechanisms, and the occurrence of lysosomally localized azo dye, were also in keeping with the predictions of quantitative structure–activity relationship models of this system. Accepted: 24 April 1997     

Regio- and stereo-specific nitrile hydrolysis by the nitrile hydratase from Rhodococcus AJ270

Abstract Acid phosphatase activity was measured in individual cells by determining their optical densities through a scanning confocal laser microscope. The naphthol AS-TR (3-hydroxy-2-naphtoic acid 4'-chloro-2'-methylanilide) phosphate-hexazotized para-rosanilin method was used to visualise the acid phosphatase content in the light microscope. Evidence was obtained that the amount of enzyme varied in exponential growth phase cells as the fission age increased. By comparing the acid phosphatase activity with the rate of food vacuole formation, it appeared that the amount of enzyme inside the cells decreased in early clonal life, whereas the rate of food uptake increased. It was assumed that the reduction of acid phosphatase content could lead to a more extended life of vacuoles and to a decreased membrane recycling rate. In turn, the reduced supply of membrane available for food vacuole formation could partly be responsible for the decrease of the food uptake rate observed after the initial increase.     

Kinetics of thermal inactivation of penaeus penicillatus acid phosphatase

The kinetics of thermal inactivation of Penaeus penicillatus acid phosphatase have been studied using a kinetic method related to the substrate reaction during irreversible inhibition of the enzyme activity as previously described by Tsou (Adv. Enzymol. Relat. Areas Mol. Biol. (1988) 61, 381-436). The kinetics of thermal inactivation of the enzyme show that the reaction is irreversible. The microscopic rate constants were determined for thermal inactivation of free enzyme and the enzyme--substrate complex. The results show that the presence of substrate has a significant protective effect against thermal inactivation of the enzyme.     

Histochemical study of a number of hydrolases,including a new acid phosphatase,in various tissues of men and mice

Summary Two acid phosphatases have been demonstrated histochemically in mouse ventral prostate, seminal vesicles, coagulating glands, and liver and in human prostate. The first is the lysosomal acid phosphatase demonstrable by the Gomori technique. The second differs from thisβ-glycerophosphatase in that it splits naphthol AS phosphates but notβ-glycerophosphate; it has a different histochemical pH optimum and it is not inhibited by MoO₄ or NaF. The enzyme does not represent the “tail” of alkaline phosphatase activity as it is not inhibited by inhibitors of alkaline phosphatase and it has a different localization in liver and in human prostate. The enzyme may be membrane-bound but a lysosomal localization has still to be confirmed.     

LOCALIZATION OF ACID PHOSPHATASE IN LIPOFUSCIN GRANULES AND POSSIBLE AUTOPHAGIC VACUOLES IN INTERSTITIAL CELLS OF THE GUINEA PIG TESTIS

The intracellular localization of acid phosphatase in guinea pig testicular interstitial cells was investigated by incubating nonfrozen thick sections of glutaraldehyde-perfused testis in a modified Gomori medium and preparing the tissue for electron microscopy. Lipofuscin pigment granules in these cells contain dense pigment, granular matrix, and often a lipid droplet. Reaction product is seen in the matrix of the pigment granules, and they may therefore be called residual bodies. At least some of the dense pigment appears to be derived from myelin figures and membrane whorls, since suitable intermediates can be seen. Lipid droplets found free in the cytoplasm are another possible source of pigment. In both cases the chemical mechanism is presumed to be autoxidation of unsaturated lipid. Acid phosphatase is present in the inner cisterna of Golgi elements. Enzyme activity also appears in possible autophagic vacuoles bounded by double membranes; the reaction product lies between the membranes. Consideration of the enzyme as a tracer suggests that the autophagic vacuoles are derived from the Golgi complex. Possible stages in the formation of these vacuoles by the inner Golgi cisternae are observed.     

Stability and catalytic kinetics of acid phosphatase immobilized on composite beads of chitosan and activated clay

The stability of acid phosphatase immobilized on composite beads was studied. The beads were prepared from equal weights of cuttlebone chitosan and activated clay and were cross-linked with glutaraldehyde. The immobilized enzyme maintained 90% of its original activity after 50 times of reuse. The immobilized acid phosphatase revealed acceptable thermal and pH stabilities over a broad experimental range. Thermal deactivation of immobilized enzyme was also examined by first-order kinetics and the deactivation energy was determined. The kinetics of a model reaction catalyzed by the immobilized acid phosphatase was finally investigated by the Michaelis–Menten equation.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques. 3. The substrate specificity of isoenzymes of acid phosphatase in m.gastrocnemius of rabbits.

Three distinct isoenzymes of acid phosphatase have been separated from extracts of m.gastrocnemius of normal and of vitamin E deficient rabbits by gel filtration and polyacrylamide gel electrophoresis. These isoenzymes, termed I, II and III, have molecular weights of: 110,000--130,000, 60,000--78,000 and 12,500--14,500. Isoenzymes I and II split the substrates 4-methylumbelliferyl phosphate and naphthol AS-BI phosphate and the activity is strongly increased in the muscles of vitamin E deficient rabbits. Isoenzyme III splits only 4-methylumbelliferyl phosphate and the activity is not increased in the muscles of vitamin E deficient rabbits. The pH-optimum for isoenzymes I and II is 4.8 and for isoenzyme III 5.5. It has been shown that the histochemical semipermeable membrane technique, using substrate naphthol AS-BI phosphate, is a very reliable technique for demonstrating activity of the isoenzymes I and II in tissue sections. On the other hand, activity of isoenzyme III cannot be demonstrated with this histochemical technique. In pathologically altered muscles, the activity of the isoenzymes I and II is greatly increased whilst the activity of isoenzyme III is not significantly altered.     

A quantitative cytochemical assay for osteoclast acid phosphatase activity in foetal rat calvaria

Summary Acid phosphatase activity is prominent in osteoclasts (bone resorbing cells) and has been implicated in the process of bone resorption, although its precise role is not understood. To study the distribution and activity of this enzyme, a quantitative cytochemical method has been developed using undecalcified fresh frozen sections of foetal rat calvariae. Sections were allowed to react with 3mm naphthol ASBI phosphate at pH 5.0, and the reaction was stopped by rinsing in ice-cold tap water containing 50mm sodium fluoride. The reaction product was post-coupled to Fast Garnet at 4°C. The absorbance of areas of reaction product in the cytoplasm was measured using scanning and integrating microdensitometry. The initial velocity rate was maintained for up to 2 min at pH 5.0 with a substrate concentration of 3mm and a section thickness of 5 µm. Under these conditions reaction product was localized to osteoclasts and the surface of bone matrix beneath these cells. Activities in osteoblasts and chondrocytes were negligible. Osteoclastic acid phosphatase was almost totally inhibited by 10mm fluoride and reduced by 70% by 100mm tartrate.     

The histochemical differentiation of various phosphatases in a population of osteoclasts by a simultaneous coupling method using different diazonium salts, with observations on the presence of inhibitors in stable diazonium salts

Synopsis The numerous osteoclasts in a giant cell tumour of bone were found to possess at least two distinct phosphatases capable of hydrolysing naphthol AS-TR phosphate. An acid phosphatase, with optimum activity about pH 4.7, could be demonstrated by simultaneous coupling with Fast Bordeaux OL or Red Violet LB, but not with Fast Red TR. The last-named salt, on the other hand, could be used for demonstrating a phosphatase with an optimum pH of activity about 7.3, showing some activity as an alkaline phosphatase at pH 8.3. This enzyme was markedly inhibited by zinc ions and could not be demonstrated by simultaneous coupling with diazonium salts stabilized with zinc chloride. The acid phosphatase was much less sensitive to zinc, but showed marked inhibition by aluminium, which had comparatively little effect on the other enzyme. Some discrepancies between the published formulae of stable diazonium salts and the substances found to be present in them are discussed.     

Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1.) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.     

Changes of lysosomal enzymes during hereditary degeneration and histogenesis of retina in mice

Summary In the normal histogenesis of mouse retina localized distribution of acid phosphatase positive granules has been seen around the photoreceptor cell nuclei along the outer limiting membrane. These granules disappear during the development of the rod elements. Temporarily increased activity is also seen along the nuclei of the inner layer adjacent to and in the course of the development of the outer and the inner plexiform layers. Within the inner nuclear layer, the cells at the outer and inner rows develop localized acid phosphatase positive granules which persist in the adult retina. Ganglion cells and the layer of nerve fibres show little change. In the pigment epithelium the enzyme gradually increases. In mice, homozygous for the retinal degeneration gene, degenerating photoreceptor cell nuclei, characterized by perinuclear acid phosphatase staining, can be detected before morphological signs of degeneration. Increased frequency of such nuclei and intensity of staining are recorded with the progress of degeneration. Enzyme activity in the photoreceptor cells, within the inner nuclear layer and in the degenerating photoreceptor cell nuclei is demonstrable using naphthol substrates but not -glycerophosphate. Positive reaction with -glycerophosphate is obtained in these sites in the presence of dimethyl sulphoxide. Existence of differential permeability among the retinal lysosomes is tentatively suggested.