Secretion of acid phosphatase in<Emphasis Type="Italic">Claviceps purpurea</Emphasis> — an ultracytochemical study

The lead phosphate precipitation method showed the reaction product of acid phosphatase (which reflects the presence of the enzyme glycoprotein) in peripheral cytoplasmic vesicles in the ascomycetous fungus Claviceps purpurea. The product appeared to diffuse from these vesicles (diameter 100-200 nm) towards the cell wall, usually to its sites covered by the capsular fibres exhibiting also acid phosphatase activity. This observation of diffusion of secretory glycoprotein in the cytoplasmic matrix and its orientation to the plasmalemma and capsular fibrils suggests an alternative to the well-described secretory mechanism of transport and exocytosis of glycoproteins via membrane-bound transport conveyors fusing with the cell membrane. It confirms and enlarges our previous finding of the reaction product of acid phosphatase performed by ultrastructural cytochemistry in vacuoles (lysosomes), in the growing cell septum, in cytoplasmic vesicles and in the fibres of the external capsule.     

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.     

Lipopolysaccharide alters ecto-ATP-diphosphohydrolase and causes relocation of its reaction product in experimental intrahepatic cholestasis

Lipopolysaccharide (LPS), a bacterial endotoxin, exerts profound inflammatory actions toward various tissues and cells. We induced intrahepatic cholestasis in rats by administration of LPS and followed ecto-ATP-diphosphohydrolase (ecto-apyrase) activity in the liver. The activity of the enzyme had decreased to 77% 2 h after injection compared with the activity in control animals. The maximum decrease was detected 24 h after administration. The activity was found to have partially recovered 1 week after injection, but had yet to reach control levels. In contrast to the decrease in ecto-apyrase activity, there were increases in alkaline phosphatase activity and bilirubin concentration, markers of cholestasis. In response to LPS, the reaction product of ecto-apyrase was found to relocate from the canalicular domain of the plasma membrane of hepatocytes, its predominant localization in the liver of intact animals, to the basolateral and sinusoidal domains. The pattern of histochemical reaction indicated modulation of the enzyme activity and changes in trafficking of intracellular proteins. Taken together, our findings showed that LPS administration alters ecto-apyrase and causes relocation of its reaction product from the canalicular domain of the plasma membrane of hepatocytes in the rat. It is suggested that relocation of the reaction product may be a protective mechanism to enable the hepatocytes to withstand the cytokine-induced metabolic perturbations.     

Leishmania donovani: generation of monospecific antibody reagents to soluble acid phosphatase

Four monoclonal antibodies (McAbs) were generated against the soluble extracellular acid phosphatase (EC 3.1.3.2) (S-AcP) of Leishmania donovani. These were detected in the primary screen using an ELISA with promastigote culture supernatants as antigen. Three of the McAbs demonstrated bound S-AcP from such culture supernatants in an enzyme activity binding assay. All immunoprecipitated metabolically labeled S-AcP but none showed any binding to the promastigote surface by indirect immunofluorescence. Moreover, none reacted with Triton X-100 solubilized plasma membranes by immunoprecipitation or Western blotting. These results demonstrated that the McAbs did not recognize the surface membrane bound acid phosphatase, but were specific for the extracellular soluble enzyme. Further, none of the antibodies immunoprecipitated any of the five human acid phosphatase isozymes or reacted with them in Western blots or the enzyme activity binding assay. Therefore, they are specific for the parasite-derived enzyme. One of these was used to affinity purify sufficient L. donovani S-AcP to immunize a rabbit and generate a specific, polyvalent antiserum. This polyvalent antibody immunoprecipitated S-AcP activity but did not cross-react with the surface membrane acid phosphatase, indicating that these two parasite enzymes are separate gene products.     

Changes in acid phosphatase activity in rat liver after ischemia

The effect of ischemia on the stability, i.e. the permeability of the lysosomal membrane of rat liver has been studied using quantitative histochemical analysis of acid phosphatase activity. Ischemia in vitro was performed for 0-240 min at 37 degrees C and ischemia in vivo for 60 min was followed by 1, 5, 24 and 48 h of reperfusion. Acid phosphatase activity was demonstrated in cryostat sections using naphthol AS-BI phosphoric acid as substrate and polyvinyl alcohol was added to the incubation medium to counteract diffusion phenomena. Ischemia in vitro up to 240 min did not affect the localization nor the total activity of acid phosphatase activity. After 60-min ischemia in vivo followed by 1-h reperfusion distinct areas showed decreased acid phosphatase activity. A further decrease in activity was observed after 5 h reperfusion. Final reaction product generated by acid phosphatase activity was rather diffusely distributed in border zones between normal and damaged tissue after 24 and 48 h of reperfusion following 60 min ischemia in vivo. It is concluded that not ischemia itself but rather reperfusion affects the stability of the lysosomal membrane due to the occurrence of oxygen-derived free radicals and/or imbalanced Ca2+ concentration. Restoration of the blood flow causes leakage of acid phosphatase from the lysosomes into the cytoplasm of liver parenchymal cells and from there to the blood.     

Localization of Acid and Alkaline Phosphatases in Staphylococcus aureus

The localization of acid and alkaline phosphatases in Staphylococcus aureus was studied by fractionation of cells after treatment with the L-11 enzyme and by electron microscopic histochemistry. The two enzyme activities were located in distinctly different positions at the surface of the cells. Acid phosphatase appeared to be localized around the cell membrane of the bacteria, because the enzyme was recovered exclusively in the membrane fraction and because deposition of lead phosphate was detected by electron microscopic histochemistry on the inner surface of the cell membrane of intact bacteria and spheroplasts. The highest specific activity of alkaline phosphatase was also associated with the membrane fraction. However, on electron microscopic histochemistry of intact cells, the deposition of lead phosphate was only seen on the outer surface of the cell wall.     

Cytochemical localisation of acid phosphatase in the phagocytising conjunctival epithelium of the guinea pig

The ultrastructural localisation of acid phosphatase activity was investigated on the guinea pig conjunctival epithelium incubated in vivo with a suspension of latex spheres. Deposits of acid phosphatase reaction product were concentrated on the elements of GERL, the phagocytic vacuoles, and the cell membrane. Acid phosphatase activity in GERL was intense in basal and suprabasal cells and decreased towards the superficial cells. Phagosomes containing latex spheres and reaction product of acid phosphatase were observed mainly in the centrospheral region of the superficial and intermediate epithelial cells. Acid phosphatase activity in phagocytising cells was not increased as compared to that in non-phagocytising cells. The observations indicate that existing acid phosphatase in unstimulated conjunctival epithelial cells is released into heterophagosomes brought within the lysosomal compartment. The number of secondary phagosomes seems to be increased by intercellular transport of latex spheres to the acid phosphatase rich cells in the deep layers of the epithelium.     

Fine structural localization of alkaline phosphatase in the granular pneumonocytes of hamster lung.

The fine structural localization of nonspecific alkaline phosphatase was studied in the granular pneumonocytes (type II alveolar epithelial cells) of hamster lung by incubating sections of glutaraldehyde-fixed tissues in a medium containing lead ions and sodium beta-glycerophosphate or alpha-naphthyl acid phosphate. The specificity of the reaction was tested by exposing the sections to inhibitors of alkaline phosphatase. The results showed that alkaline phosphatase activity was present in the inclusion bodies of granular pneumonocytes. The enzyme reaction was strong in the membrane lining the inclusion bodies and a weaker reaction was generally detectable in the inclusion contents. Although only a proportion of the inclusion bodies showed enzyme activity, there was no obvious correlation between the reactivity of the inclusions and their intracellular position or size. The other organelles were unreactive. The finding of alkaline phosphatase activity within the inclusion bodies of granular pneumonocytes is an enigma as these organelles are generally considered to be lyosomes.     

Cytochemical localisation of acid phosphatase in the phagocytising conjunctival epithelium of the guinea pig

Summary The ultrastructural localisation of acid phosphalase activity was investigated on the guinea pig conjunctival epithelium incubated in vivo with a suspension of latex spheres. Deposits of acid phosphatase reaction product were concentrated on the elements of GERL, the phagocytic vacuoles, and the cell membrane. Acid phosphatase activity in GERL was intense in basal and suprabasal cells and decreased towards the superficial cells. Phagosomes containing latex spheres and reaction product of acid phosphatase were observed mainly in the centrospheral region of the superficial and intermediate epithelial cells. Acid phosphatase activity in phagocytising cells was not increased as compared to that in non-phagocytising cells. The observations indicate that existing acid phosphatase in unstimulated conjunctival epithelial cells is released into heterophagosomes brought within the lysosomal compartment. The number of secondary phagosomes seems to be increased by intercellular transport of latex spheres to the acid phosphatase rich cells in the deep layers of the epithelium.     

Changes in acid phosphatase activity in rat liver after ischemia

Summary The effect of ischemia on the stability, i.e. the permeability of the lysosomal membrane of rat liver has been studied using quantitative histochemical analysis of acid phosphatase activity. Ischemia in vitro was performed for 0–240 min at 37° C and ischemia in vivo for 60 min was followed by 1, 5, 24 and 48 h of reperfusion. Acid phosphatase activity was demonstrated in cryostat sections using naphthol AS-BI phosphoric acid as substrate and polyvinyl alcohol was added to the incubation medium to counteract diffusion phenomena. Ischemia in vitro up to 240 min did not affect the localization nor the total activity of acid phosphatase activity. After 60-min ischemia in vivo followed by 1-h reperfusion distinct areas showed decreased acid phosphatase activity. A further decrease in activity was observed after 5 h reperfusion. Final reaction product generated by acid phosphatase activity was rather diffusely distributed in border zones between normal and damaged tissue after 24 and 48 h of reperfusion following 60 min ischemia in vivo. It is concluded that not ischemia itself but rather reperfusion affects the stability of the lysosomal membrane due to the occurrence of oxygen-derived free radicals and/or imbalanced Ca²⁺ concentration. Restoration of the blood flow causes leakage of acid phosphatase from the lysosomes into the cytoplasm of liver parenchymal cells and from there to the blood.     

Light microscope and electron microscope alkaline phosphatase cytochemistry of rat bone marrow leukocytes.

An enzyme cytochemical method yielding an osmiophilic reaction product, visible at both the light and electron microscope levels, has been applied to the study of alkaline phosphatase in rat bone marrow cells. The enzyme is present in both eosinophils and, in much smaller amounts, in neutrophils. In both cases it is present on the plasma membrane, and in eosinophils intracellular aggregations of reaction product are also seen. The specific granules in both cell types fail to react and the enzyme is first detectable at the promyelocyte stage. Thus the enzyme is demonstrable before specific granule formation begins in the neutrophil, indicating that they are not a significant site of alkaline phosphatase activity in the rat.     

Dynamic reorganization of the alkaline phosphatase-containing compartment during chemotactic peptide stimulation of human neutrophils imaged by backscattered electrons

Scanning electron microscopy (EM) and cytochemical techniques were used to examine the alkaline phosphatase-containing compartment in human neutrophils after stimulation with nanomolar concentrations of N-formylmethionyl-leucyl-phenylalanine (10^–8M fMLP). Alkaline phosphatase (AlkPase) activity was demonstrated with a lead-based metal capture cytochemical method. The reaction product was visualized with the backscattered electron imaging mode of scanning EM, and analyzed by electron probe X-ray microanalysis. Alkaline phosphatase activity was detected only in fMLP-stimulated neutrophils; unstimulated neutrophils displayed no activity. Stimulation of human neutrophils with 10^–8 M fMLP induced a time-dependent intracellular redistribution of irregular round or tubular granules containing alkaline phosphatase activity, as seen by backscattering. The intracellular redistribution of alkaline phosphatase activity was accompanied by increased cytochemical activity on the cell surface. The reaction product was localized preferentially on ridges and folds of polar neutrophils. Reorganization of the AlkPase-containing compartment correlated with changes induced by fMLP in cell shape, ie, membrane ruffling and front-tail polarity, as observed with the secondary electron image mode of scanning EM. These findings demonstrate the intracellular reorganization, increase, and asymmetric distribution of alkaline phosphatase activity on the plasma membrane of human neutrophils after stimulation by chemotactic peptides.     

Quantitative histochemical investigations of semipermeable membrane techniques for the assay of acid phosphatase in skeletal muscle

Summary Meijer's semipermeable membrane technique for acid phosphatase was modified by treating sections immediately after incubation with 70% ethanol for 30 min at room temperature instead of with formalin. The modification improved the validity of the technique considerably, by stabilising the specific final reaction product formed in presumed enzyme-containing sites. The modified technique thus seems promising for assaying the activity of acid phosphatase in sections of skeletal muscle.     

Ultrastructural localisation of alkaline phosphatase in the intertubular tissue of the testis in the domestic fowl

Alkaline phosphatase activity in the intertubular tissue of the testes of the domestic fowl was examined using an ultracytochemical technique based on the lead capture method. In the interstitial tissue, the Leydig cells, transitional cells and the fibroblasts displayed enzyme activity on their cell membranes. Vacuoles located in the transitional cells were lined by reaction products of enzyme activity, whereas the vacuoles representing extracted lipid droplets and present mainly in the Leydig cells were free of enzyme activity. In the peritubular tissue the cell processes of fibroblasts showed enzyme activity on the cell membranes and in pinocytotic vesicles. Cell processes lying adjacent to blood vessels showed pronounced activity. In the blood vessel itself some activity was present in the basement membrane and the endothelium. The surface of the red blood cell showed moderate activity. The possible role of alkaline phosphatase in the transfer of hormone from the Leydig cells to the seminiferous tubules and from the seminiferous tubules to the interstitium is discussed. The myoid cells and their processes were devoid of enzyme activity.     

PROGRAMMED CELL DEATH IN THE HONEY-BEE (APIS MELLIFERA L.) LARVAE MIDGUT

The histochemical and cytochemical localization of acid phosphatase has been used in an attempt to map the sites of cellular lysis and death. Reaction product was found both in the brush border of the midgut epithelium and in the basal membrane. Vacuolar acid phosphatase activity was found in the regenerative epithelial cells. Extra-cisternal reaction product was associated with the endoplasmic reticulum which was dilated in lysed areas of the cytoplasm. Free acid and alkaline phosphatase activity was found in the basal area of the midgut epithelial cells and the former also occurred in the haemocoel. In the tracheoblastic cells only vacuolar acid phosphatase activity was seen. Chromatin aggregates were distributed throughout the nucleus and the nuclear envelope showed some infolding. Certain mature epithelial cells proved positive for anti-histone associated DNA fragmentation indicative of programmed cell death.     

Cytochemical Localization of Certain Phosphatases in Escherichia coli

Cytochemical studies of Escherichia coli at the light and electron microscopic levels have revealed alkaline phosphatase, hexose monophosphatase, and cyclic phosphodiesterase reaction products in the periplasmic space and at the cell surface. In preparations for both light and electron microscopy, reaction product filled polar caplike enlargements of the periplasmic space, such as those described in plasmolyzed cells, indicating significant terminal concentrations of these enzymes; dense substance was often seen within these polar caps in morphological specimens. Staining of the bacterial surface was commonly encountered, but could represent artifactual accumulation of precipitate along the cell wall. Alkaline phosphatase was demonstrated with several substrates (ethanolamine phosphate, glycerophosphate, p-nitrophenylphosphate, and glucose-6-phosphate) over a wide pH range in a bacterial strain (C-90) known to be constitutive for this enzyme, whereas strains deficient in this enzyme (U-7, repressed K-37), showed no activity with these substrates. Hexose monophosphatase and cyclic phosphodiesterase activities were characterized by reaction-product deposition with specific substrates at acid or neutral, but not at alkaline, pH in strains of E. coli lacking alkaline phosphatase (U-7 and repressed K-37). Fixation in Formalin or the use of calcium as a capture reagent seemed to interfere with periplasmic staining in cells prepared for electron microscopy. Formalin fixation had little effect on biochemical assays of the phosphatase activity of intact cells in suspension, but partially reduced the activity evident in sonically treated extracts or in suspensions of dispersed cryostat sections. Glutaraldehyde treatment impaired enzyme activity more drastically.     

Concanavalin A and the in vitro induction in macrophages of vacuolation and lysosomal enzyme synthesis

Concanavalin A (ConA) induced extensive vacuolation in mouse peritoneal macrophages. Electron microscopic observations on thin sections reveal that the vacuoles are essentially empty except for minute vesicles attached to their inner periphery. The vacuoles consist of irregular structures and are heterogeneous in size distribution. ConA-induced vacuoles exhibit high acid phosphatase activity, suggesting fusion between vacuoles and lysosomes. Induction of acid phosphatase in ConA-treated macrophages was studied under several cultivation conditions. ConA-treated macrophage cultures responded in increase in acid phosphatase activity early after exposure to the lectin, a significant increase recorded already after 1 h. When cultivated in 1% serum medium for 48 h, ConA-treated macrophages exhibit twice the activity of acid phosphatase at zero time as well as that of non-treated control cultures. The effect of ConA on thioglycolate-stimulated mouse peritoneal macrophages was also studied. Vacuole formation resulting from lectin binding and internalization is discussed in terms of possible lectin effects on membrane fluidity, fusion capacity, surface to volume conservation during vacuole formation, fusion of vacuoles with lysosomes and intravacuolar lysosomal enzyme activities. The phenomenon of lysosomal enzyme induction as a result of ConA treatment is being correlated with enzyme induction due to other stimuli.     

Induction of plasma membrane alkaline phosphatase in rat liver

We have determined alkaline phosphatase activity in total liver plasma membrane fractions from rats subjected to a partial hepatectomy and sham operated with or without manipulation of the liver. In all these cases, an increase of the enzyme activity was observed. Kinetic studies of alkaline phosphatase activity performed on plasma membrane fractions from rats subjected to a partial hepatectomy suggest that alkaline phosphatase increase is produced by de novo biosynthesis of enzyme molecules. Determination of alkaline phosphatase activity in purified plasma membrane subfractions corresponding to each of the three functional regions of the hepatocyte surface (blood sinusoidal, lateral and bile canalicular), indicates that the increase of the enzyme activity observed after partial hepatectomy is selectively induced in the bile canalicular domain of the hepatocyte plasma membrane.     

Ultracytochemical characterization of non-specific acid phosphatase activities in Saccharomyces cerevisiae.

Glutaraldehyde prefixation causes a considerable inactivation of the acid phosphatase of yeast protoplasts in dependence on the duration of aldehyde influence. Lead ions necessary for ultracytochemical demonstration effect a still stronger inhibition of enzymatic activity. Prefixation, however, protects the enzyme from further inhibition by lead. At pH 4.4 in intact cells acid phosphatase activities are mainly localized in the periplasmic space and in vesicles fused with the plasma membrane. The cell wall and cytoplasm usually remain free of reaction products. On the cell surface activities are found in form of globular lead deposits. At pH 5.2 and 6.3 the periplasmic activity appears decreased compared to that at lower pH values and the intracellular activity is increased. The plasma membrane of protoplasts is completely free of precipitates. The intracellular activity sites of protoplasts (cisternae of endoplasmic reticulum and/or Golgi-like system, small vesicles, central vacuole, nuclear envelope) are the same as for intact cells. The occurrence of at least two forms of acid phosphatase in S. cerevisiae id deduced.     

Alkaline phosphatase biosynthesis in the endoplasmic reticulum and its transport through the Golgi apparatus to the plasma membrane: cytochemical evidence

Enzyme induction of HeLa cell placental alkaline phosphatase with various agents such as prednisolone, sodium butyrate, hyperosmolality (NaCl), or combination of these inducers resulted in the appearance of enzyme activity in the rough endoplasmic reticulum, nuclear envelope, Golgi apparatus, and plasma membrane. In the Golgi apparatus, intense reaction product deposits tended to be concentrated on its trans side, with small vesicles and granules also being positively stained. Inhibition of protein synthesis with cycloheximide was followed by the disappearance of enzyme activity from these cytoplasmic organelles but not from the plasma membrane. Treatment with monensin, a secretory protein transport inhibitor, uniformly increased activity in the rough endoplasmic reticulum while causing marked dilatation of the intensely positive Golgi cisternae. These results suggest that intracellular alkaline phosphatase is newly synthesized in the endoplasmic reticulum and then passes en route through the Golgi apparatus to the plasma membrane. Accordingly, the present system could represent the biosynthesis, transport, and incorporation of the model cell surface enzyme protein to add to the vesicular stomatitus virus glyco-1 (VSV-G) protein and acetylcholine receptor model systems for studying the dynamics of cell surface protein genesis, transport, and membrane integration.