Radiation effects on rat testes. V. Studies on lysosomal enzymes (acid phosphatase and acid DNAse) and their physiological significance following partial body gamma-irradiation.

Acid phosphatase is present in the nucleus and cytoplasm of cells in the seminiferous tubules and the interstitium of rat testes. The effect of irradiation on acid phosphatase is dependent on the environmental temperature and the dose of irradiation. It appears that initial rise in the enzyme at a low radiation dose and a high environmental temperature or at a high dose and low temperature is associated with a lysosomal breakdown of the germinal cells of the testes. A decrease in acid phosphatase in the advanced stages of radiation injury is a secondary radiation effect which may lead to decreased metabolic synthesis of phosphate esters owing to the unavailability of orthophosphate in the testicular tubules. The reduced acid phosphatase activity can be detected in the seminiferous tubules, suggesting that the enzyme activity is related to the state of the germ cell population. An initial increase in acid phosphatase is matched by an initial rise in acid DNAse within hours of irradiation, further suggesting that there is radiation interaction with the cells of the germinal epithelium. The enhanced activity of DNAse following a 2nd week of irradiation at 2000 R confirms the phagocytic activity of the non-germinal cells.     

Effect of repeated intraperitoneal exposure to picrotoxin on rat liver lysosomal function

Effect of repeated (20 days) exposure to picrotoxin (PTX) on rat liver lysosomal function was evaluated by measuring the free and total activities of acid phosphatase, cathepsin D, ribonuclease II (RNAse II) and deoxyribonuclease II (DNAse II). The free activities of the nucleases (both RNAse II and DNAse II) were increased following PTX exposure. The total DNAse II activity was increased by 2.2-fold whereas the total acid phosphatase activity was decreased by 28%. Consequently, the ratios of total activity / free activity were low in the PTX exposed groups, implying loss of membrane integrity. Cathepsin D activity was completely abolished. The results show that repeated exposure to PTX can lead to lysosomal dysfunction in liver.     

Cytochemical localization of acid phosphatases in the dimorphic fungusSporothrix schenckii

A modified Gomori procedure at the electron microscopic level revealed a multiplicity of acid phosphatase activity sites in both yeast-like and mycelial phase cells. Vacuoles and the periplasmic space contained electron opaque deposits (lead phosphate) that were absent in control incubations either lacking the substrate (-glycerophosphate) or fortified with an inhibitor (sodium fluoride). The outermost region of the cell envelope was also active and, in contradistinction to previous examples with other yeasts, deposition of lead phosphate in this locale occurred even when the rate of orthophosphate generation was drastically reduced by lowering the substrate concentration. When mechanically disrupted yeast-like cells were washed and then subjected to the cytochemical procedure, pieces of broken cell envelope gave a positive reaction. The reaction product was invariably restricted to one side of cell wall cross sections. A specific and novel association of acid phosphatase with a microfibrillar zone was indicated.     

Factors affecting lead capture methods for the fine localization of rat lung acid phosphatase

Synopsis Gomori's lead capture method for acid phosphatase localization was adapted for the electron microscope by Holt & Hicks (1961a). The method gave good results in rat liver, but poor tissue preservation with no reaction product in rat lung, and was, therefore, investigated in order to find the optimum conditions for the ultrastructural localization of rat lung acid phosphatase. The conditions investigated included the use of glutaraldehyde or depolymerized paraformaldehyde as the fixative, with and without dimethylsulphoxide; the effect of freezing the tissue; the pH of the incubation medium; and the use of glycerophosphate, naphthol AS-BI phosphate or -naphthyl phosphate as substrates. Improved preservation of ultrastructure with increased yield of reaction product was obtained by prefixing lung in glutaraldehyde containing 10% dimethylsulphoxide, freezing the tissue and incubating at pH 5.7 with -naphthyl phosphate. Tissue preservation was acceptable and dense deposits of reaction product occurred in lysosomal elements of all the alveolar cells and especially in macrophages. Deposits were also found closely associated with the lamellae of the inclusion bodies of Type II cells.     

Kinetic limitations on the trapping of nascent phosphate for cytochemical localization of yeast acid phosphatase

The location of extracytoplasmic acid phosphatase in haploid, diploid, and polyploid cells ofSaccharomyces cerevisiae was examined by cytochemical electron microscopy. In all cases, in the presence of lead nitrate and low concentrations of glycerophosphate, the reaction product (lead phosphate) was restricted to the periplasmic space. With higher substrate concentrations (which are typical of those previously employed by others) precipitates also appeared on the cell wall surface of some specimens; a result previously reported by three other laboratories. The surface deposits are deemed artifacts from incomplete lead capture under high enzymatic rates of orthophosphate generation. A model system that supports this is presented.     

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.     

The root surface phosphatases ofEriophorum vaginatum: Effects of temperature,pH, substrate concentration and inorganic phosphorus

Eriophorum vaginatum L. subsp.spissum (Fern.) Hult., a dominant plant in arctic tundra ecosystems, has acid phosphatase activity evenly distributed along its root surface from the root tip to a distance at least 16 cm from the tip. These root surface phosphatases have optimal activity from pH 3.5 to 4.0; mean soil pH for soil samples collected with roots was 3.69. Apparent energy of activation and Q₁₀ values (14.0 kcal mol⁻¹ and 2.2, respectively) do not provide evidence for temperature acclimation, but substantial phosphatase activity was measured at 1°C. Kinetic parameters determined for this root surface phosphatase were as follows: Km=9.23 mM, Vmax=1.61×10⁻³ μmoles mm⁻²h⁻¹. The presence of inorganic phosphorus in the assay medium did not inhibit root surface phosphatase activity except at very high concentrations (100 mM); even then, only slight inhibition was detected (7 to 19%). A comparison of hydrolysis rates with inorganic phosphate assimilation rates measured forE. vaginatum indicates that organic phosphate hydrolysis may occur at approximately one third the rate of inorganic phosphate absorption. Calculations show that inorganic phosphate produced by root surface phosphatase activity may satisfy 65% of the annual phosphate demand ofE. vaginatum. Since arctic tundra soils are typically higher in dissolved organic phosphorus compounds than in inorganic phosphate, root surface phosphatase activity may make a considerable contribution to the phosphate nutrition of this widespread and abundant arctic plant.     

Localization of Phosphatases in Trypanosoma rhodesiense1

ABSTRACT. Phosphatase activity in Trypanosoma rhodesiense has been examined histochemically by light and electron microscopy and by enzymatic assay in homogenate fractions. Using a method with lead as capture ion, acid phosphatase was found in lysosome-like vesicles and in the flagellar pocket. No alkaline adenosine triphosphatase (ATPase) was detectable by this method. Direct assay of p-nitrophenylphosphatase activity in homogenate fractions showed that acid phosphatase activity was strongly membrane-bound, but that activity at pH 9 was minimal in both soluble and particulate fractions. “Endogenous” ATPase activity was localized specifically and reproducibly in the mitochondrial membranes and under the plasma membrane of the flagellum. This nonenzymic reaction product could not be eradicated by glycerol extraction or glucose depletion. Unlike the membrane staining, which was manifest only after lead treatment, heat-resistant electron-dense material was found in the matrix of lysosomal vesicles in trypanosomes fixed in glutaraldehyde only and not subjected to further treatment with heavy metal reagents. X-ray emission analysis showed the presence of calcium and phosphorus, indicating that the matrix might have a phosphate storage function.     

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.     

Apatite genesis: A biologically induced or biologically controlled mineral formation process?

Apatite formation from organic matter (ribonucleic acid) and calcium carbonate (cuttlebone) requires intervention of microorganisms. We have attempted to characterize this mineral formation process by locating the alkaline phosphatase and the crystals formed. Alkaline phosphatase, which is important for the liberation of the necessary components, was localized in the periplasmic space of Providencia rettgeri in the same manner as in Escherichia coli. Accordingly, the release of inorganic phosphate and the formation of apatite may occur at this site. However, electron microscope observations revealed the presence of extracellular apatite; moreover, apatite particles that were formed with or without bacteria (with alkaline phosphatase from hydrolyzed ribonucleic acid as phosphorus source) were closely similar in size and appearance. The formation of apatite can thus be qualified as biologically induced mineralization. Nevertheless, a bacterial cell can also act as a nucleator for apatite crystallization, but this would appear exceptional.     

The digestion ofSaccharomyces cerevisiae byAcanthamoeba castellanii

Summary The digestion ofSaccharomyces cerevisiae byAcanthamoeba castellanii, at different times after feeding, has been examined by cytochemical techniques at electron microscope level and by measurement of yeast viability. The measurement of viability, combined with cytochemistry is presented as a novel method of examining the progress of digestion. Particular attention has been given to the temporal development of digestion.Vacuoles, probably primary lysosomes, have been identified containing acid phosphatase activity within minutes of feeding and these accumulate around and fuse with phagocytic vacuoles. Acid phosphatase levels in the digestive vacuoles appeared highest at 20 to 40 minutes. Yeast digestion was observed and yeast viability began to decline at this time. Mixing of autophagic and heterophagic material was also observed. At least half of the yeast population was still viable after 90 minutes.Our method (p-nitrophenyl phosphate) of enzyme localization has demonstrated plasma membrane associated acid phosphatase activity.     

Localization of gibberellic acid-induced acid phosphatase activity in the endoplasmic reticulum of barley aleurone cells with the electron microscope

A metal-salt precipitation method with p-nitrophenyl phosphate as substrate has been used to localize in the electron microscope acid phosphatase activity in isolated aleurone layers of barley (Hordeum vulgare L.), treated for 16 h in the presence or absence of gibberellic acid (GA₃). The paper confirms results obtained earlier with an azo-dye precipitation method of enzyme localization. In addition the results show for the first time that in GA₃-treated tissue enzyme activity is associated with the endoplasmic reticulum (ER), there being reaction product deposited in the ER cisternae. It is suggested that this activity represents new enzyme synthesized on ER in response to GA₃ and probably destined for secretion.Abbreviation ER endoplasmic reticulum     

Different tartrate sensitivity and pH optimum for two isoenzymes of acid phosphatase in osteoclasts. An electron-microscopic enzyme-cytochemical study

Summary By differentiation of substrate specificity, pH optimum range, and sensitivity to various inhibitors, 2 isoenzymes of acid phosphatase in bone cells have been studied at the electron-microscopic level. When p-nitrophenyl phosphate was used for the substrate, the demonstrable enzyme activity was affected by neither tartrate nor sodium fluoride. The reaction product, when incubated at pH 5–6, was detected in all sites along the pathway for the biosynthesis of acid phosphatase in the osteoclast, including the perinuclear space, cisternae of the endoplasmic reticulum, Golgi complex, various vesicles, and vacuoles. In the osteoclasts attached to bone, the enzymatic activity was demonstrated at the extracellular ruffled border and on the eroded bone surface. Reaction products became confined to lysosomes and extracellular ruffled border when incubated at pH 6–7. Unattached osteoclasts showed a similar intracytoplasmic localization of enzyme as the attached ones, except for the absence of the extracellular enzyme activity. The mononuclear, immature type of osteoclast also resembled the mature osteoclast in terms of enzymatic localization. Except for the osteoclasts, the acid p-nitrophenyl phosphatase activity was restricted to lysosomal vesicles in various bone cells, monocytes, and macrophages. Such activity was inhibited by adding 50 mM tartrate to the p-nitrophenyl phosphate medium. When -glycerophosphate or p-nitrocatechol sulfate was the substrate, most of the reaction product was localized intracellularly. Unlike the acid p-nitrophenyl phosphatase, the acid -glycerophosphatase or arylsulfatase activity in osteoclasts and other bone cells was inhibited completely by 10 mM tartrate or 10 mM sodium fluoride. Even preincubation of 100 mM tartrate in the buffer inhibited -glycerophosphatase activity completely, but p-nitrophenyl phosphatase activity was inhibited incompletely. Consequently, our results suggest that acid p-nitrophenyl phosphatase is a useful cytochemical marker for identification of the osteoclast family at electron-microscopic levels of resolution.     

Mycelial forms of <Emphasis Type="Italic">Pseudallescheria boydii</Emphasis> present ectophosphatase activities

Phosphatase activities were characterized in intact mycelial forms of Pseudallescheria boydii, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 41.41 ± 2.33 nmol p-NP per h per mg dry weight, linearly with increasing time and with increasing cell density. MgCl₂, MnCl₂ and ZnCl₂ were able to increase the (p-NPP) hydrolysis while CdCl₂ and CuCl₂ inhibited it. The (p-NPP) hydrolysis was enhanced by increasing pH values (2.5-8.5) over an approximately 5-fold range. High sensitivity to specific inhibitors of alkaline and acid phosphatases suggests the presence of both acid and alkaline phosphatase activities on P. boydii mycelia surface. Cytochemical localization of the acid and alkaline phosphatase showed electron-dense cerium phosphate deposits on the cell wall, as visualized by electron microscopy. The product of p-NPP hydrolysis, inorganic phosphate (Pi), and different inhibitors for phosphatase activities inhibited p-NPP hydrolysis in a dose-dependent manner, but only the inhibition promoted by sodium orthovanadate and ammonium molybdate is irreversible. Intact mycelial forms of P. boydii are also able to hydrolyze phosphoaminoacids with different specificity.     

Phosphatase production byAspergillus ficuum

Summary Effects of nutritional and cultural conditions on cell growth and phosphatase production byAspergillus ficuum were studied.A. ficuum produced high levels of phosphatases when grown on a basal medium that contained a minimal amount (2 mg/100 ml) of phosphorus in an acidic growth medium. The organism produced a nonspecific acid phosphomonoesterase rather than phytin-specific phosphatase. The enzyme hydrolyzed a variety of phosphates and produced orthophosphate. The rate of phosphate hydrolysis was dependent on the pH of the reaction, where the pH optimum for acid phosphatase was 2.5 and that for phytase was 5.0. The organism slowly released the phosphatase, and the enzyme activity in the growth medium increased continually during a one-month growth period. For a high level of phosphatase production, low levels (1–5 mg%) of initial phosphorus were necessary and polyphosphates were the desired form rather than the monophosphate. The addition of surfactants, such as polyoxyethylene ethers and sodium oleate, to fungal culture medium markedly increased the level of phosphatase production.     

Vertical Profile of Phosphatase Activity in the Sundarban Mangrove Forest,North East Coast of Bay of Bengal,India

Impact of phosphate solubilizing bacteria along with soil phosphatase activity on phosphorous cycle was found to be quiet interesting in the Sundarban mangrove ecosystem. Soil phosphatase activity showed a decreasing pattern with increase in depth [soil phosphatase activity (μg pnp produced g^?1 dry wt of soil) = 906.85 – 5.6316 Depth (cm)] from the deep forest region of the Sundarban forest ecosystem. Soil salinity showed a very little effect on soil phosphatase activity whereas soil temperature and pH was found to show significant impact on the soil phosphatase activity. This ensured that the microbes associated with phosphate mineralization present in the Sundarban forest ecosystem are more tolerant to fluctuation in salinity than that of temperature and pH. A direct correlation was perceptible between the number of phosphate solubilizing bacteria and phosphatase activity in the soil during the study period from 2007 to 2012. Soil phosphate concentration was found to be directly governed by the soil phosphatase activity [The regression equation is: avg PO₄^?3-P (μg g^?1 dry wt of soil) = 0.0311 + 0.000606 soil phosphatase activity (μg pnp produced g^?1 dry wt of soil); R² = 63.2%, p < 0.001, n = 62].     

An 18 kDa Acid Phosphatase from Chicken Heart Possesses Phosphotransferase Activity

A low molecular weight acid phosphatase was purified to homogeneity from chicken heart with a specific activity of 42 U/mg and a recovery of about 1%. Nearly 800 fold purification was achieved. The molecular weight was estimated to be 18 kDa by SDS-polyacrylamide gel electrophoresis. Para-nitrophenyl phosphate, phenyl phosphate and flavin mononucleotide were efficiently hydrolysed by the enzyme and found to be good substrates. Fluoride and tartrate had no inhibitory effect while phosphate, vanadate and molybdate strongly inhibited the enzyme. The acid phosphatase was stimulated in the presence of glycerol, ethylene glycol, methanol, ethanol and acetone, which reflected the phosphotransferase activity. When phosphate acceptors such as ethylene glycol concentrations were increased, the ratio of phosphate transfer to hydrolysis was also increased, demonstrating the presence of a transphosphorylation reaction where an acceptor can compete with water in the rate limiting step involving hydrolysis of a covalent phospho enzyme intermediate. Partition experiments carried out with two substrates, para-nitrophenyl phosphate and phenyl phosphate, revealed a constant product ratio of 1.7 for phosphotransfer to ethylene glycol versus hydrolysis, strongly supporting the existence of common covalent phospho enzyme intermediate. A constant ratio of K _cat/K _m, 4.3×10⁴, found at different ethylene glycol concentrations, also supported the idea that the rate limiting step was the hydrolysis of the phospho enzyme intermediate.     

ULTRASTRUCTURE AND DEVELOPMENT OF THE NEXINE AND INTINE IN THE POLLEN WALL OF SILENE ALBA (CARYOPHYLLACEAE)

Nexine and intine development in Silene alba (Caryophyllaceae) was investigated by electron microscopy and enzyme cytochemistry. Nexine-2 forms by deposition of sporopollenin along unit membrane lamellae closely associated with the microspore plasma membrane in the late tetrad stage. After the callose wall dissolves, electron density increases along the tangentially oriented fibers of the proximal primexine, forming nexine-1. When the exine is essentially complete, the intine begins to develop. In the nearly mature microspore, acid phosphatase activity appears in the peripheral cytoplasm just prior to its extrusion into the intine of the mature pollen grain.     

Non-destructive measurement of acid phosphatase activity in the rhizosphere using nitrocellulose membranes and image analysis

We developed a method using nitrocellulose membranes and image analysis to localise and quantify acid phosphatase activity in the rhizosphere of two plant species, one with cluster roots (Dryandra sessilis (Knight) Domin) and another with ectomycorrhizal roots (Pinus taeda L.). Membranes were placed in contact with roots and then treated with a solution of x, α-naphthyl phosphate and Fast Red TR. Acid phosphatase activity was visualised as a red imprint on the membrane. We quantified acid phosphatase activity by image analysis of scanned imprints. The method was used to estimate the spatial distribution of acid phosphatase activity within particular root classes (lateral roots, mycorrhizal roots, root clusters). Over 95% of the acid phosphatase activity of the root system of D. sessilis was associated with cluster roots, and between 20 and 32% of the root surface active. About 26 % of the acid phosphatase activity of the root system of P. taeda was associated with mycorrhizal roots and unsuberised white root tips and less than 10% of the root surface was active, irrespective of root type. This non-destructive method can be used for rapid, semi-quantitative assessment of acid phosphatase activity in the laboratory and in situ. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evaluation of pink-pigmented facultative methylotrophic bacteria for phosphate solubilization

Thirteen pink-pigmented facultative methylotrophic (PPFM) strains isolated from Adyar and Cooum rivers in Chennai and forest soil samples in Tamil Nadu, India, along with Methylobacterium extorquens, M. organophilum, M. gregans, and M. komagatae were screened for phosphate solubilization in plates. P-solubilization index of the PPFMs grown on NBRIP—BPB plates for 7 days ranged from 1.1 to 2.7. The growth of PPFMs in tricalcium phosphate amended media was found directly proportional to the glucose concentration. Higher phosphate solubilization was observed in four strains MSF 32 (415 mg l^−l), MDW 80 (301 mg l^−l), M. komagatae (279 mg l^−l), and MSF 34 (202 mg l^−l), after 7 days of incubation. A drop in the media pH from 6.6 to 3.4 was associated with an increase in titratable acidity. Acid phosphatase activity was more pronounced in the culture filtrate than alkaline phosphatase activity. Adherence of phosphate to densely grown bacterial surface was observed under scanning electron microscope after 7-day-old cultures. Biochemical characterization and screening for methanol dehydrogenase gene (mxaF) confirmed the strains as methylotrophs. The mxaF gene sequence from MSF 32 clustered towards M. lusitanum sp. with 99% similarity. This study forms the first detailed report on phosphate solubilization by the PPFMs.