Cytochemical and biochemical observations on the digestive tracts of digenetic trematodes. 8. Acid phosphatase activity in Schistosoma mansoni and Schistosomatium douthitti

At the light microscope level, nonspecific acid phosphatase (AcPase) (EC 3.1.3.2) and N-acetyl glucosaminidase (NAGase) (EC 3.2.1.29) activities are in the esophageal gland cells of Schistosoma mansoni and Schistosomatium douthitti and in the gastrodermis of S. mansoni. The gastrodermis of S. douthitti is negative for these two enzymes. At the electron microscope level, AcPase activity in the esophageal gland cells of both species is observed in cytoplasmic vesicles. In S. mansoni, AcPase activity is also observed associated with the infoldings of the basal plasma membranes of the esophagus and the gastrodermis. It is hypothesized that this enzyme(s) is involved with membrane transport. AcPase activity is also associated with “droplets” and vesicles in the gastrodermis of S. mansoni. It is believed that the digestion of foodstuffs in both species occurs extracellularly.     

Schistosoma mansoni: Radioautography of colchicine's effect on [3H]proline incorporation into adults in vitro

Adult Schistosoma mansoni were studied radioautographically in order to ascertain the effect of exposures to a fixed concentration of colchicine (5 × 10^?4M) for varying time intervals upon the incorporation of [³H]proline in the tegument. Additionally, a study was made on the effect of varying time exposures of colchicine on the cytochemical localization of alkaline phosphatase (EC 3.1.3.1) in the tegumental invaginations. Worms exposed to colchicine for more than 2 hr preceding addition of the labeled amino acid displayed significant changes in the pattern of distribution. The most profound change was noted in the male tegument where a statistically significant decrease was observed in treated worms. Female worms, on the other hand, failed to display any effect of the drug on the distribution pattern for the times utilized. The distribution of alkaline phosphatase activity was much reduced in the teguments of both sexes. Morphological effects of the drug included disappearance of microtubules from the cytoplasmic connectives, a stacking of RER in the subtegumental cells, and accumulation of discoid granules and membranous bodies in the subtegumental cells. It is hypothesized that the amino acid is associated with the discoid granule at the subtegumental cell level and is ultimately translocated, with the aid of microtubules in the cytoplasmic connectives, to the tegument. Alkaline phosphatase activity is assumed to be associated with the membranous body.     

Acid and alkaline phosphatase activity in the fat body and midgut of the beet armyworm,Spodoptera exigua (Lepidoptera: Noctuidae), infected with nuclear polyhedrosis virus

Changes in the activity of acid and alkaline phosphatase in Spodoptera exigua larvae infected with nuclear polyhedrosis virus have been investigated. Three days after per os infection, the activity of acid phosphatase in the fat body and midgut of infected larvae was significantly higher than that in normal larvae. Alkaline phosphatase activity did not show such significant changes. There were differences in the phosphatase patterns depending on whether their activities were expressed as enzyme units per milligram of fresh organ weight or per milligram of homogenate protein. The literature relevant to the subject allows us to conclude that the increase in phosphatase activities in S. exigua larvae is not specifically associated with virus infection itself, but, rather, is a reaction of the insect organism to the diminishing supply of energy sources.     

Acid phosphatase in granulocytic capsules formed in strains of Biomphalaria glabrata totally and partially resistant to Schistosoma mansoni

Cheng T. C. and Garrabrant T. A. 1977. Acid phosphatase in granulocytic capsules formed in strains of Biomphalaria glabrata totally and partially resistant to Schistosoma mansoni. International Journal for Parasitology7: 467–472. Acid phosphatase (EC 3.1.3.2, orthophosphoric monoester phosphohydrolase) has been demonstrated cytochemically in isolated granulocytes from the hemolymph of three strains of Biomphalaria glabrata. This enzyme was not detected in hyalinocytes. By employing acid phosphatase as a marker, it was determined that the cells comprising the capsule surrounding Schislosoma mansoni mother sporocysts in a totally and partially resistant strain of B. glabrata are granulocytes.The process of encapsulation of S. mansoni mother sporocysts in resistant B. glabrata was traced for 72 h post-penetration by miracidia and has been ascertained to involve two stages: (1) enlargement of the granuloma around intact sporocysts, followed by (2) disintegration of the parasite and a decrease in the size of the granuloma. There is an increase in the level of acid phosphatase activity within granulocytes comprising the granuloma during the second stage.Host cellular responses to S. mansoni mother sporocysts does not occur in susceptible snails.     

Influence of low temperature on phosphatase in roots of Verbascum thapsus,a biennial weed

The temperature peak (15 °C) of acid and alkaline phosphatase in this study coincides with a peak in alpha-amylase as seen in an earlier study of roots of Verbascum thapsus. It is speculated that one of the results of higher phosphatase activities may be increased amount of orthophosphate which can be utilized in phosphorylation of soluble carbohydrates which in turn are in greater supply due to the higher activities of the starch-degrading enzymes.A second peak in activities of acid and alkaline phosphatase was seen in plants which were returned to the greenhouse following cold treatment. This increase in enzymatic activities is also similar to increases in activities of three starch degrading enzymes studied earlier. Alkaline phosphatase showed greater activities than did acid phosphatase at lower temperatures (10 and 4 °C) and under greenhouse conditions following cold treatment.     

Enzyme levels in homogenates of liver from mice infected with Schistosoma mansoni and from uninfected mice

Enzymatic and histopathological study of the livers of 300 white mice infected with Egyptian strain of Schistosoma mansoni and of 100 noninfected control animals was carried out for a follow up period of 6 months.Significant diminution of pseudocholine esterase activity was noticed as early as the second week after infection, denoting defective synthetic function of the liver. Significant decrease of transaminases was also noticed during the fifth week, and showed correlation with the histopathological findings of cell necrosis. Glutamic oxalacetic transaminase appears to be a better index of liver cell integrity than glutamic pyruvic transaminase.Lactic dehydrogenase was only moderately diminished during the third and fourth months.Alkaline phosphatase was strikingly increased in hepatic schistosomiasis and occurred as early as the second week. Increased enzyme production and release in the circulation has been suggested.These results confirm the affection of the liver cell during infection with Schistosoma mansoni.     

Enzyme activity of cryoprotected myocardium.

The use of in vivo intravenously infused DMSO/glycerol in Krebs-Henseleit buffer solution as a cryoprotectant in rat left ventricles was investigated. Alkaline phosphatase and creatine phosphokinase activities were monitored. Data show inhibition of alkaline phosphatase by the cryoprotectant solution. No significant differences were noted for the CPK activity indicating that it may not be affected by either freezing or the cryoprotectant or both in combination.     

Boophilus microplus: characterization of larval phosphomonoesterases and isolation of subcellular fractions with high phosphatase activity.

Phosphomonoesterase activity was determined for a 115,000g pellet and soluble fractions resulting from a subcellular fractioning of a homogenate of larval Boophilus microplus. Both fractions showed maximum phosphatase activity at pH 5.5 and 10. Acid phosphatase (EC 3.1.3.2) activity was found to be greatest in the soluble fraction. When the reaction rate was plotted against homogenate concentration, the soluble acid phosphatase deviated from the linear relationship. For both fractions different thermostability patterns were obtained, inactlvation beginning for the alkaline phosphatase (EC 3.1.3.1) at 45–55 C. When the effect of substrate concentration on activity was studied, deviations from the typical hyperbolic behavior were observed. Homogenization of larvae with 5 mm EDTA buffer failed to yield a low-speed pellet with high alkaline phosphatase activity, as it is expected if absorptive structures sediment. Moreover, total alkaline phosphatase activity recovered by this method is significantly lower than activity recovered when homogenization is carried out without EDTA. Alternately, homogenization with 10 mM Tris buffer and 0.25 M sucrose gave 27,000g and 115,000g fractions with high phosphatase activity when fractioned by centrifugation. Alkaline treatment of the 115,000g fraction with 10 mM Tris buffer, pH 7.8, failed to separate endoplasmic reticulum contaminants without loss of phosphatase activity. When the 115,000g fraction was centrifuged in a sucrose density gradient, two activity peaks, coincident for both acid and alkaline phosphatases, were obtained. Antigenic analysis showed the existence of similar antigenic determinants in both peaks “immunologically” presented in different ways.     

Alkaline and acid phosphatases of the marine diatoms Chaetoceros affinis var. willei (Gran) Hustedt and Skeletonema costatum (Grev.) Cleve

Alkaline phosphatase activity in cultures of the marine diatom Chaetoceros affinis var. willei (Gran) Hustedt was higher than in Skeletonema costatum (Grev.) Cleve. The enzyme activity was localized in coarse cell particles. Acid phosphatase activity was found in the cytoplasmic fraction. Induction of alkaline phosphatase depended on the $\text{N}\text{P}$ ratio in the culture medium. A $\text{N}\text{P}$ ratio > 40 in dilution/batch culture and > 30 in large scale batch culture, respectively, induced alkaline phosphatase.Cell phosphorus showed a critical value below which alkaline phosphatase was induced. Alkaline phosphatase in natural phytoplankton from the Trondheimsfjord is unlikely to occur except possibly in special situations.     

Alkaline fixation-resistant acid phosphatases in human tissues: histochemical evidence for a new type of acid phosphatase in endothelial, endometrial and neuronal sites

The effect of pH during formalin fixation on acid phosphatases in human tissues was studied. Lysosomal-type acid phosphatase was sensitive to alkaline fixation, being completely inactive after fixation at pH 9.0. Prostatic and tartrate-resistant osteoclastic/macrophagic types were alkaline fixation-resistant, as was an acid phosphatase localized in endothelium, endometrial stromal cells and intestinal nerves. The latter activity was further separable into fluoride- and tartrate-sensitive beta-glycerophosphatase and fluoride-sensitive, tartrate-resistant alpha-naphthyl phosphatase. The activities appeared to represent either different, tightly associated enzymes or separate activity centres of a single enzyme. Alkaline fixation-resistant alpha-naphthyl phosphatase at endothelial, endometrial and neuronal sites was also well demonstrated in unfixed or neutral formalin-fixed sections as tartrate-resistant activity similar to classical tartrate-resistant acid phosphatase, but these phosphatases appear to be antigenically different. Alkaline fixation-resistant acid phosphatase showed a restricted tissue distribution both in endothelium (mainly in vessels of abdominal organs) and at neuronal sites (only in intestinal nerves). Alkaline fixation-resistant acid phosphatase appears to represent a previously unknown or uncharacterized enzyme activity whose chemical properties could not be classified as any previously known type of acid or other phosphatases.     

Seasonal activity of non-specific acid and alkaline phosphatases in beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis>) leaves

The activities of acid and alkaline phosphatases along with phosphorus content in leaves of European beech (Fagus sylvatica L.) were studied for a period from April to October. The phosphorus content of beech leaves was highest in April, at the beginning of the vegetation period; from May to October it was twofold lower than in April. Acid phosphatase activity (per unit fresh weight) in leaves collected from the middle part of the crown decreased significantly in May and July compared to the enzyme activity in April. In both the low and middle parts of the crown, the acid phosphatase activity had a peak in August, and thereafter decreased in September and October. No correlations between acid phosphatase activity and phosphorus concentrations were found. Alkaline phosphatase activity was very low and in some cases near the detection limit during the whole observation period.     

Alkaline phosphatase in KB cells: influence of hyperosmolality and prednisolone on enzyme activity and thermostability

KB cells, derived from a human nasopharyngeal carcinoma, have high alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) activity. Specific activity is 150 times higher than in HeLa S3 cells. Alkaline phosphatase activity in KB cells exhibits heterogeneity consisting of a large heat-labile and a small heat-stable component. Enzyme activity is modulated by the osmolality of the culture medium. Increasing the osmolality by the addition of NaCl results in reduction of activity. A similar effect is noted when KB cells are grown with prednisolone (Δ′-hydrocortisone). The activities of acid phosphatase, β-galactosidase and N-acetyl-β-glucosaminidase are not affected by hyperosmolality and/or prednisolone. The reduction in alkaline phosphatase activity is accompanied by an increase in the proportion of the heat-stable and a decrease of the heat-labile enzyme components. The alterations in specific activity and thermostability are independent of the basal culture medium employed. Enzyme stability at 56 °C is inversely related to the buffer concentration, but the differences in stability between the alkaline phosphatases of control cultures and of KB cells grown in hyperosmolar medium or with prednisolone can be recognized with various buffering mixtures.     

Spatial Patterns of Alkaline Phosphatase Expression within Bacterial Colonies and Biofilms in Response to Phosphate Starvation

The expression of alkaline phosphatase in response to phosphate starvation was shown to be spatially and temporally heterogeneous in bacterial biofilms and colonies. A commercial alkaline phosphatase substrate that generates a fluorescent, insoluble product was used in conjunction with frozen sectioning techniques to visualize spatial patterns of enzyme expression in both Klebsiella pneumoniae and Pseudomonas aeruginosa biofilms. Some of the expression patterns observed revealed alkaline phosphatase activity at the boundary of the biofilm opposite the place where the staining substrate was delivered, indicating that the enzyme substrate penetrated the biofilm fully. Alkaline phosphatase accumulated linearly with time in K. pneumoniae colonies transferred from high-phosphate medium to low-phosphate medium up to specific activities of 50 μmol per min per mg of protein after 24 h. In K. pneumoniae biofilms and colonies, alkaline phosphatase was initially expressed in the region of the biofilm immediately adjacent to the carbon and energy source (glucose). In time, the region of alkaline phosphatase expression expanded inward until it spanned most, but not all, of the biofilm or colony depth. In contrast, expression of alkaline phosphatase in P. aeruginosa biofilms occurred in a thin, sharply delineated band at the biofilm-bulk fluid interface. In this case, the band of activity never occupied more than approximately one-sixth of the biofilm. These results are consistent with the working hypothesis that alkaline phosphatase expression patterns are primarily controlled by the local availability of either the carbon and energy source or the electron acceptor.     

Acid and alkaline phosphatases activities in vascular smooth muscle: species differences and subcellular distribution

1. Acid and alkaline phosphatase activities were studied in rat and dog aortic muscle using p-nitrophenyl phosphate (p-NPP) as the substrate. Alkaline phosphatase activity was quite comparable to acid phosphatase activity in rat aortic microsomes as well as further purified plasma membranes, but considerably lower than acid phosphatase activity in dog aortic membranes. 2. Subcellular distribution of acid and alkaline phosphatase activities in these vascular muscles indicated that alkaline phosphatases and a large portion of acid phosphatase activities were primarily associated with plasma membranes and the distribution of acid phosphatase showed little resemblance to that of N-acetyl-beta-glucosaminidase, a lysosomal marker enzyme. 3. The rat aortic plasmalemmal acid and alkaline phosphatase activities responded very differently to magnesium, fluoride, vanadate and EDTA. The alkaline phosphatase was more susceptible to heat inactivation than acid phosphatase. 4. These results suggest that these two phosphatases are likely to be two different enzymes in the smooth muscle plasma membranes. The implication of the present findings is discussed in relation to the alteration of these phosphatases in hypertensive vascular diseases.     

Extracytoplasmic phosphatases of selected species of Saccharomyces, Kluyveromyces and Rhodotorula

Phosphatase activities of yeasts belonging to the genera Saccharomyces, Kluyveromyces and Rhodotorula were studied. Rhodotorula rubra exhibited activities at acid, neutral and alkaline pH; the other yeasts only had activity at acid pH. Growing yeasts in a constant pH (4.5) medium decreased phosphatase activities in Saccharomyces and Kluyveromyces, while neutral activity was enhanced in Rhodotorula rubra which excreted more enzyme under these conditions. Washing cells with sucrose solutions lowered phosphatase activities in all yeasts, due to enzyme liberation. Acid phosphatase activities in isolated and purified cell walls were very small. Phosphatases thus appear not to be strongly bound to yeast cell walls.     

Phosphatase synthesis in Klebsiella (Aerobacter) aerogenes growing in continuous culture

1. Phosphatase synthesis was studied in Klebsiella aerogenes grown in a wide range of continuous-culture systems. 2. Maximum acid phosphatase synthesis was associated with nutrient-limited, particularly carbohydrate-limited, growth at a relatively low rate, glucose-limited cells exhibiting the highest activity. Compared with glucose as the carbon-limiting growth material, other sugars not only altered the activity but also changed the pH–activity profile of the enzyme(s). 3. The affinity of the acid phosphatase in glucose-limited cells towards p-nitrophenyl phosphate (K_m 0.25–0.43mm) was similar to that of staphylococcal acid phosphatase but was ten times greater than that of the Escherichia coli enzyme. 4. PO₄³⁻-limitation derepressed alkaline phosphatase synthesis but the amounts of activity were largely independent of the carbon source used for growth. 5. The enzymes were further differentiated by the effect of adding inhibitors (F⁻, PO₄³⁻) and sugars to the reaction mixture during the assays. In particular, it was shown that adding glucose, but not other sugars, stimulated the rate of hydrolysis of p-nitrophenyl phosphate by the acid phosphatase in carbohydrate-limited cells at low pH values (<4.6) but inhibited it at high pH values (>4.6). Alkaline phosphatase activity was unaffected. 6. The function of phosphatases in general is discussed and possible mechanisms for the glucose effect are outlined.     

The p-nitrophenyl phosphatase activity of muscle carbonic anhydrase

Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO₂ hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a K_m of 2.8 mm. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate (K_i = 1.22 × 10^?3M), arsenate (K_i = 1.17 × 10^?3M), and molybdate (K_i = 1.34 × 10^?7M), with these inhibitors having no effect on the CO₂ hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO₂ hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO₂ hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites.     

Isolation and characterization of Xenopus soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.     

Acid and alkaline phosphomonoesterase activities in snake venoms

• 1.1. Acid and alkaline phosphatase activities of eight different snake venoms were determined quantitatively by using synthetic substrates, o-carboxyphenylphosphate and p-nitrophenylphosphate respectively.
• 2.2. It was found that most of Elapidae venoms investigated had both acid and alkaline phosphatase activities.
• 3.3. Three Crotalidae venoms investigated did not show any alkaline phosphatase activity.
• 4.4. The strength of venom acid phosphatase activity is as follows: Agkistroden acutus > Naja haje > Naja naja samarensis > Naja naja atra > Naja melanoleuca.
• 5.5. The strength of venom alkaline phosphatase activity by using p-nitrophenylphosphate is in the order of Naja hannah > Naja haje > Naja naja samarensis > Naja naja atra > Naja melanoleuca.When o-carboxyphenylphosphate was used as a substrate, the order of enzyme activity is Naja hannah > Naja haje > Naja naja samarensis > Naja melanoleuca > Naja naja atra.
• 6.6. Acid phosphatase activity of all the Elapidae venoms was inhibited completely by fluoride. The alkaline phosphatase activity of Elapidae venoms was not inhibited by fluoride either using p-nitrophenylphosphate or o-carboxyphenylphosphate.
• 7.7. The acid phosphatase of all the Elapidae venoms was not inhibited by zinc ion. However, most of the venom alkaline phosphatases were inhibited by zinc ion.
• 8.8. Ethylenediaminetetraacetic acid (EDTA) had inhibitory action on venom phosphatase activity. However, tris-(hydroxymethyl)-aminoethane had a counter effect on the inhibitory action of EDTA.
• 9.9. Optimum pH studies of the snake venom phosphatases showed that the acid phosphatases of the snake venoms had their highest activity in the range of pH 4–5. The alkaline phosphatases of the snake venoms had their optimum pH at 9.
• 10.10. Comparable experiments were also conducted by using chicken intestine alkaline phosphatase and wheat germ acid phosphatase.