Electron-cytochemical localization of alkaline phosphatase to G cells of Necturus maculosus antrum

Summary Electron-cytochemical localization of alkaline phosphatase activity was performed on G cells of Necturus maculosus antral mucosa. Alkaline phosphatase activity was localized to the nuclear membrane, the Golgi/endoplasmic reticulum, and the limiting membranes of G cell peptide-secretion vesicles. There was no specific localization of alkaline phosphatase activity to the plasma membrane. Treatment of the tissues with levamisole (an alkaline phosphatase inhibitor) did not markedly reduce the specific alkaline phosphatase activity. Specific lead deposition was reduced by removal of the substrate from the reaction mixture. The results from this study on N. maculosus G cells demonstrate that alkaline phosphatase activity can be found in a non-mammalian gastric endocrine cell and that specific activity was localized primarily to those intracellular structures involved with protein biosynthesis.     

HISTOPATHOLOGICAL AND HISTOCHEMICAL CHANGES IN HONEYBEE LARVAE (APIS MELLIFERA L.) AFTER INFECTION WITH BACILLUS LARVAE,THE CAUSATIVE AGENT OF AMERICAN FOULBROOD DISEASE

Morphological, histochemical and cytochemical changes were examined in honeybee larvae after infection with the bacterium Bacillus larvae. The results indicate cell necrosis in the midgut epithelium accompanied by increasing cell vacuolization and nuclear pyknosis following per os inoculation with B. larvae. Many autolysosomes were positive for acid phosphatase. Non-vacuolar acid phosphatase activity was also found in lysed cell compartments. No such activity was found in regenerative epithelial cells. Degradation of haemocytes, salivary glands and other tissues was also observed. Histochemical analyses after per cutaneous inoculation with B. larvae of three- and five-day-old honeybee larvae show intense non-vacuolar acid phosphatase activity followed by disintegration of infected salivary glands, epithelial cell cytoplasm and haemocytes.     

Changes in phosphatase activity in phosphorus-deficient Spirodela

Summary Onset of phosphorus deficiency in Spirodela oligorrhiza was accompanied by a 50-fold increase in phosphatase activity of cell extracts. The enzyme behaved like other plant acid phosphatase, and was both inhibited and repressed by inorganic phosphate. The phosphatase activity comprised at least three isozymes. Two, of low molecular weight, were present only in P-deficient Spirodela; one of high molecular weight was also present, though in smaller amounts, in normal Spirodela. Presence of 2-thiouracil during onset of P-deficiency partly inhibited the development of phosphatase activity. The nature and role of the increased phosphatase activity in P-deficient plants are discussed.     

Localization of alkaline phosphatase inBacillus intermedius cells

Alkaline phosphatase, an enzyme secreted byBacillus intermedius S3-19 cells to the medium, was also detected in the cell wall, membrane, and cytoplasm. The relative content of alkaline phosphatase in these cell compartments depended on the culture age and cultivation medium. The vegetative growth ofB. intermedius on 0.3% lactate was characterized by increased activity of extracellular and membrane-bound phosphatases. The increase in lactate concentration to 3% did not affect the activity of membrane-bound phosphatase but led to a decrease in the activity of the extracellular enzyme. Na₂HPO₄ at a concentration of 0.01 % diminished the activity of membrane-bound and extracellular phosphatases. CoCl₂ at a concentration of 0.1 mM released membrane-bound phosphatase into the medium. By the onset of sporulation, phosphatase was predominantly localized in the medium and in the cell wall. As is evident from zymograms, the multiple molecular forms of phosphatase varied depending on its cellular localization and growth phase.     

Changes in phosphatase activity associated with cell wall defects in Chlamydomonas reinhardi

Experiments were performed to isolate mutants lacking alkaline phosphatase in Chlamydomonas reinhardi. Mutants with null enzyme activity were obtained. A cytological study of these mutants however revealed cell wall defects, suggesting that the loss of phosphatase activity in these strains is not due to the inactivation of the corresponding phosphatase structural gene but rather to the leakage of this enzyme as a consequence of the cell wall abnormality. Incidentally, this finding provides the basis of a convenient method for selecting easily cell wall mutants of Chlamydomonas.Chercheur qualifié du Fonds National Belge de la Recherche Scientifique.     

An examination of the age-related patterns of decay of acid phosphatase (ACP1) in human red cells from individuals of different phenotypes

A study has been made of the decay of acid phosphatase (ACP₁) in the human red cell using red cell fractions of different mean ages prepared by density gradient centrifugation. Red cells from acid phosphatase type A and type B individuals were used in the study. Acid phosphatase activity of the red cell fractions was determined by two different assay methods. The results obtained were comparable and have been combined. Acid phosphatase type A and type B showed a biphasic decay pattern with a rapid early loss of activity, followed by a more gradual rate of decline. Type A appeared to decay more rapidly than type B in both decay phases. It is proposed that differences in stability between type A and type B in vivo may explain the observed differences in activity between the enzyme types. There was no evidence for the generation of secondary isozymes by acid phosphatase type A or type B during red cell aging.     

Alterations in Activity of Phosphatases during the Peridinium Bloom in Lake Kinneret

Acid and alkaline phosphatases have been isolated from Peridinium cinctum f. westii (Dinophyceae) during an algal bloom in Lake Kinneret. Acid phosphatase activity was fairly constant over the entire period of the bloom, although fluctuations in activity appeared to correlate with the chlorophyll content of the cells. Histochemical studies showed that the enzyme was localized inside the cell. Alkaline phosphatase activity was very low until May, a month after the peak of the bloom, when it increased sharply. Polyacrylamide gel electrophoresis revealed one or two bands of alkaline phosphatase that increased in intensity as the bloom progressed. However, the highest activity of the enzyme (in the last sample collected) corresponded to a new, very intense band on the gels. Similarly to acid phosphatase, alkaline phosphatase was also localized inside the cell. The appearance of alkaline phosphatase is probably related to the available phosphate concentration in the lake, although the influence of other factors that may contribute to the induction of the enzyme cannot be ruled out.     

Phosphatase activity in the heterotrophic dinoflagellate Pfiesteria shumwayae

The ELF-97 phosphatase substrate was used to examine phosphatase activity in four strains of the estuarine heterotrophic dinoflagellate, Pfiesteria shumwayae. Acid and alkaline phosphatase activities also were evaluated at different pH values using bulk colorimetric methods. Intracellular phosphatase activity was demonstrated in P. shumwayae cells that were actively feeding on a fish cell line and in food limited cells that had not fed on fish cells for 3 days. All strains, whether actively feeding or food limited showed similar phosphatase activities. P. shumwayae cells feeding on fish cells showed ELF-97 activity near, or surrounding, the food vacuole. Relatively small, spherical ELF-97 deposits were also observed in the cytoplasm and sometimes near the plasma membrane. ELF-97 fluorescence was highly variable among cells, likely reflecting different stages in digestion and related metabolic processes. The location of enzyme activity and supporting colorimetric measurements suggest that, as in other heterotrophic protists, acid phosphatases predominate in P. shumwayae and have a general catabolic function.     

Effect of endocytosis upon acid phosphatase activity ofTetrahymena pyriformis

Summary Increased endocytosis inTetrahymena pyriformis, produced by presenting starved cells with either peptone-yeast extract medium or killed yeast cell suspension, results in increased cellular acid phosphatase activity.Tetrahymena, grown in peptone-yeast extract medium, showed increased acid phosphatase activity after phagocytosis of yeast cells. This increase was not apparent until about one hour after presentation and was maximal at about 2.5 hours.Tetrahymena, grown on yeast suspension, showed little increase in acid phosphatase activity on presentation with peptone-yeast extract medium. These results may indicate that endocytosis, of either particles or solutes, produces an adaptive increase in acid phosphatase activity (presumably lysosomal in nature) which is related to feeding.Histochemical examination failed to localise the increase in acid phosphatase activity cellularly, but small particles, of about 1 diameter, which showed acid phosphatase activity and were presumably lysosomes were noted. Closely orientated yeast cells showed varying intensities of lead deposition, from absence to intense staining. This suggests that newly ingested yeast cells may be ingested initially in a single phagosome and that thereafter one or more lysosomes may fuse with them.     

Intracellular localization of alkaline phosphatase in freshly isolated foetal rat hepatocytes

Summary The cytochemical localization of alkaline phosphatase activity in foetal rat hepatocytes was examined in relation to the pattern of cell to cell attachment during cell isolation and culture. In foetal hepatocytesin vivo, alkaline phosphatase was exclusively localized on the bile canalicular membrane. In freshly isolated foetal hepatocytes, however, the activity was present in the endoplasmic reticulum, nuclear envelope, Golgi apparatus, tubulo-vesicular organelles, and over the entire plasma membrane. In monolayer cells cultured for one or two days, the activity was localized on the reconstituted bile canalicular membrane, plasma membrane sites adjacent to neighbouring cells and on the bottom surface of the monolayer, but was detected in none of the intracellular organelles. Biochemical alkaline phosphatase activity did not change during isolation of the cells. These results suggest that, in foetal hepatocytes, loss of cell—cell contact may induce a temporal disturbance, or dedifferentiation, in their membrane system.     

Effect of aluminium on the activity of apoplastic acid phosphatase and the exudation of macromolecules by roots and suspension-culture cells ofZea mays L.

Aluminium accumulates predominantly in the root apoplast where it binds to the pectin matrix of the cell wall with its negative charges. In this study, we investigated whether short-term Al treatment (2 h) affects the activity of apoplastic acid phosphatase and the exudation of macromolecules by roots and suspension-culture cells of Zea mays L. The pectin content of the cell cultures was modified by long-term adaptation to NaCl stress or long-term adaptation to the cellulose-synthesis inhibitor 2,6-dichlorbenzonitrile (DCB), and by short-term treatment for up to 15 min with pectolyase. At pH 4.5, neither acid phosphatase activity of commercial enzyme preparations nor of exudates from root-tips and suspension-cells of Zea mays L. were affected directly by Al. However, the exudation and the activity of apoplastic acid phosphatase was reduced to a greater extent by Al cells with high pectin content than in cells with normal pectin content. The strongest reduction of acid phosphatase exudation was observed in pectolyase-treated cells with the lowest pectin content. Al reduced not only the release of acid phosphatase from the suspension cells, but also the release of total proteins and pectins. However, no relationship existed between the magnitude of Al-induced reduction of protein and pectin release and the cell pectin contents. These results support the assumption that Al modifies cell-wall and plasma-membrane transport-properties for macromolecules and the activity of apoplastic enzymes thus modifying Al sensitivity.     

Acid phosphatase activity during spore differentiation of the red algaeGigartina teedii andChondria tenuissima

The acid phosphatase activity during carposporogenesis inGigartina and tetrasporogenesis inChondria was studied using the Gomori technique. During the first steps of gonimoblast maturation ofGigartina, portions of cytoplasm are ensheathed by ER cisternae with acid phosphatase activity, giving rise to autolysosomal concentric membrane bodies. In a similar way large mucilage sacs are severed. They extrude their contents in a kind of exocytosis. Multivesicular bodies, concentrically arranged cisternae and extracytoplasmic compartments, each with acid phosphatase activity, remain in young carpospores for some time, probably as remnants of the autophagocytotic and exocytotic events. The Golgi apparatus is poorly developed in gonimoblast cells and young carpospores. It becomes a prominent cell component in maturing carpospores and then participates in cell wall formation. Only some of the dictyosomal cisternae contain acid phosphatase; these are irregularly distributed in the dictyosome. — In pre- and postmeiotic tetraspore mother cells ofChondria massive lead deposits are found in the dictyosomes and in adjacent Golgi vesicles. Finer lead precipitates occur in ER cisternae, especially in those which are sequestering starch-grain-containing portions of the cytoplasm to give rise to autolysosomes. During cell cleavage, the dictyosomes aggregate. They become devoid of acid phosphatase activity with the exception of vesicles at the trans face. Later, Golgi stacks associate and have common, Gomori positively reacting, narrow cisternae at the cis face. The Golgi apparatus derived cored vesicles do not contain lead precipitates whereas the Golgi cisternae in the final stage of tetrasporogenesis show acid phosphatase activity. Variations in acid phosphatase distribution are explained in the light of current models of membrane flow.Dedicated to Univ.-Prof. DrO. Härtel on the occasion of his 80th birthday.     

Observations on acid phosphatase in Mayorella palestinensis

Acid phosphatase activity has been studied in the ameba Mayorella palestinensis. Optimum activity of the enzyme was found to be at a pH of 3.2. The enzyme is inhibited by fluoride ion, but is not sensitive to Mg⁺⁺. The activity was found to be correlated with age of culture. Two maxima have been obtained, one from cultures in the logarithmic phase, and the other during the period of maximal cell encystation. These results suggest that acid phosphatase play an important role in cell metabolism during growth and differentiation processes of this ameba.     

Localization of acid phosphatase activity in the liver of pregnant rats

Synopsis In the liver of pregnant rats, fedad libitum, there was an increase in acid phosphatase specific activity which occurred in two peaks, one at the 15th day and the other at the end of gestation. By light and electron microscopic histochemistry, the activity was found to be localized in parenchymal cell peribiliary dense bodies and also in phagosomes present in macrophages and parenchymal cells. There was an increase in liver weight which reached a peak at the 17th day of gestation. Total DNA also rose to the 17th day; there was a high rate of cell division in the hepatic parenchyma at the 17th and 18th days of gestation. During this period single cell deletion by apoptosis was relatively frequent and in late pregnancy there was evidence of cell deletion by lysis.During pregnancy there was a slight increase in sinusoidal macrophages as a proportion of the total cell population but there did not appear to be significant changes in macrophage enzymic activity. It is suggested that the acid phosphatase activity present in macrophages makes a minor contribution to total liver activity, most of which is present in parenchymal cells. Acid phosphatase activity associated with single cell deletion appears to be quantitatively negligible.There was a direct relationship between total hepatic acid phosphatase activity and the numbers of peribiliary dense bodies, which were most numerous at the 15th day and at the end of gestation. It is suggested that these residual bodies contain products of detoxification processes and also cell structural elements resulting from enhanced liver metabolism and intracellular turnover during pregnancy.     

Dependence of yeast permeabilization with Triton X-100 on the cell age

Summary Acid phosphatase activity and protein release were determined in cell suspensions ofYarrowia lipolytica andTorulospora delbrueckii at different stages of their growth by permeabilization with Triton X-100. The effect of the surfactant on the cell permeability did not depend on the cell age.     

Differential regulation of the active and inactive forms of Saccharomyces cerevisiae acid phosphatase

Summary Acid phosphatase in S. cerevisiae exists as an enzymatically active, cell wall associated form and as an enzymatically inactive, probably membrane-bound form (Schweingruber and Schweingruber, in press). Orthophosphate dependent and independent regulation determines the level of acid phosphatase activity. To deduce the regulation mechanisms we purified and quantified active and inactive acid phosphatase from cells grown under different physiological conditions and displaying variable levels of enzyme activity. Orthophosphate dependent regulation does not include significant changes in the amount of total (active and inactive) acid phosphatase protein synthesized. Under the experimental conditions chosen increased activity is achieved by preferential synthesis of the active form and by increasing the specific activity of the active enzyme. Orthophosphate independent regulation seems to occur by similar mechanisms.     

Acid phosphatase activity in Pisolithus arrhizus mycelium treated with cadmium dust

The influence of cadmium dust (containing lead, cadmium, copper, zinc, silicium and other elements) on acid phosphatase activity of Pisolithus arrhizus was observed by means of electron microscopy. Dust-treated mycelium showed increased activity of the enzyme, especially on the surface of the cell wall. There was an increase in abundance of autophagic vacuoles marked by a strong phosphatase reaction. An increase in the number of hyphae with diffuse enzyme activity within the cytoplasm coincided with a decrease of lifespan of the fungus, rapid changes in the mictoplasm stage, earlier closing of the dolipori and presumably the earlier autolysis of cell cytoplasm. Hyphae showing strong autolytic activity were separated from other hyphae by the material deposited within the doliporus and this whole area was devoid at that stage of acid phosphatase activity. The role of the enzyme in the mechanism of resistance to toxic elements is discussed.     

Involvement of WalK (VicK) phosphatase activity in setting WalR (VicR) response regulator phosphorylation level and limiting cross‐talk in Streptococcus pneumoniae D39 cells

WalRK (YycFG) two‐component systems (TCSs) of low‐GC Gram‐positive bacteria play critical roles in regulating peptidogylcan hydrolase genes involved in cell division and wall stress responses. The WalRK (VicRK) TCSs of Streptococcus pneumoniae (pneumococcus) and other Streptococcus species show numerous differences with those of other low‐GC species. Notably, the pneumococcal WalK sensor kinase is not essential for normal growth in culture, unlike its homologues in Bacillus and Staphylococcus species. The WalK sensor kinase possesses histidine autokinase activity and mediates dephosphorylation of phosphorylated WalR～P response regulator. To understand the contributions of these two WalK activities to pneumococcal growth, we constructed and characterized a set of walK kinase and phosphatase mutants in biochemical reactions and in cells. We identified an amino acid substitution in WalK that significantly reduces phosphatase activity, but not other activities. Comparisons were made between WalRK regulon expression levels and WalR～P amounts in cells determined by Phos‐tag SDS‐PAGE. Reduction of WalK phosphatase activity resulted in nearly 90% phosphorylation to WalR～P, consistent with the conclusion that WalK phosphatase is strongly active in exponentially growing cells. WalK phosphatase activity was also shown to depend on the WalK PAS domain and to limit cross‐talk and the recovery of WalR～P from walK⁺ cells.     

Use of the p-nitrophenyl phosphate method for the demonstration of acid phosphatase during starvation and cell autolysis in the planarian Polycelis tenuis Iijima.

Synopsis Acid phosphatase activity is demonstrated employingp-nitrophenyl phosphate as substrate and lead acetate as coupler. The fine structural localization of the enzyme in starved planarian tissues is described. The method is used to pin-point starvation-induced acid phosphatase activity in relation to autophagy and crinophagy in the gland cells; autophagy, autolysis and cell death in parenchymal and gastrodermal cells and basement membrane lysis. Attention is also payed to the demonstration of muscle lysis. The histochemical implications of the method are discussed.     

A Toxoplasma type 2C serine-threonine phosphatase is involved in parasite growth in the mammalian host cell

Toxoplasma gondii is a human protozoan parasite that belongs to the phylum of Apicomplexa and causes toxoplasmosis. As the other members of this phylum, T. gondii obligatory multiplies within a host cell by a peculiar type of mitosis that leads to daughter cell assembly within a mother cell. Although parasite growth and virulence have been linked for years, few molecules controlling mitosis have been yet identified and they include a couple of kinases but not the counteracting phosphatases. Here, we report that in contrast to other animal cells, type 2C is by far the major type of serine threonine phosphatase activity both in extracellular and in intracellular dividing parasites. Using wild type and transgenic parasites, we characterized the 37 kDa TgPP2C molecule as an abundant cytoplasmic and nuclear enzyme with activity being under tight regulation. In addition, we showed that the increase in TgPP2C activity significantly affected parasite growth by impairing cytokinesis while nuclear division still occurred. This study supports for the first time that type 2C protein phosphatase is an important regulator of cell growth in T. gondii.