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.     

Redox modulation of a phosphatase from Anacystis nidulans

Ascorbic acid (AA) increased the phosphatase activity (pH 6.8) in 10,000 g supernatants from Anacystis nidulans. The enzyme activated by AA was deactivated by dehydroascorbic acid (DHAA). The modulation by AA/DHAA of phosphatase activity in Anacystis appears to be specific; a number of other redox compounds, known to modulate other enzymes, had no effect on the Anacystis phosphatase. A purified phosphatase preparation from Anacystis was also deactivated by DHAA. In contrast, the purified enzyme was not activated by AA, suggesting that a factor mediating the effect of AA was lost during purification. Another factor was found to protect the purified phosphatase against deactivation by DHAA. The enzyme was characterized as a phosphatase with a broad substrate specificity, an apparent molecular weight of 19,000, and a pH optimum of 6.0–7.0. Dialysis of the enzyme preparation against EDTA abolished the phosphatase activity which could be restored by Zn²⁺ ions and partially restored by Co²⁺ ions. Crude extracts also contained a latent enzyme, the phosphatase activity of which could be detected in the presence of Co²⁺ ions only. Zn²⁺ ions did not activate this enzymatically inactive protein. The Co²⁺-dependent phosphatase had an apparent mol. wt. of 40,000, a broad substrate specificity, and an alkaline pH-optimum. Infection of Anacystis cultures by cyanophage AS-1 resulted in a decrease in phosphatase activity. The enzyme present in 10,000 g supernatants from infected cells could not be modulated by the AA/DHAA system.Abbreviations AA ascorbic acid - DEAE diethylamino ethyl - DHAA dehydroascorbic acid - EDTA ethylene-diaminetetra-acetate - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG oxidized glutathione - HMP hexose monophosphate - P _i inorganic phosphorus - pNPP p-nitrophenylphosphate - pNP p-nitrophenol - Tris Tris(hydroxymethyl)-aminomethane     

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.     

Chromatographic characteristics and subcellular localization of synthase phosphatase,phosphorylase phosphatase and histone phosphatase in human polymorphonuclear leukocytes

Summary Synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity in a leukocyte homogenate were found to have different sedimentation charcteristics: both synthase phosphatase and phosphorylase phosphatase activity are associated with the microsomal fraction, while the majority of histone phosphatase activity (75–85%) was found in the cytosol. Synthase phosphatase, phosphorylase phosphatase and histone phosphatase activities accompanying the microsomal fraction are readily solubilized by 0.3% Triton X-100.When the solubilized microsomal enzymes were chromatographed on Sephadex G-200, the majority of synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity migrated in single peaks corresponding to apparent molecular weights of 380 000, 250 000 and 68 000, respectively. A minor peak of 30 000, which had phosphatase activity against all three substrates was also obtained.Ethanol treatment resulted in solubilization and dissociation of the three phosphatase activities. It was found that although ethanol treatment resulted in a 4-fold increase of phosphorylase phosphatase activity, histone phosphatase activity was decreased (by 60%), while synthase phosphatase activity remained stable. Similar results were obtained when ethanol treatment was performed on the 17 000 × g supernatant.Chromatography of the ethanol-treated microsomes (or homogenate) on Sephadex G-200 showed that the phosphatase activity towards synthase D, phosphorylase a and phosphohistone coincided a M_r 30 000 species. Heat treatment of the M_r 30 000 peak resulted in dissociation of synthase phosphatase and phosphorylase phosphatase activity.Synthase phosphatase was inhibited by phosphorylase a in a kinetically non-competitive manner while histone phosphatase activity was notinhibited by synthase D (8.5 unit/ ml) orby phosphorylase a(12 unit/ ml).     

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 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.     

Acid phosphatase activity in plastids (plastolysomes) of senescing embryo-suspensor cells

Autolysis of the suspensor, an embryonal haustorium, starts in the basal cells and proceeds in the direction of the embryo. InPhaseolus vulgaris, acid phosphatase activity is first found in transforming plastids, similar to the acid phosphatase activity inPh. coccineus [Nagl (1977) Z. Pflanzenphysiol.85, 45–51], although the ultrastructural details are different. InTropaeolum majus, autolysis begins in the most distal part of the suspensor, i.e., the chalazal or carpel haustorium. First the endoplasmic reticulum shows acid phosphatase activity, but neither the mitochondria, which undergo transformation similar to that observed in plastids ofPhaseolus, nor the leucoplasts show such activity. Later, however, the plastids exhibit low activity. Contrarily, the plastids in the suspensor cells adjacent to the embryo show increasing activity during senescence of the suspensor. During final autolysis, activity is found in all cytoplasmic membranes, while it is reduced in plastids. The visible ultrastructural transformations of various organelles into cytolysomes does not necessarily coincide with acid phosphatase activity. Our findings are a further indication of the high diversification and specialization of plastids during plant embryogenesis.     

Primary production in ten reservoirs in southern Brazil

The growth and phosphatase activity during phosphorus starvation of cultures of Nodularia spumigera Mertens were examined. Stationary phase was reached much sooner in phosphorus-deficient cultures than in phosphorus-sufficient cultures; the growth rate did not change. Phosphatase activities were greatly increased in stationary phase. Diurnal patterns were established for phosphorus-sufficient cultures, but they were not light related. In phosphorus-deficient cultures, an increase in phosphatase activities over a 24 h period was superimposed on a diurnal pattern. Both phosphorus and nitrogen additions lowered the relative phosphatase activities in long term studies, but the effect of phosphorus was much more pronounced. In short term studies, phosphorus appeared to cause an immediate decrease in phosphatase activity, but did not affect phosphatase activity after that for up to 24 h. Nitrogen did not have any short term effect on phosphatase. Phosphatase activity was correlated with changes in the proportion of TCA-insoluble phosphorus (polyphosphates).     

Liming and nitrogen fertilization affects phosphatase activities, microbial biomass and mycorrhizal colonisation in upland grassland

We have studied the effects of factorial combinations of lime and N additions on soil microbial biomass, respiration rates and phosphatase activity of an upland grassland. We also used an Agrostis capillaris seedling bioassay to assess the effect of the treatments on the activity of arbuscular-mycorrhizal (AM) fungi and root surface phosphatase enzymes and the concentrations of N and P in the bioassay plant shoots. In the F and H horizons, soil microbial biomass carbon (C^mic) decreased in response to the liming, while addition of lime and N together reduced basal respiration rates. In the Ah horizon, C^mic was unaffected by the treatments but basal respiration rates decreased in the plots receiving nitrogen. Soil phosphatase activity decreased only in the Ah horizon in plots receiving lime, either in combination with N or alone. The mass of root fwt. colonized by AM fungi increased in response to the treatments in the order nitrogenR²=28.7%, P=0.004). The results demonstrate the sensitivity of both free-living heterotrophic microorganisms and symbiotic mycorrhizal fungi to short-term (2 years) applications of lime and N to long-term upland grassland, particularly in relation to the key P cycling activities undertaken by these organisms.     

Acid phosphatase synthesis inAspergillus flavus

Proteins with phosphatase activity were produced during the growth ofAspergillus flavus in a phosphate-supplemented liquid synthetic medium. The best carbon and nitrogen sources for the synthesis of phosphatase were glucose and ammonium sulfate, respectively. The proteins were separated by molecular exclusion and ion exclusion chromatography (IEC) into three components one of which showed phosphatase activity. The molar mass of the enzyme was approximately 62 kDa. The purified enzyme exhibited an optimum activity at pH 4.0 and at 45°C. The activity of the enzyme was stimulated by Ca²⁺ and Mg²⁺ but inhibited by fluoride, iodoacetic acid, ethylenediaminetetraacetic acid and 2,4-dinitrophenol, and exhibited an apparentK _M of approximately 420 μmol/L.     

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.     

Expression,purification, and characterization of a novel acid phosphatase that displays protein tyrosine phosphatases activity from Metarhizium anisopliae strain CQMa102

The protein tyrosine phosphatase (PTPase) plays an important role in insect immune system. Our group has purified a type of acid phosphatase that could specifically dephosphorylate trans-Golgi p230 in vitro. In order to study this phosphatase further, we have identified and cloned the phosphatase gene from a locust specific Metarhizium anisopliae Strain CQMa102. The CQMa102 phosphatase was expressed in Pichia pastoris to verify its protease activity. The molecular weight (M_W) and the isoelectric point (pI) of the phosphatase were about 85 kDa and 6.15, respectively. Substrate specificity evaluation showed that the purified enzyme exhibited high activity on O-phospho-L-tyrosine. At its optimal pH of 6.5 and optimum temperature of 70 °C, the protein showed the highest activity respectively. It can be activated by Ca²⁺, Mg²⁺, Mn²⁺, Ba²⁺, Co²⁺ and phosphate analogs, but inhibited by Zn²⁺, Cu²⁺, fluoride, dithiothreitol, β-mercaptoethanol and N-ethylmaleimide.     

Dual 4‐ and 5‐phosphatase activities regulate SopB‐dependent phosphoinositide dynamics to promote bacterial entry

Salmonella are able to invade non‐phagocytic cells such as intestinal epithelial cells by modulating the host actin cytoskeleton to produce membrane ruffles. Two type III effector proteins SopB and SopE play key roles to this modulation. SopE is a known guanine nucleotide exchange factor (GEF) capable of activating Rac1 and CDC42. SopB is a phosphatidylinositol 4‐phosphatase and 5‐phosphatase promoting membrane ruffles and invasion of Salmonella through undefined mechanisms. Previous studies have demonstrated that the 4‐phosphatase activity of SopB is required for PtdIns‐3‐phosphate (PtdIns(3)P) accumulation and SopB‐mediated invasion. We show here that both the 4‐phosphatase as well as the 5‐phosphatase activities of SopB are essential in ruffle formation and subsequent invasion. We found that the 5‐phosphatase activity of SopB is likely responsible for generating PtdIns‐3,4‐bisphosphate (PtdIns(3,4)P₂) and subsequent recruitment of sorting nexin 9 (SNX9), an actin modulating protein. Intriguingly, the 4‐phosphatase activity is responsible for the dephosphorylation of PtdIns(3,4)P₂ into PtdIns(3)P. Alone, neither activity is sufficient for ruffling but when acting in conjunction with one another, the 4‐phosphatase and 5‐phosphatase activities led to SNX9‐mediated ruffling and Salmonella invasion. This work reveals the unique ability of bacterial effector protein SopB to utilize both its 4‐ and 5‐phosphatase activities to regulate phosphoinositide dynamics to promote bacterial entry.     

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.     

Increased plasma pyridoxal-5′-phosphate when alkaline phosphatase activity is reduced in moderately zinc-deficient rats

It is generally believed that the zinc metalloenzyme alkaline phosphatase is required to hydrolyze phosphorylated forms of vitamin B-6 prior to their use. To test this hypothesis, rats were fed a liquid diet containing either adequate or moderately low zinc during gestation and lactation. Zinc deficiency was produced in dams evidenced by significant reductions in zinc concentration of plasma (49%), liver (25%), and femur (24%), and plasma alkaline phosphatase activity (48%). Plasma pyridoxal-5′-phosphate (PLP), which significantly increased (61%) in these same rats, was negatively correlated (r=−0.74,P<0.02) with plasma alkaline phosphatase activity. Maternal liver PLP concentration was unaffected by zinc status. The zinc and vitamin B-6 relationship seen in dams was less observable in offspring. Stimulation of erythrocyte alanine aminotransferase activity by exogenously added PLP in vitro tended to be higher in both moderately zinc-deficient mothers and their offspring, but the difference was not significant. Our results support the hypothesis that alkaline phosphatase activity is required for the hydrolysis of plasma PLP. Our results also suggest that zinc status as alkaline phosphatase activity should be defined in an individual if plasma PLP is to be used as an indicator of vitamin B-6 status.     

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.     

ACID AND ALKALINE PHOSPHATASES IN A BRAIN TUBULIN PREPARATION

The hydrolysis of p-Nitrophenylphosphate has been studied in vitro in a tubulin preparation from bovine brain. The activity at pH 6.8 was 16.4 ± 2.2 nmol/mg protein, h. At least two phosphatases were responsible for this activity. They were found to have pH-optima at 5.1 and 10.4. respectively, and their apparent K_M values were 1.23 ± 0.10 mm and 0.17 ± 0.03 mm . respectively. Mg²⁺ was found to stimulate activity at both pHs while Zn²⁺ inhibited at pH 5.1 and stimulated activity at pH 10.4. All of the alkaline and part of the acid phosphatase activity were found to be closely associated with microtubules/tubulin. Tubulin purified by phosphocellulose chromatography contained phosphatase activity, and it is suggested that such activity is an intrinsic property of tubulin itself. Phosphatase activity was also found in association with the microtubule-associated proteins that co-purify with tubulin. Two proteins of high molecular weight constituted the major part of the associated material. The results indicate an association of phosphatase activity with the larger of these two proteins.     

Studies of vegetative compatibility-incompatibility in higher plants

Summary In order to elucidate the events that lead to cellular autolysis, and thus better understand the mechanism of cellular incompatibility betweenSedum telephoides andSolanum pennellii stems, we have followed the appearance and fate of the hydrolytic enzyme acid phosphatase in both the compatibleSedum autograft and the incompatibleSedum/Solanum heterograft. Acid phosphatase was localized by a modified Gomori-type reaction. Following an initial association with the endoplasmic reticulum and dictyosomes by 6–10 hours after grafting, acid phosphatase activity in the compatibleSedum autograft was associated primarily with the plasmalemma, tonoplast, and vacuole. This strict compartmentation in membranes or organelles and absence of enzyme from the cytosol was maintained throughout the development of the compatible autograft inSedum. Although acid phosphatase activity in the incompatible heterograft betweenSedum andSolanum was initially similar to the compatible autograft inSedum, a marked difference in enzyme localization occurred in the two graft partners over time.Solanum cells accumulated increased amounts of acid phosphatase, but the enzyme remained sequestered in the plasmalemma, tonoplast, and vacuole. In comparableSedum cells, however, there was a dramatic increase in acid phosphatase activity in the cytosol, often without any prior compartmentation within the vacuole. This high activity of acid phosphatase in theSedum cytosol was correlated with cellular autolysis, death, and eventual cell collapse to form the characteristic necrotic layer that insulates the stock from the scion. These results suggest that the lethal cellular senescence associated withSedum cells of the incompatible heterograft is correlated with a cytoplasmic release of acid phosphatase. A similar release of the enzyme does not occur in theSolanum stock or in the compatibleSedum autograft. Thus, while acid phosphatase synthesis and/or activation is induced in both the compatible and incompatible grafts, incompatibility betweenSedum andSolanum involves a failure ofSedum cells to isolate hydrolytic enzymes from the cytosol, which subsequently leads to cellular necrosis.Supported in part by grants from the Academic Senate of UCLA, Sigma Xi, the American Philosophical Society, and the URC of Baylor University.     

Platelet-Activating Factor Modulates a Secreted Phosphatase Activity of the Trypanosomatid Parasite Herpetomonas muscarum muscarum

In the present work we characterized the secreted phosphatase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This housefly parasite hydrolyzed p-nitrophenylphosphate at a rate of 10.26 nmol Pi/mg protein/min. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (N_aVO₃), sodium fluoride (NaF), and ammonium molybdate promoted a decrease in this phosphatase activity. When the parasites were assayed in the presence of sodium tartrate, an inhibitor of Leishmania spp-secreted acid phosphatases, this activity was drastically diminished. Cytochemical analysis showed the localization of this enzyme on the external surface and in the flagellar pocket of these parasites. Sodium tartrate inhibited this reaction, confirming the biochemical data. Platelet-activating factor (PAF) inhibited the phosphatase activity determined in the supernatant of living H. m. muscarum. Received: 2 February 2001 / Accepted: 5 March 2001     

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.