An Inhibition and Kinetic Study of Acid Phosphatase in Paramecium caudatum and Paramecium tetraurelia1

ABSTRACT. Inhibition, inactivation, pH, and kinetic studies using both homogenates and purified lysosomal fractions of Paramecium caudalum and of P. tetraurelia were carried out to examine the lysosomal acid phosphatase (AcPase) and its relationship to p-nitrophenylphosphatase (pNPPase), glucose-6-phosphatase (G6Pase), and 5′-nucleotidase (AMPase). The results generally support the idea that Paramecium cells contain a distinct lysosomal AcPase with a broad substrate specificity. The hydrolysis of glucose-6-phosphate (G6P) and adenosine 5′-monophosphate (AMP) was shown to be due to this enzyme, suggesting that true G6Pase and AMPase may be lacking in these two species; however, some hydrolysis of AMP at pH 7.5 catalyzed by an unknown soluble enzyme distinct from alkaline phosphatase and Na⁺-K⁺-ATPase was observed. Since the hydrolysis of p-nitrophenylphosphate (pNPP) at acid pH was also shown to be due to AcPase alone, pNPPase could be used as a rapid assay for Paramecium AcPase. At an alkaline pH, however, this activity was catalyzed by an alkaline phosphatase located in the cytosol fraction. P. caudatum AcPase was shown to have kinetic properties similar to those of purified rat liver and human prostatic AcPase and to have relative substrate affinities in the order of G6P < β-glycerophosphate < pNPP < AMP. These different substrate affinities might account for the observed differences in the inhibition of the four lysosomal activities by NaF, L(+)-tartrate, and molybdate, all of which inhibited the hydrolysis of G6P, β-glycerophosphate, and pNPP competitively, but which exhibited a noncompetitive inhibition of a mixed type with the hydrolysis of AMP.     

A new repressible alkaline phosphatase inNeurospora crassa EM 5297a

Neurospora crassa Em 5297a can utilize sodium Β-glycerophosphate as a sole phosphorous source (in the place of KH2PO4). Under these conditions a repressible alkaline phosphatase is elaborated which has different pH optimum towards Β-glycerophosphate (10.2) and pyrophosphate (9.0) as substrates. This enzyme does not require any metal ion for its activity and could be assayed in the presence of EDTA. However, under conditions of cobalt toxicity, the activity of this enzyme is high and is decreased in copper and nickel toxicities.     

Alkaline phosphatase of <Emphasis Type="Italic">Physarum polycephalum</Emphasis> is insoluble

The plasmodia of Physarum polycephalum grow as multinucleated cells in the presence of sufficient humidity and nutriment. Under non-illuminating conditions, stresses such as low temperature or high concentrations of salts transform the plasmodia into spherules whereas dehydration induces sclerotization. Some phosphatases including protein phosphatase and acid phosphatase have been purified from the plasmodia, but alkaline phosphatase remains to be elucidated. Phosphatase of the plasmodia, spherules and sclerotia was visualized by electrophoresis gel-staining assay using 5-bromo-4-chloro-3-indolyl phosphate. Insoluble fractions of the sclerotia were abundant in phosphatase activity. The phosphatase which was extracted by nonionic detergent was subjected to column chromatography and preparative electrophoresis. Purified phosphatase showed the highest activity at pH 8.8, indicating that this enzyme belongs to alkaline phosphatase. The apparent molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition was estimated to be 100 kDa whereas that under reducing was 105 kDa. An amount of 1% sodium dodecyl sulfate or 0.5 M NaCl had no effects on the activity although the phosphatase showed heat instability, Mg²⁺-dependency and sensitivity to 2-glycerophosphate or NaF. The extracting conditions and enzymatic properties suggest that this alkaline phosphatase which is in a membrane-bound form plays important roles in phosphate metabolism.     

Histochemical study of a number of hydrolases,including a new acid phosphatase,in various tissues of men and mice

Summary Two acid phosphatases have been demonstrated histochemically in mouse ventral prostate, seminal vesicles, coagulating glands, and liver and in human prostate. The first is the lysosomal acid phosphatase demonstrable by the Gomori technique. The second differs from thisβ-glycerophosphatase in that it splits naphthol AS phosphates but notβ-glycerophosphate; it has a different histochemical pH optimum and it is not inhibited by MoO₄ or NaF. The enzyme does not represent the “tail” of alkaline phosphatase activity as it is not inhibited by inhibitors of alkaline phosphatase and it has a different localization in liver and in human prostate. The enzyme may be membrane-bound but a lysosomal localization has still to be confirmed.     

Acid and Neutral Hydrolases in Trypanosoma cruzi. Characterization and Assay*

Twelve acid hydrolases, 4 near-neutral hydrolases and alkaline phosphatase were demonstrated in 0.34 M sucrose homogenates of Trypanosoma cruzi strain Y: p-nitrophenylphosphatase and α-naphthylphosphatase, with optimum pH at ? 6.0; α-galactosidase, β-galactosidase, β-glucosidase, N-acetyl-β-glucosaminidase, cathepsin A and peptidase I and III, with optimum pH between 5.0 and 6.0: and arylsulfatase cathepsin D, α-arabinase and α-mannosidase with optimum pH at ? 4.0 α-Glucosidase, gluccse-6-phosphatase and peptidase II had optimum pH at ? 7.0. β-Glycerophcsphatase had a broad pH-activity curve from 4.0 to 7.4, with maximum activity at pH 7.0. The main kinetic characteristics of these enzymes and their quantitative assay methods were studied. No activity was detected for α-fucosidase, β-xylosidase, β-glucuronidase, elaidate esterase. acid lipase, and alkaline phospho-diesterase.     

Trypanosoma rangeli: An alkaline ecto-phosphatase activity is involved with survival and growth of the parasite

The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using β-glycerophosphate (β-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of β-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of β-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca²⁺ present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. β-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (P_i). Levamisole at the IC₅₀ spared significantly parasite growth when β-GP was the sole source of P_i and stopped it in the absence of β-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.     

Effect of detergents on the alkaline phosphatase of Cuscuta reflexa

The non-ionic detergents, in particular Tween 20, Tween 80 and Triton X100, stimulated the alkaline phosphatase activity of Cuscuta reflexa homogenates with fructose-1,6-diphosphate and β-glycerophosphate as substrates. The order of activation was usually less than 100%, suggesting that a true latency was not involved. A differential response was found towards the two substrates, indicating the existence of two enzyme activities.     

Histochemistry of non-specific phosphatases: the use of p-nitrophenyl phosphate and -glycerophosphate as substrates

Synopsis It has been found that the acid and alkaline phosphatases in homogenates of respectively intestine and hypodermis ofAscaris suum hydrolyse sodiump-nitrophenyl phosphate at about twice the rate of sodium -glycerophosphate. This difference was also observed histochemically. Thus, when sections of intestine were incubated for acid phosphatase withp-nitrophenyl phosphate as substrate, the intensity of staining was about twice as great as that obtained after incubation in -glycerophosphate. Further, alkaline phosphatase was evident in sections of hypodermis after only 2 hr incubation inp-nitrophenyl phosphate but was not apparent after 10 hr incubation with -glycerophosphate. Hence biochemical assays and histochemical studies both indicate thatp-nitrophenyl phosphate is a superior substrate to -glycerophosphate for the visualization of acid and alkaline phosphatases in tissues.This paper was presented in part at the 1969 Aberdeen meeting of the British Society for Parasitology.     

Phosphatases in the rice stem borer, Chilo suppressalis Walker (Lepidoptera; Pyralidae): Some properties and changes of the activities during hibernation

To elucidate the regulation of glycerol synthesis from α-glycerophosphate (α-GP) in larvae of the rice stem borer, Chilo suppressalis Walker, phosphatase activities were examined during hibernation. The presence of alkaline and acid phosphatase was demonstrated in hibernating larvae. The activity of the alkaline type decreased during diapause entry while the activity of the acid type increased at the initiation of diapause and then decreased during postdiapause stage. This pattern of acid type activity change coincided with that of glycerol accumulation in hemolymph during hibernation. Thus, the acid type phosphatase may largely be involved in glycerol synthesis.     

Multiple Hydrolases in Bean Leaves (Phaseolus vulgaris L.) and the Effect of the Halo Blight Disease Caused by Pseudomonas phaseolicola (Burkh.) Dowson

Multiple forms of hydrolytic enzymes were demonstrated in extracts of healthy bean leaves (Phascolus vulgaris L.) and bean leaves infected with the halo blight organism [Pseudomonas phaseolicola (Burkh.) Dowson] by polyacrylamide disc electrophoresis.Bean leaves contained up to 4 acid phosphatase bands, 9 esterase bands active towards alpha-naphthyl acetate, and 7 esterase bands towards alpha-naphthyl butyrate. Only low or no activity was found for alkaline phosphatase, lipase, and aminopeptidase.Two artifacts are described which were observed with the lead phosphate method for acid phosphatase and the Tween method for demonstration of lipase.After infection with the halo blight organism the major acid phosphatase of the host increased during early and decreased at later infection stages. An acid phosphatase of bacterial origin with a more neutral pH optimum could be demonstrated in infected leaves. It is suggested that the bacterial acid phosphatase plays a role in uptake of metabolites by the pathogen.Several esterase bands decreased after infection. One host band with activity towards alpha-naphthyl butyrate increased. Also the pathogen showed an esterase band with high activity towards alpha-naphthyl butyrate.     

Kinetic properties, and location of nonspecific phosphomonoesterases in subcellular fractions of fasciola hepatica

Optimal activity was recorded at pH 4.5–5 and pH 9.0–9.5 and specific activity was seen to be 0.013 μmoles of p-nitrophenyl phosphate/min/mg protein at 37 C at pH 4.5 and 0.00169 μmoles at pH 9.0. The ratio of acid to alkaline phosphatase was 7.7:1.0. The K_m for acid phosphatase (EC 3.1.3.2) was 0.5 mM with a V_max of 0.0128 units/mg protein and 0.2mM for alkaline phosphatase (EC 3.1.3.1) with a V_max of 0.00175 units/mg protein. Acid phosphatase activity was optimal at 60 C and alkaline at 37 C. Linearity of enzyme activity was observed with time after the first 15 min of incubation and with homogenate concentration. KCN at 20 mM inhibited 82% of activity at pH 9.0 but also 91.5% activity at pH 4.5. NaF at 10^?2M inhibited 92% of activity at pH 4.5 but had no effect at pH 9.0. The two flukicides rafoxanide and nitroxynil at 20mM had little effect on activity at pH 9.0 and pH 4.5. Enzyme activity at pH 4.5 was found to be greatest in the microsomal fraction with high activity in the lysosomal and soluble fractions. Histochemically, alkaline phosphatase was restricted to the excretory system, vitellaria, and uterus while acid phosphatase was found in the integument and gastrodermis.     

Comparative study on hydrolases in five species of Ochromonas (Chrysomonadina)

Acid phosphatase and β-glucosidase were shown to be present in five species of Ochromonas grown in organic media (O. danica, O. malhamensis, O. minuta, O. sociabilis and Ochromonas sp. 933/4). Acid phosphatase was found to have a pH optimum at 4.0 in O. danica, and at 5.1 in the four other species. No alkaline phosphatase was found in any of the above mentioned species. β-glucosidase in the species studied has a pH optimum at 4.6 Low α-glucosidase activity was found only in O. danica. Acid phosphatase in all the five species shows an increase in activity during the logarithmic phase of growth and a decrease during the early stationary phase. β-glucosidase shows a similar behavior only in O. danica.     

Further characterization of phosphatase activities using non-specific substrates

Synopsis The demonstration of non-lysosomal acid phosphatase has been the subject of a number of recent investigations. In the present study we compared the enzyme activities in rat liver and kidney that are revealed after incubation in the presence of either -glycerophosphate,p-nitrophenylphosphate or phenylphosphate at varying pH. As seen by others, the activity towardsp-nitrophenylphosphate at pH 5–6 was confined to lysosomes, Golgi apparatus, endoplasmic reticulum (ER), nuclear envelope and plasmalemma. The reactivity of the plasmalemma and the ER was increased at pH 7. The ER of Küpffer cells in the liver stained intensely in contrast to the ER of the parenchymal cells, which stained only weakly. In the presence of NaF, all sites except the plasmalemma became negative. Addition of a levamisole-analogue,l-p-bromotetramisole, which is a specific inhibitor of alkaline phosphatase, resulted in the disappearance of the plasmalemmal activity whereas the activity at the other sites appeared unaltered. The rather unusual locations of activities with so-called non-specific substrates were further compared with those obtained with specific substrates such as glucose-6-phosphate and thiamine pyrophosphate. The possible implication of these data in relation to the specificity of marker-enzymes for subcellular organelles is discussed.     

Characteristics of lysosomes in the rat placental cells

Six acid hydrolases, cytochrome oxidase, and alkaline phosphatase were demonstrated in 0.25 m sucrose homogenates of rat chorioallantoic placenta. The acid hydrolases were: acid phosphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, β-galactosidase, and acid deoxyribonuclease, showing optimum activity near pH 4.5; cathepsin, with optimum activity near pH 3.6. The free acid hydrolases present in cytoplasmic extracts expressed 20–40% of their total activity. “Total” activity was defined as the enzyme activity observed in the presence of Triton X-100, while “free” activity denoted enzyme activity measured under similar assay conditions except in the presence of sucrose and absence of Triton X-100. The decreased activity or latency in the assays for the free activity of acid phosphatase, acid deoxyribonuclease, and cathepsin persisted after incubation at pH 5 and 37 ° up to an hour. In contrast, this latency did not persist after incubation of the β-glycosidases. Additionally, the free activity of all the designated enzymes of the cytoplasmic extract was in excess of the nonsedimentable activity observed.     

Directed Evolution of Metabolic Pathways in Microbial Populations II. a Repeatable Adaptation in SACCHAROMYCES CEREVISIAE

A selection experiment was conducted for approximately 1,000 generations in a chemostat population of 10⁹ cells of the haploid yeast, S. cerevisiae. The experiment was designed to enhance genetically the rate at which the external enzyme acid phosphatase catalyzed the hydrolysis of very low concentrations of β-glycerophosphate at an unfavorably high pH. The observed genetic adaptation in this experiment consisted of a mutation (ACP-2) in the acid phosphatase structural gene which effected a shift in the pH optimum of the enzyme and incremented its activity. The effects of ACP-2 and a similar mutation, ACP-1, on acid phosphatase substrate specificity are also reported.     

Comparison of phosphatase localization in the intraradical hyphae of arbuscular mycorrhizal fungi,Glomus spp. and Gigaspora spp.

The localization of acid and alkaline phosphatases in the intraradical hyphae of the arbuscular mycorrhizal fungi, Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (Gm), Gl. etunicatum Becker and Gerd. (Ge) and Gigaspora rosea Nicol. and Schenck (Gir) were compared. Marigold (Tagetes patula L.) and leek (Allium porrum L.) were inoculated with each of the three fungi. The mycorrhizal roots were harvested at 3, 4, 5 and 6 weeks after sowing (WAS), treated with a digestion solution containing cellulase and pectinase, and then stained for phosphatase activities at pH 5.0 and pH 8.5. The development of fungal structures in the host root was also examined. Gm formed fine-branched (mature) arbuscules only at the early phase of infection (3 to 4 WAS). Mature arbuscules of Ge and Gir were observed from the early phase (4 WAS) up to the end of experiment. At pH 5.0, the localization of the phosphatase activities of the three fungi were similar irrespective to host plant species. The activity appeared in mature arbuscules and intercellular hyphae, whereas the collapsed arbuscules were inactive. Ten millimolar NaF, an acid phosphatase inhibitor, inhibited the phosphatase activities of Gm and Ge but did not affect that of Gir. At pH 8.5, a difference among the fungal species was found in the localization of phosphatase activity while that between host species was not. The mature arbuscules were also the active sites in all three species. Only Gir showed the activity in the intercellular hyphae while the two Glomus spp. did not. Five millimolar EDTA inhibited the activity of Gir at pH 8.5 while the activities of Ge and Gm were not affected by either 5 mM EDTA or 10 mM KCN (both are alkaline phosphatase inhibitors).     

Effect of 2-deoxyglucose on lysosomal enzymes in cultured human skin fibroblasts

Addition of 2-deoxyglucose, an inhibitor of glycosylation of proteins, to the medium of confluent cultures of human skin fibroblasts prevents the increase in specific activity of lysosomal enzymes that normally occurs after confluence. Maximal inhibition is obtained at a concentration of about 1 mM 2-deoxyglucose. The inhibition by 2-deoxyglucose is reversible. The K_m, pH dependence and electrophoretic mobility of the acid hydrolases tested was the same in cells cultured with or without 2-deoxyglucose. In homogenates of cultured human skin fibroblasts, about 95% of the β-hexosaminidase and α-galactosidase activity and about 65 % of the acid phosphatase activity with β-glycerolphosphate as substrate binds to concanavalin A (ConA); 2-deoxyglucose affects only the activity able to bind to ConA. In cells cultured in the presence of 2-deoxyglucose, the specific activity of alkaline phosphodiesterase I, a plasma membrane glycoprotein is lowered. 2-Deoxyglucose has no effect on the specific activity of succinate dehydrogenase, lactate dehydrogenase or total cellular protein.     

Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum

A phosphatase was purified through a combination of ion‐exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340‐fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The K _m of the enzyme for PMLC was 10 μM, and the V _max was 1.17 nkat/mg of protein. Ca²⁺ (10 μM) inhibited the activity of the enzyme, and Mg²⁺ (8.5 μM) activated the dephosphorylation of PMLC. Mn²⁺ (1.6 μM) highly stimulated the enzyme's activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.     

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