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.     

Wall-bound enzymes in callus of Convolvulus arvensis

Isolated cell walls of Convolvulus callus contain α- and β-galactosidase, α- and β-glucosidase, α- and β-mannosidase, acid invertase and acid phosphatase activities. No neutral invertase or alkaline phosphatase activities could be detected. Acid invertase activity per mg cell wall increased considerably during incubation of callus fragments in nutrient solution, as opposed to the activities of the other enzymes mentioned.     

Symbiotic interactions between arbuscular mycorrhizal (AM) fungi and male papaya plants: Its status,role and implications

Experiments were conducted to study the arbuscular mycorrhizal (AM) status and its role in P-uptake through assay of root phosphatases activities in four varieties of male Carica papaya L. viz. CO-1, CO-2, Honey Dew and Washington during flowering stages. In the present study, mean total root colonization of AM fungi recorded peak increase in flowering stage-II while mean root phosphatase (acid and alkaline) activities recorded peak increase in flowering stage-I. Unlike root colonization and root phosphatase activities, spore density did not exhibit any definite patterns and recorded a narrow range of fluctuation during different flowering stages of male C. papaya. The study brought out the fact that root colonization and spore density of AM fungi along with root phosphatase activities varied significantly within the four varieties of male C. papaya plants during each flowering stage. The study also recorded consistently higher acid root phosphatase activity than alkaline root phosphatase activity under P-deficient, acidic soil conditions during all flowering stages of male C. papaya plants. Studies revealed that the root colonization of AM fungi influenced root phosphatase activities (acid and alkaline) positively and significantly during all flowering stages of male C. papaya plants. A total of twelve species of AM fungi belonging to five genera viz. Acaulospora, Dentiscutata, Gigaspora, Glomus, and Racocetra were recovered from the rhizosphere of male C. papaya plants.     

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.     

Isolation and characterization of a tartrate-sensitive splenic acid phosphatase in Gaucher's disease

The acid phosphatase activity that is increased in the spleens of patients with Gaucher's disease can be separated into two principal isoenzymes by chromatography on sulphopropyl-Sephadex. The acid phosphatase species that is resistant to inhibition by l-(+)-tartrate is retained by the cation-exchange resin while the tartrate-sensitive species passes through. We have isolated and characterized the tartrate-sensitive acid phosphatase (designated SP_I) from the spleen of a patient with the adult (type 1) form of Gaucher's disease. SP_I acid phosphatase, representing approximately 30 to 50% of the total acid phosphatase activity in a detergent (Triton X-100) extract of spleen tissue, has been purified approximately 400-fold to a specific activity of 48 units/mg of protein (substrate, 4-methylumbelliferyl phosphate). The final preparation of acid phosphatase contains at least two protein components—each with phosphatase activity—when analyzed by polyacrylamide gel electrophoresis at pH 8.9 or isoelectric focusing. SP_I acid phosphatase exhibits a broad substrate specificity and catalyzes the hydrolysis of a variety of artificial and natural phosphate-containing compounds including p-nitrophenyl phosphate, α-naphthyl phosphate, phosphoenolpyruvate, and CMP. The enzyme is inhibited by l-(+)-tartrate, sodium fluoride, and ammonium molybdate and has the following properties: pH optimum, 4.5; K_m on 4-methylumbelliferyl phosphate, 44 μm; pI, 3.8–4.1; M_r, 177,400; s_20,w, 6.8.     

Stimulation of liver glycogen particle synthase D phosphatase activity by caffeine, AMP, and glucose 6-phosphate

Caffeine stimulates synthase and phosphorylase phosphatase activities by independent mechanisms. Both effects of caffeine are concentration-dependent with different apparent A_0.5 for each reaction. Stimulation of the synthase phosphatase reaction was independent of the initial phosphorylase a concentration, was immediate, and did not follow in sequence the depletion of phosphorylase a. Glucose 6-phosphate also was stimulatory to the synthase phosphatase reaction (A_0.5 = 0.14 mM) with little effect on phosphorylase phosphatase. In combination glucose 6-phosphate and caffeine effects were additive suggesting the existence of separate binding sites. The synthase phosphatase reaction also was stimulated by AMP (binding affinity 2.3 mm) but with no effect on phosphorylase phosphatase activity. Together caffeine and AMP effects were not additive suggesting that they share a common binding site or closely interrelated sites. The location of the AMP and caffeine site(s) has not yet been determined.     

Acid phosphatase activity during the interaction of the nematophagous fungus Duddingtonia flagrans with the nematode Panagrellus sp

The use of Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as a biological control method against free living larvae of gastrointestinal nematodes of livestock animals. This fungus captures and infects the nematode by cuticle penetration, immobilization and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. This report characterizes the acid phosphatase activity during the interaction of D. flagrans with the free-living nematode Panagrellus sp. The optimum pH for the hydrolysis of the acid phosphatase substrate p-nitrophenyl phosphate was 2.2, 2.8 and 5.4 from D. flagrans alone and 2.2 and 5.4 for Panagrellus sp alone, fungus-nematode interaction in liquid medium and fungus-nematode interaction in solid medium. Different acid phosphatase activity bands were detected by SDS-PAGE. Maximum acid phosphatase activity of the fungus or nematode alone and of the fungus-nematode interaction occurred within 70 min of incubation in the presence of the substrate 4-methylumbelliferyl phosphate. The activity of this enzyme was significantly higher for the fungus-nematode interaction when compared to the organisms alone, indicating a synergistic response. Furthermore, structures appeared in the hyphae after 30 min, nematodes were observed adhered after 40 min and many were captured by the typical fungus traps after 70 min of interaction. The participation of acid phosphatase activity and its importance during the interaction of the fungus with the nematode were discussed.     

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.     

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.     

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.     

Quantification of acetylcholinesterase,acid and alkaline phosphatases in the central nervous system of Schizodactylus monstrosus, D. (schizodactylidae:orthoptera). Effect of topical application of the pyrethrum

The newly emerged adult male and female Schizodactylus monstrosus D. were treated with an insecticide, pyrethrum or with petroleum ether in the case of controls. At selected intervals of 6 h, 12 h and 18 h after treatment, the brain and ventral nerve cord and ganglia were homogenized to estimate the activity of acetyl-cholinesterase, and acid and alkaline phosphatase. The resultant activities of these three enzymes showed that: acetylcholinesterase decreased rapidly, and acid and alkaline phosphatase increased significantly after pyrethrum treatment in both brain and ventral nerve cord with ganglia compared with the controls. The activities of alkaline phosphatase showed a marked fluctuation at different post-treatment periods in both the tissues. The results have been discussed in relation to the impact caused by the treated insecticide and its metabolism.     

有机碳和无机碳对3种真菌胞外酸性磷酸酶和蛋白酶活性的影响

多数研究表明外生菌根真菌能够促进植物养分吸收并提高植物生长,但是对其发生的原因研究较少。本文在室内控制条件下,研究了真菌菌丝分泌N、P相关胞外酶及其受土壤有机碳（胡敏酸）和无机碳（碳酸钙）添加的影响,结果表明：1）3种真菌——松乳菇(Lactarius deliciosus)、变色红菇（Russula integra）、铆钉菇（Gomphidius viscidus）菌丝均能够分泌酸性磷酸酶和蛋白酶,而且多数情况下,MMN培养基培养14 d时,各个酶活性较高,而不同菌的胞外酶活性存在较大的差异,平均值来看铆钉菇酸性磷酸酶活性最低而蛋白酶活性最高,其它2个真菌菌丝的胞外酶活性差异不大;2）添加胡敏酸后,3种菌丝的酸性磷酸酶活性都是随着胡敏酸添加量的增加而逐渐增加;但蛋白酶活性存在差异：松乳菇的蛋白酶活性随着胡敏酸添加量的增加而逐渐增加;变色红菇的蛋白酶活性对胡敏酸不敏感,受其影响不大;铆钉菇的蛋白酶活力在少量的胡敏酸作用下最强,但浓度过高反而抑制其蛋白酶的活性。3）添加碳酸钙后,总体来看,3种菌丝胞外酸性磷酸酶和蛋白酶活性都是添加少量碳酸钙时酶活性最强,随着浓度的增加(如0.1 g),其酶活性开始受到抑制。综上所述,真菌菌丝能够分泌酸性磷酸酶和蛋白酶,这可能是因为这些外生菌根真菌能够促进植物养分吸收和快速生长的原因;有机碳和无机碳的加入可以直接影响真菌菌丝胞外酶的分泌,进而影响土壤内有机磷和有机氮化合物的分解,显示其在土壤碳循环中的作用。     

Schistosoma mansoni: distribution and characteristics of alkaline and acid phosphatase

The biochemical distribution and characteristics of the alkaline and acid phosphatase activities in both sexes of Schistosoma mansoni are reported. Alkaline and acid phosphatase activities were found in the epidermis as well as in the internal tissues. Alkaline activity is mostly located within the epidermis in both sexes. The acid activity is high in the epidermis of males and more or less equally distributed in females.Starch gel electrophoresis of worm homogenates revealed two sites of activity for both phosphatases. One is an anodic band which is a soluble phosphatase, the other, a membrane bound phosphatase, which remains at the origin under the electrophoretic conditions used. These findings are discussed in relation to previous results and to the possible roles played by these enzymes in adult S. mansoni.     

Effect of Bacteria and Amoebae on Rhizosphere Phosphatase Activity

The contributions of various components of soil microflora and microfauna to rhizosphere phosphatase activity were determined with hydroponic cultures. Three treatments were employed: (i) plants alone (Bouteloua gracilis (H.B.K.) Lag. ex Steud.) (ii) plants plus bacteria (Pseudomonas sp.), and (iii) plants plus bacteria plus amoebae (Acanthamoeba sp.). No alkaline phosphatase was detected, but an appreciable amount of acid phosphatase activity (120 to 500 nmol of p-nitrophenylphosphate hydrolyzed per h per plant) was found in the root culture solutions. The presence of bacteria or bacteria and amoebae increased the amount of acid phosphatase in solution, and properties of additional activity were identical to properties of plant acid phosphatase. The presence of bacteria or bacteria and amoebae increased both solution and root phosphatase activities at most initial phosphate concentrations.     

Effects of taurolipids on lysosomal enzymes in Tetrahymena

Effects of novel taurolipid A and B localized in Tetrahymena lysosomes on the activities of lysosomal enzymes purified from Tetrahymena were investigated. Both taurolipids activated acid phosphatase, while they did not affect α-glucosidase and β-hexosaminidase. The acid phosphatase activity was activated approximately 3-fold by both taurolipids A and B, with the half-maximum activations for taurolipid A and B being at approximately 1.03·10⁻⁴ and 0.72·10⁻⁴ M, respectively. When the purified acid phosphatase was incubated at 37°C in citrate-phosphate buffer (pH 5.0) its activity was rapidly inactivated, but the inactivation was prevented to a remarkable extent by the addition of taurolipids to the incubation medium. These results thus suggest that the taurolipids may be involved in activating and stabilizing acid phosphatase in Tetrahymena lysosomes.     

The effect of fasting and refeeding on the composition and synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines in rat liver

Refeeding a high-sucrose, fat-free diet to fasted rats caused drastic alterations in the fatty acid composition of hepatic diacylglycerols, triacylglycerols, and phosphatidylcholines. However, the fatty acid profile of phosphatidylethanolamines did not change significantly. These results suggest that the fatty acid composition of diacylglycerols may influence the distribution of diacylglycerols among triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. Fasting and refeeding also affected the activities in vitro of a number of enzymes responsible for the formation of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. The activity of hepatic phosphatidate phosphatase increased fourfold upon refeeding. However, fasting the rats did not affect the activity of this enzyme despite the reduced triacylglycerol synthesis in the fasted liver in vivo. Fasting and refeeding induced alterations in the activities of diacylglycerol acyltransferase, cholinephosphotransferase, and ethanolaminephosphotransferase which correlated reasonably well with the changes observed in the synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines in vivo, although the changes in diacylglycerol acyltransferase were too moderate. The changes in the activity of cholinephosphate cytidylyltransferase, which is suggested to catalyze the rate-limiting step in the formation of CDP-choline, ran parallel with the alterations in the synthesis of phosphatidylcholines in vivo. No such correlation was found between the activity of ethanolaminephosphate cytidylyltransferase and the rate of phosphatidylethanolamine synthesis. The present results indicate that the synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines is controlled by the availability of the various substrates as well as by the activities of several enzymes involved in these processes.     

Pulmonary phosphatidic acid phosphatase: evidence for a membrane-bound phosphatidic acid-dependent activity associated with the high speed supernatant of rat lung.

The 104,000 × g supernatant fraction from rat lung contains a greater proportion of the phosphatidic acid phosphatase activity toward membrane-bound phosphatidic acid than the microsomal fraction. The microsomal fraction is more effective in hydrolyzing aqueously dispersed phosphatidic acid. The effects of various ions and chelators, particularly Mg²⁺ and EDTA, suggest that these two activities are distinct. These results indicate that the supernatant fraction of rat lung contains a phosphatidic acid phosphatase activity which may play an important role in pulmonary glycerolipid synthesis.     

Fractionation of Hevea brasiliensis latex on ficoll density gradients

Fractionation of diluted Hevea brasiliensis latex on iso-osmotic Ficoll density gradients by sedimentation produces a sharp acid phosphatase-rich lutoid band at the density of ca1.04 g/cm³. Polyphenol oxidase (PPO) is not exclusively located in the Frey-Wyssling complexes as shown earlier by others; its distribution is variable. A complete separation of acid phosphatase and PPO activities can be obtained by flotation in a Ficoll density gradient. Ethephon stimulation of the tree reduces the acid phosphatase and PPO activities. A transient change in lutoid density soon after stimulation is observed. The occurrence of some new enzyme activities and their subcellular localization are reported.     

Multiple forms of phosphotyrosyl- and phosphoseryl-protein phosphatase from cardiac muscle: Partial purification and characterization of an EDTA-stimulated phosphotyrosyl-protein phosphatase

Chromatography of cardiac muscle and brain extracts on DEAE-cellulose resolved phosphotyrosyl-protein phosphatase activity into three fractions, termed Y-1, Y-2 and Y-3. These were eluted at 0.05, 0.15 and 0.3 m KCl, representing about 33, 55 and 12%, respectively, of the enzymatic activity recovered from the resin. Comparative studies demonstrated that the properties of phosphatases Y-1, Y-2 and Y-3 were distinctly different from those of previously identified phosphoseryl-protein phosphatases-1, -2, -3, and -4. Phosphatases Y-1, Y-2 and Y-3 were stimulated by EDTA and exhibited optimal activity at neutral pH. These properties were different from those of the two minor phosphotyrosyl-protein phosphatase activities associated with phosphoseryl-protein phosphatases-3, and -4, which were divalent cation dependent and exhibited optimal activity at alkaline pH. Further purification of phosphatase Y-2 from bovine heart has been carried out. The enzyme had a M_r = 65,000 (Stokes radius = 3.8 nm; s_20,w⁰ = 4.1). Its activity was stimulated by 5- to 10-fold in the presence of EDTA (K_a = 15 μM) and was strongly inhibited by micromolar concentrations of vanadate. Phosphatase Y-2 was highly specific for phosphotyrosyl-IgG and -casein, and showed little activity toward phosphoseryl-casein, -phosphorylase a, phosphothreonyl-inhibitor-1 and p-nitrophenyl phosphate. The present studies indicate that phosphotyrosyl-protein phosphatase activity in animal tissues exists in multiple forms. The major active species are specific for phosphotyrosyl proteins and represent enzymes different from the known phosphoseryl-protein phosphatases and p-nitrophenyl phosphatases.     

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.