Purified Acid Phosphatase Research Articles

Investigating the Impact of Various Parameters On the Activity of Acid Phosphatases from Seedlings of Coronopus didymus.

The thermal stability of purified acid phosphatase from the germinating seedlings of Coronopus didymus (Jangli halon) was investigated by studying the impact of various thermodynamic parameters [t1/2, Ed, ΔH° (enthalpy change), ΔG° (free energy change), and ΔS° (entropy change)] of heat treatment in the temperature range of 55-75 °C. The thermal denaturation of acid phosphatase, assessed by loss in activity, was evidently followed by first-order kinetics, which varies with time and yield during the process of denaturation. The half-life of the enzyme was 693 min at 55 °C. The Ed (activation energy of denaturation) was calculated by the Arrhenius plot (30 kcal mol-1), and the Z-value was 17.3 °C. The various thermodynamic parameters studied were as follows: ΔH°, the change in enthalpy of inactivation, was 121.93 kJ mol-1 at 55 °C; ΔG°, the change in free energy of inactivation, was 110.65 kJ mol-1 at 55 °C; and ΔS°, the change in entropy of inactivation, was 34.39 J mol-1 k-1 at 55 °C. This suggests that acid phosphatase activity is thermostable to long heat treatment up to 60 °C.

Effect of acid phosphatase produced by Trichoderma asperellum Q1 on growth of Arabidopsis under salt stress

Salt stress is a major environmental factor that inhibits crop growth. Trichoderma spp. are the most efficient biocontrol fungi and some of the strains can stimulate plant growth. Phosphate solubilization is known as one of the main mechanisms in promoting plant growth, but the underlying mechanisms of phosphate solubilization in the salinity still need to be explored. The Trichoderma asperellum Q1 isolated and identified in our lab is a beneficial rhizosphere biocontrol fungus with a high phosphate solubilization activity. It could produce acid and alkaline phosphatases when using insoluble organic phosphorus as the sole phosphorus source, the salt stress increased the phosphorus-solubilization ability of the strain and the activities of the two enzymes. Furthermore, an acid phosphatase was purified from the fermentation broth by ammonium sulphate precipitation, ion-exchange, and gel filtration chromatography. Its molecular weight was 55 kDa as determined by SDS-PAGE. The purified acid phosphatase was used to investigate growth performance of Arabidopsis thaliana by plate assay and the result showed that it contributed to Arabidopsis growth by transforming organic phosphate into a soluble inorganic form under salt stress. To our knowledge, this is the first report on acid phosphatase purification from T. asperellum and its function in regulation of plant growth under salt stress.

Phosphate solubilization and acid phosphatase activity of Serratia sp. isolated from mangrove soil of Mahanadi river delta, Odisha, India.

Phosphorus is an essential element for all life forms. Phosphate solubilizing bacteria are capable of converting phosphate into a bioavailable form through solubilization and mineralization processes. Hence in the present study a phosphate solubilizing bacterium, PSB-37, was isolated from mangrove soil of the Mahanadi river delta using NBRIP-agar and NBRIP-BPB broth containing tricalcium phosphate as the phosphate source. Based on phenotypic and molecular characterization, the strain was identified as Serratia sp. The maximum phosphate solubilizing activity of the strain was determined to be 44.84 μg/ml, accompanied by a decrease in pH of the growth medium from 7.0 to 3.15. During phosphate solubilization, various organic acids, such as malic acid (237 mg/l), lactic acid (599.5 mg/l) and acetic acid (5.0 mg/l) were also detected in the broth culture through HPLC analysis. Acid phosphatase activity was determined by performing p-nitrophenyl phosphate assay (pNPP) of the bacterial broth culture. Optimum acid phosphatase activity was observed at 48 h of incubation (76.808 U/ml), temperature of 45 °C (77.87 U/ml), an agitation rate of 100 rpm (80.40 U/ml), pH 5.0 (80.66 U/ml) and with glucose as a original carbon source (80.6 U/ml) and ammonium sulphate as a original nitrogen source (80.92 U/ml). Characterization of the partially purified acid phosphatase showed maximum activity at pH 5.0 (85.6 U/ml), temperature of 45 °C (97.87 U/ml) and substrate concentration of 2.5 mg/ml (92.7 U/ml). Hence the present phosphate solubilizing and acid phosphatase production activity of the bacterium may have probable use for future industrial, agricultural and biotechnological application.

MALDI mass sequencing and biochemical characterization of Setaria cervi protein tyrosine phosphatase

A 30-kDa acid phosphatase with protein tyrosine phosphatase activity was identified in Setaria cervi (ScPTP). The enzyme was purified to homogeneity using three-step column chromatography. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of purified ScPTP yielded a total of eight peptides matching most closely to phosphoprotein phosphatase of Ricinus communis (RcPP). A hydrophilicity plot of RcPP revealed the presence of these peptides in the hydrophilic region, suggesting their antigenic nature. The substrate specificity of ScPTP with ortho-phospho-L-tyrosine and inhibition with sodium orthovanadate and ammonium molybdate affirmed it as a protein tyrosine phosphatase. ScPTP was also found to be tartrate resistant. The Km and Vmax were 6.60mM and 83.3μM/ml/min, respectively, with pNPP and 8.0mM and 111μM/ml/min, respectively, with ortho-phospho-L-tyrosine as the substrate. The Ki value with sodium orthovanadate was calculated to be 16.10mM. Active site modification with DEPC, EDAC and pHMB suggested the presence of histidine, cysteine and aspartate at its active site. Thus, on the basis of MALDI-TOF and biochemical studies, it was confirmed that purified acid phosphatase is a PTP.

Purification and characterization of three low- molecular-weight acid phosphatases from peanut (Arachis hypogaea) seedlings

th day of germination. At least, three acid phosphatases were identified and purified by successive chromatography separations on DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, Sephacryl S-200 HR, and PhenylSepharose HP to apparent homogeneity from developing five days old peanut seedlings. These enzymes designated acid phosphatase PI, PIIa and PIIb had native molecular weights of approximately 25.3, 22.4 and 24 kDa, respectively by gel permeation. SDS-PAGE of the purified acid phosphatase PI resolved two closely protein bands that migrated to approximately 14 and 12 kDa. Thus, this acid phosphatase likely functions as a heterodimer. Acid phosphatases PIIa and PIIb migrated as single band (each) with a similar molecular weight estimated to 21 kDa. The three enzymes had a similar optima pH (5.0) and temperature (55°C), and appeared to be stable in the presence of non-ionic detergents such as Triton X-100, Nonidet P 40 as well as Na + and K + . Substrate specificity indicated that the three acid phosphatases hydrolyzed a broad range of phosphorylated substrates. However, natural substrates such as ADP and ATP were the compounds with highest rate of hydrolysis for acid phosphatase PI, while acid phosphatase PIIa exhibited phytasic activity. These results indicate that each purified acid phosphatase from peanut seedlings played a peculiar role during germination.

Oocyte fertilization triggers acid phosphatase activity during Rhodnius prolixus embryogenesis

Acid phosphatase activity, previously identified in Rhodnius prolixus oocytes, was studied during egg development. Fertilized eggs exhibited a five fold increase of total acid phosphatase activity during the first days of development. In contrast non-fertilized oviposited eggs showed no activation of this enzyme. An optimum pH of 4.0 for pNPP hydrolysis in a saturable linear reaction and a strong inhibition by lysosomal acid phosphatase inhibitors such as NaF (10 mM) and Na +/K + tartrate (0.5 mM) are the major biochemical properties of this enzyme. Fractionation of egg homogenates through gel filtration chromatography revealed a single peak of activity with a molecular mass of 94 kDa. The role of this enzyme in VT dephosphorylation was next evaluated. Western blots probed with anti-phosphoserine polyclonal antibody demonstrated that VT phosphoaminoacid content decreases during egg development. In vivo dephosphorylation during egg development was confirmed by following the removal of 32P from 32P-VT in metabolically labeled eggs. Vitellin was the only phosphorylated molecule able to inhibit pNPPase activity of partially purified acid phosphatase. These data indicate that acid phosphatase activation follows oocyte fertilization and this enzyme seems to be involved in VT dephosphorylation.

Purification and properties of pi-repressible acid phosphatases from Aspergillus nidulans

Two forms of the pacA-encoded acid phosphatase (designated acid phosphatases I and II) secreted by the mold Aspergillus nidulans grown in low-Pi medium at 37°, pH 5.0, were purified to apparent homogeneity by PAGE. The M r of the purified enzyme forms were ca 115 000 (60 000) and 113 000 (62 000) respectively for forms I and II secreted by strain biA1 and ca 118 000 (60 000) and 121 000 (61 000) respectively for forms I and II secreted by strain biA1 pacA1, as determined by exclusion chromatography (number between brackets are the M r as determined by SDS-PAGE). All of these purified enzyme forms showed an apparent optimum pH ranging from 6.0 to 6.5 and no deviation from Michaelis kinetics for the hydrolysis of both p-nitrophenylphosphate and α-naphthylphosphate. Heat inactivation at 60° and at pH 6.0 showed half-lives of 14 min ( k=0.033 min −1) and 10 min ( k=0.069 min −1), respectively, for the purified acid phosphatases I and II secreted by biA1 strain and half-lives of 0.8 min ( k=0.92 min −1) and 0.6 min ( k=0.95 min −1), respectively, for the purified forms I and II secreted by the biA1 pacA1 strain. The neutral sugar content of purified acid phosphatases I and II secreted by strain biA1 was 48% and 37% (w/w), respectively, whereas the content of forms I and II secreted by strain biA1 pacA1 was 18% and 11%, respectively.

Acid phosphatase/glucose oxidase-based biosensors for the determination of pesticides

This work presents new amperometric bienzymatic bioelectrodes for the determination of organophosphorus and carbamic acid type pesticides. Two different kinds of bienzymatic bioelectrodes are presented: a classical bienzymatic electrode, obtained by physicochemical immobilization of purified acid phosphatase (AP) and glucose oxidase (GOD) on the tip of an amperometric H 2O 2 electrode; and a hybrid biosensor, in which AP has been employed in the form of a thin layer of potato ( Solanum tuberosum) tissue, endowed with a high content of enzyme activity. Both the biosensors can selectively detect glucose-6-phosphate (G6P), in the 5.0 × 10 −5 −1.2 × 10 −3M concentration range. Pesticides are detected, thanks to their high inhibition power towards AP, evaluated by adding the sample stepwise to a buffered solution of G6P, and recording the corresponding current change. The detection limit is therefore a function of the type of pesticide, but it can be as low as 1 μg 1 −1 in the case of organophosphorus compounds. The detection limit is generally higher for carbamates, as a consequence of their weaker inhibition power towards acid phosphatase. Both bioelectrodes presented comparable values of the main physicochemical and analytical parameters evaluated for assessing their overall performance; nonetheless the plant tissue based bioelectrode exhibited a longer shelf life and a better reliability of the amperometric results.

Purification and partial characterization of acid phosphatase from Candida lipolytica.

Non-specific acid phosphatase from Candida lipolytica cells was purified 111-fold by chromatography on DEAE-cellulose and gel filtration on Sephadex G-100 and Sepharose 4B. The enzyme is a glycoprotein containing 67% neutral sugars. The molecular mass of the highly purified acid phosphatase was found to be approximately 95 kDa by both SDS-PAGE and gel filtration. The pH and temperature optima were 5.8 and 55 degrees C, respectively. The enzyme was stable at pH values between 3.5 and 5.5 and at temperatures up to 60 degrees C. The purified phosphatase had a Km value of 3.64 mM for p-nitrophenyl phosphate and showed broad substrate specificity.

Properties of a partially purified acid phosphatase from pathogenic Nocardia brasiliensis.

Like many other bacteria, Nocardia sp. possess acid phosphatase activity. In N. brasiliensis, a human and animal pathogen, this activity was resolved into two enzyme forms by native gel electrophoresis. One (isozyme I) was partially purified and characterized. It exhibited an estimated molecular weight on SDS-PAGE of 50 kDa, a pH optimum of 5.2, and a Km value of 1.25 mM for p-nitrophenylphosphate. The N. brasiliensis enzyme was stable at 4 degrees C for at least 24 h, but readily inactivated at 60 degrees C. Ammonium molybdate, sodium fluoride and L-(+)-tartrate were found to be potent inhibitors of the enzyme. Although its function is presently unknown, by analogy to other bacterial systems it could be envisioned to play an important role in the physiology and pathogenicity of the microorganism.

Development of an Immunoassay for Human Serum Osteoclastic Tartrate-Resistant Acid Phosphatase*

A tartrate-resistant acid phosphatase (TrACP), which has been suggested to be very similar to the osteoclastic TrACP, was partially purified from the spleen of a patient with hairy cell leukemia. The purification procedure consisted of carboxymethyl-Sepharose, phosphocellulose, Sephacryl S-200, and phenyl-Sepharose chromatographies. Polyclonal antibodies were generated in guinea pigs with a titer of at least 1:6000. Immunohistochemical staining of fetal rat tibia with the antisera revealed that only the lysosomes of osteoclasts, but not osteoblasts, were stained. An enzyme-linked immunosorbent assay (ELISA) was developed with the antisera. There was no cross-reactivity with 1) partially purified acid phosphatases (ACPs) from normal human and beef spleens, 2) ACPs in extracts of human osteoblastic cells, 3) purified bovine bone matrix TrACP, or 4) commercial prostatic ACP. However, extracts of giant cell bone tumors, containing large amounts of bona fide osteoclasts, showed large amounts of cross-reactive material, which diluted in parallel with the partially purified hairy cell leukemic TrACP in the ELISA. Commercial serum band 5b TrACP also displaced in parallel with the partially purified hairy cell leukemic TrACP. Immunoblotting studies revealed that the antiserum, but not nonimmune guinea pig serum, reacted with the homogeneous hairy cell leukemia splenic band 5 TrACPs, which were recently purified by our laboratory. Preliminary application of the ELISA to sera of patients with metabolic bone diseases revealed that normal healthy individuals had measurable amounts of the immunoreactive material, and patients with Paget's disease or hyperparathyroidism, who should have high bone turnover, had elevated levels of this immunoreactive material in their sera. In contrast, the level of serum osteoclastic TrACP in a patient with an acute lymphatic leukemia was normal. In summary, 1) we have shown that hairy cell leukemia splenic TrACP shares significant immunological similarity with the osteoclastic TrACP and with the serum band 5b TrACP, and 2) the ELISA holds promise for a sensitive and specific assay for bone resorption.

Acid phosphatases from Lupinus angustifolius and their immunological relationship with grass acid phosphatases

In seeds, cotyledons and stems of blue lupine ( Lupinus angustifolius) two groups of acid phosphatases has been found which differ in their affinity to concanavalin A (Con A)-Sepharose and binding to LcA. The purified Con A-binding acid phosphatases are glycoproteins containing mannose or mannose and fucose residues in their carbohydrate moiety. They retained the enzyme activity after binding to Con A, moreover some of them were activated by binding to this lectin. The purified acid phosphatases from all tissues cross-react with monospecific antibodies raised against grass seed acid phosphatase Ba 1 indicating an immunological relationship between the enzymes of the blue lupine and grass.

On the acid phosphatase isoenzymes existing in American Leishmania promastigotes.

1. Two partially purified acid phosphatase activities present in American Leishmania promastigote homogenates were characterized by biochemical methods. 2. One isoenzyme acts preferentially on p-nitrophenyl phosphate, is strongly inhibited by 30 mM alloxan, citrate, maleate, malonate and succinate, and strongly stimulated by 3 mM spermine. Its pI is 4.8. 3. The other isoenzyme acts preferentially on beta-glycerophosphate and is resistant to 30 mM alloxan, citrate, maleate, malonate and succinate and also to 3 mM spermine. Its pI is 5.7. 4. Both acid phosphatase isoenzymes have an optimum pH of 5.2, are tartrate-sensitive and strongly membrane-bound, as shown by differential centrifugation and density gradient equilibration. 5. Both isoenzymes were separated by using homogenates prepared in 2% Triton X-100, differential centrifugation, Sepharose 4B/CL-4B gel filtration, ion exchange chromatography and electrofocusing. After this procedure, they were still contaminated with several different proteins. 6. Purification was around 150-fold, with a 32% yield. 7. When these acid phosphatase activities were measured in total homogenates from 12 different Leishmania isolates, p-nitrophenyl phosphatase specific activity values were quite close; beta-glycerophosphatase-specific activity had around a 2-fold variation. 8. This variation was independent from taxonomic classification or infectivity of susceptible hosts.

Acid phosphatases of Escherichia coli: molecular cloning and analysis of agp, the structural gene for a periplasmic acid glucose phosphatase.

Several unknown Escherichia coli genes for different species of acid phosphatase were cloned in vivo with the plasmid Mu dII4042. When present in a multicopy state, each gene promoted hydrolysis of p-nitrophenyl-phosphate at acidic pH. Among seven recombinant clones that encoded periplasmic acid phosphatase activities, five different genes could be distinguished by the pH optimum and substrate preference for the enzyme and by the restriction enzyme pattern. A 1.7-kilobase recombinant DNA fragment, common to two clones, was inserted into plasmid pBR322 and shown to contain a new gene, agp, which leads to the overexpression of the periplasmic acid glucose-1-phosphatase, a dimer of a 44-kilodalton polypeptide. Fusions of agp to gene phoA deprived of its own signal sequence conferred an alkaline phosphatase-positive phenotype to bacteria, showing the presence of an export signal on agp. The resulting hybrid proteins were characterized by immunoprecipitation with an antiserum directed against purified acid phosphatase or against alkaline phosphatase, showing that agp is the structural gene of the acid phosphatase. The beginning, the orientation, and the end of gene agp on the cloned DNA fragment were determined by the characteristics of such hybrid proteins.

Antibodies to porcine uteroferrin used in measurement of human tartrate-resistant acid phosphatase.

The immunological similarity between human tartrate-resistant acid phosphatase (EC 3.1.3.2) and porcine uteroferrin previously reported for the isoenzyme from spleens of patients with leukemic reticuloendotheliosis (Ketcham et al., J Biol Chem 1985;260:5768-76) has been confirmed for partly purified acid phosphatase found in the spleen of a patient with Gaucher's disease, and for the corresponding isoenzyme in other tissues and serum. Anti-uteroferrin antibodies raised in rabbits have been used to demonstrate the feasibility of their application in an immunoassay for tartrate-resistant acid phosphatase in serum.

Induction of acid phosphatase in cotton seedlings: Enzyme purification, subunit structure and kinetic properties

About a 16-fold rise in acid phosphatase (EC 3.1.3.2) activity was observed during the early stages of germination of cotton embryos. Administration of cyclobeximide to the germinating embryos significantly blocked the enhancement of acid phosphatase activity. This indicated that translational activity was essential for the induction of enzyme activity. Conclusive proof for the de novo synthesis of the enzyme was obtained by showing the incorporation of 35S from 35SO 2− 4 into the cysteine residues of the purified acid phosphatase. The enzyme was purified (1046-fold) to electrophoretic homogeneity by ammonium sulphate fractionation, CM-Sephadex C-50 and affinity chromatography on concanavalin A-Agarose. PAGE gave two isozyme bands. The M, of the phosphatase was 200 k as determined by molecular sieving on Sephadex G-200. SDS-PAGE of acid phosphatase revealed a single band of M 55 k. Thus the native enzyme is a tetramer of four identical subunits. The K m of the enzyme with p-nitrophenyl phosphate was 0.5 mM. Optimal enzyme activity was observed at pH 5.0, using p-nitrophenyl phosphate as substrate. The enzyme activity remained linear for 105 min at 37° and was proportional to the concentration of protein within the range 0.6–2.4 μg.

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.

Some properties and immunological relationship of acid phosphatases from gramineae

Two acid phosphatases have been found in crude extracts of seeds, coleoptiles and leaves of various grass species by means of crossed immunoelectrophoresis. The enzymes, cross-reacting with antibodies raised against proteins of Poa pratensis seeds differ in their binding to con A. The use of affinity chromatography on con A-Sepharose has separated the acid phosphatases into two fractions: the non-binding (acid phosphatase A) and the con A-binding (acid phosphatase B). The con A-binding acid phosphatase B from all tissues was further purified by gel filtration on Biogel P-100 and hydrophobic interaction chromatography on phenyl-Sepharose. Two isoenzymes: acid phosphatase B 1 and B 2 were obtained. The isoenzymes are glycoproteins containing D-mannose or D-glucose in their carbohydrate moiety. They retained the enzyme activity after binding in macromolecular complex with antibodies or con A. The purified acid phosphatases from all tissues cross-react with monospecific antibodies raised against P. pratensis seeds acid phosphatase B 1 indicating the antigenic relationship between the enzymes of various grass species.

Synthesis of acid phosphatase and other proteins by human prostatic tissue in vitro.

We have studied the synthesis of proteins by normal and hyperplastic human prostatic tissue incubated in vitro in the presence of [35S]methionine. The overall pattern of newly synthesized proteins was similar in individuals with an age ranging from 15 to 68 years. The pattern of labeled proteins was quite different from that of total proteins stained with Coomassie blue in two-dimensional polyacrylamide gels, since the major stained proteins was not labeled. Among the most heavily labeled proteins (about a dozen) were several spots representing charge isoforms with molecular weights ranging from 46 000 to 51 000, and these were the only proteins immunoprecipitated by a polyclonal antibody developed against purified acid phosphatase. The other heavily labeled proteins had molecular weights ranging from 26 000 to 72 000. These results show that tissue slices can be used to study the synthesis and processing of acid phosphatase, the major secretory product of the prostate, and of other unidentified proteins.

Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis

An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH 4) 2SO 4 precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The K m values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10 −4 M, 3.9·10 −4 M and 1.6·10 −3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg 2+ and Fe 3+ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF ( K i=5.6·10 −4 M) and by L-(+)-tartaric acid ( K i = 8.5·10 −5 M), while it was inhibited noncompetitively by molybdate K i = 5.0·10 −6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, K m, thermal stability and inhibition by NaF.