INTRODUCTION: A phosphate, an inorganic chemical, is a salt of phosphoric acid.

Inorganic phosphates are mined to obtain phosphorus for use in agriculture and industry. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry Phosphorus is essential for the growth and development of organisms and is one of the most important but least available mineral nutrients required by plant, in part because it is commonly bound to many soil constituents. For organic phosphorus sources to be used they must be first hydrolyzed by phosphatases. Plant acid phosphatase (ACP) examined include the enzyme from seeds, leaves, bulbs, root nodules, coleoptiles, cotyledons, tubers, roots and plumules. ACPs (ortophosphoric-monoester phosphohydrolase, EC 3.1.3.2) are ubiquitous in a broad variety of animals, plants and microorganisms. ACPs are widely distributed in plants and exist as compartment and/or tissues-speci.c isoforms that can signi.cantly differ in subunit molecular mass, substrate speci.city, and their susceptibility to inhibition by various compounds. ACPs catalyze the hydrolysis of phosphate esters, releasing inorganic phosphorus from a phosphorylated substrate in vitro, making their cellular role(s) difficult to define. They tend to display little substrate specificity and appear to be important in the production, transport and recycling of inorganic phosphorus. Due to low substrate specificity, ACPs are presumed to be involved in non-specific hydrolysis of organic phosphorus resulting in elevation of the (Pi) phosphorus pool and appear to function in response to phosphate deficiency. However, changes in specific isoforms of phosphatases under Pi starvation are commonly observed. Related to this, it has been observed that common bean (Phaseolus vulgaris) root nodules export nitrogen in the form of ureides allantoin and allantoic acid, which are derived from de novo-synthesized purines. Even though the first step in the conversion of purines to ureides is the removal of the 5'-phosphate group by a non-specific ACP or by a 5'-nucleotidase, a comprehensive understanding of the metabolic function of ACPs in nodules and leaves from legumes is lacking partly because of the heterogeneityand large

number of phosphatases. In the present work we describe the partial purification and characterization of a soluble ACP from nodules and leaves of common bean.

The hydrolysis of phosphate monoesters in biological system is a very important process that occurs in energy metabolism, metabolic regulation and various cellular signal transduction pathways (Tonks et.al; 1989). This hydrolytic reaction in cells is catalysed by an extremely diverse group of enzymes collectively termed phophatases. These enzymes also play an important role in the diagnosis of various pathological conditions (Bodansky, 1972). Based upon the optimal pH, the involvement of metal ion cofactors and the substrate specificity, phosphatases have been partially subclassified as alkaline phosphatases, acid phosphatases, purple acid phosphatases and protein phosphatases (Vincent, 1992). In term of such a classification, the alkaline phosphatases can be referred to as serine phosphatases (Schwartz and Lipmann, 1961), whlie the protein tyrosine phosphatases and low molecular weight acid phosphatases are cysteine phosphatases (Guan and Dixon, 1991; Wo et.al; 1992; Cho et al; 1992). Acid phosphatases (orthophosphoric monoester phosphohydrolases or phosphomonoesterases), EC 3.1.3.2, are ubiquitous in nature.

Alkaline phosphatase (ALP)(EC 3.1.3.1) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. Phosphate is an essential nutrient for all organisms. Plants can utilize only inorganic phosphate (Pi) as phosphate source. Phosphates are divided in to acid and alkaline phosphates. Acid phosphatase (EC 3.1.3.2) catalyses the hydrolysis of a wide variety of phosphate esters in an acid environment to produce Pi. Acid phosphatase is a phosphatase, a type of enzyme, used to free attached phosphate groups from other molecules during digestion.

Acid phosphatases (EC: 3.1.3.2) are a heterogeneous group of proteins that hydrolyse phosphate esters, optimally at low pH. It is basically a phosphomonoesterase. It is stored in lysosomes and functions when these fuse with endosomes, which are acidified while they function; therefore, it has an acid pH optimum. A phosphatase with an optimum pH of less than 7 (for several isozymes, it is 5.4), notably present in the prostate gland; demonstrable in lysosomes with Gomori nonspecific acid phosphatase stain; it hydrolyzes many orthophosphoric monoesters. Different forms of acid phosphatase are found in different organs, and their serum levels are used as a diagnostic for disease in the corresponding organs. For example, elevated prostatic acid phosphatase levels may indicate the presence of prostate cancer.

Acid Phosphatase:
I.U.B.: 3.1.3.2 Orthophosphoric-monoester phosphohydrolase (acid optimum) Acid phosphatase catalyzes the following reaction at an optimal pH below 7: Orthophosphoric monoester + H2O alcohol + H3PO4 Acid phosphatase is ubiquitous in nature, perhaps one of the most concentrated sources being human prostate gland, a fact exploited by the clinical chemist who measures the serum enzyme level as an index of prostatic cancer. It has also been associated with Gaucher's disease, patients exhibiting unique peaks in their electrophoresed sera (Goldberg et al. 1966). Acid phosphatases have been reviewed by Hollander (1971) although the wheat germ enzyme is not included. Acid phosphatase activity was observed by Teller in 1954 in preparations of a wheat germ lipase described by Singer in 1948. Subsequent work confirmed that the nonspecific esterase activity of the wheat germ preparation may be measured both as lipase (tracetin as substrate) and phosphatase.

The enzyme has been purified and its properties described by at least three different laboratories. Joyce and Grisolia (1960) purified it some 3000-fold and measured its activity on a variety of phosphate esters. Brouillard and Ouellet (1965) obtained four separate forms of the enzyme on ion-exchange and gave evidence for the presence of ferric iron in the molecule. Verjee (1969) obtained three active peaks and showed them to have individual properties. Characteristics of Acid Phosphatase: Molecular weight: 55,000 5,000 (Verjee 1969). Composition: Three isozymes of closely similar molecular weights have been reported by Verjee (1969): EI, EII, and EIII. See also Brouillard and Ouellet (1965). Optimal pH: EI - 5.5, EII - 4.5, and EIII - 4.0. (Verjee 1969). Specificity: The enzyme has a broad esterase activity. See Joyce and Grisolia (1960). It shows highest activity for pyrophosphate. Inhibitors: Fluoride, molybdate and orthophosphate (Verjee 1969). Alternative names: Acid acid monophosphatase; phosphomonoester acid phosphohydrolase; acid

phosphomonoesterase;

hydrolase;

phosphomonoesterase;

glycerophosphatase; uteroferrin Phosphatase enzymes are also used by soil microorganisms to access organically bound phosphate nutrients. An assay on the rates of activity of these enzymes may be used to ascertain biological demand for phosphates in the soil. The physiological function of this acid phosphatase is to provide inorganic phosphate to the growing wheat seedling during germination. Many different phosphate esters of sugars and substrates are stored in the wheat seed and these need to be hydrolyzed during germination, which makes the carbohydrates available as an energy source and the

phosphate to be used as building blocks in making new cells (new RNA and DNA all need phosphate in their backbones). It is also possible to use artificial phosphate esters since the enzyme is rather non-specific and will catalyze phosphate ester hydrolysis on many different substrates. Phosphate ester of p-nitrophenol is a good substrate to use since the product formed after ester hydrolysis, p-nitrophenol, can easily be measured. Here is the reaction catalyzed by wheat germ phosphatase with p-nitrophenylphosphate (pNPP):

The first step is the enzyme catalyzed part of this graphic. In this reaction, the catalyst is the phosphatase and the products are p-nitrophenol and inorganic phosphate or Pi. The second part of the graphic illustrates the way to detect the p-nitrophenol formed as product in the phosphatase catalyzed reaction shown in part 1.

Acid phosphatases play a key role in phosphate acquisition by plants but, except for a few enzymes performing specific metabolic functions, it is difficult to ascribe a precise role to the majority of them. Research on plant acid phosphatases has a long history yet the available data do not allow generalizing on their structure and function. This is a result of different aspects considered by researchers (Duff et al., 1994). Some of them elaborate on physiology, while other on biochemistry or molecular biology. There are a very limited number of comprehensive reports on the overall aspects of acid phosphatases. The most complete data have been published on the largest group of plant phoshatases known as purple acid phosphatases (PAPs) which occur also in microorganisms and animals (Schenk et al., 2000; Oddie et al., 2000; Vogel et al., 2001). The animal enzymes were purified and characterized from bovine spleen (Campbell & Zerner, 1973) and pregnant pig uterine fluid (uteroferrin) (Schlosnagle et al., 1974). In humans, they were isolated from the spleen (Yam et al., 1971), bones (Allen et al., 1989), alveolar macrophages (Efstratiadis & Moss, 1985) and placenta (Ketcham et al., 1989). The level of these enzymes increases in such pathological states as Gaucher disease (Robinson & Glew, 1980), hairy cell leukemia (Yam et al., 1971), osteoclastoma cells (Hayman et al., 1989) and AIDS (Schindelmeiser et al., 1989). All PAPs are tartrate-resistant and contain a binuclear metal site. The pink/purple color of their concentrated water solution is the result of a charge-transfer transition between "chromophoric" ferric ion and the tyrosine residue (Vincent & Averill, 1990). The second "nonchromophoric" metal ion, occupying the binuclear site, is iron in mammalian PAPs, and zinc or manganese in plant enzymes (Vogel et al., 2001). STRUCTURE OF ACID PHOSPHATES: Plant Protein Binuclear metal centre

Animal Protein Binuclear metal centre

APPLICATIONS: Medical use: Phosphoric acid is used in dentistry and orthodontics as an etching
solution, to clean and roughen the surfaces of teeth where dental appliances or fillings will be placed. Phosphoric acid is also an ingredient in over-the-counter anti-nausea medications that also contain high levels of sugar (glucose and fructose). It should not be used by diabetics without consultation with a doctor. This acid is also used in many teeth whiteners to eliminate plaque that may be on the teeth before application. Biological effects on bone calcium and kidney health: Phosphoric acid, used in many soft drinks (primarily cola), has been linked to lower bone density in epidemiological studies. For example, a study[2] using dual-energy X-ray absorptiometry rather than a questionnaire about breakage, provides reasonable evidence to support the theory that drinking cola results in lower bone density. This study was published in the American Journal of Clinical Nutrition. A total of 1672 women and 1148 men were studied between 1996 and 2001. Dietary information was collected using a food frequency questionnaire that had specific questions about the number of servings of cola and other carbonated beverages and that also made a differentiation between regular, caffeine-free, and diet drinks. The paper cites significant statistical evidence to show that women who consume cola daily have lower bone density. Total phosphorus intake was not significantly higher in daily cola consumers than in nonconsumers; however, the calcium-to-phosphorus ratios were lower. The study also suggests that further research is needed to confirm the findings.

Processed food use: Food-grade phosphoric acid (often labeled as E number E338.) is used to acidify foods and beverages such as various colas, but not without controversy regarding its health effects. It provides a tangy or sour taste and, being a mass-produced chemical, is available cheaply and in large quantities. The low cost and bulk availability is unlike more expensive natural seasonings that give comparable flavors, such as citric acid which is obtainable from lemons and limes. (However most citric acid in the food industry is not extracted from citrus fruit, but fermented by Aspergillus niger mold from scrap molasses, waste starch hydrolysates and phosphoric acid.) Rust removal: Phosphoric acid may be used by direct application to rusted iron, steel tools, or surfaces to convert iron(III) oxide (rust) to a water-soluble phosphate compound. It is usually available as a greenish liquid, suitable for dipping (acid bath), but is more generally used as a component in a gel, commonly called naval jelly. As a thick gel, it may be applied to sloping, vertical, or even overhead surfaces. Care must be taken to avoid acid burns of the skin and especially the eyes, but the residue is easily diluted with water. When sufficiently diluted, it can even be nutritious to plant life, containing the essential nutrients phosphorus and iron. It is sometimes sold under other names, such as "rust remover" or "rust killer." It should not be directly introduced into surface water such as creeks or into drains, however. After treatment, the reddish-brown iron oxide will be converted to a black iron phosphate compound coating that may be scrubbed off. Multiple applications of phosphoric acid may be required to remove all rust. The resultant black compound can provide further corrosion resistance (such protection is somewhat provided by the superficially similar Parkerizing and blued electrochemical conversion coating processes). After application and removal of rust using phosphoric acid compounds, the metal should be oiled (if to be used bare, as in a tool) or appropriately painted, by using a multiple coat process of primer, intermediate, and finish coats.

Physiology: In humans, alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in liver, bile duct, kidney, bone, and the placenta. The optimal pH for the activity of the E. coli enzyme is 8.0 while the bovine enzyme optimum pH is slightly higher at 8.5. Diagnostic use: The normal range is 20 to 140 IU/L. High ALP levels can show that the bile ducts are blocked. Levels are significantly higher in children and pregnant women.Lowered levels of ALP are less common than elevated levels.

OBJECTIVES: Isolation of acid phosphatases from the plant sources i.e.,. Production, detection and quantative estimation of acid phosphates. Optimum production of acid phosphates as influenced by environmental factors like temperature and pH. To study the effect of nutrients, metal ions, surfactants and mineral oils on acid phosphates production. Purification and characterization of extracellular acid phosphates from both plant and animal sources.

REVIEW OF LITERATURE: Phosphate esters are widely distributed in any organism. Nucleic acids, metabolic intermediates like glucose-6-phosphate, energy-rich substrates (AMP, creatine phosphate) are some obvious examples. While many metabolic intermediates are activated through the transfer of phosphate groups (e.g., by kinases) it is equally important that phosphate esters can also be rapidly broken down. The hydrolytic removal of phosphate groups from phosphoesters is catalyzed by phosphatases. Many phosphatases are highly substrate-specific, like those enzymes involved in signal transduction. A number of phosphatases, however, cleave virtually any phosphate ester. Such unspecific enzymes function mainly in the catabolic breakdown of metabolites or nutrients. Depending on the pH at which such phosphatases have optimal activity, we distinguish between acidic phosphatases (also called acid phosphatases) and alkaline phosphatases. The latter enzymes require divalent metal ions as cofactors and are common in animal tissues and bacteria. Acid phosphatases (orthophosphoric monoester phosphorrylase, E. C. 3.1.3.2) are enzymes that catalyze the hydrolysis of orthophosphate esters at acidic pH values. Acid

phosphatases occur ubiquitously among plants and animals (Hollander, 1971). They can be divided into groups based on their substrate type: non-specific phosphatases catalyze the hydrolysis of almost any phosphate ester whereas specific ones such as protein phosphatases prefer phosphoproteins or phosphopeptides as substrates. Alkaline and acid phosphatases are non-specific, and are only differentiated based on their optimal pH for catalysis and are thought to recycle phosphate in metabolic reactions. Little is known about their functional significance in plants. They can be further classified as prostatic, lysosomal, erythrocytic, and macrophagic acid phosphatases.

Acid phosphatases

Acid phosphatases (APs) are a family of enzymes that are widespread in nature, and can be found in many animal and plant species. Mystery surrounds the precise functional role of these molecular facilitators, despite much research. Yet, paradoxically, human APs have had considerable impact as tools of clinical investigation and intervention. One particular example is tartrate resistant acid phosphatase, which is detected in the serum in raised amounts accompanying pathological bone resorption. This article seeks to explore the identity and diversity of APs, and to demonstrate the relation between APs, human disease, and clinical diagnosis.

Purification of acid phosphatase I from germinating seeds of Vigna sinensis

Acid phosphatase I (AP-I) is the major isoform of Vigna acid phosphatase. It is constitutively expressed in seed cotyledons during germination. AP-I was separated from other isoforms and purified to homogeneity by three simple purification steps; (NH 4)2SO4 precipitation, and phosphocellulose and DEAE-cellulose column chromatography. The activity of AP-I was not affected by 1 mM Mg2+, Mn2+, Ca2+, Co2+ or Pb2+, but severely inhibited by 1 mM Cu2+, Fe3+, Hg2+, Mo6+ or Zn2+. AP-I has both phosphatase and pyrophosphatase activities, and is highly stable even at 50.

Biochemical Development

Changes

Associated

With

Brassica

juncea

Seed

Changes in acid and alkaline phosphatase activities in cytoplasmic and wall-bound fractions of developing mustard (Brassica juncea) seed were studied. Growth was measured by seed dry weight and water content. Seed dry weight data were fitted to a cubic polynomial equation. Seed water content and dry matter accumulation was significantly correlated. Cytoplasmic acid and alkaline phosphatase activities were substantially less in the cytoplasmic fraction than the wall-bound fraction. Wall-bound acid phosphatase activity was low initially, but high levels were maintained after day 25, indicating a relationship with dry matter accumulation. The results suggest that acid phosphatase plays an important role during mustard seed development.

Partial Purification and Characterization of an Acid Phosphatase from Papaya

A phosphatase in papaya was extracted, partially purified, and characterized. With pnitrophenyl phosphate as substrate, the enzyme had a pH optimum of 6.0, which categorized it as an acid phosphatase, a temperature optimum of 37C, and a Km of 1.0 mM. Heat inactivation of papaya acid phosphatase was biphasic, and the kinetics of both phases were first order reactions. D values at 60, 65, 70C for the heat resistant phase were 21.0, 11.7, and 4.0 min, respectively. For the heat labile and heat resistant isozymes of papaya acid phosphatase, the activation energies, Ea, for thermal inactivation were 60.0 Kcal/mole and 37.8 Kcal/mole, respectively. The apparent molecular weight of the enzyme as determined by gel filtration was 120,000 daltons.

Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.
Two secreted acid phosphatases (SAP1 and SAP2) were markedly up-regulated during Pi-starvation of tomato suspension cells. SAP1 and SAP2 were resolved during cation-

exchange FPLC of culture media proteins from 8-day-old Pi-starved cells, and purified to homogeneity and final p-nitrophenylphosphate hydrolyzing specific activities of 246 and 940 micro mol Pi produced.min-1 mg.protein-1, respectively. SDS/PAGE, periodic acidSchiff staining and analytical gel filtration demonstrated that SAP1 and SAP2, respectively, exist as 84 and 57 kDa glycosylated monomers. SAP1 and SAP2 are purple acid phosphatases (PAPs) as they displayed an absorption maximum at 518 and 538 nm, respectively, and were not inhibited by l-tartrate. The respective sequence of a SAP1 and SAP2 tryptic peptide was very similar to a portion of the deduced sequence of several putative Arabidopsis thaliana PAPs. CNBr peptide mapping indicated that SAP1 and SAP2 are structurally distinct. Both isozymes displayed a pH optimum of approximately pH 5.3 and were heat stable. Although they exhibited wide substrate specificities, the Vmax of SAP2 with various phosphate-esters was significantly greater than that of SAP1. SAP1 and SAP2 were activated by up to 80% by 5 mm Mg2+, and demonstrated potent competitive inhibition by molybdate, but mixed and competitive inhibition by Pi, respectively. Interestingly, both SAPs exhibited significant peroxidase activity, which was optimal at approximately pH 8.4 and insensitive to Mg2+ or molybdate. This suggests that SAP1 and SAP2 may be multifunctional proteins that operate: (a) PAPs that scavenge Pi from extracellular phosphate-esters during Pi deprivation, or (b) alkaline peroxidases that participate in the production of extracellular reactive oxygen species during the oxidative burst associated with the defense response of plants to pathogen infection.

Purification and characterization of secreted acid phosphatase in phosphorus-deficient Arabidopsis thaliana

Arabidopsis roots responded to the absence of an exogenous phosphate source with an increase in the specific activities of secreted acid phosphatases. Increases in enzyme activity were revealed beginning 2 days after P-withdrawal, reaching a maximum at 6 days. We characterized the secreted acid phosphatase. Two proteins, migrating at 52 and 63 kDa in SDS-PAGE, co-purified with the activity. Purified enzyme had a pH optimum of 5 and a pI of 5.9. In addition to the general phosphatase substrate, p-nitrophenylphosphate, the enzyme readily hydrolysed pyrophosphate, polyphosphate, ATP and PEP.

Low or negligible activity was observed with glucose-1P, fructose-1P and phytic acid. The activity of the purified secreted acid phosphatase was stimulated by calcium and inhibited by molybdate, phosphate, fluoride, vanadate and nitrate. Activity was not inhibited by tartrate. The substrate profile and the biochemical properties suggest that Arabidopsis secreted acid phosphatase may have a role in mobilizing organic phosphate in the soil. ISOLATION, CHARACTERIZATION AND POTENTIAL APPLICATION OF DEOXYRIBONUCLEASE-FREE PHOSPHATASE FROM CASSAVA LEAVES A deoxyribonuclease-free acid phosphatase from cassava leaves was prepared by ammonium sulfate precipitation, chromatofocusing and hydrophobic interaction chromatography with phenyl Sepharose. The enzyme was purified 36-fold and had a specific activity of 16 U/ mg protein. The enzyme preparation revealed a major phosphatase band of 77 kDa and three minor activity bands. The pH and temperature optima for enzyme activity were 5.2 and 60C, respectively. The enzyme was inactivated by heating at 80C for 15 minutes and could be stored at -20C for up to two months. The enzyme exhibited broad substrate specificity and had a Km (p-nitrophenyl phosphate) value of 1.7 mM. It was strongly inhibited by zinc and copper ions, molybdate and arsenate. Aluminium ions, ferrous ions, dithiothreitol and mercaptoethanol stimulated enzyme activity within 0.5-5.0 mM range.

Acid phosphatase activity and phosphorus efficiency in tomato plants (Lycopersicon esculentum Mill. Var. "Ro Grande") grown in nutrient solutions.
Growth, phosphorus efficiency, acid phosphatase activity and their kinetic properties KM and Vmax, were evaluated in root exudates collected in vivo from tomato plants previously grown under greenhouse conditions in solutions P-deficient (-P: 0.005 mM) or sufficient (+P: 1.0 mM). Plants were grown from seed and 19-day-old individual plants transplanted into 900-ml pots containing the nutrient solutions. Three plants per Ptreatments were sampled at 45 days of age for growth and P-efficiency determinations,

enzyme activity and kinetic constants in root exudates collected in dialysis tubes containing 100-mM NaCl in which the roots of intact plants were immersed. Additionally, changes in rhizosphere pH were determined in the nutrient solutions of -P and +P plants. Results showed that under P-deficiency, biomass accumulation and total leaf area were decreased up to 76.2 and 86.5%, P use efficiency was increased and P absorption efficiency decreased. The pH of the nutrient solution in plants grown in -P decreased to near 4.0 and increased to near 7.5 in +P. Differences were observed in the Na-soluble-acid phosphatase activity in root exudates of -P and +P plants and in their KM and Vmax with values 0.55 mM p-NPP and 1477.05 moles PNP h -1 g-1 Dw roots and 0.62 mM p-NPP and 279.5 moles PNP h-1 g-1 Dw roots, respectively. The results corroborate that tomato plants are adequate probes to study plant mechanisms under Pdeficient conditions and that kinetic constants can be used as appropriate physiological markers of P-tolerance in greenhouse experiments for selection genotypes under P deficient conditions STUDIES ON ACID PHOSPHATES IN RICE TISSUES Acid phosphates in dilicrcnt Ussuea of rice {Oryz'i sui iva) was studied with Polyacrylamide, gel electrophoresis and enzyme assay. The zymogram of acid phosphates showed different isozyme patterns in roots, leaves, seedlings and calli. One of the medium mobility isozymes, eel 1-w all-eon la in in g enzyme, commonly occurred in all tissues. Some iso-zymes had tissue-specificity. The optimal pH of acid phosphates was around 5. Enzyme acvity was strongly inhibited by HgCl;, (NHi)iMoyOn, NaF, CuS4 and KHsPOt- in germinating seeds, the increasing activity of acid phosphates related with Iran s por tat ton of phosphates and store food had been suggested.

MATERIALS & METHODS: Enzyme from plant source: Germinated green gram Germinated mustard 0.1M phosphate buffer pH 5.0

PROCEDURE: Enzyme extraction from plant source: 1. Weigh 50 gms of germinated seedlings of green gram and add approximately 25ml of 0.1M phosphate buffer to it and grind it properly and filter it through cheese cloth. 2. Then centrifuge the filtrate at 6000 rpm for 10 min and collect the supernatant. Make up to 50 ml with 0.1M phosphate buffer. 3. The above obtained solution is nothing but crude enzyme. Standard protein concentration must be estimated by Bradfords method: MATERIALS:

Bradfords Reagent Dissolve 5mg of Coommassie brilliant Blue G-250 in 2.5ml of 95% Ethanol. To this add 5ml of OrthoPhosphoric Acid. Make it upto 50ml with Distilled water.

Preparation of Standard Protein (BSA) Dissolve 5mg of BSA in 50ml of Distilled water in standard flask. This can be taken as standard to calculate the concentration of Protein. Procedure: Take varying amounts of protein in different test tubes. To each of the tubes add 1ml of Bradfords Reagent Vortex well and take the readings at 600nm.

Determination

of

protein

concentration

with

the

help

of

UV

Visible

spectrophotometer: Protein content was determined by Spectrophotometry measurement using this formula (1.45 X Abs. 280 nm) - (0.74 X Abs. 260 nm) Preparation of DNS reagent: Composition: 44 mM DNS 4 mM sodium sulfite 375 mM sodium hydroxide Weigh all the above chemicals according to the given concentrations and add to the distilled water (ml). = ______ mg/ml.

ENZYME ACTIVITY Pipette out 0.5ml of 1% substrate solution (10mg/ml of suitable buffer) in a test tube and add 0.5ml of enzyme to it and incubate this at 55c for 15 minutes. Then add 0.5ml of DNS reagent to the solution and keep this in boiling water bath for 5 minutes. Then make this solution to 3ml with deionized water. Then read the optical density (O.D) of the solution at 540 nm

Enzyme activity may be defined, the amount of the glucose produced per ml in the reaction mixture per unit time Specific activity determination: Specific activity can be determined by Enzyme activity / concentration = specific activity (U / mg).

AMMONIUM SULPHATE FRACTIONATION: Many cytosolic proteins are water soluble and their solubility is a function of the ionic strength and pH of the solution. The commonly used salt for this purpose is Ammonium Sulphate, due to its high solubility even at lower temperatures. Proteins in aqueous solutions are heavily hydrated, and with the addition of salt, the water molecules become more attracted to the salt than to the protein due to the higher charge. This competition for hydration is usually more favorable towards the salt, which leads to interaction between the proteins, resulting in aggregation and finally precipitation. The precipitate can then be collected by centrifugation and the protein pellet is re-dissolved in a low salt buffer. Since different proteins have distinct characteristics, it is often the case that they precipitate (or salt out) at a particular concentration of salt. Requirements: Ammonium sulphate Magnetic bead and stirrer Swing-out rotor centrifuge

PROCEDURE: Clarify the protein solution by centrifugation. Transfer the supernatant into an ice cold beaker with a magnetic bead. Transfer the beaker onto a magnetic stirrer. Weigh the amount of ammonium sulfate to be added. The amount depends on the volume of the solution and the percentage saturation of the salt needed. Slowly add the ammonium sulphate with stirring. One needs to be careful as the addition of the salt should be very slow. Add a small amount at a time and then allow it to dissolve before further addition. Keep it on the stirrer for 1hr and keep the beaker at 4C for 1hr or whole night according to the protein to be isolated. Centrifuge at 10,000g for 15min at 4C. The pellet contains the precipitated protein which could be dissolved in a suitable buffer for further analysis and purification. For a second round of precipitation of a different protein, the supernatant is again used and the above same steps are follow. DIALYSIS: Dialysis bag

Procedure: Cut desired length of dialysis tubing; take note of the MW cut-off of the tubing. Boil tubing for 2 in dH2O to sterilize. Clamp up one end, open the other end, add some distilled water into tubing, and check for leaks. Remove distilled water and add the protein soln. Leave some space at top and clamp up. Mix gently and let spin by stir bar slowly for over night in the orbital shaker at 100rpm. Transfer tubing to an eppendorf an spin it at 8000rpm for 10min. Discard the pellet and collect the supernatant.

COLUMN CHROMATOGRAPHY: Suspend the gel (for instance, Sephadex G 100) in a large volume of water or preferably in elution buffer until the gel is fully swollen. The swelling can be

done by over night suspension or by heating in a water bath for 2-4hr ( follow the manufacturer s instructions for this purpose, carefully) Plug the bottom of column tube with glass wool or sintered filter and stand upright the column. Make a good slurry of the gel (stationary phase) in a suitable buffer after proper swelling of the gel. Pour a small volume of buffer into the column to avoid trapping of any air bubbles in the plug immediately followed by the slurry to the full of column. The top protion may be carefully, gently stirred prior to pouring additional slurry to the growing column, if necessary. Wait until the gel settle down to the desired heigh by gravitational force. Place a suitable filter circle on top of the gel bed. Equilibrate the column thoroughly by passing through the column buffer. Apply the sample in column buffer onto the top of bed. The sample volume should preferably limited to 1-3% of the total bed volume. The sample can be applied to the top by careful pipetting or conveniently through the buffer pipeline. Now connect the bufferline to the elution buffer to develop the chromatogram. Protein molecules pass through the gel space while small molecules distribute between the solvent inside and outside the gel and then pass through the column at a slower rate. The effluent emerging out of the column can be routed through a suitable spectrophotometer to monitor the absorbance at a particular wavelength (for protein 280, 230 or 210 nm) and the data recorded. The effluent is then collected using a fraction collector. The effluent is manually collected in the absence of a collector in a fixed time or volume intervals in tubes and measured subsequently. The volume of mobile phase required to elute a particular solute is known as the elution volume while the corresponding time for elution of the solute at a given flow rate is known as the retention time. The elution is continued (usually 2-3 times bed volume of buffer) until the absorbance monitored reached baseline value.

Thereafter the column is extensively reequilibrated with the column buffer for subsequent run. The reference proteins are loaded onto the bed and the chromatography is carried out as above. Plot the logarithms of molecular weight of marker proteins against their respective ratios of elution volume to column void volume (Ve /Vo) the column volume being the elution volume of a very molecule such as blue dextran.

Compute the elution volume of protein of interest and deduce its molecular weight from the above linear graph.

SDS-PAGE Materials: Separating Gel composition (10%) Acrylamide (40%)-2.5ml Distilled water-3.7ml 1.5M Tris (pH-8.8)-3.75ml

20% Sodium Dodecyl sulfate-50ul 10%Ammonium persulfate-100ul TEMED-6ul Stacking gel composition: Acrylamide (40%)-281ul Distilled water-2.39ml 1M Tris (pH-6.8)-0.375ml 20%Sodium Dodecyl sulfate-15ul 10%Ammonium per sulfate -30ul TEMED-5ul 10X Electrophoresis Buffer: Tris-30gms Sodium dodecyl sulfate-10gms Glycine-145gms All the above components were dissolved in 1 liter of Distilled water. 6X gel loading Dye: 1M Tris-HCl (pH-6.8)-3ml 2-Mercaptoethanol-600ul Glycerol-6ml 0.5MEDTA- 240 ul Sodium dodecyl sulfate-1.2gms Bromophenol Blue-Pinch Staining solution: Commasie Brilliant Blue (G-250)-0.1gms Methanol: 40ml Acetic acid: 10ml Distilled Water: 50ml Destaining solution: Methanol: 40ml Acetic acid: 10ml Distilled Water: 50ml

Procedure: Assemble two glass plates (one notched) with two side spacers, clamps, grease, etc. Stand assembly upright using clamps as supports, on glass plate. Pour some pre-heated 1% agarose onto glass plate, place assembly in pool of agarose: this seals the bottom of the assembly. Mix ingredients of Separating gel gently in the order shown above, ensuring no air bubbles form. Pour into glass plate assembly carefully. Overlay gel with isopropanol to ensure a flat surface and to exclude air. Wash off isopropanol with water after gel has set. Mix the ingredients of stacking gel , then pour onto top of set resolving gel, insert comb, allow to set, Remove comb, fill with electrophoresis buffer. Assemble top tank onto glass plate assembly. Fill with1X electrophoresis buffer. Sample preparation: Take 50ul of protein sample and add 25ul of sample buffer. Boil the samples for 3 mins at 100C. Load the samples into the wells created by the comb. Allow the samples to electrophorese at 100Volts. Staining and Destaining: After Electrophoresis remove the gel by separating the glass plates and transfer the gel to Staining solution. Let it stand for 2-3hrs After this transfer the stained gel to destaining solution to remove the excess of stain. Observe the gel for the formation of sharp thick bands.

OPTIMIZATION OF PHOSPHATES PRODUCTION: EFFECT OF CARBON SOURCE ON ENZYME ACTIVITY Pipette out 0.5ml of 1% carbon source (glucose; sucrose; starch; CMC) dissolved in respective buffer solution in a test tube and add 0.5ml of enzyme to it and incubate this at 55c for 15 minutes. Then add 0.5ml of DNS reagent (terminating agent) to the solution and keep this in boiling water bath for 5 minutes. Then make this solution to 3ml with deionized water. Then read the optical density (O.D) of the solution at 540 nm

EFFECT OF NITROGEN SOURCE ON ENZYME ACTIVITY Pipette out 0.5ml of 1% nitrogen source (yeast extract; tryptone/peptone; ammonium sulphate) dissolved in respective buffer solution in a test tube and add 0.5ml of enzyme to it and incubate this at 55c for 15 minutes. Then add 0.5ml of DNS reagent (terminating agent) to the solution and keep this in boiling water bath for 5 minutes. Then make this solution to 3ml with deionized water. Then read the optical density (O.D) of the solution at 540 nm

EFFECT OF METAL IONS ON THE ENZYME ACTIVITY Pipette out 0.5ml of 3mM; 5mM concentrations of metal ions (ZnCl2; MgCl2; COCl2; KCl; NH4Cl; MnSO4; EDTA; NaN3) dissolved in respective buffer solution in a test tube and add 0.5ml of enzyme to it and incubate this at 55c for 15 minutes. Then add 0.5ml of DNS reagent (terminating agent) to the solution and keep this in boiling water bath for 5 minutes.

Then make this solution to 3ml with deionized water. Then read the optical density (O.D) of the solution at 540 nm

EFFECT OF SURFACTANTS ON ENZYME ACTIVITY Pipette out 0.5ml of 1% surfactants (SDS; Triton-X100;Tween-20; Tween-80) dissolved in respective buffer solution in a test tube and add 0.5ml of enzyme to it and incubate this at 55c for 15 minutes. Then add 0.5ml of DNS reagent to the solution and keep this in boiling water bath for 5 minutes. Then make this solution to 3ml with deionized water. Then read the optical density (O.D) of the solution at 540 nm

EFFECT OF MINERAL OILS ON ENZYME ACTIVITY Pipette out 0.5ml of 1% mineral oils (vegetable oil; castor oil; groundnut oil; olive oil) dissolved in respective buffer solution in a test tube and add 0.5ml of enzyme to it and incubate this at 55c for 15 minutes. Then add 0.5ml of DNS reagent (terminating agent) to the solution and keep this in boiling water bath for 5 minutes. Then make this solution to 3ml with deionized water. Then read the optical density (O.D) of the solution at 540 nm

EFFECT OF AMINO ACIDS ON ENZYME ACTIVITY Pipette out 0.5ml of 1% amino acid (arginine; histidine; cysteine; tryptophan ) dissolved in respective buffer solution in a test tube and add 0.5ml of enzyme to it and incubate this at 55c for 15 minutes. Then add 0.5ml of DNS reagent (terminating agent) to the solution and keep this in boiling water bath for 5 minutes.

Then make this solution to 3ml with deionized water. Then read the optical density (O.D) of the solution at 540 nm

EFFECT OF TEMPERATURE ON ENZYME ACTIVITY Pipette out 0.5ml of 1% substrate solution in a test tube and add 0.5ml of enzyme to it and incubate this at different temperatures (350C; 450C; 550C; 650C; 750C) for 10 minutes. Then add 0.5ml of DNS reagent (terminating agent) to the solution and keep this in boiling water bath for 5 minutes. Then make this solution to 3ml with deionized water. Then read the optical density (O.D) of the solution at 540 nm

EFFECT OF pH ON ENZYME ACTIVITY Pipette out 0.5ml of 1% substrate solution in a test tube and add 0.5ml of enzyme to it and add different pH solutions (pH 3; pH 5; pH 7; pH 9) incubate this at 55c for 15 minutes. Then add 0.5ml of DNS reagent (terminating agent) to the solution and keep this in boiling water bath for 5 minutes. Then make this solution to 3ml with deionized water. Then read the optical density (O.D) of the solution at 540 nm

RESULTS: Plant enzymes are most important bio products and are being utilized for large number of processes in the areas of industrial, environmental and food biotechnology. Moreover current developments in biotechnology are yielding new applications for enzymes. Plants are usually the most convenient source as they can be isolated and cultivated from various natural sources. method. Bradfords method:Extra cellular protein content was calculated by Bradfords estimation. It is useful when the amount of the unknown protein is limited. The protein concentrations were measured by Bradfords

S.No

Protein (ml))

Conc (ug)

Distilled water (ml)

Bradford reagent (ml)

Optical densityat 595nm 0.28 0.33 0.38 0.41 0.42 0.45 0.48 0.5 0.54 0.5

1 2 3 4 5 6 7 8 9 10

100 200 300 400 500 600 700 800 900 1000

10 20 30 40 50 60 70 80 90 100

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 _

1 1 1 1 1 1 1 1 1 1

11

Unknown (green gram) 200 Unknown (mustard) 200

0.8

0.34

12

0.8

0.37

Determination of protein concentration; Enzyme activity and specific activity for phosphates enzyme: s.no PHOSPHATES ENZYME Crude Vigna radiata 1. Concentration 1.2 mg/ml Brassica juncea 2.6 mg/ml Dialysis Vigna radiata 0.32 mg/ml Brassica juncea 0.138 mg/ml

2. 3.

Activity Specific activity

0.24 U/ml 0.2 U/mg

1.31 U/ml 0.5 U/mg

0.20 U/ml 1.6 U/mg

0.25 U/ml 1.8 U/mg

The Crude enzyme was first precipitated with 40% in case of bacteria and 35-60% in case of fungus ammonium sulfate saturation and column chromatography was conducted to purify the enzyme. Purification step yielded pure lipase enzyme and Purity was checked by 10 % SDS-PAGE Electrophoresis technique. The phosphates enzyme obtained by both plant and animal were separated on 10% SDSPAGE

Optimization:
Effect of carbon sources on enzyme activity Among different carbon sources used NaH2PO4 showed its maximum phosphates activity in plant source and in animal glucose has showed its maximum activity Sl.no 1. 2. Carbon sources Glucose sucrose Enzyme activity IU/ml Vigna radiata Brassica juncea

3. 4.

Na2HPO4 NaH2PO4

Effect of nitrogen sources on enzyme activity

Among different nitrogen sources used activity Sl.no 1. 2. 3. Nitrogen sources Ammonium sulphate Tryptone Yeast extract

showed its maximum phosphates

Enzyme activity IU/ml Vigna radiata Brassica juncea

Effect of Metal ions (3mM, 5mM) on enzyme activity Among different metal ions used in Vigna radiata NaCl and in Brassica juncea CaCl2 has showed their maximum phosphates activity Sl.no 1. 2. 3. 4. Metal ions (1mM) Zncl2 MgCl2 NaCl CaCl2 Enzyme activity IU/ml Vigna radiata Brassica juncea 1.0 0.95 1.0 1.2 1.4 0.10 1.0 1.48

Among different metal ions used Sl.no 1. 2. Metal ions (3mM)

showed its maximum phosphates activity Enzyme activity IU/ml Brassica juncea 0.80 0.1

Vigna radiata Zncl2 MgCl2 1.0 1.2

3. 4.

NaCl CaCl2

1.5 1.0

0.09 1.0

Effect of surfactants on enzyme activity Among different surfactants used Sl.no 1. 2. 3. 4. surfactants SDS Triton-X100 Tween-20 Tween-80 showed its maximum phosphates activity Enzyme activity IU/ml Vigna radiata Brassica juncea

Effect of mineral oils on enzyme activity Among different mineral oils used Sl.no 1. 2. 3. 4. mineral oils Olive oil Castor oil Groundnut oil Ginger oil showed its maximum phosphates activity

Enzyme activity IU/ml Vigna radiata

Brassica juncea

Effect of amino acids on enzyme activity Among different amino acids used in phosphates activity and showed its maximum

Sl.no 1. 2. 3. 4.

Amino acids Histidine Cysteine Arginine Tryptophan

Enzyme activity IU/ml Vigna radiata Brassica juncea

Effect of temperature on enzyme activity (line graph) Among different temperatures used Sl.no 1. 2. 3. 4. 5. 6. Temperature (c) 35c 45c 55c 65c 75c 85c showed its maximum phosphates activity Enzyme activity IU/ml Vigna radiata Brassica juncea

Effect of PH on enzyme activity (line graph) Among different pH solutions used activity Sl.no
P

showed its maximum phosphates

Enzyme activity IU/ml Vigna radiata Brassica juncea 3 5 7 9

1. 2. 3. 4.

Protein elution profile for phosphates by column chromatography (line graph)

Sl.No

Fractions

OD. at 280 nm Vigna radiata 0.229 0.249 0.258 0.300 0.491 0.554 0.841 0.658 0.350 0.154 Brassica juncea 0.213 0.259 0.356 0.429 0.550 0.569 0.542 0.319 0.223 0.109

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6 Fraction 7 Fraction 8 Fraction 9 Fraction 10

SUMMARY & CONCLUSION: Acid phosphatase is a phosphatase, a type of enzyme, used to free attached phosphate groups from other molecules during digestion. It is basically a phosphomonoesterase. It is stored in lysosomes and functions when these fuse with endosomes, which are acidified while they function; therefore, it has an acid pH optimum

To purify the enzyme extracted from plant as well as animal, plant crude enzyme was first precipitated with 40% ammonium sulfate saturation and in the case of animal crude enzyme there is a range i.e., 35-60%. Proteins can be separated on the basis of size/molecular weight. The purified phosphates enzymes (both plant and animal) are separated on 10% SDS-PAGE. The purified enzyme thus obtained was stored at 4C.

Optimization of phosphates production was carried out with various carbon, nitrogen sources, metal ions, surfactants, mineral oils, organic acids and amino acids. Of all the carbon sources tested in this study, the case of nitrogen sources different metals activity mineral oils, organic acids amino acids, showed maximum activity. and the enzymes are active produces maximum enzyme

production in terms of its activity and other sources are less in enzyme production and in produces maximum enzyme production. Among showed maximum showed maximum activity, showed maximum activity and yields maximum enzyme activity and other metal ions decreases

activity. Like wise among different surfactants, in case of

Effect of temperature and pH on enzyme indicates in pH7 and in pH5.

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