DIFFERENTIAL ACTIVITIES OF ACID PHOSPHATASE FROM ADAXIAL AND ABAXIAL REGIONS OF LUPINUS LUTEUS (FABACEAE) COTYLEDONS

Acid phosphatases of abaxial and adaxial regions in the cotyledons of the Lupinus luteus which possess structurally distinct protein bodies were examined. Acid phosphatase activity was investigated by enzyme assays and by gel electrophoresis and was localized by cytochemical methods in the cotyledons of Lupinus luteus L. during germination and seedling development. Acid phosphatase activity was significantly higher in the adaxial (heterogeneous protein body) region as compared to the abaxial (homogeneous protein body) region of the cotyledon. The pH optimum of acid phosphatase from the abaxial region and from the adaxial region was 4.5 and 5.0, respectively. There were significant differences in substrate specificity and isoenzymic composition of the enzyme between the two regions. Isoenzymic composition changed during the course of germination and seedling development. Acid phosphatase was localized in the matrix of the homogeneous protein bodies and in the globoids of the heterogeneous protein bodies at imbibition. After germination (d 3, d 4, d 7) acid phosphatase was localized primarily in the inner cell walls and intercellular spaces of both regions. These results show that different isoenzymes of acid phosphatase show differential localization and the rate of acid phosphatase activation or synthesis differs in cells from the two regions of the cotyledon.     

Acid phosphatase activities during the germination of Glycine max seeds.

In this paper, we describe a study concerning the determination of some characteristics of soybean seedlings and the detection of acid phosphatase activities towards different substrates during the germination. Enzyme activities with p-nitrophenylphosphate (pNPP) and inorganic pyrophosphate (PPi) as substrates were detected from the 5th and 7th days after germination, respectively. Acid phosphatase activities with tyrosine phosphate (TyrP), glucose-6-phosphate (G6P) and phosphoenol pyruvate (PEP) were also observed but to a lesser extent. Under the same conditions, no enzyme activity was detected with phytic acid (PhyAc) as substrate. The appearance of phosphatase activity was coincident with the decrease of inorganic phosphate content during germination; over the same period, the protein content increased up to the 5th day, decreased until the 8th day, and remained constant after this period. Relative to phosphatase activity in the cotyledons, the activities detected in the hypocotyl and roots were 82% and 38%, respectively. During storage the enzyme maintained about 63% of its activity for 3 months at 5 degrees C. The specificity constant (Vmax/Km) values for pNPP and PPi were 212 and 64 mu kat mM-1 mg-1, respectively. Amongst the substrates tested, PPi could be a potential physiological substrate for acid phosphatase during the germination of soybean seeds.     

Induction of acid phosphatase activity during germination of maize (Zea mays) seeds.

Acid phosphatase activity (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) increased during the first 24 h of maize (Zea mays) seed germination. The enzyme displayed a pH optimum of 4.5-5.5. Catalytic activity in vitro displayed a linear time course (60 min) and reached its half maximum value at 0.47 mM p-nitrophenyl phosphate (pNPP). Phosphatase activity towards phosphoamino acids was greatest for phosphotyrosine. The phosphatase activity was strongly inhibited by ammonium molybdate, vanadate and NaF and did not require divalent cations for the catalysis. The temperature optimum for pNPP hydrolysis was 37 degrees C. Under the same conditions, no enzyme activity was detected with phytic acid as substrate. Western blotting of total homogenates during seed germination revealed proteins/polypeptides that were phosphorylated on tyrosine residues; a protein of approximately 14 kDa is potentially a major biological substrate for the phosphatase activity. The results presented in this study suggest that the acid phosphatase characterized under the tested conditions is a member of the phosphotyrosine phosphatase family.     

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 (32)P from (32)P-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.     

Changes and induction of aminopeptidase activities in response to pathogen infection during germination of pigeonpea (Cajanas cajan) seeds

Aminopeptidases play important role in the mobilization of storage proteins at the cotyledon during seed germination. It is often referred as inducible component of defense against herbivore attack. However the role of aminopeptidase in response to pathogen attack in germinating seeds is remained to be unknown. An attempt was made to analyze change in the aminopeptidase (EC 3.4.11.1) activity during germination of pigeonpea (Cajanus cajan L.) seeds by infecting the seeds with fungi. Two aminopeptidase activity bands (AP1 and AP2) were detected in control as well as infected pigeonpea seeds. During latter stages of germination in control seeds, AP1 activity was replaced by AP2 activity. However AP1 activity was significantly induced in germinating seeds infected with Fusarium oxysporum f.sp. ciceri and Aspergillus niger var. niger. The estimated molecular weights of AP1 and AP2 were ∼97 and 42.8 kDa respectively. The induced enzyme was purified up to 30 fold by gel filtration chromatography. The purified enzyme was preferentially cleaved leucine p-nitroanilide than alanine p-nitroanilide. The enzyme was strongly inhibited by bestatin and 1,10-phenanthroline. Almost 50% of enzyme activity was inhibited by ethylene diamine tetra acetate. The purified enzyme showed broad pH optima ranging from pH 6.0 to 9.0 and optimum at pH 8.5. The induction of aminopeptidase activity during pigeonpea seed germination and in response to pathogen attack indicates significant involvement of these enzymes in primary as well as secondary metabolism of the seeds. These findings could be helpful to further dissect defensive role of aminopeptidases in seed germination which is an important event in plant's life.     

Phytase activity in tobacco (Nicotiana tabacum) root exudates is exhibited by a purple acid phosphatase

Phytases are enzymes that catalyze liberation of inorganic phosphates from phytate, the major organic phosphorus in soil. Tobacco (Nicotiana tabacum) responds to phosphorus starvation with an increase in extracellular phytase activity. By a three-step purification scheme, a phosphatase with phytase activity was purified 486-fold from tobacco root exudates to a specific activity of 6,028 nkat mg(-1) and an overall yield of 3%. SDS-PAGE revealed a single polypeptide of 64 kDa, thus indicating apparent homogeneity of the final enzyme preparation. Gel filtration chromatography suggested that the enzyme was a ca. 56 kDa monomeric protein. De novo sequencing by tandem mass spectrometry resulted in a tryptic peptide sequence that shares high homology with several plant purple acid phosphatases. The identity of the enzyme was further confirmed by molybdate-inhibition assay and cDNA cloning. The purified enzyme exhibited pH and temperature optima at 5.0-5.5 and 45 degrees C, respectively, and were found to have high affinities for both p-nitrophenyl phosphate (pNPP; K(m)=13.9 microM) and phytate (K(m)=14.7 microM), but a higher kcat for pNPP (2,056 s(-1)) than phytate (908 s(-1)). Although a broad specificity of the enzyme was observed for a range of physiological substrates in soil, maximum activity was achieved using mononucleotides as substrates. We conclude that the phytase activity in tobacco root exudates is exhibited by a purple acid phosphatase and its catalytic properties are pertinent to its role in mobilizing organic P in soil.     

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 -nitrophenyl-phosphate, 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.     

The localization of enzymes in the cotyledons of Pisum arvense L. during germination

Summary Enzyme activity is not uniformly distributed through the cotyledon of Pisum arvense. Initially the peripheral region, certain scattered cells of the storage tissue and the procambium show a high level of activity of succinic dehydrogenase, cytochrome oxidase, acid phosphatase and esterase. Activity of acid phosphatase declines sharply after the first day of germination; activity of the other enzymes declines after about three days. In the storage tissue, where activity is lower initially, it declines after about five days and is correlated with the disappearance of the reserves. The pattern of alkaline phosphatase activity is similar except that activity is lower in the procambium but increases in the sieve-elements during differentiation of the phloem. 5-nucleotidase and glucose-6-phosphatase activity is low throughout the cotyledon but it also increases to a significant level in the sieve-elements. Activity of starch synthesizing enzymes is high in the parenchymatous bundle sheath, where they may be involved in the pathway from lipids to soluble carbohydrates.     

Purification, properties and alternate substrate specificities of arginase from two different sources: Vigna catjang cotyledon and buffalo liver

Arginase was purified from Vigna catjang cotyledons and buffalo liver by chromatographic separations using Bio-Gel P-150, DEAE-cellulose and arginine AH Sepharose 4B affinity columns. The native molecular weight of an enzyme estimated on Bio-Gel P-300 column for Vigna catjang was 210 kDa and 120 kDa of buffalo liver, while SDS-PAGE showed a single band of molecular weight 52 kDa for cotyledon and 43 kDa for buffalo liver arginase. The kinetic properties determined for the purified cotyledon and liver arginase showed an optimum pH of 10.0 and pH 9.2 respectively. Optimal cofactor Mn++ ion concentration was found to be 0.6 mM for cotyledon and 2 mM for liver arginase. The Michaelis-Menten constant for cotyledon arginase and hepatic arginase were found to be 42 mM and 2 mM respectively. The activity of guanidino compounds as alternate substrates for Vigna catjang cotyledon and buffalo liver arginase is critically dependent on the length of the amino acid side chain and the number of carbon atoms. In addition to L-arginine cotyledon arginase showed substrate specificity towards agmatine and L-canavanine, whereas the liver arginase showed substrate specificity towards only L-canavanine.     

Carbon metabolism in germinating Ricinus communis cotyledons. Purification, characterization and developmental profile of NADP-dependent malic enzyme

The possible implication of NADP-dependent malic enzyme (NADP-ME; L-malate:NADP oxidoreductase [oxaloacetate-decarboxylating], EC 1.1.1.40) in fatty acid synthesis was examined in Ricinus communis L. cotyledons, NADP-ME catalyses the conversion of L-malate to pyruvate and NADPH, potential substrates for fatty acid synthesis. NADP-ME activity and protein levels were monitored during germination, up to 20 days postimbibition. The developmental profile showed a peak in activity (6 times with respect to the basal value) and immunoreactive protein (a single 72-kDa band using anti-maize NADP-ME antibodies) around day 7. The enzyme was partially purified (41-fold) and its kinetics characterized. The optimum pH was around 7.1. K_m values for L-malate and NADP⁺ were 0.68 m M and 8.2 μ M respectively. The enzyme used Mg²⁺ or Mn²⁺ as essential cofactors. Several metabolites were assayed as potential enzyme modulators. Succinate, CoA, acetyl-CoA and palmitoyl-CoA were activators of NADP-ME, at saturating or sub-saturating substrate concentrations, K² values for CoA and derivative compounds were in the micromolar range (i.e., 0.8 μ M for acetyl-CoA). No significant effects were obtained with other Krebs cycle intermediates and amino acids (i.e. 2-oxoglutarate, glutamate, glutamine, fumarate). The activity was 29 times higher in the forward (decarboxylating) direction compared to the reverse direction. These results hint at cotyledon NADP-ME behaving as a regulatory enzyme in R. communis . Its activity is responsive to metabolites of the fatty acid synthesis pathway, and thus a role in this metabolism is suggested.     

The development of ribonuclease and acid phosphatase during germination of Pisum arvense

1. Development of ribonuclease activity in the cotyledons of germinating peas is biphasic, the time of appearance of the two phases depending on the conditions of growth. 2. Acid phosphatase exhibits a single phase of development. 3. Cycloheximide inhibits development of ribonuclease activity in phase II but not in phase I. 4. (14)C-labelled amino acids are not incorporated into ribonuclease isolated during phase I. 5. The buoyant density of ribonuclease isolated during phase I is not affected by imbibition of the seed in 80% deuterium oxide. 6. Acid phosphatase was isolated from the supernatant fraction of the cotyledons of germinating peas and partially purified. 7. Development of acid phosphatase activity during germination is inhibited by treatment of the seed with cycloheximide or actinomycin D. 8. Partial purification of acid phosphatase from peas germinated in the presence of (14)C-labelled amino acids suggests that the enzyme is radioactively labelled. 9. Germination of peas in the presence of 80% deuterium oxide results in an increase in the buoyant density of acid phosphatase. 10. The results suggest that increase in ribonuclease activity during the first 4 days of germination does not result from synthesis of protein de novo, but that the corresponding increase in acid phosphatase activity does result from synthesis de novo.     

Effect of an endogenous factor on arginase activity in germinating Bengal gram (Cicer arietinum) seeds

Arginase (EC 3.5.3.1) activity increased in the cotyledon, while it declined rapidly in the embryonic axis of Bengal gram ( Cicer arietinum L. cv. Desi type) seeds during germination. The decrease in enzyme activity in the embryonic axis was accompanied by changes in the properties of arginase in vitro such as decreased stability, increased heat lability and failure to bind to DEAE-cellulose. These alterations were due to the presence of a low-molecular-weight factor in the extract, which was purified and identified as a hexose derivative. During germination the concentration of the factor increased in the embryonic axis, while no detectable level of the factor was present in the cotyledons. We postulate that the factor may have a role in the regulation of arginase activity in vivo.     

Occurrence and characterization of a UDP-glucose:hydroxamic acid glucosyltransferase isolated from wheat (Triticum aestivum) seedlings

Cyclic hydroxamic acid glucosides are present at high concentrations immediately after germination in wheat (Triticum aestivum L.). Changes in the activity of UDP-Glucose:cyclic hydroxamic acid glucosyltransferase (EC 2.4.1.-) in wheat were investigated using the cyclic hydroxamic acids 2.4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and its 7-methoxy derivative (DIMBOA) as sugar acceptors. Glucosyltransferase activity on both substrates was detected in dry seeds, with activity increasing after imbibition, peaking in shoots and roots 36-48 hours after imbibition and decreasing thereafter. The transience of glucosyltransferase activity was concurrent with the transient occurrence of the hydroxamic acid glucosides [Nakagawa E., Amano T., Hirai N., and Iwamura H. (1995) Phytochemistry 38, 1349-1354], suggesting that glucosyltransferases regulate the accumulation of hydroxamic acid glucosides in wheat seedlings. Two peaks in activity of UDP-Glucose:DIMBOA glucosyltransferase were detected using a Mono Q column, indicating the presence of at least two isozymes of this glucosyltransferase. The enzyme in the major peak was purified about 1500-fold and shown to be in a monomeric form with a molecular mass of 47 or 49 kDa. The enzyme reacted strongly with DIMBOA, less so with DIBOA. The enzyme of the minor peak on the Mono Q chromatogram, which was also a monomeric enzyme with a molecular mass of 47 kDa, showed similar substrate specificity to that of the major peak enzyme.     

Properties of acid phosphatase from scutella of germinating maize seeds

One acid phosphatase (optimum pH at 5.4) was purified from maize scutellum after 96 hr of germination. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE) with or without sodium dodecyl sulfate (SDS). The enzyme has a MW of 65 000 ± 4000 as determined by Sephadex G-200 gel filtration and SDS-PAGE. The enzyme contained 16% neutral sugars, and cations are not required for activity. The purified enzyme was not inactivated by DTNB at pH 8. The hydrolysis of glucose-6-phosphate in the presence of 4 mM fluoride and 4 mm EDTA, at pH 6.7 (optimum pH), seems to be catalysed by this acid phosphatase.     

Inositol-1 (or 4)-monophosphatase from Glycine max embryo axes is a phosphatase with broad substrate specificity that includes phytate dephosphorylation

A phosphate-hydrolyzing activity from Glycine max embryo axes was purified by a series of chromatographic steps and electroelution from activity gels, and demonstrated to be an inositol-1 (or 4)-monophosphatase by partial internal amino acid sequence. This enzyme hydrolyzed ATP, sodium pyrophosphate (NaPPi), inositol hexakisphosphate, and inositol 1-monophosphate, but not p-nitrophenyl phosphate, ADP, AMP or glucose 6-P. Using NaPPi as substrate, the highly purified protein hydrolyzed up to 0.4 mmol phosphate min(-1) mg(-1) protein and had a Km(avg) of 235 microM for NaPPi. Since NaPPi is relatively inexpensive and readily available, we used this as substrate for the subsequent characterization. We observed the following: (a) specific inhibition by Li and NaF but not by butanedione monoxime, or orthovanadate; (b) activation by Cu(2+) and Mg(2+); (c) optimum activity at pH 7.4; and (d) temperature stability after 1-h incubations at 37-80 degrees C, with maximum activity at 37 degrees C. The partially purified protein was detected by in-gel activity assays and the band was electroeluted to yield a highly purified protein. Analysis by SDS-PAGE and native IEF-PAGE yielded a single major polypeptide of 29 kDa and pI approximately 5.9, respectively. In addition, in-gel activity from embryo axes and whole hypocotyls at early germination times revealed one high and one intermediate molecular weight isoform, but only the intermediate one corresponded to IMPase. Throughout the post-imbibition period, the activity of the high molecular weight isoform disappeared and IMPase increased, indicating an increasing expression of the enzyme as germination and growth proceeded. These data indicate that the inositol-1 (or 4)-monophosphatase present in the embryo axis of G. max has a wide phosphate substrate specificity, and may play an important role in phosphate metabolism during the germination process.     

Alkaline phosphatase of Blastocladiella emersonii: partial purification and characterization.

Alkaline phosphomonoesterase (EC 3.1.3.1) activity from Blastocladiella emersonii, while displaying typically broad substrate specificity for phosphorylated organic compounds, exhibited nearly complete substrate preference for N-acetylglucosamine-6-phosphate over N-acetylglucosamine-1-phosphate. Enzyme in zoospore extracts was purified 43-fold by differential centrifugation followed by gel filtration (Sephadex G-200) and then by ion-exchange chromatography (diethylaminoethyl-cellulose). The partially purified enzyme displayed an apparent molecular weight (Sephadex G-200) of approximately 170,000. The activity of partially purified enzyme exhibited a pH optimum of pH 8.5, did not require a metal divalent cation, but was inhibitable by ethylenediaminetetraacetic acid. During the life cycle of the organism, the specific activity of the phosphatase decreased slightly during germination and early exponential growth but then increased about 4.5-fold during sporulation. B. emersonii alkaline phosphatase does not appear to be a repressible enzyme.     

Purification and characterization of multiple S6 phosphatases from the rat parotid gland

S6 phosphatase activities, which dephosphorylate the phosphorylated S6 synthetic peptide, RRLSSLRASTSKSESSQK, were purified to near homogeneity from the membrane and cytosolic fractions of the rat parotid gland. Multiple S6 phosphatases were fractionated on Mono Q and gel filtration columns. In the cytosolic fraction, at least three forms of S6 phosphatase, termed peaks I, II, and III, were differentially resolved. The three forms had different sizes and protein compositions. The peak I enzyme, which had an approximately Mr of 68 kDa on gel filtration, appears to represent a dimeric form of the 39 kDa protein. This S6 phosphatase showed the high activity in the presence of EGTA and was completely inhibited by nanomolar concentrations of either okadaic acid or inhibitor 2. The peak II S6 phosphatase enzyme, with an Mr of 35 kDa, was activated by Mn²⁺. This form could be a proteolytic product of the catalytic subunit of type 1 phosphatase, due to its sensitivities to okadaic acid and inhibitor 2. The peak III enzyme, with an Mr of 55 kDa, is a Mn²⁺-dependent S6 phosphatase. This S6 phosphatase can be classified as a type 1 phosphatase, due to its sensitivity to okadaic acid, since the IC₅₀ of okadaic acid is 4 nM. However, the molecular mass of this S6 phosphatase differs from that of the type 1 catalytic subunit (37 kDa) and showed less sensitivity to inhibitor 2. On the other hand, the membrane fraction contained one form of the S6 phosphatases, termed peak V (Mr 34 and 28 kDa), which could be classified as a type 1 phosphatase. This S6 phosphatase activity was greatly stimulated by Mn²⁺.Abbreviations PP1-C catalytic subunit of type 1 protein phosphatase - SDS sodium dodecyl sulfate - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PMSF phenylmethylsulfonyl fluoride - Mops 4-morpholine propanesulfonic acid - EDTA ethylenediaminetetraacetate - EGTA [ethylenbis (oxyethylenenitrilo)]-tetra acetic acid     

Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma

The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5-7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K(m)=2.1x10(-3)M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase. The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis.     

Production, purification, and properties of a lipase from a bacterium (Pseudomonas aeruginosa YS-7) capable of growing in water-restricted environments.

An extracellular lipase from the low-water-tolerant bacterium P. aeruginosa YS-7 was produced, purified, and characterized with respect to its functional properties in aqueous solutions and organic solvents. The enzyme was partially released from the cells during fermentation in defined medium with 5% (wt/vol) soybean oil. Approximately one-half of the total culture activity remained in solution after removal of cells. More than 95% of the activity was found in culture supernatant after mild detergent treatment (10 mM sodium deoxycholate) or after shifting the carbon source during the fermentation from triglyceride to a free fatty acid. The enzyme was recovered from an acetone precipitate of the whole culture and purified by hydrophobic interaction chromatography, yielding a preparation having a specific activity of about 1,300 mumol of fatty acid mg-1 h-1. The lipase (molecular size, approximately 40 kDa) hydrolyzes a variety of fatty acid esters and has an optimum pH of about 7. The enzyme retained its full activity at 20 to 55 degrees C, even after prolonged exposure (more than 30 days) to different concentrations of water-miscible organic solvents such as alcohols, glycols, pyridine, acetonitrile, dimethyl formamide, and dimethyl sulfoxide. The hydrolysis of 4-nitrophenyl laurate ester and of triglyceride emulsified in water was slightly accelerated with increasing concentrations of alcohols and glycols up to about 20% but was abolished with a further increase in alcohol concentration or in the presence of acetonitrile. In contrast, the rate of hydrolysis of these substrates in concentrated solutions of dimethyl formamide or dimethyl sulfoxide was markedly increased, by more than twofold and more than fivefold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of diphosphonucleotide phosphatase/phosphodiesterase from yellow lupin (Lupinus luteus) seeds

A phosphatase cleaving the pyrophosphate bond in diphosphonucleotides and phosphodiester bond in various phosphodiesters (pH optimum at 6.25) was purified from yellow lupin (Lupinus luteus L.) seeds. The enzyme is 75 kDa monomeric glycoprotein (pI=6.4) with 4.4% of carbohydrate (mannose, N-acetylglucosamine, fucose and xylose). Analysis of its partial amino acid sequence (8 peptides, 101 amino acid residues) together with no divalent cation requirements for catalysis points out that the purified enzyme is different from known plant pyrophosphate cleaving enzymes (apyrases and inorganic pyrophosphatases). Its physiological role could be related to a regulation of diphosphonucleotides level in plant metabolism.