BACKGROUND: Prostate cancer (PCa) is the most commonly diagnosed solid tumor and the second leading cancer death in the United States, and also one of the major cancer-related deaths in Chinese. Androgen deprivation therapy (ADT) is the first line treatment for metastatic PCa. PCa ultimately relapses with subsequent ADT treatment failure and becomes castrate-resistant (CR). It is important to develop effective therapies with a surrogate marker towards CR PCa. METHOD: Histone deacetylase (HDAC) inhibitors were examined to determine their effects in androgen receptor (AR)/ cellular prostatic acid phosphatase (cPAcP)-positive PCa cells, including LNCaP C-33, C-81, C4-2 and C4-2B and MDA PCa2b androgen-sensitive and androgen-independent cells, and AR/cPAcP-negative PCa cells, including PC-3 and DU 145 cells. Cell growth was determined by cell number counting. Western blot analyses were carried out to determine AR, cPAcP and PSA protein levels. RESULTS: cPAcP protein level was increased by HDAC inhibitor treatment. Valproic acid, a HDAC inhibitor, suppressed the growth of AR/cPAcP-positive PCa cells by over 50% in steroid-reduced conditions, higher than on AR/cPAcP-negative PCa cells. Further, HDAC inhibitor pretreatments increased androgen responsiveness as demonstrated by PSA protein level quantitation. CONCLUSION: Our results clearly demonstrate that HDAC inhibitors can induce cPAcP protein level, increase androgen responsiveness, and exhibit higher inhibitory activities on AR/cPAcP-positive PCa cells than on AR/cPAcP-negative PCa cells. Upon HDAC inhibitor pretreatment, PSA level was greatly elevated by androgens. This data indicates the potential clinical importance of cPAcP serving as a useful biomarker in the identification of PCa patient sub-population suitable for HDAC inhibitor treatment.

By immumolabeling and electronmicroscopy, the subcellular localization of GmPAP3 has been proved to be mainly localized in mitochondria, a primary site for ROS production. Ectopic expression of GmPAP3 in transgenic tobacco BY-2 cells mimicked the protective effects exhibited by the antioxidant ascorbic acid by: (1) increase the percentage of cells with active mitochondria; (2) reduce the percentage of dead cells; and (3) lower the accumulation of ROS under NaCl and osmotic stress treatments. However, when ectopically express a truncated GmPAP3 with the mitochondria transit peptide removed, such protective effect was not observed. This provides evidences on the significance of mitochondria localization to the physiological function of GmPAP3. In addition, when GmPAP3 transgenic Arabidopsis thaliana seedlings were subjected to NaCl, osmotic stress, and oxidative stress treatments, the growth performance of the transgenic lines was significantly better than the wild type. To summarize, these studies has demonstrate that the mitochondrial localized GmPAP3 may play a role in stress tolerance by enhancing ROS scavenging. / Mitochondrion is one of the major sites for the production of reactive oxygen species (ROS). Abiotic stresses such as salinity and osmotic stress can cause oxidative damage to organelle membranes due to excess accumulation of ROS. The inducibility of GmPAP3 gene expression by salinity and oxidative stresses and the putative mitochondrial localization of GmPAP3 prompt us to further investigate the possible physiological roles of GmPAP3 under abiotic stress-induced oxidative stress. / My Ph.D. study has been focused on the detailed functional analysis of the GmPAP3 gene. The objectives of my research include: (i) to verified the subcellular localization of GmPAP3; (ii) to investigate the physiological functions of GmPAP3 under NaC1 and osmotic stress in both cellular level and in planta level. and (iii) to examine the significance of mitochondria] localization of GmPAP3 in relationship to its protective roles. / Purple acid phosphatases (PAPs) represent a diverse group of acid phosphatases in animals and plants. While the mammalian PAPs were found to be related to Reactive Oxygen Species (ROS) evolution in important physiological functions, the roles of plant PAPs remain largely unknown. / Recently, we have isolated a novel PAP-like gene (GmPAP3) from soybean that is induced by NaC1 and oxidative stresses. Subcellular localization prediction programs suggested that GmPAP3 may be a novel PAP that localized in mitochondria. Most other PAPs are extracellularly located and membrane localization of PAPs was only verified in a few cases. / by Li, Wing Yen Francisca. / "December 2009." / Adviser: Lam Hon-Ming. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 123-134). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

This thesis examines the influence of nutritional phosphate (Pi) deprivation on extracellular proteins secreted by the model plant Arabidopsis thaliana. Initial studies compared the secretome of Pi-sufficient (+Pi) versus Pi-deficient (-Pi) Arabidopsis cell cultures by 2-dimensional gel electrophoresis. Mass spectrometry identified 18 different secreted proteins that were upregulated by at least 2-fold by –Pi Arabidopsis. They were predicted to function in Pi scavenging, cell wall and ROS metabolism, proteolysis, and pathogen responses. The relationship between mRNA levels and relative amounts of selected secretome proteins was assessed. The results indicate that transcriptional control is but one of many factors contributing to Arabidopsis Pi starvation responses and highlight the importance of parallel biochemical and proteomic studies of –Pi plants. Three purple acid phosphatase (APase) isoforms were fully purified from the culture media of –Pi Arabidopsis cells and identified as AtPAP12 (At2g27190) and two AtPAP26 (At5g34850) glycoforms. As each purple APase exhibited broad substrate specificities and pH-activity profiles, it is hypothesized that their combined activities facilitate Pi scavenging from soil-localized organophosphates during nutritional Pi deprivation. AtPAP26 is dual-targeted during Pi stress since an earlier report demonstrated that it is also the principal intracellular (vacuolar) APase upregulated by -Pi Arabidopsis. The results indicate that differential glycosylation influences AtPAP26’s substrate specificity and subcellular targeting. An atpap26 T-DNA insertional mutant lacking AtPAP26 transcripts and immunoreactive AtPAP26 polypeptides exhibited: (i) 9- and 5-fold lower shoot and root APase activity, respectively, which did not change in response to Pi starvation, (ii) a 40% reduction in secreted APase activity during Pi deprivation, (iii) 35 and 50% reductions in free and total Pi concentration, respectively, in shoots of –Pi plants, and (iv) impaired shoot and root development when subjected to Pi deficiency. By contrast, no deleterious influence of AtPAP26 loss of function was apparent in +Pi plants. The results establish a firm role for AtPAP26 in the acclimation of Arabidopsis to Pi deficiency. The identification and functional characterization of secreted proteins upregulated by –Pi Arabidopsis is relevant to applied efforts to engineer Pi-efficient transgenic plants, needed to minimize the input of expensive, unsustainable, and polluting Pi fertilizers in crop production. / Thesis (Ph.D, Biology) -- Queen's University, 2010-04-28 17:20:46.892

Upregulation of intracellular and secreted acid phosphatases (APases) is a universal response of orthophosphate-starved (-Pi) plants. APases hydrolize Pi from a broad spectrum of phosphomonoesters at an acidic pH. Plant APases belong to a relatively large multigene family whose specific functions in Pi metabolism are poorly understood. This study focuses on the identification and characterization of cell wall (CW) localized purple acid APases (PAPs) upregulated by -Pi Arabidopsis thaliana. Three glycosylated PAP isozymes secreted into the CW of -Pi Arabidopsis suspension cells were purified and identified by peptide mass fingerprinting using mass spectrometry (MALDI-TOF MS) and N-terminal microsequencing as AtPAP12 (At2g27190; subunit size 60-kDa), AtPAP25 (At4g36350; subunit size 55-kDa) and AtPAP26 (At5g34850; subunit size 55-kDa). Both AtPAP12 and AtPAP26 were previously shown to be upregulated and secreted by –Pi Arabidopsis to scavenge Pi from extracellular organic-P. However, the novel AtPAP25 has never been suggested to be involved in the plant Pi-starvation response. Biochemical characterization of AtPAP25 revealed a monomeric 55 kDa protein. Similar to other PAPs it was purple-in-solution and insensitive to tartrate. Glycoprofiling via LC MS/MS revealed highly complex NXS/T glycosylation motifs at Asn172, Asn367 and Asn424. I hypothesize that these motifs play a role in AtPAP25 targeting and function. Kinetic characterization revealed a broad pH optimum centered at 5.6 and inhibition of activity by several common APase inhibitors. AtPAP25 exhibited broad substrate selectivity, low Vmax, and a Km (phosphoenolpyruvate) value of 0.52 mM. Immunoblot and semi-quantitative RT-PCR transcript analysis indicated that AtPAP25 is exclusively synthesized under –Pi conditions. Deduced amino acid sequences were compared using multiple sequence alignment and phylogenetic analysis. Growth of atpap25 T-DNA insertion mutant knockout seedlings was completely arrested when transferred to a soluble Pi deficient organic-P containing soil mix, pointing to a potential regulatory function of AtPAP25 during nutritional Pi stress. Overall, this research is helping to shed light on the functional importance of specific PAP isozymes in facilitating plant acclimation to nutritional Pi deficiency. This is important because there is an urgent need to engineer Pi-efficient transgenic crops to minimize the huge input of expensive, non-renewable, and polluting Pi fertilizers in agriculture. / Thesis (Master, Biology) -- Queen's University, 2012-09-10 08:28:21.631

Acid phosphatases (apase) are important hydrolytic enzymes that function in the acquisition, production; transport and recycling of inorganic phosphate (Pi), thus making a significant contribution towards nutrients dynamic of many ecological niches. The aim of this study was to characterize the apase enzyme found in the lichen Cladonia portentosa at the molecular level. The initial experiment entailed cloning the apase gene by PCR using degenerate primers designed from close relatives of C. portentosa from the Ascomycete family. The isolation of apase gene from Cladonia portentosa using PCR was not successful. Attempts were then made to purify the secreted apase and to determine its biochemical and molecular properties and to allow comparison with already characterized secreted phosphatases from other fungal sources existing in the NCBI database. It was anticipated that the partial sequence of the purified enzymes would provide a corresponding apase gene. The acid phosphatase enzyme was partial purified to 45 fold by a gel filtration with a yield of 18%. It gave a single, broad glycoprotein band on native PAGE and SDS-PAGE corresponding in size to 250 and 148 kDa, respectively. Under reducing conditions, the purified enzyme migrated as two bands of 116 and 32 kDa, indicating the heterodimer nature of this enzyme. Only one distinct band, (pI 6.4) was observed after electrofocusing. The optimum temperature for the enzyme was 65 °C where an optimal pH was detected at 2.5. The enzyme was inhibited by known acid phosphatase inhibitors (fluoride, molybdate, orthovanadate and tartrate) and the metals (Cu²⁺ and Zn²⁺). The purified enzyme demonstrated broad substrates selectivity and had a KM of 31.2±0.25 μM for phytic acid. Peptide analysis by Mass Spectrometry (MS) MALDI-TOF indicated the presence of two apase proteins. The amino sequences of purified apase/s from Cladonia portentosa were FLAETNPAPFGH, AVGLGYVEELLAR and AQGLGYVQEVLAR. Comparing the amino acids of the sequenced protein with that of already known proteins confirmed the enzyme to be a secreted histidine acid phosphatase, resembling other acid phosphatases and phytase from several filamentous fungi with respect to amino acid composition. To investigate the effect of phosphorus on C. portentosa apase, the mycelium was grown under different concentrations of Pi [0.05, 1.0, 3.0, 10 and 100 mM (KH₂PO₄)]. The aim was to localize the apase enzyme and to screen for the occurrence of the gene coding for the acid phosphatase enzyme. A treatment of 3.0 mM Pi induced high levels of apase compared to all other treatments. In addition, cultures of C. portentosa were grown in axenic cultures to study the effect of pH and Pi versus menadione on the production of acid phosphatase and mycelia growth. A culture media of pH 4.8 and 6.0 resulted in higher apase secretion than when compared with pH 2.5 medium. The presence of 2.0 μM menadione marginally increased levels of the apase compared to the control treatment. Apase was further localized cytochemically using fluorescent substrate-enzyme-labelled fluorescence (ELF-97) which forms a fluorescent crystalline precipitate at the site of phosphate activity. Fluorescent microscope revealed that the enzyme was present in all treatments, irrespective of Pi concentration, however, the fluorescence signals were intense in low Pi concentrations (0.05 and 1.0 and 3.0 mM Pi). Ultrastructure localization using live mycelium under confocal microscopy using Vector blue III substrate revealed that the enzyme was localized in the cytoplasm, cell membrane, vacuole and small organelles, presumed to be endosomes. Co-staining with FM4-64, confirmed the punctuate structure to be secretory vesicles or a vacuolar network. To investigate the effect of P starvation on C. portentosa at a molecular level, the effect of Pi on the gene expression profile was examined. The generation of a cDNA library from axenic grown mycelium treated with P provided a foundation for the identification and characterization of genes expressed in the P treated mycelium through expressed sequence tags (ESTs). Several genes were identified whose transcriptional profiles have been significantly changed by phosphorus treatment and menadione. They include genes required for signal transduction and vesicular transport, cell biosynthesis and protein metabolism and stress response. In conclusion, this study constitutes the first step towards understanding the molecular mechanism governing acid phosphatase in C. portentosa. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Bloodstream forms of African trypanosomes, which endocytose macromolecules exclusively through their flagellar pockets, contain an acid phosphatase (AcP) activity in this organelle. In the present thesis, AcP activity was demonstrated cytochemically in some intracellular vesicles and on the surface of Trypanosoma congolense as well as in the flagellar pocket. Unlike other trypanosomatids such as Leishmania spp. and Trichomonas spp., these trypanosomes, while viable, did not release this enzyme into the surrounding medium. In contrast to mammalian cells, the AcP in T. congolense was shown by cell fractionation to be a non-lysosomal enzyme. The enzyme was mostly recovered in the microsomal and cytosolic fractions which had 52.7% and 44.4% of the total activity, respectively. Further separation of the microsomal fraction showed an association of AcP activity with vesicles derived from the plasma membrane, Golgi apparatus and endoplasmic reticulum. After ammonium sulfate precipitation and chromatography on a succession of columns containing Sephacryl S-300, DEAE-cellulose and Sephadex G-75, two acid phosphatases (AcPi and ACP2) were produced from the cytosolic fraction. A membrane-bound acid phosphatase (ACP3) was isolated from the microsomal pellets extracted with Triton X-l 14 and subjected to the above chromatographic procedures. The molecular mass of AcP 1 was higher than 700 kDa. It had an isoelectric point of 4.7. AcP2 (pi 5.3) and AcP3 (pi 6.5) had molecular masses of 33 and 320 kDa, respectively. AcPi and ACP3 were strongly inhibited by vanadate while ACP2 was strongly inhibited by p-chloromercuribenzoate. None of the enzymes was inhibited by tartrate but all were inhibited by NaF. The Km values for each of the various substrates differed widely between the three AcPs indicating that the binding site of each enzyme was distinct. The best of all the substrates tested was para-nitrophenyl phosphate. On non-denaturing gels the enzymes exhibited very high molecular masses but on denaturing SDS-PAGE, two similar bands of activity, localised at 62 and 65 kDa, were observed in all three AcP preparations. Thus the three isolated enzymes may be derived from the same base 62 and 65 kDa units. Differences between enzymes may be derived from differential processing of the isoenzymes for different functions at different locations. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-29Bitstream added on 2014-06-13T19:35:22Z : No. of bitstreams: 1 sato_vs_me_jabo.pdf: 522350 bytes, checksum: 34c42a9a6f174fa4f2002e3d48b07c50 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A capacidade de bactérias endofíticas em solubilizar fosfato inorgânico é alvo de grande interesse por parte dos microbiologistas, uma vez que as fosfatases são responsáveis por hidrolisar compostos orgânicos produzindo fósforo solúvel. Dessa forma, a fosfatase ácida ligada à membrana (MBAP) foi obtida a partir de Enterobacter sp. isolada de raízes de Cattleya walkeriana (Orchidaceae) e identificada pelo seqüenciamento do gene 16S rRNA. A expressão da enzima mostrou-se estritamente regulada pelo fósforo (expressão ideal em 7 mm). O pH ótimo aparente (3,5) não foi afetado pela concentração de p-nitrofenilfosfato. Em pH 3,5, a enzima é uma fosfomonidrolase inespecífica capaz de hidrolisar os substratos PNPP (61,2 U/mg), ATP (19,7 U/mg), e o pirofosfato (29,7 U/mg), com K0.5 de 0,06 mM, 0,11 mM e 0,08 mM, respectivamente. A enzima exibi cinética Michaelina para o pNPP (n=1,2). Para o ATP e o pirofosfato interações sítio-sítio foram observadas com n=1,6 e 2,3, respectivamente. Os íons de magnésio foram potentes estimuladores (K0.5=2,2 mM), enquanto o arsenato e o fosfato foram potentes inibidores competitivos. A atividade PNPPase foi inibida pelo EDTA, mas não pelo cálcio, levamisol, zinco, cobalto e phidroximercuribenzoato. A entalpia de inativação térmica foi da ordem de 77,5 kcal.mol- 1. Os resultados sugerem que a produção da fosfatase ácida ligada à membrana representa um mecanismo de solubilização do fosfato mineral aumentando a disponibilidade de nutrientes para as plantas / The ability of endophytic bacteria in solubilizing inorganic phosphate is of great interest by microbiologists since phosphatases are responsible for catalyzing the hydrolysis of organic compounds producing soluble phosphorus. Thus, the membranebound acid phosphatase (MBAP) was obtained from Enterobacter sp. isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis. The enzyme expression was demonstrated to be strictly regulated by phosphorus (optimal expression at 7 mM). The enzyme was obtained by centrifugation at 100.000g for 1 h at 4ºC. The apparent optimal pH (3.5) was not affect by p-Nitrophenyl phosphate concentration. At pH 3.5, the enzyme showed a broad substrate specificity hydrolyzing different substrates such as PNPP (61.2 U/mg), ATP (19.7 U/mg), and pyrophosphate (29.7 U/mg), with K0.5 values of 0.06 mM, 0.11 mM and 0.08 mM, respectively. The hydrolysis of PNPP by the enzyme exhibited Michaelian kinetics with n= 1.2. For ATP and pyrophosphate site-site interactions were observed with n= 1.6 and 2.3, respectively. Although magnesium ions were stimulatory (K0.5= 2.2 mM), arsenate and phosphate were a powerful competitive inhibitor. The PNPPase activity was inhibited EDTA but not by calcium, levamisole, zinc, cobalt and phydroxymercurybenzoate. The ΔH for thermal inactivation was 77.5 kcal.mol-1. Our results suggest that the production of a membrane-bound acid phosphatase might be one mechanism of mineral phosphate solubilization turn it´s nutrients availability to plants

Semen is one of the most common biological fluids encountered by a forensic serologist on varying substrate types. Seminal fluid contains many enzymes, proteins, and cellular material such as acid phosphatase (AP), prostate-specific antigen (PSA), semenogelin (Sg), and spermatozoa; detection of these components can aid in the forensic identification of body fluids. Forensic laboratories usually follow a prescribed testing workflow (from visual examination including an alternate light source (ALS) and AP testing to PSA or Sg testing followed by a microscopic examination for sperm cells) to ensure laboratory resources are being used in a proper manner with minimal waste of both time and resources. However, this approach can be problematic when degradation of semen stains results in the inability to detect the presence of certain seminal components. When a stain yields negative results for an AP reaction, no further analyses for semen may be performed and analysis comes to an end. In common practice, evidentiary items containing biological fluids may not be immediately recovered following an incident and/or may not be stored properly, causing contamination or exposure of these biological fluids to harsh environments, potentially degrading the sample. This study investigates how exposure to different environmental conditions and packaging types affects the degradation of the four most common semen components targeted in forensic testing: AP, PSA, Sg, and spermatozoa. Semen stains were prepared and exposed to ten different storage and/or environmental conditions to compare their effects on the detectability of seminal components (fluorescence, AP, PSA, Sg, and spermatozoa as well as deoxyribonucleic acid (DNA) testing) for a period of approximately four months. Samples from each condition were tested on select days throughout the study. Minimal changes in detection of the seminal components were observed under the five conditions in which the stains remained dry: packaged in paper stored at room temperature, packaged in paper stored at high temperatures, exposed to sunlight, exposed to ultraviolet light, and stored in high humidity. Two of the conditions involved exposure to outdoor environments. The stains openly exposed to the elements or buried in soil exhibited the most notable degradation of all components when compared to other conditions. Negative results were obtained for nearly all seminal components (AP, PSA, or Sg) on Day 8 for stains openly exposed outdoors and Day 32 for buried samples. The remaining three conditions exposed the stains to damp or wet conditions and gave variable results throughout the study. DNA quantification was performed for select samples from each condition to assess DNA degradation. Most samples did not exhibit DNA degradation on quantification results up to Day 112; however, two samples exposed to outdoor environments exhibited DNA degradation as early as Day 8 (earliest day quantified). More notably, two samples from Day 112 demonstrated the presence of non-degraded DNA in sufficient quantity for profiling, while the presumptive semen analyses (AP, PSA, and Sg) for the same samples exhibited negative results when using an AP reaction cut-off time of 2 minutes. These results suggest that an allotted time of 2 minutes for AP detection may not be sufficient in some samples, and that valuable DNA evidence may go undiscovered, especially when other presumptive tests show negative results. Overall, the results revealed variation in the sequence and rate of degradation for seminal components in semen stains exposed to different environmental conditions. It was not possible to predict which of the remaining components of semen would be detectable based on the outcome of any one of the tests. Therefore, it is recommended that comprehensive testing of possible semen stains is performed, even after negative presumptive results are obtained, when the case scenario suggests exposure to damp/wet or otherwise less than ideal conditions.

<p> Preliminary experiments indicated that lysosomes are present in rat liver and onion embryos. A differential centrifugation study was made of the intracellular distribution of acid phosphatase in pea embryo tissue in an attempt to show that this enzyme is enclosed by a membrane forming granules similar to the lysosomes of hepatic tissue. The results reveal that acid phosphatase is soluble under the conditions employed, but it is believed that this may well have resulted from excessive damage to the subcellular bodies during homogenization.</p> / Thesis / Master of Science (MSc)

Les « Purple Acid Phosphatases » (PAPs) sont des phosphatases acides présentes dans le règne végétal et animal. Elles sont caractérisées à la fois par la présence de sept acides aminés conservés impliqués dans la structure d’un centre bimétallique et par leur couleur rose/violète lorsqu’elles se trouvent en solution. Chez les plantes, certaines PAPs présenteraient à la fois une activité phosphatase sur des métabolites (PEP, G6P…) et sur des phospho-peptides (glycosidases…), d’une part, et une activité peroxydase, de l’autre. Ces PAPs pourraient donc, non seulement être impliquées dans le métabolisme carboné, mais aussi dans la détoxication des espèces réactives de l’oxygène. On compte à ce jour 29 gènes codant pour des PAPs chez Arabidopsis thaliana , dont un certain nombre est induit par la carence en Pi et par le stress oxydatif ou la senescence. Parmi eux, le gène de l’AtPAP26 (At5g34850) présente de très fortes homologies de séquence avec des orthologues présents chez d’autres plantes. La protéine AtPAP26, qui présente in vitro une activité phosphatase sur le PEP et le G6P et une activité peroxydase, est sécrétée même si de nombreux indices laissent penser qu’elle pourrait également être localisée dans la vacuole.Notre objectif est d’étudier le rôle d’AtPAP26 dans le métabolisme et le stockage des sucres dans la vacuole. Notre hypothèse de départ est que la protéine AtPAP26 pourrait intervenir via son activité phosphatase dans un cycle de substrat vacuolaire impliquant le glucose-6-phosphate et le glucose. Nous avons montré que chez le mutant pap26, l’activité glucose-6-phosphatase, mesurée in vitro et in organello, chute de manière importante. Parallèlement, chez le mutant gpt2, le transport tonoplastique des hexoses phosphates diminue de façon significative. Des expériences de marquages isotopiques sur du tissu racinaire en culture ont montré qu’une part importante du glucose et du fructose ne provient pas de l’hydrolyse du saccharose et que AtPAP26 est en partie responsable de cette synthèse. Enfin, par une approche in-silico, nous avons modélisé le métabolisme des sucres dans un système compartimenté impliquant la vacuole et ses transporteurs. La confrontation du modèle avec deux jeux de données expérimentales (concentrations à l’état stationnaire et cinétiques de marquage) a permis de confirmer l’existence d’une activité hydrolytique sur les hexoses phosphates vacuolaires. / «Purple Acid Phosphatases» (PAPs) are acid phosphatases found both in animal and vegetal kingdoms. They are characterized by the presence of seven conserved amino acids involved in a di-metal center and by their pink/purple color in solution. In plants, some PAPs exhibit an acid phosphatase activity on various phospho-esters and on phospho-peptides. An alcalin peroxidase activity has been demonstrated in vitro. Therefore, PAPs could be implicated both in the carbon metabolism and in the scavenging of reactive oxygen species. To date, 29 different PAP-encoding genes were identified in the Arabidopsis thaliana genome. Some of them are induced by phosphate deprivation, oxidative burst or senescence. The gene encoding the protein AtPAP26 (At5g34850) exhibits a high sequence similarity with orthologous from other plants. The AtPAP26 protein, showing both a phosphatase activity on PEP, G6P and F6P, and a peroxidase activity, is one of the most abundant root-secreted acid phosphatases but also appears to be localized in the vacuole. Our aim is to study the implication of AtPAP26 in the metabolism and storage of sugars in the vacuole. Our first hypothesis is that AtPAP26, via its phosphatase activity, could be involved in a glucose / glucose-6-phosphate cycle localized in the vacuole. We showed that the glucose-6-phosphatase activity in the pap26 mutant is highly affected both in vitro and in organello compared to the wild type. At the same time, the hexose phosphate transport capacity of the vacuole is highly affected in the gpt2 mutant compared to the wild-type. Moreover, isotopic labeling experiments performed on cultured root tissues have shown that a significant part of the intracellular glucose and fructose pool does not originate from sucrose and that AtPAP26 is, at least in part, responsible for this synthesis. Finally, we build a kinetic model of sugar metabolism in a compartmented system which has been validated by using two independent experimental data sets (steady state concentrations and kinetics of labeling of soluble sugars). This in silico modeling approach confirmed the involvement of a hexose phosphatase activity localized in the vacuole.