Alkaline and acid phosphatases of the silkworm, Bombyx mori L.

The variations in the activities of the alkaline and acid phosphatases of the silkworm, Bombyx mori, were studied in all stages of the life cycle. From hatching until the spinning stage a steady increase was recorded in the activity of both the enzymes followed with a conspicuous decrease at each moult. During the pupal stage the alkaline phosphatase was almost absent, whereas the acid phosphatase maintained a high and constant value. Increase or decrease of the activity of the enzymes during larval development was reflected in a decrease or increase in the acid-soluble phosphorus content. Acid phosphatase activity slowly increased from laying of the eggs to hatching of the larvae with a concomitant decrease in the acid-soluble phosphorus. Tissue analysis showed a high concentration of the alkaline enzyme in the intestines, but the haemolymph was almost free of both enzymes. Feeding of inorganic phosphate increased the alkaline enzyme in the intestines, whereas glucose had no effect on either of the enzymes in the intestines.

Modulation of Catalytic Activity in Multi-Domain Protein Tyrosine Phosphatases

Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs) containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1) domains, while the membrane-distal (D2) domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR) and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A). While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.

Bi-Domain Protein Tyrosine Phosphatases Reveal an Evolutionary Adaptation to Optimize Signal Transduction

Significance: The bi-domain protein tyrosine phosphatases (PTPs) exemplify functional evolution in signaling proteins for optimal spatiotemporal signal transduction. Bi-domain PTPs are products of gene duplication. The catalytic activity, however, is often localized to one PTP domain. The inactive PTP domain adopts multiple functional roles. These include modulation of catalytic activity, substrate specificity, and stability of the bi-domain enzyme. In some cases, the inactive PTP domain is a receptor for redox stimuli. Since multiple bi-domain PTPs are concurrently active in related cellular pathways, a stringent regulatory mechanism and selective cross-talk is essential to ensure fidelity in signal transduction. Recent Advances: The inactive PTP domain is an activator for the catalytic PTP domain in some cases, whereas it reduces catalytic activity in other bi-domain PTPs. The relative orientation of the two domains provides a conformational rationale for this regulatory mechanism. Recent structural and biochemical data reveal that these PTP domains participate in substrate recruitment. The inactive PTP domain has also been demonstrated to undergo substantial conformational rearrangement and oligomerization under oxidative stress. Critical Issues and Future Directions: The role of the inactive PTP domain in coupling environmental stimuli with catalytic activity needs to be further examined. Another aspect that merits attention is the role of this domain in substrate recruitment. These aspects have been poorly characterized in vivo. These lacunae currently restrict our understanding of neo-functionalization of the inactive PTP domain in the bi-domain enzyme. It appears likely that more data from these research themes could form the basis for understanding the fidelity in intracellular signal transduction.

Extracellular phosphatases produced by phytoplankton and other sources in shallow eutrophic lakes (Wuhan, China): taxon-specific versus bulk activity

Extracellular phosphatases are an important part of the phosphorus cycle in aquatic environments. Phosphatase activity (PA) in plankton was studied in seven subtropical shallow lakes of different exploitation management and trophic status in the urban area of Wuhan City. Bulk PA was rather high (range 1.1-11 mu mol l(-1) h(-1)), although concentrations of soluble reactive phosphorus (SRP) were also high (range 27 mu g P l(-1) to similar to 1.5 mg P l(-1)) in all lakes. Cell-associated extracellular PA in phytoplankton was detected using the fluorescence-labelled enzyme activity technique. Phytoplankton species partly contributed to the bulk PA. We found explicit differences in the presence of cell-associated phosphatase within the main phytoplankton groups; species belonging to Chlorophyta and Dinophyta were regularly phosphatase-positive, while Cyanophyta and Bacillariophyceae were phosphatase-negative in all but one case. Furthermore, there is a certain potential of extracellular phosphatases produced by heterotrophic nanoflagellates in most of the lakes. This new finding compromises the 'traditional' interpretation of bulk phosphatase data as being due to overall phytoplankton or bacterial P regeneration.

The role of cell-surface-bound phosphatases in species competition within natural phytoplankton assemblage: an in situ experiment

Despite it is widely acknowledged that the ability to hydrolyze dissolved organic matter using extracellular phosphatases is diverse in fresh water phytoplankton, the competition within single species related to presence and quantity of cell-surface-bound phosphatases has not been examined in natural conditions yet. Here, we studied phytoplankton species competition in a freshwater reservoir during an in situ experiment. A natural plankton community, with the exclusion of large zooplankton, was enclosed in permeable dialysis bags inside two large containers of different bioavailable phosphate concentrations. Phytoplankton species biomass and the abundance of bacteria were determined in purpose to compare the development of enclosed microbial communities. Total and cell-surface-bound phosphatase activities in the phytoplankton were investigated using the Fluorescently Labelled Enzyme Activity (FLEA) technique that allows for direct microscopic detection of phosphatase-positive cells and, with image cytometry, enables quantification of phosphatase hydrolytic capacity. Production of extracellular phosphatases was not completely inhibited or stopped in the phosphate-enriched environment, phytoplankton cells only showed the activity less often. Under the phosphate-nonenriched conditions, the production of phosphatases was enhanced, but active species did not proliferate amongst phytoplankton assemblage. Further, specific growth rates of the phosphatase-positive species in the non-enriched environment were lower than the same phosphatase-positive species in phosphate-enriched environment. Interestingly, the phosphatase-positive cells of Ankyra ancora increased their size in both treatments equally, although the population in phosphate-enriched environment grew much faster and the cell-specific phosphatase activity was lower. We hypothesize that brand new daughter cells had sufficient phosphorus reserves and therefore did not employ extracellular phosphatases until they matured and needed extra bioavailable phosphorus to support their metabolism before cell division. Based on presented in situ experiment, we propose that the ability to hydrolyze organic polymers and particles with cell-surface-hound phosphatases is advantageous for longer persistence of given population in a phosphate-scarce environment; although phosphatase-positive species cannot dominate the reservoir phytoplankton solely because of specific phosphorus-scavenging strategy.

Physico-chemical properties of alkaline phosphatases released by a planktonic crustacean, Daphnia magna (Cladocera)

Our studies investigated the physico-chemical properties of alkaline phosphatase excreted by D. magna. This cladoceran mainly released alkaline phosphatase, though it also released a small amount of acid phosphatase. The alkaline phosphatase showed a broad pH optimum (8.05-10.0), and had a broad optimum temperature (30-35 degrees C) with a temperature coefficient (Q(10)) of 2.45. The K-m of the enzyme is 0.15 +/- 0.02 mM when p-nitrophenyl phosphate is used as a substrate, and the V-max is 0.43 +/- 0.01 mu M pNP mg(-1) DW h(-1). Even though alkaline phosphatase had been incubated in chloroform saturated with WC medium for 13 days, its activity was 54% that of the original. The enzyme was strongly inactivated by EDTA, and appeared to be zinc dependent. The alkaline phosphatase activity remained constant when D. magna was fed different quantities of Chlorella sp. The sensitivity of D. magna phosphatase activity to phosphate was time-dependent. During the first 16 hrs, the enzyme was insensitive to phosphate addition, after 24 hrs incubation the enzyme became sensitive to phosphate addition.

Detection of extracellular phosphatases in natural spring phytoplankton of a shallow eutrophic lake (Donghu, China)

The species-specific production of extracellular phosphatases in phytoplankton of a subtropical polymictic take was investigated from March to May 2004. Phosphatase activity was detected directly at the site of enzyme action using the enzyme-labelled fluorescence (ELF) technique. Size fractionation of bulk phosphatase activity (PA), concentrations of soluble reactive phosphorus (SRP), chlorophyll a, and phytoplankton composition were determined in parallel. Phosphatase-positive cells were present in every phytoplankton sample; labelled cells were detected in 33 algal taxa, including many chlorophytes, dinoflagellates and some diatoms, but never among cyanobacteria. We recorded an unusual dinoflagellate bloom (Peridiniopsis sp.), of which similar to 25% of the cells were phosphatase-positive. Several populations were partly phosphatase-positive whenever present, while some other species never showed any activity. The production of extracellular phosphatases was not primarily regulated by ambient P concentrations; algae produced these enzymes even if SRP concentrations were high. Moreover, heterotrophic nanoflagellates most probably contributed to the pool of particle-bound PA in some samples.

Comparative study on in vitro inhibition of grass carp (Ctenopharyngodon idellus) and mouse protein phosphatases by microcystins

Microcystins isolated from toxic cyanobacteria are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). The inhibitory effects of three structural variants of microcystins (microcystin-LR, -YR, and -RR) on protein phosphatases isolated and purified from the liver and kidney of grass carp (Ctenopharyngodon idellus) were investigated using the P-32 radiometric assay. The relationships between percentage inhibition of protein phosphatase activity and microcystin levels followed a typical dose-dependent sigmoid curve. These results were compared to those obtained from mouse PP2A. The degree and pattern of inhibition of both fish and mouse protein phosphatases by microcystins were similar. Protein phosphatases in crude fish tissue homogenates showed similar inhibition patterns as purified fish PP2A toward microcystins. (C) 2000 by John Wiley & Sons, Inc.

Protein phosphatases acting on the replication checkpoint

A génese de um cancro está dependente da acumulação de mutações genéticas que dão origem a instabilidade genómica, que por sua vez resulta na proliferação descontrolada. Para prevenir a acumulação destas mutações, as células têm mecanismos de controlo (checkpoints) que suspendem o ciclo celular e accionam as vias de reparação do ADN. Estes eventos são muitas vezes regulados por dinâmicas de (des)fosforilação de proteínas. As proteínas fosfatases (PPs), enzimas responsáveis pela remoção do grupo fosfato de resíduos fosforilados, desempenham funções cruciais na regulação de muitos mecanismos celulares. Enquanto que no início do projecto as cinases envolvidas no checkpoint da replicação estavam bem estabelecidas, as PPs envolvidas não eram conhecidas. A Chk1, um componente da maquinaria do checkpoint da replicação, é exemplo dessa regulação por (des)fosforilação, como sejam nos resíduos Ser317 e Ser345. Assim, como primeira abordagem para determinar quais os grupos de PPs envolvidos na regulação do checkpoint da replicação, decidimos investigar o seu papel na regulação da fosforilação da Chk1. A primeira conclusão é que a desfosforilação da Chk1 ao longo do tempo, tanto in vivo como in vitro, ocorre com uma dinâmica bi-fásica. Em segundo, a abordagem in vitro sugere que as famílias PP1, PP2A e PP2C estão envolvidas na desfosforilação da Chk1. Uma vez que a família PP2A foi a que mostrou a maior acção nesta reacção, decidimos investigar outros membros da família in vivo, primeiro com uma abordagem geral (tratando com OA ou sobreexpressando a PME-1), e depois com o knockdown específico da PP4 e PP6 (através de siRNA). Os resultados mostram que a inibição das PPs afectam tanto a desfosforilação como o estado de activação da Chk1 em resposta a tratamento com Hidroxiureia (HU). Todas as PPs testadas in vivo pareceram ser capazes de regular, a níveis diferentes, tanto a fosforilação como a desfosforilação da Chk1. A função das PPs foi também investigada ao nível: da regulação do disparo das origens de replicação, e da recuperação da suspensão da replicação, induzida pela HU. No último caso, os dados indicam que na situação simultânea de knockdown da PP4 com tratamento de HU, há um atraso do ciclo celular na resolução da transição de G2/M. No ensaio de replicação por pulse-chase, os resultamos mostram que tanto o tratamento com OA, como a sobre-expressão de I-2 ou PME-1, atrasam a cronologia do disparo programado das origens de replicação. No entanto, nenhum dos tratamentos efectuados parece desregular o início do checkpoint da replicação. Um rastreio de 2-híbrido de levedura com uma biblioteca de cDNA de testículo humano foi realizado, usando a Chk1 como isco, no sentido de descobrir novos interactores e definir novas possíveis funções para a Chk1 no contexto da meiose. Com base nos resultados do rastreio, duas novas funções são sugeridas: a interacção com a GAGE12 sugere uma função na recombinação genómica/vigilância do genoma durante a meiose, e as interacções com a EEF1α1 e a RPS5 sugerem uma função na regulação da síntese proteíca. Estas experiências fornecem um visão geral para a compreensão da diversidade de funções das proteínas fosfatases envolvidas no checkpoint da replicação, bem como, abre novos caminhos para o desenvolvimento de novas drogas para o tratamento do cancro.

RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3.

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling.

Phosphatases from Bacteria Isolated from the Arabian Sea and Cochin Estuary

This study aims to reveal the ability of bacteria isolated from Cochin estuary and the Arabian Sea to produce phosphatases, important characters of the enzymes, its ability to utilize organophosphorus compounds as source of phosphate and also their role in degradation of organophosphorus pesticides. It deals with isolation, identification and screening of bacteria for phosphatase production, and it describes the effect of cultural conditions on growth and phosphatase production. The effect of various factors like pH, NaCl concentration, temperature of incubation, carbon source, period of incubation etc. on growth and phosphatase production by the two selected species were studied to establish suitable environment for phosphatase production by these bacteria. In this study regulation of phosphatase synthesis, characteristics of acid and alkaline phosphatases are discussed.

STUDIES ON HAEMOLYMPH CONSTITUENTS OF INDOPLANORBIS EXUSTUS (DESHAYES) AND LYMNAEA ACUMINATA (LAMARCK) F.RUFESCENS (GRAY) AND THE EFFECTS OF COPPER ON THE ACTIVITY PATTERN OF SELECTED TRANSAMINASES AND PHOSPHATASES

The problem investigated is on the haematological aspects of two freshwater pulmonate snails, Indoplanorbis exustus (Deshayes),and Lymnaea acuminata f.rufescens (Gray). An important aspect of the present investigation is to emphasize the utilization of freshwater organisms as models for research directed at understanding the basic biomedical problems that remain unresolved. Another aspect is to demonstrate how haemolymph can be treated as a tissue because of late, it has been shown that several parameters of blood can be taken as reliable indicators for diagnostic purposes, and also to monitor environmental pollution. The various haematological parameters studied are total haemocyte number,packed cell volume, haemoglobin, and inorganic as well organic constituents in three size groups of both the snail species. The effect of copper toxicity was measured in terms of total haemocyte count, and the activity pattern of selected phosphatases and transaminases.The study concluded that enzyme activity levels can be taken as reliable indicators to monitor pollution. Age is a factor that determines several of the physiological, biochemical and metabolic activities. This study also indicates that haemolymph can be taken as an organ system to study the various changes taking place at organ systems levels.

Dual-specificity phosphatases in the hypo-osmotic stress response of keratin-defective epithelial cell lines

Although mutations in intermediate filament proteins cause many human disorders, the detailed pathogenic mechanisms and the way these mutations affect cell metabolism are unclear. In this study, selected keratin mutations were analysed for their effect on the epidermal stress response. Expression profiles of two keratin-mutant cell lines from epidermolysis bullosa simplex patients (one severe and one mild) were compared to a control keratinocyte line before and after challenge with hypo-osmotic shock, a common physiological stress that transiently distorts cell shape. Fewer changes in gene expression were found in cells with the severely disruptive mutation (55 genes altered) than with the mild mutation (174 genes) or the wild type cells (261 genes) possibly due to stress response pre-activation in these cells. We identified 16 immediate-early genes contributing to a general cell response to hypo-osmotic shock, and 20 genes with an altered expression pattern in the mutant keratin lines only. A number of dual-specificity phosphatases (MKP-1, MKP-2, MKP-3, MKP-5 and hVH3) are differentially regulated in these cells, and their downstream targets p-ERK and p-p38 are significantly up-regulated in the mutant keratin lines. Our findings strengthen the case for the expression of mutant keratin proteins inducing physiological stress, and this intrinsic stress may affect the cell responses to secondary stresses in patients' skin.