Giardia lamblia: biochemical characterization of an ecto-ATPase activity

In this work, we describe the ability of living trophozoites of Giardia lamblia to hydrolyze extracellular ATP. In the absence of any divalent cations, a low level of ATP hydrolysis was observed (0.78 ± 0.08 nmol Pi × h⁻¹ × 10⁻⁶ cells). The ATP hydrolysis was stimulated by MgCl₂ in a dose-dependent manner. Half maximum stimulation of ATP hydrolysis was obtained with 0.53 ± 0.07 mM. ATP was the best substrate for this enzyme. The apparent K_m for ATP was 0.21 ± 0.04 mM. In the pH range from 5.6 to 8.4, in which cells were viable, this activity was not modified. The Mg²⁺-stimulated ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A₁ (V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The impermeant agent DIDS and suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases, decreased the enzymatic activity in a dose-dependent manner, confirming the external localization of this enzyme. Besides ATP, trophozoites were also able to hydrolyse ADP and 5´ AMP, but the hydrolysis of these nucleotides was not stimulated by MgCl₂. Our results are indicative of the occurrence of a G. lamblia ecto-ATPase activity that may have a role in parasite physiology.     

Development and characterization of a potent producer of penicillin G amidase by mutagenization

Summary The penicillin G amidase (PGA) activity of a parent strain of E. coli (PCSIR-102) was enhanced by chemical mutagenization with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). After screening and optimization, a penicillinase deficient mutant (MNNG-37) was isolated and found effective for the production of penicillin G amidase as compared to the parent strain of E. coli (PCSIR-102). Penicillin G amidase activity of MNNG-37 appeared during an early stage of growth, whereas PCSIR-102 did not exhibit PGA activity due to the presence of penicillinase enzyme which inhibits the activity of enzyme PGA. However, MNNG-37 gave a three-fold increase in enzyme activity (231 IU mg⁻¹) as compared to PCSIR-102 (77 IU mg⁻¹) in medium containing 0.15 and 0.1% concentrations of phenylacetic acid, respectively which was added after 6 h of cultivation. The difference in K _m values of the enzyme produced by parent strain PCSIR-102 (0.26 mM) and mutant strain MNNG-37 (0.20 mM) is significant (1.3-fold increase in K _m value) which may show the superiority of the latter in terms of better enzyme properties.     

Metabolic energy utilization during development of Antarctic naked dragonfish (Gymnodraco acuticeps)

We have capitalised on the availability of eggs and adults of the naked dragonfish Gymnodraco acuticeps (Sub-order Notothenioidei, F. Bathydraconidae) near McMurdo Station, Antarctica to examine metabolic energy utilization at different stages of its life cycle. Average egg respiration rates were found to increase from 2.17±1.02 nmol O₂ h⁻¹ ind⁻¹ at about 17 h post-fertilization (hpf) to 5.72±0.56 nmol h⁻¹ ind⁻¹ at about 24 hpf, during which time the eggs underwent first cleavage. The respiration rates of embryos from 2–20 days post-fertilization (dpf) averaged 4.11±1.47 nmol O₂ h⁻¹ ind⁻¹. About 10 months post-fertilization, oxygen consumption rates of 27.14±3.92 nmol O₂ h⁻¹ ind⁻¹ were recorded immediately prior to hatching, with a peak of 112.41±31.38 nmol O₂ h⁻¹ ind⁻¹ at the time of hatch. Larvae aged 46–63 days post-hatch had an average respiration rate of 64.4±15.11 nmol O₂ h⁻¹ ind⁻¹. Mass-specific respiration rates of hatched larvae (approximately 1–2 months old) were calculated using dry weights (DW) and averaged 16.1±3.4 nmol O₂ h⁻¹ mg⁻¹ DW. Adult dragonfish respiration rates (corrected for a 100 g fish and using a 0.8 scaling exponent) averaged 0.91±0.36 mmol O₂ kg⁻¹ h⁻¹ after a 48 h acclimatization period, which is not indicative of significant metabolic cold adaptation. The energy contents of dragonfish eggs and larvae were also measured by microbomb calorimetry and used, along with the respiration data, in an initial approach to estimate an energy budget. In order to balance the budget, the bulk of the available post-gastrulation respiratory energy (during 213 days of embryonic incubation) must be consumed at a relatively low average rate (7.1 nmol O₂ h⁻¹ ind⁻¹), which supports the possibility that advanced dragonfish embryos overwinter in a relatively quiescent metabolic state while awaiting a suitable stimulus (such as the return of the sun) to initiate hatching.     

Characterization of the Mn2+-stimulated (di)adenosine polyphosphate hydrolase encoded by the Deinococcus radiodurans DR2356 nudix gene

The DR2356 nudix hydrolase gene from Deinococcus radiodurans has been cloned and the product expressed as an 18 kDa histidine-tagged protein. The enzyme hydrolysed adenosine and diadenosine polyphosphates, always generating ATP as one of the initial products. ATP and other (deoxy)nucleoside triphosphates were also substrates, yielding (d)NDP and Pi as products. The DR2356 protein was most active at pH 8.6–9.0 and showed a strong preference for Mn²⁺ as activating cation. Mg²⁺ ions at 15 mM supported only 5% of the activity achieved with 2 mM Mn²⁺. K _m and k _cat values for diadenosine tetra-, penta- and hexaphosphates were 2.0, 2.4 and 1.1 μM and 11.4, 28.6 and 12.0 s⁻¹, respectively, while for GTP they were 20.3 μM and 1.8 s⁻¹, respectively. The K _m for adenosine 5′-pentaphosphate was <1 μM. Expression analysis showed the DR2356 gene to be induced eight- to ninefold in stationary phase and in cells subjected to slow dehydration plus rehydration. Superoxide (but not peroxide) treatment and rapid dehydration caused a two-to threefold induction. The Mn-requirement and induction in stationary phase suggest that DR2356 may have a specific role in maintenance mode metabolism in stationary phase as Mn²⁺ accumulates.     

Nitrogen fixation by the benthic freshwater cyanobacterium Lyngbya wollei

The ability of the benthic cyanobacterium Lyngbya wollei to fix nitrogen was studied using field samples and axenic cultures. L. wollei was collected and isolated from Lake Okeechobee, Florida, where it forms extensive mats. Rates of acetylene reduction up to 39.1 nmol mg dry wt⁻¹ h⁻¹ were observed for field samples. The maximum observed rate of acetylene reduction in axenic laboratory cultures was 200 nmol mg dry wt⁻¹ h⁻¹. Aerobic conditions limited nitrogen fixation activity, but dark/light cycles promoted the development of activity. Reduced oxygen levels appeared to be required for the development of significant levels of nitrogenase activity. The level of irradiance also had a significant impact on the level of activity. The potential significance of nitrogen fixation to Lyngbya production is discussed.     

Purification and characterization of dihydroorotase fromPseudomonas putida

Dihydroorotase was purified to homogeneity fromPseudomonas putida. The relative molecular mass of the native enzyme was 82 kDa and the enzyme consisted of two identical subunits with a relative molecular mass of 41 kDa. The enzyme only hydrolyzed dihydro-l-orotate and its methyl ester, and the reactions were reversible. The apparentK _m andV _max values for dihydro-l-orotate hydrolysis (at pH 7.4) were 0.081 mM and 18 μmol min⁻¹ mg⁻¹, respectively; and those forN-carbamoyl-dl-aspartate (at pH 6.0) were 2.2 mM and 68 μmol min⁻¹ mg⁻¹, respectively. The enzyme was inhibited by metal ion chelators and activated by Zn²⁺. However, excessive Zn²⁺ was inhibitory. The enzyme was inhibited by sulfhydryl reagents, and competitively inhibited byN-carbamoylamino acids such asN-carbamoylglycine, with aK _i value of 2.7 mM. The enzyme was also inhibited noncompetitively by pyrimidine-metabolism intermediates such as dihydrouracil and orotate, with aK _i value of 3.4 and 0.75 mM, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates and that dihydroorotase plays a role in the control of pyrimidine biosynthesis.     

Nitrite-driven anaerobic ATP synthesis in barley and rice root mitochondria

Mitochondria isolated from the roots of barley (Hordeum vulgare L.) and rice (Oryza sativa L.) seedlings were capable of oxidizing external NADH and NADPH anaerobically in the presence of nitrite. The reaction was linked to ATP synthesis and nitric oxide (NO) was a measurable product. The rates of NADH and NADPH oxidation were in the range of 12–16 nmol min⁻¹ mg⁻¹ protein for both species. The anaerobic ATP synthesis rate was 7–9 nmol min⁻¹ mg⁻¹ protein for barley and 15–17 nmol min⁻¹ mg⁻¹ protein for rice. The rates are of the same order of magnitude as glycolytic ATP production during anoxia and about 3–5% of the aerobic mitochondrial ATP synthesis rate. NADH/NADPH oxidation and ATP synthesis were sensitive to the mitochondrial inhibitors myxothiazol, oligomycin, diphenyleneiodonium and insensitive to rotenone and antimycin A. The uncoupler FCCP completely eliminated ATP production. Succinate was also capable of driving ATP synthesis. We conclude that plant mitochondria, under anaerobic conditions, have a capacity to use nitrite as an electron acceptor to oxidize cytosolic NADH/NADPH and generate ATP.     

Nitrogen fixation by the woody legumeLeucaena leucocephala in Tanzania

Summary The nitrogen fixation rate in a 4-year-old stand of the woody legumeLeucaena leucocephala (Lam.) de Wit. was estimated in the field at a rather dry site in Tanzania by use of an acetylene reduction technique. The diurnal mean value during April–May was 35 nmol C₂H₄ mg^–1 (dry weight) nodules h^–1, with a variation between 22±8 and 48±12 nmol C₂H₄ mg^–1 (dry weight) nodules h^–1 in early morning and at midday, respectively. The nodule biomass was determined by auger sampling to be 51±16 kg (dry weight) ha^–1. Most of the nodules were found at the 10–30 cm soil depth level. A rough calculation of the amount of nitrogen fixed annually arrived at 110±30 kg ha^–1. The results give strong support for the use ofL. leucocephala for soil enrichment in less humid areas of tropical Africa.     

Improvement of artemisinin production by chitosan in hairy root cultures of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

Artemisinin production by hairy roots of Artemisia annua L. was increased 6-fold to 1.8 μg mg⁻¹ dry wt over 6 days by adding 150 mg chitosan l⁻¹. The increase was dose-dependent. Similar treatment of hairy roots with methyl jasmonate (0.2 mM) or yeast extract (2 mg ml⁻¹) increased artemisinin production to 1.5 and 0.9 μg mg⁻¹ dry wt, respectively.     

Impact of oxygen supply on rtPA expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 (DE3): ammonia effects

A phytase with high activity at neutral pH and typical water temperatures (∼25°C) could effectively hydrolyze phytate in aquaculture. In this study, a phytase-producing strain, Pedobacter nyackensis MJ11 CGMCC 2503, was isolated from glacier soil, and the relevant gene, PhyP, was cloned using degenerate PCR and thermal asymmetric interlaced PCR. To our knowledge, this is the first report of detection of phytase activity and cloning of phytase gene from Pedobacter. PhyP belongs to beta-propeller phytase family and shares very low identity (∼28.5%) with Bacillus subtilis phytase. The purified recombinant enzyme (r-PhyP) from Escherichia coli displayed high specific activity for sodium phytate of 24.4 U mg⁻¹. The optimum pH was 7.0, and the optimum temperature was 45°C. The K _m, V _max, and k _cat values were 1.28 mM, 71.9 μmol min⁻¹ mg⁻¹, and 45.1 s⁻¹, respectively. Compared with Bacillus phytases, r-PhyP had higher relative activity at 25°C (r-PhyP (>50%), B. subtilis phytase (<8%)) and hydrolyzed phytate from soybean with greater efficacy at neutral pH. These characteristics suggest that r-PhyP might be a good candidate for an aquatic feed additive in the aquaculture industry.     

Ouabain-insensitive Na-ATPase activity of Malpighian tubules from Rhodnius prolixus

In the present paper, we show the existence of a furosemide-sensitive Na⁺-stimulated, Mg²⁺-dependent ATPase activity in cell lysates of Malpighian tubular cells from Rhodnius prolixus, which could be the biochemical expression of the Na⁺-pump. The main characteristics of this activity are: (1) K_0.5 for Na⁺=1.49±0.18 mM, (2) V_max=2.8±0.1 nmol inorganic orthophosphate (Pi)·mg prot⁻¹·min⁻¹, (3) it is fully abolished by 2 mM furosemide, (4) it is insensitive to ouabain concentrations up to 10⁻² M, (5) it is sensitive to the presence of vanadate in the incubation medium indicating it to be a P-type ATPase, and (6) it is stimulated by nanomolar concentrations of Ca²⁺ in the incubation medium.     

Influence of agitation and aeration on growth and antibiotic production by <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

The effect of agitation and aeration on the growth and antibiotic production by Xenorhabdus nematophila YL001 grown in batch cultures were investigated. Efficiency of aeration and agitation was evaluated through the oxygen mass transfer coefficient (K _L a). With increase in K _L a, the biomass and antibiotic activity increased. Activity units of antibiotic and dry cell weight were increased to 232 U ml⁻¹ and 19.58 g l⁻¹, respectively, productivity in cell and antibiotic was up more than 30% when K _L a increased from 115.9 h⁻¹ to 185.7 h⁻¹. During the exponential growth phase, DO concentration was zero, the oxygen supply was not sufficient. So, based on process analysis, a three-stage oxygen supply control strategy was used to improved the DO concentration above 30% by controlling the agitation speed and aeration rate. The dry cell weight and activity units of antibiotic were further increased to 24.22 g l⁻¹ and 249 U ml⁻¹, and were improved by 24.0% and 7.0%, compared with fermentation at a constant agitation speed and a constant aeration rate (300 rev min⁻¹, 2.5 l min⁻¹).     

Freshwater to seawater acclimation of juvenile bull sharks (<Emphasis Type="Italic">Carcharhinus leucas</Emphasis>): plasma osmolytes and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in gill,rectal gland,kidney and intestine

This study examined the osmoregulatory status of the euryhaline elasmobranch Carcharhinus leucas acclimated to freshwater (FW) and seawater (SW). Juvenile C. leucas captured in FW (3 mOsm l^–1 kg^–1) were acclimated to SW (980–1,000 mOsm l^–1 kg^–1) over 16 days. A FW group was maintained in captivity over a similar time period. In FW, bull sharks were hyper-osmotic regulators, having a plasma osmolarity of 595 mOsm l^–1 kg^–1. In SW, bull sharks had significantly higher plasma osmolarities (940 mOsm l^–1 kg^–1) than FW-acclimated animals and were slightly hypo-osmotic to the environment. Plasma Na⁺, Cl^–, K⁺, Mg²⁺, Ca²⁺, urea and trimethylamine oxide (TMAO) concentrations were all significantly higher in bull sharks acclimated to SW, with urea and TMAO showing the greatest increase. Gill, rectal gland, kidney and intestinal tissue were taken from animals acclimated to FW and SW and analysed for maximal Na⁺/K⁺-ATPase activity. Na⁺/K⁺-ATPase activity in the gills and intestine was less than 1 mmol Pi mg^–1 protein h^–1 and there was no difference in activity between FW- and SW-acclimated animals. In contrast Na⁺/K⁺-ATPase activity in the rectal gland and kidney were significantly higher than gill and intestine and showed significant differences between the FW- and SW-acclimated groups. In FW and SW, rectal gland Na⁺/K⁺-ATPase activity was 5.6±0.8 and 9.2±0.6 mmol Pi mg^–1 protein h^–1, respectively. Na⁺/K⁺-ATPase activity in the kidney of FW and SW acclimated animals was 8.4±1.1 and 3.3±1.1 Pi mg^–1 protein h^–1, respectively. Thus juvenile bull sharks have the osmoregulatory plasticity to acclimate to SW; their preference for the upper reaches of rivers where salinity is low is therefore likely to be for predator avoidance and/or increased food abundance rather than because of a physiological constraint.     

Heterologous expression of polyhydroxyalkanoate depolymerase from <Emphasis Type="Italic">Thermobifida</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and catalytic analysis by surface plasmon resonance

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His₆-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V _max and K _m of 3.63 ± 0.16 μmol min⁻¹ mg⁻¹ and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm⁻² h⁻¹ for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.     

High production of laccase by a new basidiomycete, <Emphasis Type="Italic">Trametes</Emphasis> sp

A new basidiomycete, Trametes sp. 420, produced laccase at 6,810 U l⁻¹ (268 mg, 25.4 U mg⁻¹ protein for guaiacol) in glucose medium and 7,870 U l⁻¹ (310 mg) in cellobiose medium with induction by 0.5 mM Cu²⁺ and 6 mM o-toluidine. Laccase isozyme E (LacE) was the sole laccase in the fermentation products. It was stable at pH 5–9 and below 70°C over 30 min. The K _m values of LacE for four substrates (guaiacol ABTS, 2,6-dimethoxyphenol and syringaldazine) varied from 5 to 245 μM. The activity of LacE was strongly inhibited by NaN₃ but not by EDTA or dimethylsulfoxide. LacE at 0.5 U l⁻¹ could decolorize industrial dyes. The open reading frame of the lacE gene was 2,130 bp and was interrupted by 10 introns. It displayed a high homology to laccases from other fungi. Pingui Tong and Yuzhi Hong contributed equally to the study     

Respiration of individual honeybee larvae in relation to age and ambient temperature

The CO₂ production of individual larvae of Apis mellifera carnica, which were incubated within their cells at a natural air humidity of 60–80%, was determined by an open-flow gas analyzer in relation to larval age and ambient temperature. In larvae incubated at 34 °C the amount of CO₂ produced appeared to fall only moderately from 3.89±1.57 µl mg^–1 h^–1 in 0.5-day-old larvae to 2.98±0.57 µl mg^–1 h^–1 in 3.5-day-old larvae. The decline was steeper up to an age of 5.5 days (0.95±1.15 µl mg^–1 h^–1). Our measurements show that the respiration and energy turnover of larvae younger than about 80 h is considerably lower (up to 35%) than expected from extrapolations of data determined in older larvae. The temperature dependency of CO₂ production was determined in 3.5-day-old larvae, which were incubated at temperatures varying from 18 to 38 °C in steps of 4 °C. The larvae generated 0.48±0.03 µl mg^–1 h^–1 CO₂ at 18 °C, and 3.97±0.50 µl mg^–1 h^–1 CO₂ at 38 °C. The temperature-dependent respiration rate was fitted to a logistic curve. We found that the inflection point of this curve (32.5 °C) is below the normal brood nest temperature (33–36 °C). The average Q₁₀ was 3.13, which is higher than in freshly emerged resting honeybees but similar to adult bees. This strong temperature dependency enables the bees to speed up brood development by achieving high temperatures. On the other hand, the results suggest that the strong temperature dependency forces the bees to maintain thermal homeostasis of the brood nest to avoid delayed brood development during periods of low temperature.Abbreviations m body mass - R rate of development or respiration - T_I inflexion point of a logistic (sigmoid) curve - T_L lethal temperature - T_O temperature of optimum (maximum) developmentCommunicated by G. Heldmaier     

Production of lipase by high cell density fed-batch culture of Candida cylindracea

Candida cylindracea NRRL Y-17506 was grown to produce extracellular lipase from oleic acid as a carbon source. Through flask cultures, it was found that the optimum initial oleic acid concentration for cell growth was 20 g l⁻¹. However, high initial concentrations of oleic acid up to 50 g l⁻¹ were not inhibitory. The highest extracellular lipase activity obtained in flask culture was 3.0 U ml⁻¹ after 48 h with 5 g l⁻¹ of initial oleic acid concentration. Fed-batch cultures (intermittent and stepwise feeding) were carried out to improve cell concentration and lipase activity. For the intermittent feeding fed-batch culture, the final cell concentration was 52 g l⁻¹ and the extracellular lipase activity was 6.3 U ml⁻¹ at 138.5 h. Stepwise feeding fed-batch cultures were carried out to simulate an exponential feeding and to investigate the effects of specific growth rate (0.02, 0.04 and 0.08 h⁻¹) on cell growth and lipase production. The highest final cell concentration obtained was 90 g l⁻¹ when the set point of specific growth rate (μ_set) was 0.02 h⁻¹. High specific growth rate (0.04 and 0.08 h⁻¹) decreased extracellular lipase production in the later part of fed-batch cultures due to build-up of the oleic acid oversupplied. The highest extracellular lipase activity was 23.7 U ml⁻¹ when μ_set was 0.02 h⁻¹, while the highest lipase productivity was 0.31 U ml⁻¹ h⁻¹ at μ_set of 0.08 h⁻¹.     

Production of inulinase by solid-state fermentation: effect of process parameters on production and preliminary characterization of enzyme preparations

This work was aimed at producing inulinase by solid-state fermentation of sugarcane bagasse, using factorial design to identify the effect of corn steep liquor (CSL) and soybean bran concentration, particle size of bagasse and size of inoculum. Maximum inulinase activity achieved was 250 U per g of dry substrate (gds) at 20% (w/w) of CSL, 5% (w/w) of soybean bran, 1 × 10¹⁰ cells mL⁻¹ and particle size of bagasse in the range 9/32 mesh. The use of soybean bran decreased the time to reach maximum activity from 96 to 24 h and the maximum productivity achieved was 8.87 U gds⁻¹ h⁻¹. The maximum activity was obtained at pH 5.0 and 55.0°C. Within the investigated range, the enzyme extract was more thermostable at 50.0°C, showing a D-value of 123.1 h and deactivation energy of 343.9 kJ gmol⁻¹. The extract showed highest stability from pH 4.5 to 4.8. Apparent K _m and V _max are 7.1 mM and 17.79 M min⁻¹, respectively.     

Prospects of using marine actinobacteria as probiotics in aquaculture

In the present study, optimum culture conditions for the production of extracellular polysaccharides (EPS) in submerged culture of an edible mushroom, Laetiporus sulphureus var. miniatus and their stimulatory effects on insulinoma cell (RINm5F) proliferation and insulin secretion were investigated. The maximum mycelial growth (4.1 g l⁻¹) and EPS production (0.6 g l⁻¹) in submerged flask culture were achieved in a medium containing 30 g l⁻¹ maltose, 2 g l⁻¹ soy peptone, and 2 mM MnSO₄·5H₂O at an initial pH 2.0 and temperature 25°C. In the stirred-tank fermenter under optimized medium, the concentrations of mycelial biomass and EPS reached a maximum level of 8.1 and 3.9 g l⁻¹, respectively. Interestingly, supplementation of deep sea water (DSW) into the culture medium significantly increased both mycelial biomass and EPS production by 4- and 6.7-fold at 70% (v/v) DSW medium, respectively. The EPS were proved to be glucose-rich polysaccharides and were able to increase proliferation and insulin secretary function of rat insulinoma RINm5F cells, in a dose-dependent manner. In addition, EPS also strikingly reduced the streptozotocin-induced apoptosis in RINm5F cells indicating the mode of the cytoprotective role of EPS on RINm5F cells.     

Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions

Background

The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes.

Methods

We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-³²P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation.

Results

This activity was linear with time up to 20 min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5 mM MgCl₂ was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1 mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN₃) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN₃. The dose–response of ATP revealed a hyperbolic profile with maximal velocity of 25.2 ± 1.2 nmol Pi x mg^− 1 x min^− 1 and K_0.5 of 0.07 ± 0.01 mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60 min of ischemia.

Conclusion

Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia.

General Significance

This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.