Extracellular Alkaline Phosphatase Activity Research Articles

TheLPB1Gene Is Important for Acclimation ofChlamydomonas reinhardtiito Phosphorus and Sulfur Deprivation

Organisms exhibit a diverse set of responses when exposed to low-phosphate conditions. Some of these responses are specific for phosphorus limitation, including responses that enable cells to efficiently scavenge phosphate from internal and external stores via the production of high-affinity phosphate transporters and the synthesis of intracellular and extracellular phosphatases. Other responses are general and occur under a number of different environmental stresses, helping coordinate cellular metabolism and cell division with the growth potential of the cell. In this article, we describe the isolation and characterization of a mutant of Chlamydomonas reinhardtii, low-phosphate bleaching (lpb1), which dies more rapidly than wild-type cells during phosphorus limitation. The responses of this mutant to nitrogen limitation appear normal, although the strain is also somewhat more sensitive than wild-type cells to sulfur deprivation. Interestingly, depriving the cells of both nutrients simultaneously allows for sustained survival that is similar to that observed with wild-type cells. Furthermore, upon phosphorus deprivation, the lpb1 mutant, like wild-type cells, exhibits increased levels of mRNA encoding the PHOX alkaline phosphatase, the PTB2 phosphate transporter, and the regulatory element PSR1. The mutant strain is also able to synthesize the extracellular alkaline phosphatase activity upon phosphorus deprivation and the arylsulfatase upon sulfur deprivation, suggesting that the specific responses to phosphorus and sulfur deprivation are normal. The LPB1 gene was tagged by insertion of the ARG7 gene, which facilitated its isolation and characterization. This gene encodes a protein with strong similarity to expressed proteins in Arabidopsis (Arabidopsis thaliana) and predicted proteins in Oryza sativa and Parachlamydia. A domain in the protein contains some similarity to the superfamily of nucleotide-diphospho-sugar transferases, and it is likely to be localized to the chloroplast or mitochondrion based on programs that predict subcellular localization. While the precise catalytic role and physiological function of the putative protein is not known, it may function in some aspect of polysaccharide metabolism and/or influence phosphorus metabolism (either structural or regulatory) in a way that is critical for allowing the cells to acclimate to nutrient limitation conditions.

Modulation of extracellular matrix synthesis and alkaline phosphatase activity of periodontal ligament cells by mechanical stress

Loss of occlusal function has been reported to induce atrophic changes in the periodontal ligament. It is likely that mechanical stress triggers the biological response of periodontal ligament. However, there have been few reports studying the correlation between mechanical stress of varying magnitude and periodontal ligament cell activities such as extracellular matrix (ECM) synthesis. The objective of this study is to clarify the influence of the mechanical stress on changes in mRNA expression levels of type I collagen and decorin genes, as well as alkaline phosphatase (ALP) activity in response to mechanical stress of varying magnitude. Bovine periodontal ligament cells were cultured on flexible-bottomed culture plates and placed on the BioFlex Loading Stations. Cells were elongated at 6 cycles/min (5 s on and 5 s off) at each of six levels of stretch (0.2, 1.0, 2.0, 3.0, 10, 18% increase in the surface area of the bottom) for 48 h. We measured mRNA expression levels of type I collagen and decorin genes using quantitative reverse transcription-polymerase chain reaction (RT-PCR), and ALP activity in periodontal ligament cell culture under cyclic mechanical stretching. Mechanical tensional stress of low magnitude induced the increase of both type I collagen and decorin mRNA expression without changing ALP activity in periodontal ligament cells. Mechanical tensional stress of high magnitude induced the increase of type I collagen and decorin mRNA expression while decreasing ALP activity. These results suggest that different magnitude of tensional force induces different responses from periodontal ligament cells, and that mechanical stress plays an important role in remodeling and functional regulation of periodontal ligament.

Improved use of organic phosphate by Skeletonema costatum through regulation of Zn2+ concentrations.

The maximum growth rate (1.4-2 x 10(5) cells ml(-1) d(-1)), cell final yields (2.6-5.2 x 10(5) cells ml(-1)) and extracellular alkaline phosphatase activity (2.4-10.6 microg phosphate released ml(-1) h(-1)) of the red tide alga, Skeletonema costatum, increased when Zn2+ was increased from 0 to 24 pM, but decreased with 66 pM Zn2+ in growth medium with glycerophosphate as the sole phosphorus source. Extracellular carbonic anhydrase activity and the affinity for HCO3- and CO2 uptake increased when Zn2+ was increased from 0 to 12 pM, but then decreased at higher concentrations. The results suggested that utilization of organic phosphate required more Zn2+ than the uptake of inorganic carbon did, while utilization of dissolved inorganic carbon by Skeletonema costatum was very sensitive to Zn2+ concentration variations.

Phosphatase activity with reference to bacteria and phosphorus in tropical freshwater aquaculture pond systems

The bio-geochemical cycle of phosphorus is significantly influenced by microbes in the aquatic environment. Organic phosphorus compounds are decomposed and mineralized by enzymatic complexes such as phosphatases produced by microbes. Enzymatic catalysis results in the production of orthophosphate, which can be used readily by primary producers. Even the smallest concentration of phosphate in water has an influence over the production process in aquaculture systems. Extracellular alkaline phosphatase activity was observed in water and sediment media of aquaculture ponds with different management practices. Heterotrophic bacterial populations as well as phosphatase-producing bacterial populations were higher in sediments compared with water. In the freshwater fish ponds, Bacillus spp. were the dominant forms of bacteria producing phosphatase. The alkaline phosphatase activity of sediment was always higher than that of water. The partitioning of extracellular alkaline phosphatase in pond water by a 0.22-µm membrane filter revealed that a proportion was often free rather than cell associated and might have originated as free enzymes released by enriched sediments or by fish or microbes. In the case of water, although the dissolved alkaline phosphatase activity was lower than the total alkaline phosphatase activity, the former was nevertheless unimportant, as it constituted about 20% of the ‘total’ activity. Free alkaline phosphatase activity shared a negative correlation with the orthophosphate concentration of water, whereas gross alkaline phosphatase activity was positively correlated with the total phosphorus and bacterial population of water.

DsbA and DsbC affect extracellular enzyme formation in Pseudomonas aeruginosa.

DsbA and DsbC proteins involved in the periplasmic formation of disulfide bonds in Pseudomonas aeruginosa were identified and shown to play an important role for the formation of extracellular enzymes. Mutants deficient in either dsbA or dsbC or both genes were constructed, and extracellular elastase, alkaline phosphatase, and lipase activities were determined. The dsbA mutant no longer produced these enzymes, whereas the lipase activity was doubled in the dsbC mutant. Also, extracellar lipase production was severely reduced in a P. aeruginosa dsbA mutant in which an inactive DsbA variant carrying the mutation C34S was expressed. Even when the lipase gene lipA was constitutively expressed in trans in a lipA dsbA double mutant, lipase activity in cell extracts and culture supernatants was still reduced to about 25%. Interestingly, the presence of dithiothreitol in the growth medium completely inhibited the formation of extracellular lipase whereas the addition of dithiothreitol to a cell-free culture supernatant did not affect lipase activity. We conclude that the correct formation of the disulfide bond catalyzed in vivo by DsbA is necessary to stabilize periplasmic lipase. Such a stabilization is the prerequisite for efficient secretion using the type II pathway.

Extracellular alkaline phosphatase activity as a possible marker for wound healing: A preliminary report

Purpose: This study evaluated the use of extracellular alkaline phosphatase activity as a marker of wound healing. Materials and Methods: Fifteen 8-week-old Wistar rats, weighing 180 to 220 g and of both sexes, made up the material of this study. Histopathologic aspects of the healing of full-thickness of wounds created on the dorsal skin of these rats were studied by means of alkaline phosphatase (ALP) enzyme histochemistry and routine microscopy. Results: The current study showed an exclusive localization of extracellular ALP activity in the regions of granulation tissue formation. This localization was time related, and decreased as healing progressed. Conclusions: Although the biologic significance of ALP is still obscure, use of extracellular ALP activity as a simple and reliable histochemical process marker of the skin wound healing is proposed.

Extracellular alkaline phosphatase activity as a possible marker for wound healing: A preliminary report

Extracellular alkaline phosphatase activity as a possible marker for wound healing: A preliminary report

Biochemical characterization of the extracellular phosphatases produced by phosphorus-deprived Chlamydomonas reinhardtii.

We have examined the extracellular phosphatases produced by the terrestrial green alga Chlamydomonas reinhardtii in response to phosphorus deprivation. Phosphorus-deprived cells increase extra-cellular alkaline phosphatase activity 300-fold relative to unstarved cells. The alkaline phosphatases are released into the medium by cell-wall-deficient strains and by wild-type cells after treatment with autolysin, indicating that they are localized to the periplasm. Anion-exchange chromatography and analysis by nondenaturing polyacrylamide gel electrophoresis revealed that there are two major inducible alkaline phosphatases. A calcium-dependent enzyme composed of 190-kD glycoprotein subunits accounts for 85 to 95% of the Alkaline phosphatase activity. This phosphatase has optimal activity at pH 9.5 and a Km of 120 to 262 microns for all physiological substrates tested, with the exception of phytic acid, which it cleaved with a 50-fold lower efficiency. An enzyme with optimal activity at pH 9 and no requirement for divalent cations accounts for 2 to 10% of the alkaline phosphatase activity. This phosphatase was only able to efficiently hydrolyze arylphosphates. The information reported here, in conjunction with the results of previous studies, defines the complement of extracellular phosphatases produced by phosphorus-deprived Chlamydomonas cells.

P depletion and activity of phosphatases in the rhizosphere of mycorrhizal and non-mycorrhizal cucumber ( cucumis sativus L.)

An experiment was set up to test the ability of arbuscular mycorrhizal (AM) roots and hyphae to produce extracellular phosphatases and to study the relationship between phosphatase activity and soil organic P (P o). Non-mycorrhizal cucumber and cucumber in symbiosis with either of two mycorrhizal fungi were grown in a sandy loam-sand mixture in three-compartment pots. Plant roots were separated from two consecutively adjoining compartments, first by a 37 m mesh excluding roots and subsequently by a 0.45 m membrane excluding mycorrhizal hyphae. Soil from the two root-free compartments was sectioned in a freezing microtome and analyzed for extracellular acid (pH 5.2) and alkaline (pH 8.5) phosphatase activity as well as depletion of NaHCO 3-extractable inorganic P (P i) and P o. Roots and mycorrhizal hyphae depleted the soil of P i but did not influence the concentration of P o in spite of increased phosphatase activity in soil influenced by roots. Phosphatase activity at both pH values was highest in soil influenced by uncolonized roots, but this was attributed to higher root length densities as compared to mycorrhizal roots. Mycorrhizal hyphae showed no influence on soil phosphatase activity in spite of high hyphal length densities (> 22 m cm −3). Hyphae were also able to deplete soil of P i beyond the membrane interface.

Comparison of phosphorus deficiency indices during a spring phytoplankton bloom in a eutrophic reservoir

SUMMARY1. Phosphorus limitation was studied along the eutrophic, canyon‐type Římov reservoir (Czech Republic) during a spring phytoplankton bloom. Concentration of soluble reactive phosphorus (SRP), C:P molar ratio in seston, extracellular alkaline phosphatase activity (APA), and P limitation (bioassay) were used as indices for phosphorus deficiency in the phytoplankton.2. SRP, C:P, APA, and P limitation indicated a moderate P deficiency in the downstream, but not upper, part of the reservoir.3. Significant correlations between these parameters were found in the downstream part. Chlorophyll a concentration correlated with APA and P limitation in the upper part.4. APA was significantly enhanced in the phosphorus‐deficient phytoplankton. However, APA was apparently not related to total biomass or species composition of the phytoplankton.5. Generally, APA was closely correlated with pH in the reservoir. However, extracellular alkaline phosphatases, with a pH optimum above 9.0, were induced and active only during the phytoplankton bloom, whereas low background activity of extracellular phosphatases was found at low chlorophyll a concentrations (winter, clear‐water phase).

Extracellular alkaline phosphatase activity in mineralizing matrices of cartilage and bone: ultrastructural localization using a cerium-based method.

The ultrastructural localization of alkaline phosphatase (A1P) activity has been demonstrated in epiphyseal growth cartilage and metaphyseal bone of rats. Epiphyso-metaphyseal specimens were decalcified with EDTA and treated with MgCl2 to regenerate the enzymatic activity before incubation in a medium containing beta-glycerophosphate, MgCl2 and CeCl3. A1P activity was present on the outer surface of the plasmamembrane of maturing and hypertrophic chondrocytes and of osteoblasts. Moreover, the reaction product was present in chondrocyte lacunae, in matrix vesicles, and in cartilage matrix, as well as among uncalcified collagen fibrils of osteoid tissue in bone. The intensity of reaction was the lowest, or completely lacking, where the degree of matrix calcification was the highest. These results suggest that alkaline phosphatase is transported from the cells into the cartilage and bone matrix by its association with matrix vesicles and plasmamembrane components, and that its activity in cartilage and bone matrix is inhibited as it is incorporated in the mineral substance.

Phosphatase activity during development cycle of Myxococcus xanthus

Cell-bound and extracellular acid and alkaline phosphatase activity have been studied in Myxococcus xanthus strains DK101, DK1050, DK2834, and DK2836. Both phosphatases were released into a liquid medium during vegetative growth and the levels were similar in all strains. On solid media, M. xanthus DK101 showed maximum activity at the end of the developmental process, when mature myxospores appeared. An increase in phosphatase activity was also observed in glycerol-induced myxospores. A transitory increase in phosphatase activity occurred during the germination of both glycerol-induced and fruiting-body myxospores, although the activity of both phosphatases in fruiting-body myxospores was greater than that in glycerol-induced ones. Key words: acid phosphatase, alkaline phosphatase, Myxococcus xanthus.

In vivo biocompatibility of aliphatic segmented polyurethane in rabbit

An aliphatic segmented polyurethane with soft to hard segment ratio 3 was synthesised using hexamethylene diisocyanate, polypropylene glycol 400 and 1,4-butane diol.A stainless steel cage implant system has been used to study the in vivo biocompatibility of this polyurethane. United States Pharmacopoeia negative control polyethylene was used for the comparison. Three cages, one with polyurethane another with United States Pharmacopoeia polyethylene and the third control empty cage were implanted subcutaneously in the dorsal aspect of rabbits. The inflammatory exudate surrounding the material was aspirated from the cages on 4, 7, 14 and 21 days after implantation. The total protein content in the exudate aspirated from all the 3 cages was significantly higher at 7 days than in the reported normal rabbit serum of New Zealand white rabbit but equal to that of our rabbit colony. The albumin concentration was lower in the initial period but increased at 21 days post implantation period in all the cages. Concentration of α1, α2 and γ-globulin also decreased in all cages at 21 days. Neutrophils were predominant in all the exudates aspirated from polyurethane, polyethylene and empty control cages during whole implantation period. This is attributed to the profound effect of the cages on the surrounding vasculature. Macrophage was found to be seen during acute phase of inflammation due to the migration of macrophage along with neutrophil towards the inflammatory lesion. The percentage of neutrophils showed a faster decline in the cage containing polyethylene at 21 days. The extra cellular alkaline phosphatase activity, though higher in exudate from cages containing polyurethane at 14 days post implantation, was same in all 3 cages at 21 days. Leucine amino peptidase activity was found to be decreased at 21 days of post implantation time though the empty control cage exhibited an increase at 14 days post implantation. The inflammatory response at 21 days was similar in polyurethane and the control polyethylene.

Extracellular alkaline phosphatase activity in Ulva lactuca L.

Extracellular alkaline phosphatase activity (APA) was detected in the marine macroalga Ulva lactuca, cultivated in laboratory conditions under phosphate limitation. Algal discs were grown in continuous light and in darkness for a maximum of 205 h in sterilized seawater supplied with ammonium nitrate and various phosphate concentrations. APA and phosphate uptake in U. lactuca was stimulated in light whereas inhibition was detected in darkness. APA increased significantly during the cultivation in a phosphate-deficient medium but was suppressed at high external phosphate concentrations. In addition, pretreatment in penicillin over 48 h markedly enhanced enzyme activity during P limitation. The pretreatment in penicillin reduced the number of surface-associated bacteria. Our results indicate that APA in U. lactuca was dependent on the external phosphate concentration, energy from photophosphorylation and bacteria. Since intracellular polyphosphates and an orthophosphate pool still were displayed in 31P NMR spectra after cultivation at phosphate-limiting conditions, P deficiency was not suggested. Unfortunately, NMR spectra reflect total amounts of P compounds and local differences in the P status of the multicellular tissue could not be distinguished. Postulating a negative correlation between the P status and APA, a lower P status in meristematic areas during P limitation was suggested. Alkalisation of the growth medium during algal growth in light, and/or intracellular pH changes may additionally have affected enzyme production.

In vivo biocompatibility of catheter materials

The inflammatory response to four different catheter materials was quantitatively characterized using an in vivo cage implant system over a 21 d implantation period. The greatest differences between materials were observed at day 4, where the total leucocyte concentration in the exudate decreased in the following order: AE-PVC > A-PU > D-PU1 > D-PU2. Similar trends were observed for the polymorphonuclear leucocyte (PMN) and macrophage concentrations at day 4. At day 7, this trend continued, but by day 21 the total leucocyte concentration, PMN concentration and macrophage concentration had decreased to comparable values for all materials. The extracellular alkaline phosphatase activity at day 4 was lowest for the AE-PVC exudate samples. Similar results were observed for the extracellular acid phosphatase activity but after that time point no significant differences were observed. From these results, it appears that AE-PVC is least biocompatible compared with the other three catheter materials, which were polyurethanes.

Optimized extracellular production of alkaline phosphatase by lky mutants of Escherichia coli K12

Periplasmic-leaky (lky) Hfr mutant strains of Escherichia coli K12, grown in low-phosphate Tris medium, excreted alkaline phosphatase (AP) into the extracellular fluid. The lky207 mutation, which proved to induce the highest AP excretion rate, was transferred to an F- host, carrying a phoS, T mutation allowing constitutive AP biosynthesis. Use of high-phosphate LB-rich medium for growing this F- lky strain improved cell biomass, extracellular AP activity and excretion specificity in favour of the enzyme. Physiological studies helped us to develop a new culture medium (LB 8.3) giving higher enzyme and excretion yields. LB 8.3 medium also increased cell viability of lky mutants stored at 4° C. Using optimized culture conditions, the highest extracellular enzyme activity produced by lky mutant 706 was reached in the late stationary growth phase and was equal to 1,400 U/ml of culture medium (i.e., 6 times the intracellular AP content of wild-type strain, Ga15, developed in derepressed conditions); AP released into the extracellular fluid corresponded to 34% of total excreted proteins and was equivalent to a purified enzyme preparation.

Studies on the Extracellular Alkaline Phosphatase of Micrococcus sodonensis: II. FACTORS AFFECTING SECRETION

The secretion of alkaline phosphatase by Micrococcus sodonensis has been studied by observing the production and accumulation of extracellular enzyme by log phase cells which have been resuspended in fresh growth medium. The accumulation of extracellular alkaline phosphatase activity is the result of a selective permeation process and is totally dependent upon the presence of divalent cation. Although calcium is an actual component of the enzyme and is required for both catalytic activity and enzyme stability, magnesium appears to be the divalent cation required for the actual synthesis or release (or both) of alkaline phosphatase from the cell. Studies with various inhibitors of protein synthesis suggest that the bacterial cell may possess at least two distinct sites of protein synthesis, one site functioning to provide intracellular and cell-associated proteins and another for the synthesis of extracellular proteins. The over-all process of enzyme secretion is markedly temperature-dependent and is characterized by an activation energy of 55 kcal per mole. This observation suggests that the rate-limiting step in alkaline phosphatase secretion is probably not an enzyme-catalyzed event. A relatively small but significant amount of alkaline phosphatase activity exists in a cell-bound form and can be quantitatively released from cells by treatment with lysozyme. Although lysozyme-sensitive cell wall components are involved in the binding of alkaline phosphatase to the cell, the cell wall is not an obligatory component of the secretion process since lysozyme-prepared, sucrose-stabilized protoplasts are capable of extracellular enzyme production.