The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia Type 3 pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

Microbial and enzymatic activity in soil after organic composting

Microbial activity and biochemical properties are important indicators of the impact of organic composting on soil. The objective of this study was to evaluate some indicators of soil microbial and biochemical processes after application of compost (household waste). A Typic Acrustox, sampled at a depth of 10 cm under Cerrado biome vegetation, was evaluated in three treatments: control (soil without organic compost amendment) and soil with two doses of domestic organic compost (10 and 20 g kg-1 soil). The following properties were evaluated: released C (C-CO2): microbial respiration 15 days after incubation; microbial biomass C (MBC); total glucose (TG); metabolic quotient (qCO2); and enzyme activity of β-glucosidase and acid and alkaline phosphatase. The application of household compost, at doses of 10 and 20 g kg-1 Typic Acrustox, resulted in significant gains in microbial activity, organic C and C stock, as evidenced by increased MBC and TG levels. On the other hand, qCO2 decreases indicated greater microbial diversity and more efficient energy use. The addition of compost, particularly the 20 g kg-1 dose, strongly influenced the enzyme β-glucosidase and phosphatase (acid and alkaline). The β-glucosidase activity was significantly increased and acid phosphatase activity increased more than the alkaline. The ratio of β-glucosidase to MBC was greater in the control than in the composted treatments which suggests that there were more enzymes in the control than in the substrate or that the addition of compost induced a great MBC increase.

Analysis of the mechanisms controlling the activity of flp1, a chromosomal passenger protein in the fission Schizosaccharomyces pombe

ABSTRACT In S. cerevisiae, the protein phosphatase Cdc14pwt is essential far mitotic exit through its contribution to reducing mitotic CDK activity. But Cdc14pwt also acts as a mare general temporal coordinator of mid and late mitotic events by controlling the partitioning of DNA, microtubule stability and cytokinesis. Cdc14pwt orthologs are well conserved from yeasts to humans, and sequence comparison revealed the presence of three domains, A, B and C, of which A and B form the catalytic domain. Cdc14pwt orthologs are regulated (in part) through cell cycle dependent changes in their localization. Some of them are thought to be kept inactive by sequestration in the nucleolus during interphase. This is the case for flp1pwt, the single identified Cdc14pwt ortholog in the fission yeast S. pombe. In early mitosis, flp1pwt leaves the nucleolus and localizes to the kinetochores, the contractile ring and the mitotic spindle, suggesting that it has multiple substrates and regulates many mitotic processes. flp1D cells show a high chromosome loss rate and septation defects, suggesting a role for flp1wt in the fidelity of chromosome transmission and cytokinesis. The aim of this study is to characterize the mechanisms underlying flp1pwt functions and the control of its activity. A structure-function analysis has revealed that the presence of both A and B domains is required for biological function and for proper flp1pwt mitotic localization. In contrast, the C domain of flp1pwt is responsible for its proper nucleolar localization in G2/interphase. My data suggest that dephosphorylation of substrates by flp1pwt is not necessary for any changes in localization of flp1pwt except that at the medial ring. In that particular case, the catalytic activity of flp1pwt is required for efficient localization, therefore revealing an additional level of regulation. All the functions of flp1pwt assayed to date require its catalytic activity, emphasizing the importance of further identification of its substrates. As described for other orthologs, the capability of selfinteraction and phosphorylation status might help to control flp1pwt activity. My data suggest that flp1pwt forms oligomers in vivo and that phosphorylation is not essential far localization changes of the protein. In addition, the hypophosphorylated form of flp1pwt might be specifically involved in the promotion of cytokinesis. The results of this study suggest that multiple modes of regulation including localization, selfassociation and phosphorylation allow a fine-tuning regulation of flp1pwt phosphatase activity, and more generally that of Cdc14pwt family of phosphatases. RESUME Chez la levure S. cerevisiae, la protéine phosphatase Cdc14pwt est essentielle pour la sortie de mitose du fait de sa contribution dans la réduction d'activité des CDK mitotiques. Comme elle contrôle également le partage de l'ADN, la stabilité des microtubules et la cytokinèse, Cdc14pwt est en fait considérée comme un coordinateur temporel général des évènements de milieu et de fin de mitose. Les orthologues de Cdc14pwt sont bien conservés, des levures jusqu'à l'espèce humaine. Des comparaisons de séquence ont révélé la présence de trois domaines A, B et C, les deux premiers constituant le domaine catalytique. Ils sont régulés (en partie) via des changements dans leur localisation, eux-mêmes dépendants du cycle cellulaire. Plusieurs de ces orthologues sont supposés inactivés par séquestration dans le nucléole en interphase, ce qui est le cas de flp1pwt le seul orthologue de Cdc14pwt identifié chez la levure fissipare S, pombe. En début de mitose, flp1pwt quitte le nucléole et localise au niveau des kinetochores, de l'anneau contractile d'actine et du fuseau mitotique, ce qui laisse supposer de multiples substrats et fonctions. Comme les cellules délétées pour le gène flp1wt présentent un taux élevé de perte de chromosome et des défauts de septation, flp1pwt semble jouer un rôle dans la fidélité de la transmission du matériel génétique et la cytokinèse. Le but de cette étude est de caractériser les mécanismes impliqués dans les fonctions assurées par flp1pwt d'une part, et dans le contrôle de son activité d'autre part. Une analyse structure-fonction a révélé que la présence simultanée des deux domaines A et B est requise pour la fonction biologique de flp1pwt et sa localisation correcte pendant la mitose. Par contre, le domaine C de flp1pwt confère une localisation nucléolaire adéquate en G2/interphase. Mes données suggèrent que la déphosphorylation de substrats par flp1pwt est dispensable pour sa localisation correcte excepté celle à l'anneau médian, qui requiert dans ce cas, l'activité catalytique de flp1pwt, révélant ainsi un niveau de régulation supplémentaire. Toutes les fonctions de flp1 pwt testées jusqu'à présent nécessitent également son activité catalytique, ce qui accentue l'importance de l'identification future de ses substrats. Comme cela a déjà été décrit pour d'autres orthologues, la capacité d'auto-intéraction et le niveau de phosphorylation pourraient contrôler l'activité de flp1pwt. En effet, mes données suggèrent que flp1pwt forme des oligomères in vivo et que la phosphorylation n'est pas essentielle pour les changements de localisation observés pour la protéine. De plus, la forme hypophosphorylée de flp1pwt pourrait être spécifiquement impliquée dans la promotion de la cytokinèse. De multiples modes de régulation incluant la localisation, l'auto-association et la phosphorylation semblent permettre un contrôle fin et subtil de l'activité de la phosphatase flp1pwt, et plus généralement celle des protéines de la famille de Cdc14pwt.

Novel acid phosphatase in Candida glabrata suggests selective pressure and niche specialization in the phosphate signal transduction pathway.

Evolution through natural selection suggests unnecessary genes are lost. We observed that the yeast Candida glabrata lost the gene encoding a phosphate-repressible acid phosphatase (PHO5) present in many yeasts including Saccharomyces cerevisiae. However, C. glabrata still had phosphate starvation-inducible phosphatase activity. Screening a C. glabrata genomic library, we identified CgPMU2, a member of a three-gene family that contains a phosphomutase-like domain. This small-scale gene duplication event could allow for sub- or neofunctionalization. On the basis of phylogenetic and biochemical characterizations, CgPMU2 has neofunctionalized to become a broad range, phosphate starvation-regulated acid phosphatase, which functionally replaces PHO5 in this pathogenic yeast. We determined that CgPmu2, unlike ScPho5, is not able to hydrolyze phytic acid (inositol hexakisphosphate). Phytic acid is present in fruits and seeds where S. cerevisiae grows, but is not abundant in mammalian tissues where C. glabrata grows. We demonstrated that C. glabrata is limited from an environment where phytic acid is the only source of phosphate. Our work suggests that during evolutionary time, the selection for the ancestral PHO5 was lost and that C. glabrata neofunctionalized a weak phosphatase to replace PHO5. Convergent evolution of a phosphate starvation-inducible acid phosphatase in C. glabrata relative to most yeast species provides an example of how small changes in signal transduction pathways can mediate genetic isolation and uncovers a potential speciation gene.

The IBO germination quantitative trait locus encodes a phosphatase 2C-related variant with a nonsynonymous amino acid change that interferes with abscisic acid signaling.

Natural genetic variation is crucial for adaptability of plants to different environments. Seed dormancy prevents precocious germination in unsuitable conditions and is an adaptation to a major macro-environmental parameter, the seasonal variation in temperature and day length. Here we report the isolation of IBO, a quantitative trait locus (QTL) that governs c. 30% of germination rate variance in an Arabidopsis recombinant inbred line (RIL) population derived from the parental accessions Eilenburg-0 (Eil-0) and Loch Ness-0 (Lc-0). IBO encodes an uncharacterized phosphatase 2C-related protein, but neither the Eil-0 nor the Lc-0 variant, which differ in a single amino acid, have any appreciable phosphatase activity in in vitro assays. However, we found that the amino acid change in the Lc-0 variant of the IBO protein confers reduced germination rate. Moreover, unlike the Eil-0 variant of the protein, the Lc-0 variant can interfere with the activity of the phosphatase 2C ABSCISIC ACID INSENSITIVE 1 in vitro. This suggests that the Lc-0 variant possibly interferes with abscisic acid signaling, a notion that is supported by physiological assays. Thus, we isolated an example of a QTL allele with a nonsynonymous amino acid change that might mediate local adaptation of seed germination timing.

Shifts in rhizosphere microbial communities and enzyme activity of Poa alpina across an alpine chronosequence

This study quantifies the influence of Poa alpina on the soil microbial community in primary succession of alpine ecosystems, and whether these effects are controlled by the successional stage. Four successional sites representative of four stages of grassland development (initial, 4 years (non-vegetated); pioneer, 20 years; transition, 75 years; mature, 9500 years old) on the Rotmoos glacier foreland, Austria, were sampled. The size, composition and activity of the microbial community in the rhizosphere and bulk soil were characterized using the chloroform-fumigation extraction procedure, phospholipid fatty acid (PLFA) analysis and measurements of the enzymes beta-glucosidase, beta-xylosidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, acid phosphatase and sulfatase. The interplay between the host plant and the successional stage was quantified using principal component (PCA) and multidimensional scaling analyses. Correlation analyses were applied to evaluate the relationship between soil factors (C-org, N-t, C/N ratio, pH, ammonium, phosphorus, potassium) and microbial properties in the bulk soil. In the pioneer stage microbial colonization of the rhizosphere of P. alpina was dependent on the reservoir of microbial species in the bulk soil. As a consequence, the rhizosphere and bulk soil were similar in microbial biomass (ninhydrin-reactive nitrogen (NHR-N)), community composition (PLFA), and enzyme activity. In the transition and mature grassland stage, more benign soil conditions stimulated microbial growth (NHR-N, total amount of PLFA, bacterial PLFA, Gram-positive bacteria, Gram-negative bacteria), and microbial diversity (Shannon index H) in the rhizosphere either directly or indirectly through enhanced carbon allocation. In the same period, the rhizosphere microflora shifted from a G(-) to a more G(+), and from a fungal to a more bacteria-dominated community. Rhizosphere beta-xylosidase, N-acetyl-beta-glucosaminidase, and sulfatase activity peaked in the mature grassland soil, whereas rhizosphere leucine aminopeptidase, beta-glucosidase, and phosphatase activity were highest in the transition stage, probably because of enhanced carbon and nutrient allocation into the rhizosphere due to better growth conditions. Soil organic matter appeared to be the most important driver of microbial colonization in the bulk soil. The decrease in soil pH and soil C/N ratio mediated the shifts in the soil microbial community composition (bacPLFA, bacPLFA/fungPLFA, G(-), G(+)/G(-)). The activities of beta-glucosidase, beta-xylosidase and phosphatase were related to soil ammonium and phosphorus, indicating that higher decomposition rates enhanced the nutrient availability in the bulk soil. We conclude that the major determinants of the microllora vary along the successional gradient: in the pioneer stage the rhizosphere microflora was primarily determined by the harsh soil environment; under more favourable environmental conditions, however, the host plant selected for a specific microbial community that was related to the dynamic interplay between soil properties and carbon supply. (C) 2004 Elsevier Ltd. All rights reserved.

Shifts in rhizosphere microbial communities and enzyme activity of Poa alpina across an alpine chronosequence

This study quantifies the influence of Poa alpina on the soil microbial community in primary succession of alpine ecosystems, and whether these effects are controlled by the successional stage. Four successional sites representative of four stages of grassland development (initial, 4 years (non-vegetated); pioneer, 20 years; transition, 75 years; mature, 9500 years old) on the Rotmoos glacier foreland, Austria, were sampled. The size, composition and activity of the microbial community in the rhizosphere and bulk soil were characterized using the chloroform-fumigation extraction procedure, phospholipid fatty acid (PLFA) analysis and measurements of the enzymes beta-glucosidase, beta-xylosidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, acid phosphatase and sulfatase. The interplay between the host plant and the successional stage was quantified using principal component (PCA) and multidimensional scaling analyses. Correlation analyses were applied to evaluate the relationship between soil factors (C-org, N-t, C/N ratio, pH, ammonium, phosphorus, potassium) and microbial properties in the bulk soil. In the pioneer stage microbial colonization of the rhizosphere of P. alpina was dependent on the reservoir of microbial species in the bulk soil. As a consequence, the rhizosphere and bulk soil were similar in microbial biomass (ninhydrin-reactive nitrogen (NHR-N)), community composition (PLFA), and enzyme activity. In the transition and mature grassland stage, more benign soil conditions stimulated microbial growth (NHR-N, total amount of PLFA, bacterial PLFA, Gram-positive bacteria, Gram-negative bacteria), and microbial diversity (Shannon index H) in the rhizosphere either directly or indirectly through enhanced carbon allocation. In the same period, the rhizosphere microflora shifted from a G(-) to a more G(+), and from a fungal to a more bacteria-dominated community. Rhizosphere beta-xylosidase, N-acetyl-beta-glucosaminidase, and sulfatase activity peaked in the mature grassland soil, whereas rhizosphere leucine aminopeptidase, beta-glucosidase, and phosphatase activity were highest in the transition stage, probably because of enhanced carbon and nutrient allocation into the rhizosphere due to better growth conditions. Soil organic matter appeared to be the most important driver of microbial colonization in the bulk soil. The decrease in soil pH and soil C/N ratio mediated the shifts in the soil microbial community composition (bacPLFA, bacPLFA/fungPLFA, G(-), G(+)/G(-)). The activities of beta-glucosidase, beta-xylosidase and phosphatase were related to soil ammonium and phosphorus, indicating that higher decomposition rates enhanced the nutrient availability in the bulk soil. We conclude that the major determinants of the microllora vary along the successional gradient: in the pioneer stage the rhizosphere microflora was primarily determined by the harsh soil environment; under more favourable environmental conditions, however, the host plant selected for a specific microbial community that was related to the dynamic interplay between soil properties and carbon supply. (C) 2004 Elsevier Ltd. All rights reserved.

Modeling chemotaxis reveals the role of reversed phosphotransfer and a bi-functional kinase-phosphatase

Understanding how multiple signals are integrated in living cells to produce a balanced response is a major challenge in biology. Two-component signal transduction pathways, such as bacterial chemotaxis, comprise histidine protein kinases (HPKs) and response regulators (RRs). These are used to sense and respond to changes in the environment. Rhodobacter sphaeroides has a complex chemosensory network with two signaling clusters, each containing a HPK, CheA. Here we demonstrate, using a mathematical model, how the outputs of the two signaling clusters may be integrated. We use our mathematical model supported by experimental data to predict that: (1) the main RR controlling flagellar rotation, CheY6, aided by its specific phosphatase, the bifunctional kinase CheA3, acts as a phosphate sink for the other RRs; and (2) a phosphorelay pathway involving CheB2 connects the cytoplasmic cluster kinase CheA3 with the polar localised kinase CheA2, and allows CheA3-P to phosphorylate non-cognate chemotaxis RRs. These two mechanisms enable the bifunctional kinase/phosphatase activity of CheA3 to integrate and tune the sensory output of each signaling cluster to produce a balanced response. The signal integration mechanisms identified here may be widely used by other bacteria, since like R. sphaeroides, over 50% of chemotactic bacteria have multiple cheA homologues and need to integrate signals from different sources.

Isolation and partial characterisation of acid phosphatase isozymes from dormant oilseed of Corylus avellana L.

The acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) complement from dormant hazel (Corylus avellana L.) seeds was found to exhibit significant electrophoretic heterogeneity partially attributable to the presence of distinct molecular forms. In axiferous tissue, total acid phosphatase activity increased in a biphasic fashion during chilling, a treatment necessary to alleviate seed dormancy. Three acid phosphatase isozymes were isolated from cotyledons of dormant hazel seeds by successive ammonium sulphate precipitation, size-exclusion, Concanavalin A affinity, cation- and anion-exchange chromatographies resulting in 75-, 389- and 191-fold purification (APase1, APase2, APase3, respectively). The three glycosylated isoforms were isolated to catalytic homogeneity as determined by electrophoretic, kinetic and heat-inactivation studies. The native acid phosphatase complement of hazel seeds had an apparent Mr of 81.5±3.5 kDa as estimated by size-exclusion chromatography, while the determined pI values were 5.1 (APase1), 6.9 (APase2) and 7.3 (APase3). The optimum pH for p-nitrophenyl phosphate hydrolysis was pH 3 (APase1), pH 5.6 (APase2) and pH 6 (APase3). The hazel isozymes hydrolysed a variety of phosphorylated substrates in a non-specific manner, exhibiting low Km and the highest specificity constant (Vmax/Km) for pyrophosphate. They were not primary phytases since they could not initiate phytic acid hydrolysis, while APase2 and APase3 had significant phospho-tyrosine phosphatase activity. Inorganic phosphate was a competitive inhibitor, while activity was significantly impaired in the presence of vanadate and fluoride.

Variation in root-associated phosphatase activities in wheat contributes to the utilization of organic P substrates in vitro, but does not explain differences in the P-nutrition of plants when grown in soils

To understand whether genotypic variation in root-associated phosphatase activities in wheat impacts on its ability to acquire phosphorus (P), various phosphatase activities of roots were measured in relation to the utilization of organic P substrates in agar, and the P-nutrition of plants was investigated in a range of soils. Root-associated phosphatase activities of plants grown in hydroponics were measured against different organic P substrates. Representative genotypes were then grown in both agar culture and in soils with differing organic P contents and plant biomass and P uptake were determined. Differences in the activities of both root-associated and exuded phosphodiesterase and phosphomonoesterase were observed, and were related to the P content of plants supplied with either ribonucleic acid or glucose 6-phosphate, respectively, as the sole form of P. When the cereal lines were grown in different soils, however, there was little relationship between any root-associated phosphatase activity and plant P uptake. This indicates that despite differences in phosphatase activities of cereal roots, such variability appears to play no significant role in the P-nutrition of the plant grown in soil, and that any benefit derived from the hydrolysis of soil organic P is common to all genotypes.

Culture of osteogenic cells from human alveolar bone: A useful source of alkaline phosphatase

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of similar to 120 kDa that could be solubilized by phospholipase C or Polidocanol. (c) 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Inhibition of 5-alpha-reductase activity induces stromal remodeling and smooth muscle de-differentiation in adult gerbil ventral prostate

Prostatic differentiation during embryogenesis and its further homeostatic state maintenance during adult life depend on androgens. Dihydrotestosterone, which is synthesized from testosterone by 5alpha-reductase (5alpha-r), is the active molecule triggering androgen action within the prostate. In the present work, we examined the effects of 5alpha-reductase inhibition by finasteride in the ventral prostate (VP) of the adult gerbil, employing histochemical and electron microscopy techniques to demonstrate the morphological and organizational changes of the organ. After 10 days of finasteride treatment at a dose of 100 mg/kg/day, the prostatic complex (VP and dorsolateral prostate) absolute weight was reduced to about 18%. The epithelial cells became short and cuboidal, with less secretory blebs and reduced acid phosphatase activity. The luminal sectional area diminished, suggestive of decreased secretory activity. The stromal/epithelial ratio increased, the stroma becoming thicker but less cellular. There was a striking accumulation of collagen fibrils, which was accompanied by an increase in deposits of amorphous granular material adjacent to the basal lamina and in the clefts between smooth muscle cells (SMC). Additionally, the periacinar smooth muscle became loosely packed. Some SMC were atrophic and showed a denser array of the cytoskeleton, whereas other SMC had a highly irregular outline with numerous spine-like projections. The present data indicate that 5alpha-r inhibition causes epithelial and stromal changes by affecting intra-prostatic hormone levels. These alterations are probably the result of an imbalance of the homeostatic interaction between the epithelium and the underlying stroma.

MICROBIAL ACTIVITY of DARK RED LATOSOL SAMPLES STORED AT DIFFERENT TEMPERATURES

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Enzyme activity and microbial biomass in an Oxisol amended with sewage sludge contaminated with nickel

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

OSSEOUS PLATE ALKALINE-PHOSPHATASE IS ANCHORED BY GPI

Alkaline phosphatase activity was released up to 100% from the membrane by using 0.1 U of phosphatidylinositol-specific phospholipase C from B. thuringiensis. The Mr of solubilized enzyme was 145,000 by Sephacryl S-300 gel filtration and 66,000 by SDS-PAGE, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyze p-nitrophenyl phosphate (PNPP) (264.3 mu mol min(-1) mg(-1)), ATP (42.0 mu mol min(-1) mg(-1)) and pyrophosphate (28.4 mu mol min(-1) mg(-1)). The hydrolysis of ATP and PNPP by solubilized enzyme exhibited ''Michaelian'' kinetics with K-0.5 = 70 and 979 mu M, respectively. For pyrophosphate, K-0.5 was 128 mu M and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K-d = 1.5 mM) but zinc ions were powerful non-competitive inhibitors (K-d = 6.2 mu M) of solubilized enzyme. Treatment of solubilized alkaline phosphatase with Chellex 100 reduced the original PNPPase activity to 5%. Cobalt (K-0.5 = 10.1 mu M), magnesium (K-0.5 = 29.5 mu M) and manganese ions (K-0.5 = 5 mu M) restored the activity of the apoenzyme with positive cooperativity, suggesting that phosphatidylinositol-specific phospholipase C-solubilized alkaline phosphatase is a metalloenzyme. The stimulation of the apoenzyme by calcium ions (K-0.5 = 653 mu M) was lower than that observed for the other ions (26%) and exhibited site-site interactions (n = 0.7). Zinc ions had no effect on the apoenzyme of the solubilized enzyme.