Elevated Phosphatase Activity Research Articles

Incubation study on remediation of nitrate-contaminated soil by Chroococcus sp.

The possibility of using the non-nitrogen-fixing cyanobacterium (Chroococcus sp.) for the reduction of soil nitrate contamination was tested through Petri dish experiments. The application of 0.03, 0.05 and 0.08mg/cm2 Chroococcus sp. efficiently removed NO3--N from the soil through assimilation of nitrate nutrient and promotion of soil denitrification. At the optimal application dose of 0.05mg/cm2, 44.06%, 36.89% and 36.17% of NO3--N were removed at initial NO3--N concentrations of 60, 90 and 120mg/kg, respectively. The polysaccharides released by Chroococcus sp. acted as carbon sources for bacterial denitrification and facilitated the reduction of soil salinity, which significantly (p < 0.05) stimulated the growth of denitrifying bacteria (Hyphomicrobium denitrificans and Hyphomicrobium sp.) as well as significantly (p < 0.05) elevated the activities of nitrate reductase and nitrite reductase by 1.07-1.23 and 1.15-1.22 times, respectively. The application of Chroococcus sp. promoted the dominance of Nocardioides maradonensis in soil microbial community, which resulted in elevated phosphatase activity and increased available phosphorus content. The application of Chroococcus sp. positively regulated the growth of soil bacteria belonging to the genera Chitinophaga, Prevotella and Tumebacillus, which may contribute to increased soil fertility through the production of beneficial enzymes such as invertase, urease and catalase. To date, this is the first study verifying the remediation effect of non-nitrogen-fixing cyanobacteria on nitrate-contaminated soil.

A simple dynamic equilibrium model shows that high phosphatase activity in tropical forest soils could be explained by rapid microbial turnover

Microorganisms and plant roots secrete phosphatase to obtain phosphorus (P) by degrading organic P. Previous studies have reported high phosphatase activity in tropical forest soils, which has been attributed to poor soil P conditions. However, rapid microbial turnover in tropical forest soils can also contribute to the high soil phosphatase activity. To test this, a simplified dynamic equilibrium model was developed. This model was intentionally designed to exclude the pathways of P originating from rock weathering or P adsorption by soils, with the aim of assessing the specific effect of accelerated microbial turnover on soil phosphatase activity. This model showed that accelerated microbial turnover consistently leads to elevated phosphatase activity, irrespective of the parameters, under the assumed environmental condition with the same litter P input. This suggests that rapid soil microbial turnover may serve as another crucial factor contributing to the high phosphatase activity in tropical forest soils, where both moisture and temperature are high.

Sperm Competition and Its Effects on the Evolution of Prostatic Acid Phosphatase in Hominids

Selective pressures and their effects on protein structure and function can be found by comparing genes or proteins from different organisms. These pressures can take varying forms. Sexual selection, a form of natural selection, occurs when an organism acquires traits that give it an advantage in mating and producing offspring. An example can be seen in hominid seminal proteins. Hominids exhibit different mating systems which has resulted in seminal proteins being affected by varying degrees of sperm competition. This can result in variation both in the sequences of these genes and the function of the gene products. One such gene, ACPP, is used to generate the protein prostatic acid phosphatase (PAP). This protein is known to dephosphorylate a number of targets in seminal plasma, and it functions as a phosphatase in the acidic pH range. It has been suggested computationally by comparing dN/dS ratios that ACPP may be under strong positive selection in chimpanzees (a species with high sperm competition) and purifying selection in humans and gorillas (both species with weaker sperm competition). We hypothesize that varying degrees of sperm competition have resulted in sequence and functional variation at ACPP/PAP among hominids. ACPPcoding sequences for human, chimp, and gorilla homologs were each inserted into a mammalian expression vector with a C‐terminal His6 tag and transfected into HEK‐293T cells. The phosphatase activities of the PAP homologs were compared using a DiFMUP fluorescent substrate assay. Each protein was tested for pH optima, phosphatase activity at a fixed substrate concentration, and kcat/km. The PAP orthologs exhibited similar pH profiles, with a pH optimum between pH 5.0‐6.0. However, it was found that chimp PAP exhibited elevated phosphatase activity when compared to the other hominids, and this was consistent across a varying pH range. These results suggest that positive selection of chimp ACPPhas resulted in a more efficient PAP protein with elevated activity when compared to human and gorilla PAP, perhaps due to strong sperm competition. Gorilla PAP showed the lowest activity, which is interesting considering that many genes that code for male reproductive proteins have become pseudogenes in gorillas, a species with very low sperm competition. Overall, the results suggest that the varying mating systems of the hominid primates have resulted in functional differences in the PAP homologs of humans, chimpanzees, and gorillas. By bridging together functional and computational techniques such as in this research, we are beginning to understand the effects of specific selective pressures on the function of male reproductive proteins.

A multivariate snapshot of New Zealand milk seasonality in individual cows

Seasonal variation of milk composition poses a challenge for maintaining dairy product quality. Multivariate statistical techniques were used here to identify seasonal patterns in mineral levels, fatty acid profiles and selected metabolites for New Zealand raw milk collected from 24 individual cows in early-season (November), mid-season (January/February) and late-season (March). Partial least-squares discriminant analysis (PLS-DA) showed clear seasonal differences in mineral and fatty acid profiles that may have been related to supplementary feeding, dilution effects (higher total solids in late-season milk) and the lactation cycle. Fatty acid profiles showed an inverse relationship between the C6–C12 fatty acids and the branched- or long-chain fatty acids, which was thought to be related to feeding of maize in mid- and late-season. Targeted metabolite analysis suggested elevated phosphatase activity in March, and this warrants further investigation as an indicator of metabolic changes related to declining milk quality at the end of the milking season.

Molecular basis of gain-of-function LEOPARD syndrome-associated SHP2 mutations.

The Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP2) is a critical signal transducer downstream of growth factors that promotes the activation of the RAS-ERK1/2 cascade. In its basal state, SHP2 exists in an autoinhibited closed conformation because of an intramolecular interaction between its N-SH2 and protein tyrosine phosphatase (PTP) domains. Binding to pTyr ligands present on growth factor receptors and adaptor proteins with its N-SH2 domain localizes SHP2 to its substrates and frees the active site from allosteric inhibition. Germline mutations in SHP2 are known to cause both Noonan syndrome (NS) and LEOPARD syndrome (LS), two clinically similar autosomal dominant developmental disorders. NS-associated SHP2 mutants display elevated phosphatase activity, while LS-associated SHP2 mutants exhibit reduced catalytic activity. A conundrum in how clinically similar diseases result from mutations to SHP2 that have opposite effects on this enzyme’s catalytic functionality exists. Here we report a comprehensive investigation of the kinetic, structural, dynamic, and biochemical signaling properties of the wild type as well as all reported LS-associated SHP2 mutants. The results reveal that LS-causing mutations not only affect SHP2 phosphatase activity but also induce a weakening of the intramolecular interaction between the N-SH2 and PTP domains, leading to mutants that are more readily activated by competing pTyr ligands. Our data also indicate that the residual phosphatase activity associated with the LS SHP2 mutant is required for enhanced ERK1/2 activation. Consequently, catalytically impaired SHP2 mutants could display gain-of-function properties because of their ability to localize to the vicinity of substrates for longer periods of time, thereby affording the opportunity for prolonged substrate turnover and sustained RAS-ERK1/2 activation.

Changes in Soil Properties and Enzymatic Activities Following Manure Applications to a Rangeland

Manure amendments to rangelands may alter soil functions related to nutrient recycling. We investigated the influence of grazing and cattle manure on soil carbon, nitrogen, Mehlich 3 phosphorus, and activities of alkaline phosphatase and dehydrogenase. Fertilizer treatments (unamended, manure, or urea + potassium dihydrogen phosphate [KH2PO4] fertilizer) were imposed under grazed and nongrazed conditions in a short-grass native rangeland. Manure was applied at rates of ∼ 125 kg N ha−1 and ∼ 42 kg P ha−1, and urea + KH2PO4 was applied at 75 kg N ha−1 and 20 kg P ha−1, respectively. Total aboveground biomass and soil samples at 4 depths (0–200 mm) were collected throughout 2 growing seasons. A controlled environment study also evaluated fertilizer source effects on enzymatic activities at 5 P rates (0–120 mg·kg−1 P as manure or urea + KH2PO4). Amendments significantly (P < 0.05) increased extractable P following the second application for the 3 uppermost depth increments. Extractable P was greatest on manure-amended plots, increasing 44% from February 1999 to July 2000 at the surface. However, increases in P extractability as a proportion of total P applied were similar for manure and KH2PO4. Enzymatic activities were significantly (P < 0.001) influenced by sampling date and soil depth. There were no consistent grazing effects on enzyme activities. Amendments did not influence dehydrogenase activities in the field; however, in the controlled environment, activities averaged 16% greater across all rates for manure-amended soil as compared with urea + KH2PO4–amended soil (P = 0.025). Phosphatase activities increased significantly following manure applications under both field (P = 0.007) and controlled environment (P = 0.003) conditions. Elevated phosphatase activities following manure applications probably led to enhanced P mineralization and similar P extractabilities as a proportion of total P applied for manure- and KH2PO4–amended soils. Therefore, when determining applications rates, total manure P should be considered bioavailable. La aplicación de estiércol en los pastizales puede alterar las funciones del suelo y el reciclaje de nutrientes. Investigamos la influencia del apacentamiento y estiércol del ganado sobre el carbón del suelo, nitrógeno, fósforo Mehlich 3 y las actividades de la fosfatasa alcalina y la dehidrogenasa. Se aplicaron tratamientos de fertilización (control sin fertilizante o estiércol, estiércol y fertilizante a base de urea + KH2PO4) a un pastizal nativo de zacates cortos con condiciones de apacentamiento y sin apacentamiento. El estiércol se aplicó en dosis de ∼ 125 kg N ha−1 y ∼ 42 kg P ha−1 y la urea + KH2PO4 se aplicó en dosis de 75 kg N ha−1 y 20 kg P ha−1, respectivamente. Muestras de biomasa aérea y de suelo a 4 profundidades (0–200 mm) se colectaron a lo largo de 2 estaciones de crecimiento. En un estudio bajo ambiente controlado también se evaluaron los efectos de la fuente de fertilizante sobre las actividades enzimáticas a 5 dosis de P (0–120 mg·kg−1 P como estiércol o como urea + KH2PO4). Los fertilizantes incrementaron significativamente (P < 0.05) el fósforo extractable después de la segunda aplicación, en las 3 profundidades de suelo superiores. El fósforo extractable fue mayor en la superficie de las parcelas donde se aplicó el estiércol, aumentando 44% de Febrero de 1999 a Julio del 2000. Sin embargo, el aumento de la extractibilidad del P, en proporción al P total aplicado, fue similar para el estiércol y el KH2PO4. Las actividades enzimáticas fueron significativamente (P < 0.001) influenciadas por la fecha de muestreo y la profundidad del suelo. No hubo efectos consistentes del apacentamiento del ganado sobre la actividad enzimática. Los fertilizantes no influenciaron las actividades de la dehidrogenasa en el campo; sin embargo, en el ambiente controlado, las actividades promediaron 16% más, a través de todas las dosis, en los suelos con aplicación de estiércol en comparación con los que recibieron urea + KH2PO4 (P = 0.025). Las actividades de la fosfatasa aumentaron significativamente después de la aplicación de estiércol, tanto en condiciones de campo (P = 0.007) como en ambiente controlado (P = 0.003). Las elevadas actividades de la fosfatasa después de la aplicación de estiércol probablemente condujeron a un aumento en la mineralización del P y a extractibilidades del P, con relación al P total aplicado, similares para suelos con aplicaciones de estiércol y KH2PO4. Por lo tanto, al determinar las dosis de aplicación, el P total del estiércol debe ser considerado biodisponible.

Changes in Soil Properties and Enzymatic Activities Following Manure Applications to a Rangeland

Manure amendments to rangelands may alter soil functions related to nutrient recycling. We investigated the influence of grazing and cattle manure on soil carbon, nitrogen, Mehlich 3 phosphorus, and activities of alkaline phosphatase and dehydrogenase. Fertilizer treatments (unamended, manure, or urea + potassium dihydrogen phosphate [KH2PO4] fertilizer) were imposed under grazed and nongrazed conditions in a short-grass native rangeland. Manure was applied at rates of – 125 kg N ha-1 and – 42 kg P ha-1, and urea + KH2PO4 was applied at 75 kg N ha-1 and 20 kg P ha-1, respectively. Total aboveground biomass and soil samples at 4 depths (0-200 mm) were collected throughout 2 growing seasons. A controlled environment study also evaluated fertilizer source effects on enzymatic activities at 5 P rates (0-120 mg kg-1 P as manure or urea + KH2PO4). Amendments significantly (P &lt; 0.05) increased extractable P following the second application for the 3 uppermost depth increments. Extractable P was greatest on manure-amended plots, increasing 44% from February 1999 to July 2000 at the surface. However, increases in P extractability as a proportion of total P applied were similar for manure and KH2PO4. Enzymatic activities were significantly (P &lt; 0.001) influenced by sampling date and soil depth. There were no consistent grazing effects on enzyme activities. Amendments did not influence dehydrogenase activities in the field; however, in the controlled environment, activities averaged 16% greater across all rates for manure-amended soil as compared with urea + KH2PO4-amended soil (P = 0.025). Phosphatase activities increased significantly following manure applications under both field (P = 0.007) and controlled environment (P = 0.003) conditions. Elevated phosphatase activities following manure applications probably led to enhanced P mineralization and similar P extractabilities as a proportion of total P applied for manure- and KH2PO4-amended soils. Therefore, when determining applications rates, total manure P should be considered bioavailable.

Molecular studies on the protective effect of nicotine in adult-onset hypothyroidism-induced impairment of long-term potentiation

We have recently shown that chronic nicotine treatment reverses hypothyroidism-induced learning and memory impairment. Chronic nicotine treatment also reverses the hypothyroidism-induced impairment of long-term potentiation (LTP). Analysis of LTP associated key signaling molecules revealed that chronic nicotine treatment prevented the hypothyroidism-induced reduction of the basal phosphotransferase activity of CaMKII and protein levels of P-CaMKII. In addition, the failure of high frequency stimulation to increase the levels of P-CaMKII in hypothyroid rats was reversed by nicotine treatment, suggesting that the neuroprotective effect of nicotine during hypothyroidism involved activation of CaMKII. Furthermore, chronic nicotine treatment reverses the hypothyroidism-induced elevated phosphatase activity and protein levels of calcineurin, a phosphatase that regulates CaMKII activation. We conclude that the neuroprotective effects of nicotine in adult-onset hypothyroidism may result from restoration of CaMKII and calcineurin activity.

FLT3/ITD Mutation Signaling Includes Suppression of SHP-1

Mutations in the FLT3 gene are the most common genetic alteration found in AML patients. FLT3 internal tandem duplication (ITD) mutations result in constitutive activation of FLT3 tyrosine kinase activity. The consequences of this activation are an increase in total phosphotyrosine content, persistent downstream signaling, and ultimately transformation of hematopoietic cells to factor-independent growth. The Src homology (SH)2 domain-containing protein-tyrosine phosphatase (SHP)-1 is involved in the down-regulation of a broad range of growth factor and cytokine-driven signaling cascades. Loss-of-function or deficiency of SHP-1 activity results in a hyperproliferative response of myelomonocytic cell populations to growth factor stimulation. In this study, we examined the possible role of SHP-1 in regulating FLT3 signaling. We found that transformation of TF-1 cells with FLT3/ITD mutations suppressed the activity of SHP-1 by approximately 3-fold. Suppression was caused by decreased SHP-1 protein expression, as analyzed at both the protein and RNA levels. In contrast, protein levels of SHP-2, a phosphatase that plays a stimulatory role in signaling through a variety of receptors, did not change significantly in FLT3 mutant cells. Suppressed SHP-1 protein levels in TF-1/ITD cells were partially overcome after cells were exposed to CEP-701, a selective FLT3 inhibitor. SHP-1 protein levels also increased in naturally occurring FLT3/ITD expressing AML cell lines and in primary FLT3/ITD AML samples after CEP-701 treatment. Furthermore, a small but reproducible growth/survival advantage was observed in both TF-1 and TF-1/ITD cells when SHP-1 expression was knocked down by RNAi. Taken together, these data provide the first evidence that suppression of SHP-1 by FLT3/ITD signaling may be another mechanism contributing to the transformation by FLT3/ITD mutations.

Enhancement of acid phosphatase activity in Capsicum annuum L. plants in response to phosphate deficiency

This study examined acid phosphatase activity in the extracts of Capsicum annuum L. cv. Sweet Banana seedlings grown axenically on water-agar medium or plants grown in a hydroponic system with or without phosphate. Initially, no elevated phosphatase activity was detected in the root surface, in root surface extractions or cell-free tissue extracts from plants that were not showing morphological symptoms of stress. Then elevated specific phosphatase activity was evident in all organ tissue extracts of the plants that showed signs of growth inhibition. The increase in specific activity in the cell free extracts of different organs appeared to be primarily the result of increased activity of the dominant isozyme present in all these extracts, instead of due to de novo synthesis of new isozymes. These and other experiments indicate the lack of phosphate starvation-inducible (psi) excreted phosphatase in Capsicum annuum L. cv. Sweet Banana. Some limitations with the use of XP, 5-bromo-4-chloro-indolyl phosphate p-toluidine, particularly when incorporated into growth media and buffer with or without phosphate for the study of phosphate starvation-inducible excreted phosphatase activity were noted.

Identification of Protein-tyrosine Phosphatase 1B as the Major Tyrosine Phosphatase Activity Capable of Dephosphorylating and Activating c-Src in Several Human Breast Cancer Cell Lines

c-Src tyrosine kinase activity is elevated in several types of human cancer, and this has been attributed to elevated c-Src expression levels, increased c-Src specific activity, and activating mutations in c-Src. We have found a number of human breast cancer cell lines with elevated c-Src specific activity that also possess elevated phosphatase activity directed against the carboxyl-terminal negative regulatory domain of Src family kinases. To identify this phosphatase, cell extracts from MDA-MB-435S cells were chromatographed and the fractions were assayed for phosphatase activity. Four peaks of phosphatase activity directed against the nonspecific substrate poly(Glu/Tyr) were detected. One peak also dephosphorylated a peptide modeled against the c-Src carboxyl-terminal negative regulatory domain and intact human c-Src. Immunoblotting and immunodepletion experiments identified the phosphatase as protein-tyrosine phosphatase 1B (PTP1B). Examination of several human breast cancer cell lines with increased c-Src activity showed elevated levels of PTP1B protein relative to normal control breast cells. In vitro c-Src reactivation experiments confirmed the ability of PTP1B to dephosphorylate and activate c-Src. In vivo overexpression of PTP1B in 293 cells caused a 2-fold increase of endogenous c-Src kinase activity. Our findings indicate that PTP1B is the primary protein-tyrosine phosphatase capable of dephosphorylating c-Src in several human breast cancer cell lines and suggests a regulatory role for PTP1B in the control of c-Src kinase activity.

Induction of protein phosphatase type 2A in response to disruption of cell-matrix interactions.

The proliferation of most cells is strictly dependent on cell-matrix interactions, a phenomenon called anchorage dependence. Because tumor cells are often independent of this regulation, it is important to characterize the molecular components that are involved in this control. We therefore investigated a possible role of serine/threonine protein phosphatases in the regulation of anchorage-dependent cell growth. We found that the activity of serine/threonine protein phosphatase type 2A (PP2A) and, to a lesser extent, that of type 1 (PP1), was upregulated in response to the disruption of cellular attachment. In the case of PP2A, this induction was due to the transcriptional activation of the gene and increased expression of its protein. The increase in phosphatase activity corresponded with a decrease in the phosphorylation of cellular proteins that occurred in anchorage-dependent cells, but to a much lesser degree in anchorage-independent cells. At the same time, the activity of cyclin-dependent kinases was downregulated in anchorage-dependent, but not in anchorage-independent cells. Thus, our results indicate that the balance of kinase and phosphatase activity in anchorage-dependent cells is tipped in favor of phosphatase activity, which seems to dominate the extent of reversible protein phosphorylations after cellular detachment. In contrast, anchorage-independent cells appear to neutralize elevated phosphatase activity through sustained, strong kinase activity.

Induction of protein phosphatase type 2A in response to disruption of cell‐matrix interactions

The proliferation of most cells is strictly dependent on cell-matrix interactions, a phenomenon called anchorage dependence. Because tumor cells are often independent of this regulation, it is important to characterize the molecular components that are involved in this control. We therefore investigated a possible role of serine/threonine protein phosphatases in the regulation of anchorage-dependent cell growth. We found that the activity of serine/threonine protein phosphatase type 2A (PP2A) and, to a lesser extent, that of type 1 (PP1), was upregulated in response to the disruption of cellular attachment. In the case of PP2A, this induction was due to the transcriptional activation of the gene and increased expression of its protein. The increase in phosphatase activity corresponded with a decrease in the phosphorylation of cellular proteins that occurred in anchorage-dependent cells, but to a much lesser degree in anchorage-independent cells. At the same time, the activity of cyclin-dependent kinases was downregulated in anchorage-dependent, but not in anchorage-independent cells. Thus, our results indicate that the balance of kinase and phosphatase activity in anchorage-dependent cells is tipped in favor of phosphatase activity, which seems to dominate the extent of reversible protein phosphorylations after cellular detachment. In contrast, anchorage-independent cells appear to neutralize elevated phosphatase activity through sustained, strong kinase activity. J. Cell. Physiol. 182:88–96, 2000. © 2000 Wiley-Liss, Inc.

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent protein phosphatase activity in rat brain cytosol.

The effect of Ca2+-binding protein regucalcin on neutral phosphatase activity in rat brain cytosol was investigated. Phosphatase activity was assayed in a reaction mixture containing the cytosolic protein in the presence of phosphotyrosine, phosphoserine, and phosphothreonine. The presence of calcium chloride (10(-5) and 10(-4) M) in the enzyme reaction mixture caused a significant increase in phosphatase activity toward three phosphoaminoacids. The enzyme activity toward phosphoserine and phosphothreonine was significantly enhanced by the addition of calmodulin (1 or 5 microg/ml) in the presence of calcium (10(-5) M). Such an effect was not seen in the presence of phosphotyrosine. Trifluoperazine (2x10(-5) M), an antagonist of calmodulin, completely inhibited calcium (10(-5) M)-increased phosphatase activity toward phosphoserine and phosphothreonine, whereas it had no effect on the enzyme activity toward phosphotyrosine. Regucalcin (10(-9) M) significantly inhibited phosphatase activity toward three phosphoaminoacids without or with Ca2+ addition. The inhibitory effect of regucalcin (10(-10) and 10(-9) M) was also seen in the presence of Ca2+ (10(-5) M) and calmodulin (5 microg/ml). The presence of anti-regucalcin monoclonal antibody (20 or 50 ng/ml) in the enzyme reaction mixture caused a significant elevation of phosphatase activity toward three phosphoaminoacids; this effect was completely abolished by addition of regucalcin (10(-9) M). The present study suggests that the endogenous regucalcin has an inhibitory effect on Ca2+/calmodulin-dependent protein phosphatase activity in rat brain cytosol.

Effect of phytohaemagglutinin on CD45 in T cells

In this study the effect of PHA activation on the phosphatase activity of CD45 has been investigated in human leukemic T-cell lines. It has been found that in vivo activation of the cells with PHA resulted in 2–4-fold increase in enzyme activity. Addition of PHA to the postnuclear supernatant of cell lysates also resulted in elevation of phosphatase activity. Elevation of enzyme activity resulted from an increase in the amount of antigen in the immunoprecipitates. Elevation of the quantity was not the result of a de novo protein synthesis since the presence of cycloheximide, a protein synthesis inhibitor, did not modulate the effect of PHA. The effect of PHA was specific since ConA, that also bound to the CD45 molecules, or crosslinking of the antigen by antibody did not affect CD45. Since direct binding of PHA to CD45 molecules was shown in immunoblotting analysis, we suggest that the effect of PHA is a consequence of a PHA-induced conformational change of CD45 that results in up-regulation of the analyzed CD45 epitopes.

Role of the cdc25C phosphatase in G2 arrest induced by nitrogen mustard.

G2 arrest induced by nitrogen mustard in human lymphoma CA46 cells is associated with a failure to activate hyperphosphorylated cdc2/cyclin B1 complexes. We investigated the possibility that this might be due to a suppression of cdc25C phosphatase activity. cdc25C from interphase cells migrated as a 54- to 57-kDa doublet in SDS gels and exhibited basal phosphatase activity. cdc25C from mitotic cells migrated as a 66-kDa hyperphosphorylated species and exhibited elevated phosphatase activity. cdc25C hyperphosphorylation and activation were mediated by cdc2, supporting the view of a cdc2-cdc25C autocatalytic feedback loop. Immunofluorescence and cell fractionation studies suggested cdc2-cdc25C interaction occurred within the cytoplasm. Cells arrested in G2 phase following nitrogen mustard treatment or cells arrested in S phase with aphidicolin failed to dephosphorylate and activate cdc2, and this correlated with failure to convert cdc25C into the most active hyperphosphorylated species. Our findings suggest that checkpoints guarding against mitotic entry in the presence of unreplicated or damaged DNA suppress formation of the cdc2-cdc25C autocatalytic feedback loop that normally brings about rapid activation of cdc2.

Regulation of the cdc25 protein during the cell cycle in Xenopus extracts

The cdc25 protein is a highly specific tyrosine phosphatase that triggers mitosis by dephosphorylating the cdc2 protein kinase. Using Xenopus extracts, we have found that the cdc25 protein is active at a low level throughout interphase. Near the onset of mitosis, the cdc25 protein undergoes a marked elevation in phosphatase activity that coincides with an extensive phosphorylation of the protein in its N-terminal region. In vitro dephosphorylation of this hyperphosphorylated form of cdc25 reduces its phosphatase activity back to the interphase level. Moreover, treatment of interphase Xenopus extracts with okadaic acid, a phosphatase inhibitor that accelerates the entry into mitosis, elicits both the premature hyperphosphorylation of cdc25 and the stimulation of its cdc2-specific tyrosine phosphatase activity. These experiments demonstrate the existence of a cdc25 regulatory system consisting of both a stimulatory kinase that phosphorylates a putative regulatory domain of the cdc25 protein and an inhibitory serine/threonine phosphatase that counteracts this kinase activity.

Intestinal alkaline phosphatase in patients with chronic renal failure

Because of the suggestion that intestinal alkaline phosphatase was elevated in the serum of patients with chronic renal failure, we studied the serum of 42 patients undergoing hemodialysis with elevated enzyme activity. Using a sensitive and specific electroimmunoassay for the intestinal isoenzyme, 26 of 42 serum samples were positive, compared with 3 of 25 samples obtained from hospitalized patients with elevated phosphatase activity. The fractional amount of this isoenzyme was also higher, ranging from 1.5% to 41% of the total serum phosphatase, compared with 0.1%–1.2% in control sera. Kidneys removed during transplantation or postmortem contained a membranous phosphatase with immunologic activity identical to the intestinal isoenzyme in 5 of 6 patients. This enzyme accounted for 8%–21% of the total kidney phosphatase activity. By morphology the immunoreaction was localized to the apical membranes of the collecting tubules. Thus, the kidney is the likely source of the observed increase in serum intestinal-type phosphatase activity noted in patients with chronic renal failure. An elevation in the intestinal isoenzyme rather than the presence of early metabolic bone disease or hepatic disease should be considered in renal failure patients with mildly elevated (up to 50% over normal) total serum alkaline phosphatase.

Modulation of enzyme activity in azaguanine-resistant V79 cells selected by chronic drug exposure

Exposure of V79 cells to azaguanine (7–21 μM for 2–7 weeks) had little effect on growth or plating efficiency but resulted in gradual acquisition of resistance to 8-azaguanine (AZ) and 6-thioguanine (TG) and loss of ability to grow in HAT. The rate of evolution of the resistant phenotype was dependent on the concentration and duration of exposure to AZ. The increase in proportion of resistant cells was paralleled by a rise in phosphatase activity (pH optimum 7.0–7.5) expressed by intact cells and this preceded the fall in HGPRT activity. Elevated phosphatase activity and a resistant phenotype were stably expressed in clones isolated and cultured in the absence of AZ. Hypoxanthine guanine phosphoribosyl transferase (HGPRT) activity in cell extracts of three resistant clones ranged from 18 to 43% of wild-type levels but was unaltered with respect to substrate affinity and electrophoretic mobility. Mg 2+-dependent activity dephosphorylated inosine 5′monophosphate (IMP), guanine 5′monophosphate (GMP), adenosine-5-monophosphate (AMP) and p-nitrophenylphosphate (PNPP) and was also elevated with respect to wild-type levels in resistant cell extracts. Purine nucleoside phosphorylase levels were similar in sensitive and resistant cell extracts. Cross-sensitivity studies with other purine analogues suggest that the elevated phosphatase activity does not contribute to the resistant phenotype. No karyotypic changes were observed in the resistant cell lines.

Studies of intracellular distribution of acid phosphatase 5 in the spleen cells of leukemic reticuloendotheliosis by isopycnic gradient centrifugation

A fresh spleen sample obtained from a patient with leukemic reticuloendotheliosis was homogenized and subjected to centrifugation on a sucrose density gradient. A major portion of acid phosphatase band 5 was observed in the lysosome, confirming that the elevated phosphatase activity in the neoplastic spleen is a lysosomal enzyme. However, a significant amount of band 5 was also observed in the microsome. The microsomal and lysosomal enzymes have different affinity to CM-cellulose. The relationship between lysosomal and microsomal enzymes has not been established.