Kinetic properties of de-repressible alkaline phosphatase (EC 3.1.3.1) from Neurospora crassa

De-repressible alkaline phosphatase from N. crassa shows inhibition by PNP-P and a hyperbolic mixed-type inhibition by Pi. Both increasing concentrations of Pi and decreases in assay pH abolished inhibition by the substrate. Also, Pi promoted polymerization of the enzyme molecule, whose effect may account for the inhibitory behaviour shown by the enzyme in the presence of low Pi concentrations. © 1991 Rapid Communications of Oxford Ltd.

The dermatophyte Trichophyton rubrum secretes an EDTA-sensitive alkaline phosphatase on high-phosphate medium

In this communication, we show that the growth of isolate H6 of the dermatophyte Trichophyton rubrum on non-buffered medium and under saturating phosphate conditions is dependent on the initial growth pH, with an apparent optimum at pH 4.0. In addition, irrespective of the initial growth pH, the pH of the medium altered during cultivation reaching values that ranged from 8.3 to 8.9. Furthermore, this isolate synthesized and secreted almost the same levels of an alkaline phosphatase with an apparent optimum pH ranging from 9.0 to 10.0 when grown on both low- and high-phosphate medium. Also, this alkaline phosphatase is activated by Mg2+ and is EDTA-sensitive. On the other hand, the very low levels of the enzyme retained by the mycelium grown on buffered medium at pH 5.0-5.2 suggest that this enzyme is encoded by an alkaline gene, i.e., a gene responsive to ambient pH signaling.

Alkaline phosphatase localization in golden hamster epididymidis: A biochemical and histochemical investigation

The aim of this study was to determine alkaline phosphatase (ALP; E.C. 3.1.3.1) activity and major expression in homogenates obtained from different regions of Golden hamster epididymis, comprising the initial segment, head, body and tail, with concomitant research of this enzyme localization and activity in samples of tissues. These were collected from the same regions and investigated by histochemical conventional study performed on frozen histological sections. No significant differences in mean ALP activity, reported as U.100 mg-1 of tissue, were observed among the biological specimens collected from the epididymidis initial segment (0.92 ± 0.28 U.100 mg-1 tissue), head (1.07 ± 0.67 U.100 mg-1 tissue) and body (0.77 ± 0.23 U.100 mg-1 tissue). However, mean ALP activity was significantly higher in the epididymal tail (8.94 ± 0.40 U.100 mg-1 tissue) compared with the precedent segments. The findings suggested that ALP plays a significant role in the tail of the Golden hamster epididymidis, mediating androgenic segregation necessary to maintain the epithelial integrity. Furthermore, ALP acts on active transport of substances between the luminal fluid and spermatozoon membrane, and contrariwise. Thus, the high concentration of ALP in the epididymal tail helps to indicate the importance of this enzyme in the metabolism and maintenance of spermatozoa maturation and storage into the epididymidis luminal compartment, perhaps directly influencing the normal reproductive morphophysiology.

Acid and alkaline phosphatase activity in broiler chicks fed with different levels of phytase and non-phytate phosphorus

Tartrate-resistant acid (ACP) and alkaline phosphatase (ALP) activities were evaluated in the serum and bone of broiler chicks fed with various amounts of non-phytate phosphorus (NPP) or phytase. Data were analysed using a 4×3 factorial design containing four NPP levels per period. Analyses were performed in chicks aged 1-21 days (0.21; 0.29; 0.37; 0.45 ppm) and 36-42 days (0.13; 0.21; 0.29; 0.37 ppm) and under three different phytase level treatments (0, 500 and 1000 FTU/kg) for each period. In 42-day-old animals, the serum ACP and ALP activities did not differ in response to NPP and phytase levels and bone ACP activity decreased with increased phosphorus levels. We observed effects on ALP activity by approximately 70% in lower phosphorus (0.13 and 0.21) levels without phytase. The phytase addition decreased (P<0.05) ALP values in lower phosphorus levels. The bone ALP and ACP levels of 21-day-old animals were not affected by phosphorus or phytase. Pi depletion induces a significant increase in alkaline phosphatase synthesis, suggesting that the function of this enzyme is downregulated by phosphorus. © 2013 Copyright Taylor and Francis Group, LLC.

Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification

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

Platelet-rich plasma stimulates cytokine expression and alkaline phosphatase activity in osteoblast-derived osteosarcoma cells

Objective: The aim of this study was to investigate the effects of PRP on SAOS-2 cells in terms of cytokine expression, cell activity and oxidative stress. Design: Cell line SAOS-2 (1 x 10(5) cells/mL) were grown in culture medium alpha-MEM with 10% FBS for 24 h and stimulated (or not) with PRP at concentrations of 3, 10 and 20%, LPS (E. coli, 10 g/mL) and IL-1 beta (1 mg/mL) for 24 h. The supernatant was collected and analyzed for the expression of cytokines in a panel array, ALP using a commercial kit and NO2- with Griess reaction method. Also, the cells were analyzed using Western blot for RANKL and slot blotting for nitrotyrosine expression. Result: There were no significant differences amongst the groups in terms of NO2-, protein nitrotyrosine content and RANKL expression. However, all stimuli increased ALP activity and in case of PRP, it was in a dose-dependent manner (p < 0.001). Also, all stimuli induced an increase in cytokines and chemokines expression, but only PRP promoted an increase of component C5, sICAM-1 and RANTES expression. Whilst IL-1 receptor antagonist (IL-1ra) expression was down-regulated by PRP, both LPS and IL-1 beta caused up-regulation of this cytokine. Conclusions: PRP can stimulate osteoblast activity and cytokine/chemokine release, as well as indicate some of the mediators that can (and cannot) be involved in this activation. (C) 2012 Elsevier Ltd. All rights reserved.

Differential expression of the bone and the liver tissue non-specific alkaline phosphatase isoforms in brain tissues

The enzyme tissue non-specific alkaline phosphatase (TNAP) belongs to the ectophosphatase family. It is present in large amounts in bone in which it plays a role in mineralization but little is known about its function in other tissues. Arguments are accumulating for its involvement in the brain, in particular in view of the neurological symptoms accompanying human TNAP deficiencies. We have previously shown, by histochemistry, alkaline phosphatase (AP) activity in monkey brain vessels and parenchyma in which AP exhibits specific patterns. Here, we clearly attribute this activity to TNAP expression rather than to other APs in primates (human and marmoset) and in rodents (rat and mouse). We have not found any brain-specific transcripts but our data demonstrate that neuronal and endothelial cells exclusively express the bone TNAP transcript in all species tested, except in mouse neurons in which liver TNAP transcripts have also been detected. Moreover, we highlight the developmental regulation of TNAP expression; this also acts during neuronal differentiation. Our study should help to characterize the regulation of the expression of this ectophosphatase in various cell types of the central nervous system.

CA15-3 and alkaline phosphatase as predictors for breast cancer recurrence: a combined analysis of seven International Breast Cancer Study Group trials

BACKGROUND: We evaluated the ability of CA15-3 and alkaline phosphatase (ALP) to predict breast cancer recurrence. PATIENTS AND METHODS: Data from seven International Breast Cancer Study Group trials were combined. The primary end point was relapse-free survival (RFS) (time from randomization to first breast cancer recurrence), and analyses included 3953 patients with one or more CA15-3 and ALP measurement during their RFS period. CA15-3 was considered abnormal if >30 U/ml or >50% higher than the first value recorded; ALP was recorded as normal, abnormal, or equivocal. Cox proportional hazards models with a time-varying indicator for abnormal CA15-3 and/or ALP were utilized. RESULTS: Overall, 784 patients (20%) had a recurrence, before which 274 (35%) had one or more abnormal CA15-3 and 35 (4%) had one or more abnormal ALP. Risk of recurrence increased by 30% for patients with abnormal CA15-3 [hazard ratio (HR) = 1.30; P = 0.0005], and by 4% for those with abnormal ALP (HR = 1.04; P = 0.82). Recurrence risk was greatest for patients with either (HR = 2.40; P < 0.0001) and with both (HR = 4.69; P < 0.0001) biomarkers abnormal. ALP better predicted liver recurrence. CONCLUSIONS: CA15-3 was better able to predict breast cancer recurrence than ALP, but use of both biomarkers together provided a better early indicator of recurrence. Whether routine use of these biomarkers improves overall survival remains an open question.

Tenofovir use is associated with an increase in serum alkaline phosphatase in the Swiss HIV Cohort Study

BACKGROUND: Tenofovir (TDF) use has been associated with proximal renal tubulopathy, reduced calculated glomerular filtration rates (cGFR) and losses in bone mineral density. Bone resorption could result in a compensatory osteoblast activation indicated by an increase in serum alkaline phosphatase (sAP). A few small studies have reported a positive correlation between renal phosphate losses, increased bone turnover and sAP. METHODS: We analysed sAP dynamics in patients initiating (n = 657), reinitiating (n = 361) and discontinuing (n = 73) combined antiretroviral therapy with and without TDF and assessed correlations with clinical and epidemiological parameters. RESULTS: TDF use was associated with a significant increase of sAP from a median of 74 U/I (interquartile range 60-98) to a plateau of 99 U/I (82-123) after 6 months (P < 0.0001), with a prompt return to baseline upon TDF discontinuation. No change occurred in TDF-sparing regimes. Univariable and multivariable linear regression analyses revealed a positive correlation between sAP and TDF use (P < or = 0.003), but no correlation with baseline cGFR, TDF-related cGFR reduction, changes in serum alanine aminotransferase (sALT) or active hepatitis C. CONCLUSIONS: We document a highly significant association between TDF use and increased sAP in a large observational cohort. The lack of correlation between TDF use and sALT suggests that the increase in sAP is because of the bone isoenzyme and indicates stimulated bone turnover. This finding, together with published data on TDF-related renal phosphate losses, this finding raises concerns that TDF use could result in osteomalacia with a loss in bone mineral density at least in a subset of patients. This potentially severe long-term toxicity should be addressed in future studies.

Vps41p Function in the Alkaline Phosphatase Pathway Requires Homo-oligomerization and Interaction with AP-3 through Two Distinct Domains

Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p. However, unlike other adaptor protein–dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin. Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway. These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways. The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex. Sequencing analysis of these VPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3.

A molecular sensor system based on genetically engineered alkaline phosphatase.

Binding and signaling proteins based on Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1) were designed for the detection of antibodies. Hybrid proteins were constructed by using wild-type AP and point mutants of AP [Asp-101 --> Ser (D101S) and Asp-153 --> Gly (D153G)]. The binding function of the hybrid proteins is provided by a peptide epitope inserted between amino acids 407 and 408 in AP. Binding of anti-epitope antibodies to the hybrid proteins modulates the enzyme activity of the hybrids; upon antibody binding, enzyme activity can increase to as much as 300% of the level of activity in the absence of antibody or can decrease as much as 40%, depending on the presence or absence of the point mutations in AP. The fact that modulation is altered from inhibition to activation by single amino acid changes in the active site of AP suggests that the mechanism for modulation is due to structural alterations upon antibody binding. Modulation is a general phenomenon. The properties of the system are demonstrated by using two epitopes, one from the V3 loop of human immunodeficiency virus type 1 gp120 protein and one from hepatitis C virus core protein, and corresponding monoclonal antibodies. The trend of modulation is consistent for all hybrids; those in wild-type AP are inhibited by antibody, while those in the AP mutants are activated by antibody. This demonstrates that modulation of enzyme activity of the AP-epitope hybrid proteins is not specific to either a particular epitope sequence or a particular antibody-epitope combination.

Effect of salinity on growth, pigmentation, N2 fixation and alkaline phosphatase activity of cultured Trichodesmium sp

Trichodesmium sp. isolated from the Great Barrier Reef lagoon was cultured in artificial seawater media containing a range of salinities. Trichodesmium sp. actively grew over a wide range of salinities (22 to 43 psu) and hence can be classed as euryhaline. Maximum growth occurred with salinities in the range 33 to 37 psu. Chl a content and alkaline phosphatase activity were found to increase with salinity over the range 22 to 43 psu, but the N-2 fixation rate was reduced at salinities below and above the range for maximum growth. Growth in media exhibiting maximum growth was characterised by well-dispersed cultures of filaments, while significant aggregations of filaments formed in other media. It is proposed that the tendency for Trichodesmium filaments to aggregate in media with salinities outside the range for maximum growth is an opportunistic response to a deficiency of cellular nitrogen, which results from the reduced N-2 fixation rates, and the aggregation occurs in order to enhance the uptake of combined N released within the aggregates and/or the N-2 fixation within the aggregates.