Purification and some properties of tartrate-sensitive acid phosphatase from rabbit kidney cortex.
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ABSTRACT:
SUBMITTER: Helwig JJ
PROVIDER: S-EPMC1186068 | biostudies-other | 1978 Oct
REPOSITORIES: biostudies-other
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Project description:BackgroundObesity is associated with macrophage infiltration of adipose tissue, which may link adipose inflammation to insulin resistance. However, the impact of inflammatory cells in the pathophysiology of obesity remains unclear. Tartrate resistant acid phosphatase (TRAP) is an enzyme expressed by subsets of macrophages and osteoclasts that exists either as an enzymatically inactive monomer or as an active, proteolytically processed dimer.Principal findingsUsing mice over expressing TRAP, we show that over-expression of monomeric, but not the dimeric form in adipose tissue leads to early onset spontaneous hyperplastic obesity i.e. many small fat cells. In vitro, recombinant monomeric, but not proteolytically processed TRAP induced proliferation and differentiation of mouse and human adipocyte precursor cells. In humans, monomeric TRAP was highly expressed in the adipose tissue of obese individuals. In both the mouse model and in the obese humans the source of TRAP in adipose tissue was macrophages. In addition, the obese TRAP over expressing mice exhibited signs of a low-grade inflammatory reaction in adipose tissue without evidence of abnormal adipocyte lipolysis, lipogenesis or insulin sensitivity.ConclusionMonomeric TRAP, most likely secreted from adipose tissue macrophages, induces hyperplastic obesity with normal adipocyte lipid metabolism and insulin sensitivity.
| S-EPMC2248616 | biostudies-literature
Project description:Arylsulphatases A and B (EC 3.1.6.1) of rabbit kidney cortex were purified 5250- and 7720-fold respectively by a multiple-column-chromatography method. The specific activity toward 4-nitrocatechol sulphate was 42mumol/min per mg for arylsulphatase A and 62 mumol/min per mg for arylsulphatase B. Each enzyme migrated as a single band on polyacrylamide-gel electrophoresis, and the enzyme activity corresponded to the band of protein on the gel. The rate of hydrolysis of ascorbic acid 2-sulphate by arylsulphatase A was three times that for cerebroside 3-sulphate. Arylsulphatase B hydrolysed UDP-N--acetylgalactosamine 4-sulphate and glucosamine 4,6-disulphate, but not galactosamine 6-sulphate.
| S-EPMC1164877 | biostudies-other
Project description:Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we have purified this enzyme from human osteoclastomas by sequential chromatography on SP-Sephadex, CM-Sephadex, hydroxylapatite, Sephadex G-150 and concanavalin A-Sepharose. The purification over the original tumour extract was about 2000-fold, with a yield of 10%. The enzyme appeared to be homogeneous when assessed by SDS/polyacrylamide-gel electrophoresis. Both gel filtration and SDS/polyacrylamide-gel electrophoresis indicated an Mr of about 30,000. The reduced and alkylated enzyme consists of two subunits with Mrs of 15,000 and 17,500. The N-terminal amino acid sequence of both subunits indicates that there is a high degree of identity between the osteoclastoma enzyme and similar enzymes purified from spleen and uterus. Using 4-methylumbelliferyl phosphate as substrate, the specific activity of the purified enzyme was 387 units.mg-1, and the Km was 284 microns. The pH optimum was 5.7. Unlike similar enzymes purified from human and bovine bone, osteoclastoma acid phosphatase is not activated by reducing agents (2-mercaptoethanol or ascorbic acid). The enzyme contains 4.8 mol of Fe2+/3+, 0.3 mol of Mn2+ and 1.7 mol of Mg2+ per mol of enzyme. Although the enzyme loses 50% of its activity in the presence of EDTA, it is not inhibited by the iron chelator 1,10-phenanthroline. However, the enzyme is activated to a small extent by Mn2+ and Mg2+. Using a variety of substrates and inhibitors, we demonstrate that there are differences between the osteoclastoma acid phosphatase and the enzyme purified from other sources.
| S-EPMC1138867 | biostudies-other
Project description:The tartrate-resistant acid phosphatase (TRAP) of rat osteoclasts has been shown to exhibit high (85-94%) identity at the amino acid sequence level with the purple acid phosphatase (PAP) from bovine spleen and with pig uteroferrin. These iron-containing purple enzymes contain a binuclear iron centre, with a tyrosinate-to-Fe(III) charge-transfer transition responsible for the purple colour. In the present study, production of rat osteoclast TRAP could be achieved at a level of 4.3 mg/litre of medium using a baculovirus expression system. The enzyme was purified to apparent homogeneity using a combination of cation-exchange, hydrophobic-interaction, lectin-affinity and gel-permeation chromatography steps. The protein as isolated had a purple colour, a specific activity of 428 units/mg of protein and consisted of the single-chain form of molecular mass 34 kDa, with only trace amounts of proteolytically derived subunits. The recombinant enzyme had the ability to dephosphorylate bone matrix phosphoproteins, as previously shown for bone TRAP. Light absorption spectroscopy of the isolated purple enzyme showed a lambda max at 544 nm, which upon reduction with ascorbic acid changed to 515 nm, concomitant with the transition to a pink colour. EPR spectroscopic analysis of the reduced enzyme at 3.6 K revealed a typical mu-hydr(oxo)-bridged mixed-valent Fe(II)Fe(III) signal with g-values at 1.96, 1.74 and 1.60, proving that recombinant rat TRAP belongs to the family of PAPs. To validate the use of recombinant PAP in substituting for the rat bone counterpart in functional studies, various comparative studies were carried out. The enzyme isolated from bone exhibited a lower K(m) for p-nitrophenyl phosphate and was slightly more sensitive to PAP inhibitors such as molybdate, tungstate, arsenate and phosphate. In contrast with the recombinant enzyme, TRAP from bone was isolated predominantly as the proteolytically cleaved, two-subunit, form. Both the recombinant enzyme and rat bone TRAP were shown to be substituted with N-linked oligosaccharides. A slightly higher apparent molecular mass of the monomeric form and N-terminal chain of bone TRAP compared with the recombinant enzyme could not be accounted for by differential N-glycosylation. Despite differences in specific post-translational modifications, the recombinant PAP should be useful in future studies on the properties and regulation of the mammalian PAP enzyme.
| S-EPMC1218069 | biostudies-other
Project description:BackgroundBone metastases often occur in the majority of patients with advanced cancer, such as prostate cancer, lung cancer and breast cancer. Serum tartrate-resistant acid phosphatase 5b (TRACP 5b), a novel bone resorption marker, has been used gradually in the clinics as a specific and sensitive marker of bone resorption for the early diagnosis of cancer patients with bone metastasis. Here, we reported that high concentrations of uric acid (UA) lead to decrease of TRACP 5b levels and determined whether TRACP 5b level was associated with UA in interference experiment.MethodsA total of 77 patients with high concentrations of UA and 77 healthy subjects were tested to evaluate the differences in their TRACP 5b levels. Serial dilutions of UA were respectively spiked with a known concentration of TRACP 5b standard sample, then Serum TRACP 5b was detected by using bone TRAP® Assay. A correction equation was set to eliminate UA-derived TRACP 5b false-decrease. The effect of this correction was evaluated in high-UA individuals.ResultsThe average TRACP level of the high-UA individuals (1.47 ± 0.62 U/L) was significantly lower than that of the healthy subjects (2.62 ± 0.63 U/L) (t-test, p < 0.0001). The UA correction equation derived: ?TRACP 5b = -1.9751lg?UA + 3.7365 with an R2 = 0.98899. Application of the UA correction equation resulted in a statistically non-significant difference in TRACP 5b values between the healthy subjects and high-UA individuals (p = 0.24).ConclusionsHigh UA concentrations can falsely decrease TRACP 5b levels due to a method-related systematic error. To avoid misdiagnoses or inappropriate therapeutic decisions, increased attention should be paid to UA interference, when TRACP 5b is used for early diagnosis of cancer patients with bone metastasis, evaluation of the aggressiveness of osteosarcoma or prediction of survival in prostate cancer and breast cancer with bone metastases.
| S-EPMC4723035 | biostudies-literature
Project description:Biallelic mutations in ACP5, encoding tartrate-resistant acid phosphatase (TRACP), have recently been identified to cause the inherited immuno-osseous disorder, spondyloenchondrodysplasia (SPENCD). This study was undertaken to characterize the eight reported missense mutations in ACP5 associated with SPENCD on TRACP expression. ACP5 mutant genes were synthesized, transfected into human embryonic kidney (HEK-293) cells and stably expressing cell lines were established. TRACP expression was assessed by cytochemical and immuno-cytochemical staining with a panel of monoclonal antibodies. Analysis of wild (WT) type and eight mutant stable cell lines indicated that all mutants lacked stainable enzyme activity. All ACP5 mutant constructs were translated into intact proteins by HEK-293 cells. The mutant TRACP proteins displayed variable immune reactivity patterns, and all drastically reduced enzymatic activity, revealing that there is no gross inhibition of TRACP biosynthesis by the mutations. But they likely interfere with folding thereby impairing enzyme function. TRACP exists as two isoforms. TRACP 5a is a less active monomeric enzyme (35kD), with the intact loop peptide and TRACP 5b is proteolytically cleaved highly active enzyme encompassing two subunits (23 kD and 16 kD) held together by disulfide bonds. None of the mutant proteins were proteolytically processed into isoform 5b intracellularly, and only three mutants were secreted in significant amounts into the culture medium as intact isoform 5a-like proteins. Analysis of antibody reactivity patterns revealed that T89I and M264K mutant proteins retained some native conformation, whereas all others were in "denatured" or "unfolded" forms. Western blot analysis with intracellular and secreted TRACP proteins also revealed similar observations indicating that mutant T89I is amply secreted as inactive protein. All mutant proteins were attacked by Endo-H sensitive glycans and none could be activated by proteolytic cleavage in vitro. In conclusion, determining the structure-function relationship of the SPENCD mutations in TRACP will expand our understanding of basic mechanisms underlying immune responsiveness and its involvement in dysregulated bone metabolism.
| S-EPMC7098635 | biostudies-literature
Project description:Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with limited therapeutic options. Tartrate-resistant acid phosphatase 5 (ACP5) performs a variety of functions. However, its role in IPF remains unclear. Here, we demonstrate that the levels of ACP5 are increased in IPF patient samples and mice with bleomycin (BLM)-induced pulmonary fibrosis. In particular, higher levels of ACP5 are present in the sera of IPF patients with a diffusing capacity of the lungs for carbonmonoxide (DLCO) less than 40% of the predicted value. Additionally, Acp5 deficiency protects mice from BLM-induced lung injury and fibrosis coupled with a significant reduction of fibroblast differentiation and proliferation. Mechanistic studies reveal that Acp5 is upregulated by transforming growth factor-β1 (TGF-β1) in a TGF-β receptor 1 (TGFβR1)/Smad family member 3 (Smad3)-dependent manner, after which Acp5 dephosphorylates p-β-catenin at serine 33 and threonine 41, inhibiting the degradation of β-catenin and subsequently enhancing β-catenin signaling in the nucleus, which promotes the differentiation, proliferation and migration of fibroblast. More importantly, the treatment of mice with Acp5 siRNA-loaded liposomes or Acp5 inhibitor reverses established lung fibrosis. In conclusions, Acp5 is involved in the initiation and progression of pulmonary fibrosis and strategies aimed at silencing or suppressing Acp5 could be considered as potential therapeutic approaches against pulmonary fibrosis.
| S-EPMC8748833 | biostudies-literature
Project description:Prothrombin (PT) and osteopontin (OPN) promotes adhesion of different TRAP-positive multinucleated cells isolated from rat long bone (Hu et al. Exp Cell Res. 2008; 314: 638-50). The PT-adhering cell could represent either an immature precursor to the OPN-adherent osteoclast or constitute a distinct multinucleated cell population with a unique role in bone. Herein, phenotypic differences between PT- and OPN- cells were investigated with microarray- expression analysis. Characteristic for PT-cells was expression of innate immune response genes and scavenger receptors whereas OPN-cells expressed typical osteoclast proteins such as collagenases implicated in bone degradation.
2010-12-31 | GSE19950 | GEO
Project description:Alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes was solubilized from membrane precipitates by butan-1-ol at a critical pH of 7.0. The 12000-fold purification procedure included (NH(4))(2)SO(4) precipitation, DEAE-and TEAE-cellulose chromatography, Sephadex G-200 gel filtration and neuraminidase digestion followed by DEAE-cellulose chromatography. The purified protein contained 20% (w/w) carbohydrate and had mol.wt. 150000-156000 as estimated by Sephadex filtration and ultracentrifuge analysis. It was a tetrameric glycoprotein consisting of identical subunits, and it had a molecular activity at 25 degrees C of 2600s(-1) per tetramer. Its concentration in kidney was estimated to be 8.5-8.8mg/kg.
| S-EPMC1168074 | biostudies-other
Project description:BackgroundTartrate-resistant acid phosphatase (TRAP/ACP5), a metalloenzyme that is characteristic for its expression in activated osteoclasts and in macrophages, has recently gained considerable focus as a driver of metastasis and was associated with clinically relevant parameters of cancer progression and cancer aggressiveness.MethodsMDA-MB-231 breast cancer cells with different TRAP expression levels (overexpression and knockdown) were generated and characterized for protein expression and activity levels. Functional cell experiments, such as proliferation, migration and invasion assays were performed as well as global phosphoproteomic and proteomic analysis was conducted to connect molecular perturbations to the phenotypic changes.ResultsWe identified an association between metastasis-related properties of TRAP-overexpressing MDA-MB-231 breast cancer cells and a TRAP-dependent regulation of Transforming growth factor (TGFβ) pathway proteins and Cluster of differentiation 44 (CD44). Overexpression of TRAP increased anchorage-independent and anchorage-dependent cell growth and proliferation, induced a more elongated cellular morphology and promoted cell migration and invasion. Migration was increased in the presence of the extracellular matrix (ECM) proteins osteopontin and fibronectin and the basement membrane proteins collagen IV and laminin I. TRAP-induced properties were reverted upon shRNA-mediated knockdown of TRAP or treatment with the small molecule TRAP inhibitor 5-PNA. Global phosphoproteomics and proteomics analyses identified possible substrates of TRAP phosphatase activity or signaling intermediates and outlined a TRAP-dependent regulation of proteins involved in cell adhesion and ECM organization. Upregulation of TGFβ isoform 2 (TGFβ2), TGFβ receptor type 1 (TβR1) and Mothers against decapentaplegic homolog 2 (SMAD2), as well as increased intracellular phosphorylation of CD44 were identified upon TRAP perturbation. Functional antibody-mediated blocking and chemical inhibition demonstrated that TRAP-dependent migration and proliferation is regulated via TGFβ2/TβR, whereas proliferation beyond basal levels is regulated through CD44.ConclusionAltogether, TRAP promotes metastasis-related cell properties in MDA-MB-231 breast cancer cells via TGFβ2/TβR and CD44, thereby identifying a potential signaling mechanism associated to TRAP action in breast cancer cells.
| S-EPMC5602878 | biostudies-literature