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In Vivo (Athens, Greece) 2022Macrophages and biomaterial-induced multinucleated giant cells (BMGCs) are central elements in the tissue reaction cascade towards bone substitute materials (BSM). The...
Comparison of the Validity of Enzymatic and Immunohistochemical Detection of Tartrate-resistant Acid Phosphatase (TRAP) in the Context of Biocompatibility Analyses of Bone Substitutes.
BACKGROUND/AIM
Macrophages and biomaterial-induced multinucleated giant cells (BMGCs) are central elements in the tissue reaction cascade towards bone substitute materials (BSM). The enzymatic detection of the lytic enzyme tartrate-resistant acid phosphatase (TRAP) has manifoldly been used to examine the so-called "bioactivity" of BSM. The present study aimed to compare the detection validity and expression pattern of the TRAP enzyme using enzymatic and immunohistochemical detection methods in the context of biocompatibility analyses of BSM.
PATIENTS AND METHODS
Biopsies from 8 patients were analyzed after sinus augmentation with a xenogeneic bone substitute. Analysis of both macrophage and BMGC polarization were performed by histochemical TRAP detection and immunohistochemical detection of TRAP5a. Histomorphometrical analysis was used for comparison of the TRAP detection of BMGCs.
RESULTS
The enzymatic TRAP detection method revealed that in 7 out of 8 biopsies only single cells were TRAP-positive, whereas most of the cells and especially the BMGCs were TRAP-negative. The immunohistochemical detection of TRAP5a showed moderate numbers of stained mononuclear cells, while the majority of the BMGCs showed signs of TRAP5a-expression. The enzymatic TRAP detection was comparable to the results obtained via immunohistochemistry only in one case. The histomorphometrical analysis showed that significantly more mononuclear and multinucleated TRAP-positive cells were found using immunohistochemical TRAP5a-staining compared to the enzymatic TRAP detection method. Also, significantly more TRAP-negative BMGCs were found using the enzymatic TRAP detection.
CONCLUSION
The immunohistochemical detection of TRAP is more accurate for examination of the bioactivity and cellular degradability of BSM.
Topics: Acid Phosphatase; Biocompatible Materials; Bone Substitutes; Humans; Immunohistochemistry; Tartrate-Resistant Acid Phosphatase
PubMed: 36099106
DOI: 10.21873/invivo.12930 -
The Biochemical Journal Aug 19741. Development of ribonuclease activity in the cotyledons of germinating peas is biphasic, the time of appearance of the two phases depending on the conditions of...
1. Development of ribonuclease activity in the cotyledons of germinating peas is biphasic, the time of appearance of the two phases depending on the conditions of growth. 2. Acid phosphatase exhibits a single phase of development. 3. Cycloheximide inhibits development of ribonuclease activity in phase II but not in phase I. 4. (14)C-labelled amino acids are not incorporated into ribonuclease isolated during phase I. 5. The buoyant density of ribonuclease isolated during phase I is not affected by imbibition of the seed in 80% deuterium oxide. 6. Acid phosphatase was isolated from the supernatant fraction of the cotyledons of germinating peas and partially purified. 7. Development of acid phosphatase activity during germination is inhibited by treatment of the seed with cycloheximide or actinomycin D. 8. Partial purification of acid phosphatase from peas germinated in the presence of (14)C-labelled amino acids suggests that the enzyme is radioactively labelled. 9. Germination of peas in the presence of 80% deuterium oxide results in an increase in the buoyant density of acid phosphatase. 10. The results suggest that increase in ribonuclease activity during the first 4 days of germination does not result from synthesis of protein de novo, but that the corresponding increase in acid phosphatase activity does result from synthesis de novo.
Topics: Acid Phosphatase; Age Factors; Amino Acids; Carbon Radioisotopes; Centrifugation; Centrifugation, Density Gradient; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Cycloheximide; Dactinomycin; Deuterium; Electrophoresis, Polyacrylamide Gel; Plants; Ribonucleases; Seeds
PubMed: 4441377
DOI: 10.1042/bj1420211 -
High force eccentric exercise enhances serum tartrate-resistant acid phosphatase-5b and osteocalcin.Journal of Musculoskeletal & Neuronal... Mar 2014We investigated the effects of eccentric contractions (ECs) on bone metabolism markers and the relationship between bone metabolism and skeletal muscle related protein....
We investigated the effects of eccentric contractions (ECs) on bone metabolism markers and the relationship between bone metabolism and skeletal muscle related protein. Seventeen young untrained men were divided into two groups and performed either 60 or 30 maximal ECs. We measured serum levels of osteocalcin (OC), bone alkaline phosphatase, cross-linked N-telopeptide of type I collagen (NTx), and tartrate-resistant acid phosphatase 5b (TRACP-5b), growth hormone (GH), and insulin-like growth factor-1 (IGF-1). Blood samples were collected for up to five days after ECs. OC with 60 ECs were significantly higher than with 30 ECs (2 hours; p<0.05, day 1 and day 5; p<0.01). TRACP-5b with 60 ECs were significantly higher than with 30 ECs (day 3 and day 5; p<0.001). IGF-1 and OC were significantly positively correlated with 60 ECs (2 hours, day 1, and day 5; p<0.05). There were also significant positive correlations between IGF-1 and NTx with 60 ECs (2 hours, p<0.01; day 1, p<0.05). We found that one bout of severe ECs caused increases in OC and TRACP-5b, which promote increased bone metabolism. Our results suggest that contraction-induced IGF-1 may activate OC and NTx in acute response.
Topics: Acid Phosphatase; Bone and Bones; Exercise; Humans; Insulin-Like Growth Factor I; Isoenzymes; Male; Muscle Contraction; Osteocalcin; Tartrate-Resistant Acid Phosphatase; Young Adult
PubMed: 24583540
DOI: No ID Found -
Plant Physiology Dec 1970We report the effects of abscisic acid and auxin (alpha-naphthalene acetic acid) on regulation of enzyme synthesis during senescence of leaf sections of Rhoeo discolor...
We report the effects of abscisic acid and auxin (alpha-naphthalene acetic acid) on regulation of enzyme synthesis during senescence of leaf sections of Rhoeo discolor Hance. Abscisic acid always accelerates the onset of and enhances the magnitude of the increase in activity of acid phosphatase; this is followed by an acceleration of the onset of a rapid increase in free space.RNase activity increases 2- to 5-fold after cutting of leaf sections. Abscisic acid increases RNase activity and inhibits the rate of incorporation of uridine and leucine in leaf sections removed from plants grown under stress but not favorable conditions. Auxin inhibits the increase in RNase and acid phosphatase and suppresses the effects of abscisic acid. The increase in activity of RNase and acid phosphatase is inhibited by inhibitors of RNA and protein synthesis. This and other evidence suggests that the increases in hydrolase activity could result from new enzyme synthesis. The possible significance of the results in respect of hormonal regulation of enzyme activity and senescence is discussed.
Topics: Acid Phosphatase; Aging
PubMed: 5500207
DOI: 10.1104/pp.46.6.806 -
The Journal of Biological Chemistry Jan 1987An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall... (Comparative Study)
Comparative Study
An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.
Topics: Acid Phosphatase; Animals; Bone Matrix; Cattle; Chromatography; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Isoelectric Focusing; Kinetics; Lysosomes; Molecular Weight; Phosphoprotein Phosphatases; Protein Tyrosine Phosphatases; Substrate Specificity; Tartrates
PubMed: 3027088
DOI: No ID Found -
The Journal of Biological Chemistry Jan 1994The purple phosphatases catalyze hydrolysis of phosphate esters (optimum pH approximately 5) and are resistant to inhibition by dextro-rotatory tartrate; their...
Purple acid phosphatase of the human macrophage and osteoclast. Characterization, molecular properties, and crystallization of the recombinant di-iron-oxo protein secreted by baculovirus-infected insect cells.
The purple phosphatases catalyze hydrolysis of phosphate esters (optimum pH approximately 5) and are resistant to inhibition by dextro-rotatory tartrate; their distinctive color is due to Fe(III)-phenolate charge-transfer transitions at their active site. Expression of human purple phosphatase, designated type 5 acid phosphatase, is restricted to osteoclasts and other activated cells of monohistiocytic lineage, but its biological rôle in relation to bone resorption and phagocytosis is unknown. To characterize this enzyme further, we have engineered the human type 5 acid phosphatase into a baculovirus vector expression system that enabled milligram quantities of purple protein to be purified from medium containing Sf9 host cells. The phosphatase cDNA was transcribed as a single RNA species of 1.5 kilobases as in human tissues. Tartrate-resistant acid phosphatase activity reacting with uteroferrin antisera appeared in the culture medium, from which up to 8 mg/liter was purified by two-step cation-exchange chromatography at pH 8.0. Two isoforms of approximately 36 kDa were identified by SDS-polyacrylamide electrophoresis and were converted to a single species of apparent molecular size 34 kDa upon treatment with N-glycosidase F, indicating secreted glycoforms of a single polypeptide. Mass spectroscopy showed that the mean molecular mass of the active, secreted glycoprotein was 35849 Da. The recombinant enzyme (specific activity, 190 mumol p-nitrophenol/min/mg at 37 degrees C) contained 2 iron atoms/molecule and formed purple, monoclinic crystals. Exposure to the ferric chelator, 1,2-dimethyl-3-hydroxypyrid-4-one, rapidly inactivated the enzyme, which was not inhibited by alpha, alpha'-bipyridyl, a ferrous chelator. That ferric iron is essential for enzymatic catalysis, was further indicated by the synergistic effects of the reductant, dithiothreitol, and bipyridyl on phosphatase activity. The recombinant purple phosphatase catalyzed the peroxidation of 5-aminophthalhydrazide (luminol), as evidenced by the induction of chemiluminescence; this reaction was inhibited by alpha, alpha'-bipyridyl at concentrations that did not inhibit phosphatase activity. The divalent iron moiety of human type 5 phosphatase may therefore participate in the generation of free radical species by fluid-phase reactions involving Fenton chemistry that are dissociated from its phosphatase function.
Topics: Acid Phosphatase; Animals; Cells, Cultured; Cloning, Molecular; Crystallography; Genes; Humans; In Vitro Techniques; Luminescent Measurements; Macrophages; Metalloproteins; Moths; Nucleopolyhedroviruses; Osteoclasts; Recombinant Proteins
PubMed: 8288593
DOI: No ID Found -
G3 (Bethesda, Md.) Dec 2018Regulation of plant root angle is critical for obtaining nutrients and water and is an important trait for plant breeding. A plant's final, long-term root angle is the...
Regulation of plant root angle is critical for obtaining nutrients and water and is an important trait for plant breeding. A plant's final, long-term root angle is the net result of a complex series of decisions made by a root tip in response to changes in nutrient availability, impediments, the gravity vector and other stimuli. When a root tip is displaced from the gravity vector, the short-term process of gravitropism results in rapid reorientation of the root toward the vertical. Here, we explore both short- and long-term regulation of root growth angle, using natural variation in tomato to identify shared and separate genetic features of the two responses. Mapping of expression quantitative trait loci mapping and leveraging natural variation between and within species including Arabidopsis suggest a role for 27 and in determining root angle.
Topics: Acid Phosphatase; Arabidopsis; Arabidopsis Proteins; Glycoproteins; Gravitropism; Plant Roots
PubMed: 30322904
DOI: 10.1534/g3.118.200540 -
PloS One 2021Genomic sequence analysis of Acinetobacter baumannii revealed the presence of a putative Acid Phosphatase (AcpA; EC 3.1.3.2). A plasmid construct was made, and...
Genomic sequence analysis of Acinetobacter baumannii revealed the presence of a putative Acid Phosphatase (AcpA; EC 3.1.3.2). A plasmid construct was made, and recombinant protein (rAcpA) was expressed in E. coli. PAGE analysis (carried out under denaturing/reducing conditions) of nickel-affinity purified protein revealed the presence of a near-homogeneous band of approximately 37 kDa. The identity of the 37 kDa species was verified as rAcpA by proteomic analysis with a molecular mass of 34.6 kDa from the deduced sequence. The dependence of substrate hydrolysis on pH was broad with an optimum observed at 6.0. Kinetic analysis revealed relatively high affinity for PNPP (Km = 90 μM) with Vmax, kcat, and Kcat/Km values of 19.2 pmoles s-1, 4.80 s-1(calculated on the basis of 37 kDa), and 5.30 x 104 M-1s-1, respectively. Sensitivity to a variety of reagents, i.e., detergents, reducing, and chelating agents as well as classic acid phosphatase inhibitors was examined in addition to assessment of hydrolysis of a number of phosphorylated compounds. Removal of phosphate from different phosphorylated compounds is supportive of broad, i.e., 'nonspecific' substrate specificity; although, the enzyme appears to prefer phosphotyrosine and/or peptides containing phosphotyrosine in comparison to serine and threonine. Examination of the primary sequence indicated the absence of signature sequences characteristic of Type A, B, and C nonspecific bacterial acid phosphatases.
Topics: Acid Phosphatase; Acinetobacter baumannii; Amino Acid Sequence; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Molecular Weight; Proteome; Recombinant Proteins; Sequence Homology; Substrate Specificity
PubMed: 34077475
DOI: 10.1371/journal.pone.0252377 -
Journal of Bone and Mineral Research :... Oct 2003TRACP is synthesized as a latent proenzyme requiring proteolytic processing to attain maximal phosphatase activity. Excision of an exposed loop domain abolishes the... (Review)
Review
TRACP is synthesized as a latent proenzyme requiring proteolytic processing to attain maximal phosphatase activity. Excision of an exposed loop domain abolishes the interaction between the loop residue Asp146 and a ligand to the redox-sensitive iron of the active site, most likely Asn91, providing a mechanism for the enzyme repression. Both cathepsin K and L efficiently cleave in the loop domain and activate the latent enzyme, and we propose that cathepsin K acts as a physiological activator of TRACP in osteoclasts, whereas cathepsin L might fulfill a similar role in different types of macrophages. Considering the rather broad substrate specificity of TRACP, a tight regulation of its activity in the cell appears warranted. Besides proteolytic cleavage, the enzyme should need a specific local environment with a slightly acidic pH and reducing equivalents to keep the enzyme fully active. Cellular subcompartments where these required conditions prevail are potential subcellular site(s) of TRACP action. Of bone phosphoproteins shown to be substrates for TRACP, both osteopontin and bone sialoprotein are colocalized with TRACP in the resorption lacuna of the osteoclasts, and dephosphorylation of OPN impair its ability to promote adhesion as well as migration of osteoclasts in vitro. A role for TRACP as an osteopontin phosphatase in bone is therefore suggested. The expression of TRACP as well as OPN in other tissues with possible interactions between the two could suggest a more general function for TRACP as a regulator of OPN phosphorylation and bioactivity.
Topics: Acid Phosphatase; Animals; Binding Sites; Bone and Bones; Cell Adhesion; Cell Movement; Humans; Hydrogen-Ion Concentration; Ions; Isoenzymes; Macrophages; Osteopontin; Oxidation-Reduction; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Binding; Protein Structure, Tertiary; Sialoglycoproteins; Substrate Specificity; Tartrate-Resistant Acid Phosphatase
PubMed: 14584906
DOI: 10.1359/jbmr.2003.18.10.1912 -
Infection and Immunity Nov 1988Virulent and avirulent clones of Leishmania donovani promastigotes were examined for their acid phosphatase activity. The acid phosphatase activity of whole-cell lysates...
Virulent and avirulent clones of Leishmania donovani promastigotes were examined for their acid phosphatase activity. The acid phosphatase activity of whole-cell lysates of virulent clones was 1.5 to 2.0 times higher than that of avirulent clones. Pellet fractions (260,000 x g, 30 min) from sonicated promastigotes of a virulent clone and an avirulent clone contained 60 and 40% of the total enzyme activity, respectively. Membrane-bound acid phosphatase was extracted with Triton X-100 from the pellet. This membrane-bound phosphatase activity was 2.4-fold higher in virulent organisms than in avirulent organisms. The membrane acid phosphatase exhibited two distinct bands on polyacrylamide gels stained for enzyme activity. One diffuse, faster-migrating band showed identical electrophoretic mobility in both virulent and avirulent clones, although a higher enzymatic activity was observed with the extract from virulent cells. In contrast, a slower-migrating band was different between the two clones in the mobility. These results suggest that membrane-bound acid phosphatase was quantitatively and qualitatively different between virulent and avirulent promastigotes of L. donovani. In addition, virulent cells produced a relatively high level of acid phosphatase throughout the growth in culture.
Topics: Acid Phosphatase; Animals; Cell Membrane; Clone Cells; Electrophoresis, Polyacrylamide Gel; Leishmania donovani; Polyethylene Glycols; Time Factors
PubMed: 3169990
DOI: 10.1128/iai.56.11.2856-2860.1988