Tartrate-resistant acid phosphatase in human alveolar macrophages

Tartrate-resistant acid phosphatase has been extracted from human alveolar macrophages, in which its specific activity is 10-fold that in whole lung. The apparent identity of the alveolar macrophage isoenzyme with that associated with osteoclasts suggests that both types of cell belong to the mononuclear phagocyte system. Within this system, expression of tartrate-resistant acid phosphatase appears to accompany certain kinds of differentiation.     

Alkaline fixation-resistant acid phosphatases in human tissues: histochemical evidence for a new type of acid phosphatase in endothelial, endometrial and neuronal sites

The effect of pH during formalin fixation on acid phosphatases in human tissues was studied. Lysosomal-type acid phosphatase was sensitive to alkaline fixation, being completely inactive after fixation at pH 9.0. Prostatic and tartrate-resistant osteoclastic/macrophagic types were alkaline fixation-resistant, as was an acid phosphatase localized in endothelium, endometrial stromal cells and intestinal nerves. The latter activity was further separable into fluoride- and tartrate-sensitive beta-glycerophosphatase and fluoride-sensitive, tartrate-resistant alpha-naphthyl phosphatase. The activities appeared to represent either different, tightly associated enzymes or separate activity centres of a single enzyme. Alkaline fixation-resistant alpha-naphthyl phosphatase at endothelial, endometrial and neuronal sites was also well demonstrated in unfixed or neutral formalin-fixed sections as tartrate-resistant activity similar to classical tartrate-resistant acid phosphatase, but these phosphatases appear to be antigenically different. Alkaline fixation-resistant acid phosphatase showed a restricted tissue distribution both in endothelium (mainly in vessels of abdominal organs) and at neuronal sites (only in intestinal nerves). Alkaline fixation-resistant acid phosphatase appears to represent a previously unknown or uncharacterized enzyme activity whose chemical properties could not be classified as any previously known type of acid or other phosphatases.     

Purification and N-terminal amino acid sequence of the tartrate-resistant acid phosphatase from human osteoclastoma: evidence for a single structure

Tartrate-resistant acid phosphatase type-5 was purified to apparent homogeneity from human osteoclastomas by sequential chromatography on CM-Sepharose, Phenyl-Sepharose, concanavalin A-Sepharose, FPLC Superose-12, and FPLC Mono-S. The purification over the original tissue extract was 1167-fold, with a yield of 16%. An identity in the N-terminal amino acid sequence and Mr was found between this enzyme and two type-5 tartrate-resistant acid phosphatases isolated from hairy cell leukemia spleen. However, they appeared to be different as assessed by amino acid composition. In contrast to a previous report, no evidence was found for two subunits of the tartrate-resistant acid phosphatase.     

Establishment and characterization of tartrate-resistant acid phosphatase and alkaline phosphatase double positive cell lines

Morphologically macrophage-like cells were cloned from hamster bone marrow cells by coculturing bone marrow cells with hamster chondrocytes. One of the clones (CCP-2) was characterized in the present study. CCP-2 cells were positive in an osteoclast marker enzyme, tartrate-resistant acid phosphatase (TRAP), alkaline phosphatase (ALP) and non-specific esterase (NSE). We showed CCP-2 cells degraded cartilage matrix and hydroxyapatite coated on Osteologic disks. A gelatinase secreted from CCP-2 cells was observed and purified from serum-free conditioned medium of the cells. N-terminal amino acid sequencing of the purified enzyme revealed it was matrix metalloproteinase-9. However, CCP-2 cells failed to express calcitonin receptors, a mature osteoclast marker, even after coculture with osteoblast ST2 cells in the presence of 1alpha, 25-dihydroxyvitamin D3 [1alpha, 25-(OH)2D3]. The cells showed high affinity to types X and I but not to type II collagen. In addition, histochemical studies have shown the presence of tartrate-resistant acid phosphatase and alkaline phosphatase double positive cells at the secondary ossification site of the hamster humerus. From these observations, we concluded that CCP-2 cells are similar to osteoclast but not the same. CCP-2 cells are therefore important tools for investigating chondroclastogenesis/osteoclastogenesis and endochondral ossification.     

Elevated total and isoenzyme forms of acid phosphatase in falciparum malaria

The activities of total serum acid phosphatase (E.C. 3.1.3.2) and of two of its isoenzymes, tartrate-resistant acid phosphatase and erythrocyte-specific acid phosphatase were measured in 109 adult male and female patients presenting acute falciparum malaria infection, and a normal, healthy control group comprised of 82 subjects. All the three forms of acid phosphatase were found to be significantly (p<0.05) higher during infection as compared to their activity in the control group. This result suggests that the measurement of acid phosphatase, particularly the erythrocyte isoenzyme, in serum could be potentially used as a biomarker of acute falciparum malaria infection.     

Localization of tartrate-resistant acid phosphatase in human placenta

Summary Tartrate-resistant acid phosphatase is an inducible marker of cell differentiation and activation expressed by specialized cells of macrophage lineage and some activated lymphocytes. Clinically, this phosphatase is a diagnostic marker for hairy cell leukaemia and osteoclast activity. The cDNA for this enzyme has been cloned from a placental expression library, yet the cell(s) expressing the enzyme protein has not been determined with certainty. Our laboratories have developed a monoclonal antibody, 9C5, suitable for immunohistochemical localization of tartrate-resistant acid phosphatase in paraffin sections. The purpose of this study was to use antibody 9C5 to identify cells expressing tartrate-resistant acid phosphatase in sections of paraffin-embedded, normal, full-term placenta and to determine if those cells expressed other macrophage markers including CD68(PG-M1 antibody), LN5, lysozyme ₁-antitrypsin and ₁-antichymotrypsin. Histochemical localization of activity in frozen sections was compared with immunohistochemical localization in paraffin sections of the same tissue specimens. The activity and antigenicity of this enzyme were detected in decidual cells, syncytiotrophoblast, and some macrophages distributed throughout maternal and embryonic tissues, but not in neutrophils. Unlike other tissues previously examined, placenta contains significant numbers of the phosphate-positive cells that are not of macrophage origin.     

Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase

A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions.     

Localization of acid phosphatase in lactating rat mammary glands

Summary Localization of acid phosphatase in mammary glands of lactating rats was studied by both biochemical and cytochemical methods. Cytochemically, acid phosphatase activity was detected by using lead citrate as the capture agent for the inorganic phosphate released from p-nitrophenyl phosphate. The activity was predominantly localized in the lumina of the endomembrane system and in the milk that had been secreted into the alveolar lumen. Biochemically, acid phosphatase was present in all the subcellular fractions with higher activities in the membrane-associated fractions. The localization of tartrate-resistant acid phosphatases within the endomembrane system of fully lactating rat mammary tissue suggests a possible role for these enzymes in milk secretory processes.Abbreviations ASMX 3-hydroxy-2-naphthoic acid 2,4-dimethylanilide - DMSO dimethylsulfoxide - DTT dithiothreitol - EDTA ethylenedinitrilo tetra-acetic acid - FGM fat globule membranes - MES 2-(N-morpholino) ethanesulfonic acid - PCMB p-chloromercuribenzoate - p-NPP p-nitrophenyl phosphate     

Expression of tartrate-resistant acid phosphatase in bone marrow macrophages

Bone marrow macrophages were found to express tartrate-resistant acid phosphatase (TRAP) under pathological conditions. In chronic granulocytic leukemia and metastatic carcinoma in the bone marrow this phenomenon was striking, all or almost all of the marrow macrophages being reactive. In other conditions, such as hypertransfusion or chemotherapy-induced marrow aplasia, the phenomenon did occur but was clearly a minor one. These observations indicate that tissue macrophages may become TRAP positive under the effect of unknown stimuli operating in certain pathological conditions. The results further suggest that the synthesis of the isoenzyme of acid phosphatase resistant to tartrate inhibition is a marker of macrophage activation rather than of differentiation towards particular subsets of the mononuclear phagocyte system.     

Activity and thermal stability of acid phosphatase in homogenates of Amoeba proteus, acclimated to various temperatures

Activity and thermoresistance of acid phosphatase were determined in supernatant of Amoeba proteus homogenates using 1-naphthyl phosphate (pH 4.0) and p-nitrophenyl phosphate (pH 5.5). Although tartrate-resistant and tartrate-sensitive acid phosphatases hydrolyse both substrates, the former mainly hydrolyses p-nitrophenyl phosphate and the latter 1-naphthyl phosphate. A decrease in the activity of the total and tartrate-sensitive acid phosphatases, when using 1-naphthyl phosphate, and of the total and tartrate-resistant acid phosphatases, when using p-nitrophenyl phosphate, was found in amoebae acclimated to 10 degrees C (10 degrees-amoebae) compared to those acclimated to 25 degrees C (25 degrees-amoebae). Using 1-naphthyl phosphate, the thermoresistance of the total acid phosphatase was lower in 10 degrees-amoebae than in 25 degrees-amoebae, but the thermostability of tartrate-resistant enzyme was the same in both groups of amoebae. Using p-nitrophenyl phosphate, the thermoresistance of the total and tartrate-resistant acid phosphatases was lower (the latter only slightly) in 10 degrees-amoebae than in 25 degrees-amoebae. It is suggested that at least with the use of 1-naphthyl phosphate a decrease in thermostability of the total acid phosphatase may be due to a decrease in thermoresistance of tartrate-sensitive enzyme. The results obtained confirm the author's previous data on the activity and thermostability of electrophoretic forms of acid phosphatase using 2-naphthyl phosphate in 10- and 25 degrees-amoebae (Sopina, 2001). It is the first case of discovering a correlation between changes in primary cell thermoresistance of amoebae cultured at different temperatures and changes in the activity and thermostability of acid phosphatase in their homogenates, with the number of electrophoretic forms of this enzyme and their mobility being permanent.     

Immunological characterization of human acid phosphatase gene products

The immunological cross-reactivity of heterogeneous acid phosphatase isozymes from different human tissues has been studied using monospecific antisera prepared against four homogeneous acid phosphatases. The enzyme characterized as tartrate-inhibitable, prostatic acid phosphatase is also found to be present in leukocytes, kidney, spleen, and placenta. The tartrate-inhibitable (liver) lysosomal enzyme is also found in kidney, fibroblasts, brain, placenta, and spleen, but it is not detectable in erythrocytes and prostate. In several tissues, 10–20% of the tartrate-inhibitable enzyme is not precipitated by any of the antisera used; an exceptionally high amount (54%) of such an enzyme is present in human brain. Antiserum against a low molecular weight tartrate-resistant liver enzyme (14 kDa) does not cross-react with the erythrocyte enzyme. (10–20 kDa). All other tissues except placenta, prostate, and fibroblast cells show a cross-reactivity with the 14-kDa acid phosphatase antiserum. Thus, the low molecular weight human liver acid phosphatase is distinct from the erythrocyte enzyme, and there are also at least three different tartrate-inhibitable acid phosphatases in human tissues. Chromosomal assignments have been made for only two of the (at least) five acid phosphatases that are present in adult human tissues.This study was supported by DHHS Research Grant GM 27003 from the U.S. National Institute of General Medical Sciences and by Grant SFB-104 from the Deutsche Forschungsgemeinschaft.     

Effects of proteolysis and reduction on phosphatase and ROS-generating activity of human tartrate-resistant acid phosphatase

Osteoclasts and macrophages express high amounts of tartrate-resistant acid phosphatase (TRACP), an enzyme with unknown biological function. TRACP contains a disulfide bond, a protease-sensitive loop peptide, and a redox-active iron that can catalyze formation of reactive oxygen species (ROS). We studied the effects of proteolytic cleavage by trypsin, reduction of the disulfide bond by beta-mercaptoethanol, and reduction of the redox-active iron by ascorbate on the phosphatase and ROS-generating activity of baculovirus-generated recombinant human TRACP. Ascorbate alone and trypsin in combination with beta-mercaptoethanol increased k(cat)/K(m) of the phosphatase activity seven- to ninefold. The pH-optimum was changed from 5.4-5.6 to 6.2-6.4 by ascorbate and trypsin cleavage. Trypsin cleavage increased k(cat)/K(m) of the ROS-generating activity 2.5-fold without affecting the pH-optimum (7.0). These results suggest that the protease-sensitive loop peptide, redox-active iron, and disulfide bond are important regulatory sites in TRACP, and that the phosphatase and ROS-generating activity are performed with different reaction mechanisms.     

Purification and characterization of purple acid phosphatase from developing rat bone

Tartrate-resistant acid phosphatase active on nucleoside di- and triphosphate substrates was isolated from developing rat bone and purified 2500-fold. The enzyme concentration had a purple coloration and activity that was sensitive to reducing agents. Mild reducing agents such as ferrous ion and ascorbic acid caused loss of purple color and increased activity toward substrates severalfold; however, a strong reductant such as dithionite caused loss of both color and activity which were partially restored by addition of ferrous ion and ascorbic acid. Enzyme activity was homogeneous with protein during the final gel permeation steps of chromatography and gave an apparent molecular size of about 40,000 Da. Determination of iron in the most pure preparation revealed the presence of 1.3 atoms of iron per molecule of the tartrate-resistant enzyme E2. Other properties of the purified enzyme include a pI of approximately 9.5 and sensitivity to inhibition by ions of copper, zinc, fluoride, and molybdate. Antibody prepared to the pre-concanavalin A (Con A)-Sepharose purified enzyme reacted with all protein from the Con A step, but it did not react with tartrate-sensitive acid phosphatase from rat bone or with potato acid phosphatase. Purple acid phosphatase from rat bone has many properties that parallel the iron-containing purple acid phosphatases from rat spleen, bovine spleen, and pig uterine secretions.     

Histochemical investigations on the localization of the purple acid phosphatase in the bovine spleen

The localization of the purple tartrate-resistant, iron-containing acid phosphatase in the bovine spleen was studied by enzyme histochemistry at the light and electron microscopic levels as well as by immunohistochemistry. The purple phosphatase was localized only in lysosome-like-organelles of cells belonging to the reticulo-phagocytic system. The same cells were identified as containing large iron(III)-deposits as ferritin in homogeneously granular accumulations and freely in the cytoplasm, or as hemosiderin in siderosomes. The phagocytosing cells containing purple phosphatase and ferritin often had close contact with clusters of aged and deformed erythrocytes. A possible catabolic role of the purple enzyme as a phosphatase degrading phosphoproteins of the erythrocyte membrane and the cytoskeleton was assumed.     

Histochemical and immunological demonstration of purple acid phosphatase in human and bovine alveolar macrophages

A tartrate-resistant purple acid phosphatase was localized in human and bovine alveolar macrophages by enzyme- and immuno-histochemistry using an antibody to bovine spleen purple phosphatase. The enzyme could be detected in human and bovine lung tissues as well as on cytospin preparations of alveolar macrophage suspensions from bronchoalveolar lavages. The immunological identity of human and bovine purple phosphatases from alveolar macrophages was demonstrated by Western blot analysis of material separated by polyacrylamide gel electrophoresis. A possible significance of the purple phosphatase as a marker enzyme of activated cells of the mononuclear phagocyte system is discussed.     

Histochemical investigations on the localization of the purple acid phosphatase in the bovine spleen

Summary The localization of the purple tartrate-resistant, iron-containing acid phosphatase in the bovine spleen was studied by enzyme histochemistry at the light and electron microscopic levels as well as by immunohistochemistry. The purple phosphatase was localized only in lysosome-like organelles of cells belonging to the reticulo-phagocytic system. The same cells were identified as containing large iron(III)-deposits as ferritin in homogeneously granular accumulations and freely in the cytoplasm, or as hemosiderin in siderosomes. The phagocytosing cells containing purple phosphatase and ferritin often had close contact with clusters of aged and deformed erythrocytes.A possible catabolic role of the purple enzyme as a phosphatase degrading phosphoproteins of the erythrocyte membrane and the cytoskeleton was assumed.     

Histochemical and immunological demonstration of purple acid phosphatase in human and bovine alveolar macrophages

Summary A tartrate-resistant purple acid phosphatase was localized in human and bovine alveolar macrophages by enzyme- and immuno-histochemistry using an antibody to bovine spleen purple phosphatase. The enzyme could be detected in human and bovine lung tissues as well as on cytospin preparations of alveolar macrophage suspensions from bronchoalveolar lavages. The immunological identity of human and bovine purple phosphatases from alveolar macrophages was demonstrated by Western blot analysis of material separated by polyacrylamide gel electrophoresis. A possible significance of the purple phosphatase as a marker enzyme of activated cells of the mononuclear phagocyte system is discussed.     

Tartrate-resistant acid phosphatase in free-living Amoeba proteus

Tartrate-resistant acid phosphatase (TRAP) of Amoeba proteus (strain B) was represented by 3 of 6 bands (= electromorphs) revealed after disc-electrophoresis in polyacrylamide gels with the use of 2-naphthyl phosphate as a substrate at pH 4.0. The presence of MgCl2, CaCl2 or ZnCl2 (50 mM) in the incubation mixture used for gel staining stimulated activities of all 3 TRAP electromorphs or of two of them (in the case of ZnCl2). When gels were treated with MgCl2, CaCl2 or ZnCl2 (10 and 100 mM, 30 min) before their staining activity of TRAP electromorphs also increased. But unlike 1 M MgCl2 or 1 M CaCl2, 1 M ZnCl2 partly inactivated two of the three TRAP electromorphs. EDTA and EGTA (5 mM), and H2O2 (10 mM) completely inhibited TRAP electromorphs after gel treatment for 10, 20 and 30 min, resp. Of 5 tested ions (Mg2+, Ca2+, Fe2+, Fe3+ and Zn2+), only the latter reactivated the TRAP electromorphs previously inactivated by EDTA or EGTA treatment. In addition, after EDTA inactivation, TRAP electromorphs were reactivated better than after EGTA. The resistance of TRAP electromorphs to okadaic acid and phosphatase inhibitor cocktail 1 used in different concentrations is indicative of the absence of PP1 and PP2A among these electromorphs. Mg2+, Ca2+ and Zn2+ dependence of TRAP activity, and the resistance of its electromorphs to vanadate and phosphatase inhibitor cocktail 2 prevents these electromorphs from being classified as PTP. It is suggested that the active center of A. proteus TRAP contains zinc ion, which is essential for catalytic activity of the enzyme. Thus, TRAP of these amoebae is metallophosphatase showing phosphomonoesterase activity in acidic medium. This metalloenzyme differs from both mammalian tartrate-resistant PAPs and tartrate-resistant metallophosphatase of Rana esculenta.     

Cytochemical examination of acid phosphatase and beta-glucuronidase enzymes in low-grade B cell non-Hodgkin's lymphomas

The differential diagnostic significance of acid phosphatase and beta-glucuronidase were studied in 77 cases of low-grade B cell non-Hodgkin's lymphomas. In most cases the results of cytochemical enzyme studies performed on malignant cells of the bone marrow were evaluated. B cell chronic lymphocytic leukaemia, centrocytic and centroblastic/centrocytic lymphomas were characterized by a weak or a negative acid phosphatase and beta-glucuronidase activity. Stronger positivity was observed in immunocytoma and in Waldenstr?m's macroglobulinaemia, while the highest activity was found in multiple myeloma. Hairy cell leukaemia of B cell origin showed intensive tartrate-resistant acid phosphatase activity. The cytochemical examination of these lysosomal enzymes may be useful in the diagnosis of low-grade malignant lymphomas of B cell origin by completing other methods.     

Leishmania donovani: surface membrane acid phosphatase blocks neutrophil oxidative metabolite production

We show that a purified preparation of the prominent tartrate-resistant acid phosphatase (E.C.3.1.3.2), isolated from the external surface of the intracellular parasite Leishmania donovani (promastigote form), inhibits toxic oxidative metabolite production of neutrophils. Preincubation of a neutrophil suspension (2.5 X 10(6) cells/ml) for 15 min at 37 C with 250 units (1 unit equals 1 nmole of 4-methylumbelliferyl phosphate cleaved per hr at pH 5.5) of the acid phosphatase in Krebs-Ringer phosphate buffer (pH 7.4) decreased O2 consumption, O2- production, and H2O2 production of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils to 15-25% of control values. The acid phosphatase also affected concanavalin A-stimulated O2-production by neutrophils, but had no effect on the rate of phorbol myristic acetate-stimulated O2- production, chemotactic peptide binding, degranulation, or membrane depolarization. Addition of an acid phosphatase inhibitor (Complex E; (NH4)6[P2Mo18O62] X 9H2O) to suspensions of opsonized promastigotes and neutrophils resulted in a threefold or greater enhancement of O2- production. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.