RESUMEN
OBJECTIVE: LPS can induce differentiation to osteoclast-like cells independent of RANKL. In comparison with RANKL, the effects of Th1 and Th2 cytokines on LPS-induced osteoclastogenesis have not been extensively studied. In this study, we investigated the effects of IFN-γ and IL-4 on RANKL- or LPS-induced osteoclastogenesis. MATERIALS AND METHODS: RAW 264.7 cells were induced to differentiate into osteoclast-like cells by RANKL or LPS, in the absence or presence of IFN-γ or IL-4. The number of TRAP-positive, multinucleated (≥ 3 nuclei) cells (MNCs) was counted. mRNA and protein levels of TRAP and cathepsin K were determined by quantitative RT-PCR and Western immunoblot, respectively. Expression of other genes implicated in osteoclast and macrophage differentiation and inflammation was also quantitated and was subsequently assessed in bone marrow-derived macrophages (BMMs). Phagocytic capacity of differentiated RAW264.7 was investigated by the uptake of pHrodo S. aureus bioparticles conjugates. RESULTS: In contrast to the RANKL-treated cell population that gained more macrophage-like properties at the level of gene and protein expression as well as phagocytosis in the presence of IFN-γ or IL-4, the LPS-induced population gained more osteoclast-like properties by the addition of the same factors. CONCLUSION: These data suggest that the adaptive immune system, through either Th1 or Th2 cytokines, is able to modify the differentiation process of osteoclasts in inflammatory situations. Moreover, the study provides an example of different regulation of osteoclast differentiation during physiological and inflammatory conditions.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Interferón gamma/farmacología , Interleucina-4/farmacología , Lipopolisacáridos/farmacología , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Ligando RANK/farmacología , Animales , Macrófagos/citología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
R-Ras contains a proline-rich motif that resembles SH3 domain-binding sites but that has escaped notice previously. We show here that this site in R-Ras is capable of binding SH3 domains and that the SH3 domain binding may be important for R-Ras function. A fusion protein containing the SH3 domains of the adaptor protein Nck interacted strongly with the R-Ras proline-rich sequence and with the intact protein. The binding was independent of whether R-Ras was in its GDP or GTP form. The Nck binding, which was mediated by the second of the three SH3 domains of Nck, was obliterated by mutations in the proline-rich sequence of R-Ras. The interaction of Nck with R-Ras could also be shown in yeast two-hybrid assays and by co-immunoprecipitation in human cells transfected with Nck and R-Ras. Previous results have shown that the expression of a constitutively active R-Ras mutant, R-Ras(38V), converts mouse 32D monocytic cells into highly adherent cells. Introducing the proline mutations into R-Ras(38V) suppressed the effect of R-Ras on 32D cell adhesion while not affecting GTP binding. These results reveal an unexpected regulatory pathway that controls R-Ras through an SH3 domain interaction. This pathway appears to be important for the ability of R-Ras to control cell adhesion.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Integrinas/metabolismo , Prolina/metabolismo , Proteínas ras/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , GTP Fosfohidrolasas/química , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido , Proteínas ras/químicaRESUMEN
Purple acid phosphatases (PAPs) are binuclear acid metallohydrolases also referred to as tartrate-resistant acid phosphatases (TRAPs) or type 5 acid phosphatases. The cDNA sequences of TRAP/PAP enzymes from different species and organs indicate that these enzymes are translated as monomeric polypeptides of approx. 35 kDa, contrasting with the predominantly two-subunit structure observed in purified enzyme preparations. In the present study we have compared certain structural and enzyme-kinetic properties of recombinant rat PAP (monomeric) with those of the native rat bone TRAP/PAP enzyme (two-subunit), and examined effects on these parameters by cleaving the monomeric recombinant PAP with the serine proteinase trypsin or the cysteine proteinases papain or cathepsin B. Cleavage with trypsin resulted in a moderate activation of the recombinant enzyme and shifted the pH optimum to a slightly more basic value (5.0-5.5). Cleavage with papain resulted in complete activation and conferred similar properties to those of the bone PAP variant with regard to pH optimum (5.5-6.0) and sensitivity to reducing agents, as well as in the sizes of the subunits. Substrate specificity studies showed that the two-subunit bone PAP was considerably more active than the monomeric recombinant rat PAP towards a variety of serine-, threonine- and tyrosine-phosphorylated substrates. Of these substrates, bovine milk osteopontin seemed to be the most readily dephosphorylated substrate. In conclusion, the results suggest that the monomeric form of PAP represent a latent proenzyme with low enzymic activity towards both tyrosine- and serine/threonine-containing phosphorylated substrates. Besides being implicated in the catabolism of the extracellular matrix, members of the cysteine proteinase family might also exert a regulatory role in degradative processes involving the PAP enzymes by converting the newly synthesized PAPs to enzymically active and microenvironmentally regulated species.
Asunto(s)
Fosfatasa Ácida/biosíntesis , Cisteína Endopeptidasas/metabolismo , Precursores Enzimáticos/biosíntesis , Glicoproteínas/biosíntesis , Fosfatasa Ácida/metabolismo , Secuencia de Aminoácidos , Animales , Huesos/enzimología , Bovinos , Activación Enzimática , Glicoproteínas/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Tartrate-resistant acid phosphatase (TRAP) is a mammalian di-iron- containing enzyme that belongs to the family of purple acid phosphatases (PAP). It is highly expressed in a limited number of tissues, predominantly in bone-resorbing osteoclasts and in macrophages of spleen. We have determined the crystal structure of rat TRAP in complex with a phosphate ion to 2.7 A resolution. The fold resembles that of the catalytic domain of kidney bean purple acid phosphatase (KBPAP), although the sequence similarity is limited to the active site residues. A surface loop near the active site is absent due to proteolysis, leaving the active-site easily accessible from the surrounding solvent. This, we believe, gives a structural explanation for the observed proteolytic activation of TRAP. The current structure was determined at a relatively high pH and without any external reducing agents. It is likely that it represents an oxidized and therefore catalytically inactive form of the enzyme.
Asunto(s)
Fosfatasa Ácida/química , Glicoproteínas/química , Fosfatasa Ácida/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Glicoproteínas/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Ratas , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Fosfatasa Ácida/química , Huesos/enzimología , Glicoproteínas/química , Isoenzimas/química , Proteínas de Plantas/química , Proteínas Recombinantes/química , Fosfatasa Ácida/genética , Fosfatasa Ácida/aislamiento & purificación , Animales , Baculoviridae/genética , Conformación de Carbohidratos , Catálisis , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Hierro/análisis , Oligosacáridos/química , Fosfoproteínas Fosfatasas , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Espectrofotometría , Spodoptera/genética , Relación Estructura-Actividad , Fosfatasa Ácida TartratorresistenteRESUMEN
The tartrate-resistant acid phosphatase (TRAP) of skeletal osteoclasts was found to partially dephosphorylate the bone matrix phosphoproteins osteopontin (OPN) and bone sialoprotein (BSP). TRAP also partially dephosphorylated metabolically [32P]PO4-labeled OPN as well as BSP, whereas comparable amounts of either alkaline phosphatase or prostatic acid phosphatase, at their respective pH optima, were ineffective, indicating a certain preference of TRAP for these phosphoprotein substrates. It has previously (Flores, M., Norgärd, M., Heinegård, D., Reinholt, F. P., and Andersson, G. (1992) Exp. Cell Res. 201, 526-530) been shown that osteoclasts bind to OPN as well as to BSP coated onto glass. We can now show that the partially dephosphorylated proteins no longer support osteoclast binding. These results indicate that the secretion of TRAP from osteoclasts into the resorption area could exert a regulatory influence on the attachment of the cells to the bone surface. This could imply roles in the development of ruffled borders and/or in the regulation of osteoclast motility on the bone surface.
Asunto(s)
Fosfatasa Ácida/metabolismo , Osteoclastos/enzimología , Sialoglicoproteínas/metabolismo , Fosfatasa Ácida/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Adhesión Celular , Humanos , Sialoproteína de Unión a Integrina , Datos de Secuencia Molecular , Osteoclastos/citología , Osteopontina , Fosforilación , Ratas , Ratas Sprague-Dawley , Tartratos/farmacologíaRESUMEN
Tartrate-resistant acid phosphatase (TRAP) is a characteristic constituent of osteoclasts and some mononuclear preosteoclasts and, therefore, used as a histochemical and biochemical marker for osteoclasts and bone resorption. We now report the isolation of a 1397-base pair (bp) full-length TRAP/tartrate-resistant acid ATPase (TrATPase) cDNA clone from a neonatal rat calvaria lambda gt11 cDNA library. The cDNA clone consists of a 92-bp untranslated 5'-flank, an open reading frame of 981 bp and a 324-bp untranslated 3'-poly(A)-containing region. The deduced protein sequence of 327 amino acids contains a putative cleavable signal sequence of 21 amino acids. The mature polypeptide of 306 amino acids has a calculated Mr of 34,350 Da and a pI of 9.18, and it contains two potential N-glycosylation sites and the lysosomal targeting sequence DKRFQ. At the protein level, the sequence displays 89-94% homology to TRAP enzymes from human placenta, beef spleen, and uteroferrin and identity to the N terminus of purified rat bone TRAP/TrATPase. An N-terminal amino acid segment is strikingly homologous to the corresponding region in lysosomal and prostatic acid phosphatases. The cDNA recognized a 1.5-kilobase mRNA in long bones and calvaria, and in vitro translation using, as template, mRNA transcribed from the full-length insert yielded an immunoprecipitated product of 34 kDa. In neonatal rats, TRAP/TrATPase mRNA was highly expressed in skeletal tissues, with much lower (less than 10%) levels detected in spleen, thymus, liver, skin, brain, kidney, brain, lung, and heart. In situ hybridization demonstrated specific labeling of osteoclasts at endostal surfaces and bone trabeculae of long bones. Thus, despite the apparent similarity of this osteoclastic TRAP/TrATPase with type 5, tartrate-resistant and purple, acid phosphatases expressed in other mammalian tissues, this gene appears to be preferentially expressed at skeletal sites.
Asunto(s)
Fosfatasa Ácida/genética , Huesos/enzimología , Tartratos/farmacología , Fosfatasa Ácida/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Bovinos , ADN/genética , Femenino , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Placenta/enzimología , Biosíntesis de Proteínas , ARN/genética , Ratas , Homología de Secuencia de Ácido Nucleico , Bazo/enzimología , Transcripción Genética , Útero/enzimologíaRESUMEN
Certain physicochemical properties of rat bone tartrate-resistant acid ATPase (TrATPase), including the size and shape of the enzyme, potential subunit composition, and detergent binding, have been elucidated. SDS-polyacrylamide gel electrophoresis in combination with immunoblot analysis showed that the bone TrATPase has a molecular weight of 33,000 D and is composed of disulfide-linked polypeptides of 20,000 and 16,000 D. The enzyme contains 1.7 mol Fe per mol enzyme. Hydrodynamic studies allowed calculation of the Stokes radius (24 A), the sedimentation coefficient (3.19S), the partial specific volume (0.748 ml/g), the frictional ratio (0.995), and the axial ratio (1.0). The amount of detergent bound to the protein was determined to 4 mol of Triton X-100 per mol enzyme. The molecular weight of bone TrATPase derived from these parameters was 31,900 D. N-terminal amino acid sequence analysis of the Mr 20,000 subunit indicated a high degree of similarity with TRAP enzymes from spleen, uterus, placenta, hairy cell leukemia, and osteoclastoma. It is concluded that rat bone TrATPase belongs to the type 5 (tartrate-resistant and purple) acid phosphatase family. The similarities in the N-terminal amino acid sequences, iron content, and physicochemical properties of TRAP enzymes indicate a close structural relationship between type 5 acid phosphatases expressed in different tissues. The findings that TrATPase has a spherical shape and binds low amounts of detergent suggest that the enzyme is a soluble protein, compatible with the view that TrATPase is secreted by the osteoclast.
Asunto(s)
Fosfatasa Ácida/análisis , Adenosina Trifosfatasas/análisis , Huesos/enzimología , Isoenzimas/análisis , Tartratos/farmacología , Secuencia de Aminoácidos , Animales , Centrifugación por Gradiente de Densidad , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Hierro/análisis , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de Ácido NucleicoRESUMEN
Osteoclasts are effector cells in bone breakdown, and the active bone resorption is confined to the ruffled border zone of these cells. An acid milieu is maintained in this zone which is probably a prerequisite for bone resorption. Tartrate-resistant acid phosphatase (TRAP) activity has been recognized as a characteristic property of osteoclasts and in several studies proposed as a cytochemical marker of osteoclasts. We have previously isolated and characterized a tartrate-resistant and iron-activated acid ATPase (TrATPase) from rat bone, the enzyme being a member of the TRAP family. In the present study the ultrastructural localization of this enzyme was delineated by employing immunogold technique on low temperature-embedded maxillar rat bone. Intensive immunolabeling was seen on the bone surfaces facing the ruffled border zone while lower amounts of marker were seen in adjacent bone areas, that is, on the bone surfaces facing the clear zone and deeper-into the bone. Within the osteoclasts gold markers were observed mainly in vesicular structures interpreted as lysosomes. Immunolabeling was also observed in the recently described endocytic cells located near osteoblasts and osteoclasts. Also in these cells, the marker was confined to lysosomelike structures. The amount of label in bone facing osteoblasts was low, as was the amount within osteoblasts. Our observation of extracellular localization, in particular accumulation of TrATPase in bone matrix facing the ruffled border area of the osteoclasts, favors the view that the enzyme is exported to areas of active bone resorption, thereby indicating a potential role for the enzyme in this process.
Asunto(s)
Adenosina Trifosfatasas/análisis , Huesos/enzimología , Animales , Huesos/ultraestructura , Inmunohistoquímica , Microscopía Inmunoelectrónica , Ratas , Ratas Endogámicas , Tartratos/farmacologíaRESUMEN
In order to evaluate the usefulness of a recently described acid ATPase as a marker for osteoclast differentiation, we have performed histochemical and biochemical analyses of the distribution of tartrate-resistant acid phosphatase (TRAP) and tartrate-resistant acid ATPase (TrATPase) in bone, bone marrow and spleen. Histochemical studies of bone demonstrated that multinucleated osteoclasts stained for both TRAP and TrATPase. However, staining for TRAP covered the entire cytoplasm, whereas TrATPase staining was localized primarily to cytoplasmic areas next to bone and on adjacent mineralized surfaces. Occasionally TrATPase-positive mononuclear cells were observed on excavations in the bone surface. In the spleen, mononuclear TRAP-positive cells were located in the marginal zone between the white and red pulp, whereas no staining for TrATPase was observed. Comparison of the biochemically measured TRAP and TrATPase activities showed that bone had the highest specific activity for both enzymes followed by the bone marrow and spleen. However, enzyme activity in the spleen compared to bone was about 4-fold higher for TRAP compared to TrATPase. Additional evidence for a restricted expression of TrATPase to bone relative to spleen was obtained by in vitro translation studies. These data indicate that TrATPase is a more selective marker than TRAP in histochemical and biochemical studies of osteoclast differentiation and furthermore suggest that development of TrATPase is a late acquisition in osteoclast ontogeny.
Asunto(s)
Fosfatasa Ácida/análisis , Adenosina Trifosfatasas/análisis , Médula Ósea/enzimología , Huesos/enzimología , Osteoclastos/fisiología , Bazo/enzimología , Animales , Desarrollo Óseo , Diferenciación Celular , Citoplasma/enzimología , Histocitoquímica , Ratas , Tartratos/farmacologíaRESUMEN
We have used a recently described acid ATPase assay in a histochemical and biochemical analysis of osteoclast development in the three osteopetrotic mutations in the rat where bone resorption is reduced but osteoclast numbers vary, depending upon the mutation. Enzymatic activity in bone was elevated in one mutation, severely reduced in another and moderately reduced in a third. Histochemical studies confirmed that the skeletal activity resided primarily in osteoclasts and showed that the enzyme activity in the mutation with moderately reduced levels was inversely distributed between osteoclasts and mononuclear cells. Enzyme activity in spleen, liver and peritoneal macrophages was not different in mutants and normal littermates. These results represent the first biochemical correlation of previous histochemical data and underscore the heterogeneity of osteopetrotic mutations. These data suggest that acid ATPase is a characteristic but not exclusive osteoclast marker which appears in skeletal sites late in osteoclast ontogeny.
Asunto(s)
Adenosina Trifosfatasas/análisis , Mutación , Osteoclastos/enzimología , Osteopetrosis/enzimología , Animales , Resorción Ósea/enzimología , Histocitoquímica , Hígado/enzimología , Macrófagos/enzimología , Osteopetrosis/genética , Ratas , Ratas Mutantes , Bazo/enzimologíaRESUMEN
A tartrate-resistant, iron-activated and vanadate-sensitive nucleotide tri- and diphosphatase has been purified from rat bone. The purified enzyme (1,400-fold, 45% yield) has an Mr on SDS-PAGE of 30,000 Da. Hydrodynamic properties include a Stokes radius of 24A, a sedimentation coefficient of 3.2 S and a partial specific volume of 0.748 ml/g. The calculated Mr from hydrodynamic data is 32,000 and the enzyme binds 4 mol Triton X-100/mol enzyme. Substrate specificity studies demonstrate that the enzyme is active against nucleotide tri- and diphosphates and phosphotyrosine, but not against phosphoserine or phosphothreonine. Based on the purification profile and enzyme histochemistry, showing labelling of fewer mononuclear cells using ATP compared to conventional acid phosphatase substrates, it is suggested that the acid ATPase constitutes a unique form in the family of tartrate-resistant acid phosphatases and may thus have the potential as a marker for osteoclast ontogeny and function.
Asunto(s)
Adenosina Trifosfatasas/aislamiento & purificación , Osteoclastos/enzimología , Fosfatasa Ácida/metabolismo , Adenosina Trifosfatasas/análisis , Animales , Biomarcadores/análisis , Resistencia a Medicamentos , Osteoclastos/análisis , Ratas , Tartratos/farmacologíaRESUMEN
Alkaline phosphatase (APase) is a plasma membrane-integrated protein with unknown function and is found in many different tissues of the body. It is normally not present in squamous epithelium. Nevertheless, it has been demonstrated in developing rat oral epithelium, where it exists together with several different phosphatases. In this study APase was solubilized from rat buccal mucosa and partially purified by gel chromatography followed by ion-exchange chromatography. Three isoenzymes with similar kinetic characteristics but different sensitivities to heat inactivation were obtained. All three had a pH optimum of 10.2 and a Michaelis-Menten constant of 0.3 mM. Combined chromatographic/electrophoretic and histochemical tissue section analysis of APase isoenzymes indicated that the buccal mucosa of young rats contains three APase isoenzymes, two of which are of epithelial origin and the third of endothelial origin.
Asunto(s)
Fosfatasa Alcalina/aislamiento & purificación , Isoenzimas/aislamiento & purificación , Mucosa Bucal/enzimología , Animales , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Liofilización , Ratas , Ratas Endogámicas , Espectrofotometría Ultravioleta , Coloración y EtiquetadoRESUMEN
Purified rabbit antiserum to a tartrate-resistant and vanadate-sensitive acid phosphatase (nucleotide tri- and diphosphatase) prepared from rat bone was used in immunocytochemical studies. The antigen was localized in sections of fixed, decalcified tissue (head from rat) using the peroxidase-antiperoxidase bridge (PAP) or the avidin-biotin-peroxidase complex (ABC) technique. Both techniques resulted in similar and specific immunostaining in the following cells and tissues: osteoclasts situated in resorption lacunae, epithelium overlying enamel-free areas of tips of cusps of unerupted molars, cilia of respiratory epithelium, and tissue macrophages. This distribution corresponds to the cellular sites of tartrate-resistant acid phosphatase activity, as revealed by enzyme histochemistry. With the ABC method, staining in osteoclasts was obtained with antiserum dilutions of up to 1:10,000. Biochemical studies revealed that vanadate-sensitive acid ATPase activity in liver subcellular fractions was almost exclusively confined to lysosomes. Thus, the immunostaining has revealed the presence of the tartrate-resistant and vanadate-sensitive nucleotide phosphatase in many cells associated with tissue resorption and phagocytosis.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Apirasa/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Tartratos/farmacología , Vanadio/farmacología , Animales , Huesos/enzimología , Precipitación Química , Resistencia a Medicamentos , Femenino , Histocitoquímica , Inmunoquímica , Conejos , Ratas , Ratas Endogámicas , Diente/enzimología , Diente/crecimiento & desarrollo , VanadatosRESUMEN
Rat bone was extracted with KCl and Triton X-100, and a tartrate-resistant acid phosphatase activity was purified by protamine sulfate precipitation, ion-exchange chromatography (CM-cellulose), and gel filtration on Sephadex G-200 according to previously described procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining demonstrated a major band with an apparent monomer molecular size of approximately 14,000 Da. The enzyme is active with p-nitrophenylphosphate (p-NPP) but exhibits a 5- to 10-fold higher affinity towards several nucleotides of which ATP and ADP are the most readily hydrolyzed substrates based on kinetic studies. Based on sensitivity towards proteolytic treatment and detergent removal, as well as pH-optimum studies, a single enzyme was found to be responsible for activity towards nucleotide phosphates as well as p-NPP. This nucleotide tri- and diphosphatase constitutes around 15% of the total acid phosphatase activity in rat bone. The activity with ATP as substrate in contrast to that with p-NPP was inhibited in a noncompetitive fashion by MgCl2, sodium metavanadate, and p-chloromercuribenzoate. Enzyme activity with p-NPP and ATP is dependent on the presence of KCl and detergent and is activated by Fe3+ and ascorbate. The reported characteristics of the enzyme suggest that it functions as a unique membrane acid ATPase.