Showing papers on "Tartrate-resistant acid phosphatase published in 1995"

Matrix Metalloproteinases Are Obligatory for the Migration of Preosteoclasts To the Developing Marrow Cavity of Primitive Long Bones

Abstract: A key event in bone resorption is the recruitment of osteoclasts to future resorption sites, We follow here the migration of preosteoclasts from the periosteum to the developing marrow cavity of fetal mouse metatarsals in culture, and investigate the role of proteinases and demineralization in this migration, Our approach consisted in testing inhibitors of proteinases and demineralization on the migration kinetics, Migration was monitored by histomorphometry and the (pre)osteoclasts were identified by their tartrate resistant acid phosphatase (TRAP) activity, At the time of explantation, TRAP+ cells (all mononucleated) are detected only in the periosteum, and the core of the diaphysis (future marrow cavity) consists of calcified cartilage, Upon culture, TRAP+ cells (differentiating progressively into multinucleated osteoclasts) migrate through a seam of osteoid and a very thin and discontinuous layer of mineral, invade the calcified cartilage and transform it into a 'marrow' cavity; despite the passage of maturing osteoclasts, the osteoid develops into a bone collar, The migration of TRAP+ cells is completely prevented by matrix metalloproteinase (MMP) inhibitors, but not by a cysteine proteinase inhibitor, an inhibitor of carbonic anhydrase, or a bisphosphonate, The latter three drugs inhibit, however, the resorptive activity of mature osteoclasts at least as efficiently as do the MMP inhibitors, as assessed in cultures of calvariae and radii, Furthermore, in situ hybridizations reveal the expression of 2 MMPs, gelatinase B (MMP-9 or 92 kDa type IV collagenase) in (pre)osteoclasts, and interstitial collagenase (MMP-13) in hypertrophic chondrocytes, Tt is concluded that only MMPs appear obligatory for the migration of (pre)osteoclasts, and that this role is distinct from the one MMPs may play in the subosteoclastic resorption compartment, We propose that this new role of MMPs is a major component of the mechanism that determines where and when the osteoclasts will attack the bone.

Expression of the calcitonin receptor in bone marrow cell cultures and in bone: a specific marker of the differentiated osteoclast that is regulated by calcitonin

Abstract: We studied the temporal sequence of osteoclast (OC) differentiation from precursor cells in murine marrow cultures. Two markers of the OC phenotype, calcitonin (CT) receptor (CTR) and tartrate resistant acid phosphatase (TRAP), were assessed. Marrow cells from C57BL/6 mice were cultured for 3, 5, 7, and 9 days with or without 1,25-(OH)2vitamin D3 (10(-8) M). In controls only small numbers of osteoclastic multinucleated cells 9MNCs) formed per well ( 80 per well on day 7). Messenger RNA (mRNA) for TRAP was detectable by reverse transcription-polymerase chain reaction amplification in both control and 1,25-(OH)2D3 treated groups at all times. However, TRAP mRNA was detectable in MNCs by the less sensitive in situ hybridization only on days 5, 7, and 9 and only in 1,25-(OH)2D3 treated cells. In control cultures, CTR mRNA was present on day 3 only in nonadherent cells and was not present in adherent cells (where MNCs formed) at any ...

Tartrate resistant acid phosphatase as a marker for scale resorption in rainbow trout, Oncorhynchus mykiss: effects of estradiol-17β treatment and refeeding.

Abstract: In teleosts, a considerable part of the body calcium is found in the scales. Associated with the scales are osteoblasts and osteoclasts, and during periods of high calcium demand such as during sexual maturation or starvation, the scales can be resorbed and thereby act as an internal calcium reservoir. In mammalian bone tissue, the activity of an acid phosphatase (ACP) isoenzyme, tartrate resistant acid phosphatase (TRACP), can be used as a marker for osteoclastic activity. In the present study, an evaluation of TRACP as a marker for osteoclastic activity in teleost scales has been performed. ACP and TRACP was histologically localized at resorption sites around the edge of the scales as well as at resorption holes in the scales. The optimal conditions for biochemical measurements of ACP and TRACP activity were found to be pH 5.3, 10 mM paranitrophenylphosphate, incubated for 30 min at room temperature, and 10 mM tartrate added when required. Using TRACP as a marker, estradiol-17β (E2) was found to increase the proportion of scales being resorbed, as well as the number and size of resorption sites per scale. Also, the scales of E2-treated fish showed weaker staining for calcium. Together, the obtained data indicate that estradiol-17β induces osteoclastic activity in teleost scales, resulting in increased resorption of the scales. A period of refeeding following a period of starvation did not have detectable effects on the scale osteoclastic activity and scale resorption.

Immunohistochemical detection of tartrate-resistant acid phosphatase in non-hematopoietic human tissues.

Abstract: Immunohistochemical studies were done on formalin-fixed, paraffin-embedded tissues to evaluate the specificity of a newly developed monoclonal antibody (9C5) against tartrate-resistant acid phosphatase. Sections from 195 specimens were examined, which included 33 types of tissues/organs. These tissues included normal, inflammatory, and neoplastic processes. Neoplastic tissues from 14 patients with hairy cell leukemia served as positive controls. Epitope enhancement was accomplished either by microwave irradiation in citrate buffer or by boiling in water followed by trypsin digestion. Tissues were reacted with monoclonal antibody 9C5 and stained with either the avidin-biotin peroxidase method or the alkaline phosphatase anti-alkaline phosphatase method. The hairy cells of all cases of hairy cell leukemia reacted positively with 9C5. Other positively stained cells included osteoclasts, activated macrophages and giant cells. Immunohistochemical studies with 9C5, when interpreted within the context of the specificity of this antibody, are useful for the diagnosis and assessment of treatment results for hairy cell leukemia. Monoclonal antibody 9C5 also may be useful as a marker for osteoclasts and the activated macrophages and for the diagnosis of disorders involved by these cells.

Immunoassay of a tartrate-resistant acid phosphatase in serum.

Abstract: A tartrate-resistant acid phosphatase (TRACP) was purified from cord plasma by use of cation-exchange chromatography (carboxymethyl Sepharose), gel-filtration chromatography (Sephacryl S-200), and preparative isoelectric focusing. After raising polyclonal antibodies to the purified TRACP in rabbit, we used the antiserum to develop an ELISA. The antiserum cross-reacted with an extract of bone, but not with extracts of spleen, erythrocytes, platelets, or osteoblasts or with prostatic acid phosphatase. With this ELISA we determined the concentration of serum TRACP in healthy men to be 197 (61-301) micrograms/L (median and range). Serum TRACP concentrations were significantly higher in children and postmenopausal women and in patients with chronic renal failure, hyperparathyroidism, or hyperthyroidism. Estrogen replacement therapy of postmenopausal women for 3-6.5 months decreased their serum TRACP concentration by 70%.

Differentiation dependent expression of tensin and cortactin in chicken osteoclasts

Abstract: The expression and localization of tensin and cortactin were examined in osteoclast precursors in comparison with isolated osteoclasts on various substrates. Initially, the ability of hen monocytes to differentiate into osteoclasts was evaluated on plastic or glass, and compared to differentiation on bone. Specifically, monocytes were isolated from the medullary bones of egg-laying hens maintained on a Ca-deficient diet. Differentiation was monitored morphologically and by quantitation of the ability to form Howship's lacunae in bone slices or resorb radiolabeled bone particles of 20–53 mm diameter. These cells differentiated into tartrate resistant acid phosphatase (TRAP)-positive, bone resorbing, multinucleated syncytia in the presence of cytosine-1-β-D-arabinofuranoside in a time dependent manner (day 1–6). Differentiation into osteoclast-like cells was similar whether cultured on plastic, on glass, or on bone. When compared to GAP-DH control levels, tensin and cortctin mRNA levels increased by 7- and 10-fold, respectively, by day 6. Tensin and cortactin protein levels also increased by 6- and 15-fold, respectively, by day 6. Immunofluorescence of differentiating precursors showed that tensin localized between regions of cell to cell contact and colocalized with vinculin in podosomes of osteoclast-like cells and of real osteoclasts. Cortactin immunofluorescence was not detectable in monocytes but localized inside tensin/vinculin podosome structures after fusion into osteoclast-like cells and in freshly isolated osteoclasts. Both tensin and cortactin were associated with attachment complexes used by osteoclast-like cells and osteoclasts to resorb bone. Specifically, punctate cortactin staining was observed inside tensin staining which formed a double ring structure at the membrane/bone interface of resorbing osteoclasts. These data showed that tensin and cortactin can be used as osteoclast differentiation markers, that participate in attachment complexes used to resorb bone, and that tensin may participate in the fusion process of osteoclast precursors. © 1995 Wiley-Liss, Inc.

Human osteoclast and giant cell differentiation: the apparent switch from nonspecific esterase to tartrate resistant acid phosphatase activity coincides with the in situ expression of osteopontin mRNA.

Abstract: Animal model and in vitro cultures suggest that osteoclasts and cells of the mononuclear phagocyte system share a common precursor. However, the human osteoclast precursor has not been positively identified. We attempted to identify the precursor in situ by using a number of osteoclast- and macrophage-selective markers, together with the expression of osteopontin mRNA, previously shown to be abundant in human osteoclasts. Sections of osteophytic bone and a panel of inflammatory connective tissues were processed for in situ hybridization; serial sections were analyzed for tartrate-resistant acid phosphatase (TRAP) and nonspecific esterase (NSE) activity, selective cytochemical markers for the osteoclast and cells of the macrophage/monocyte lineage, respectively. The murine anti-human osteoclast monoclonal antibodies 23C6 (vitronectin receptor) and C35 (osteoclast-selective) were used to further identify the osteoclast phenotype. We compared osteoclasts, giant cells, and their respective putative mononuclea...

Targeting simian virus 40 T antigen to the osteoclast in transgenic mice causes osteoclast tumors and transformation and apoptosis of osteoclasts.

Abstract: Osteoclasts are terminally differentiated cells that express tartrate-resistant acid phosphatase (TRAP) at a higher level than other normal cells. Therefore, in an attempt to develop immortalized osteoclasts, we produced two lines of transgenic mice in which expression of the simian virus 40 T antigen oncogene was targeted to osteoclasts using the TRAP gene promoter. Osteoclasts were increased in number in bones from both lines. More than 50% of them appeared morphologically transformed, 2-5% were mitotic, but, unexpectedly, 5% were apoptotic. Osteoclast tumors were observed occasionally in one line of mice (line 4), and sheets of TRAP-positive cells (tumorlets) developed in most mice in both lines. Although cells isolated from these tumorlets formed multinucleated TRAP-positive cells that resorbed bone in vitro, to date we have been unable to develop an immortalized osteoclast cell line from them. Osteoclasts from one line (line 5) had reduced ruffled border formation and a higher level of T-antigen expr...

In vitro differentiation of the murine macrophage cell line BDM-1 into osteoclast-like cells.

Abstract: Osteoclasts are derived from hematopoietic stem cells, but details about their precursor are still obscure. We present here a mouse macrophage cell line, BDM-1 cells, that showed a high potential to differentiate into osteoclast-like multinucleate cells (MNCs) when cocultured with primary osteoblasts for 14 days in the presence of 10(-8) M 1 alpha,25-dihydroxyvitamin D3. These MNCs had tartrate-resistant acid phosphatase (TRAP) activity and strong ability to resorb dentine. In this culture system, 10(-10)-10(-8) M 12-O-tetradecanoylphorbol-13-acetate stimulated the formation of TRAP-positive MNCs, whereas salmon calcitonin inhibited it. Time-course effect studies showed that 12-O-tetradecanoylphorbol-13-acetate had an effect on the late phase of osteoclast differentiation but not on precursor proliferation. By immunocytochemical staining, all BDM-1 cells expressed Mac-1, Mac-2, and MOMA-2 antigens, and a large number of them expressed F4/80 antigen, but the rest of them were negative for this antigen. To ...

Femoral peak bone mass and osteoclast number in an animal model of age-related spontaneous osteopenia.

Abstract: Background: SAMP6 was developed as a murine model of age-related spontaneous osteopenia characterized by low peak bone mass. A morphometric study of the growing femur in SAMP6 and sex-matched SAMP2 at 10 days to 4 months of age was done to examine the pathogenic process related to osteopenia. Methods: Age-related changes in cortical bone thickness, femur score, trabecular bone volume, thickness of epiphyseal growth plate, number of osteoclasts, and osteoclast surface were measured with a computerized image analyzer. Osteoclasts were examined cytomorphometrically after TRAP (tartrate resistant acid phosphatase) staining of the femoral sections. Results: Cortical bone thickness and femur score increased significantly with age, while trabecular bone volume decreased significantly. Comparing mean values of cortical bone thickness, femur score and trabecular bone volume, we noted significantly lower mean values in SAMP6 than in SAMP2 mice. These significant inter-stain differences first became evident in 20–40-day-old mice, but there was no significant difference in thickness of the epiphyseal growth plate between the two strains. The mean values of the number of osteoclasts per unit none surface length and of the osteoclast surface in SAMP6 were significantly greater than in age- and sex-matched SAMP2. Histograms of distribution of size of osteoclasts of 40-day-old male mice revealed that larger ones were more frequently seen in SAMP6. Furthermore, the ratio of osteoclasts/TRAP positive cells free in the bone marrow cavity was significantly higher in SAMP6 than in SAMP2. Conclusion: Activated bone resorption may play a role in the osteopenia seen in SAMP6. © 1995 Wiley-Liss, Inc.

17β-estradiol suppresses gene expression of tartrate-resistant acid phosphatase and carbonic anhydrase II in ovariectomized rats

Abstract: Tartrate-resistant acid phosphatase (TRACP) and carbonic anhydrase II (CA II) are key enzymes responsible for osteoclastic bone resorption. In this study, we proposed that estrogen loss in postmenopausal osteoporosis may enhance gene expression of TRACP and CA II, and subsequently increase osteoclastic bone resorption. We have, therefore, used the ovariectomized rat model of postmenopausal bone loss to investigate changes at the gene transcripional level in osteoclastic bone-resorbing enzymes in ovariectomized (OVX) rats, sham ovariectomized (S-OVX) rats, and estrogen-treated ovariectomized (E-OVX) rats. We have demonstrated for the first time that ovariectomy in rats enhances gene expression of TRACP and CA II. The mRNA levels in OVX were approximately three- and four-fold higher, respectively, than those in S-OVX. Enhancement was observed 1 week after ovariectomy and transcripts remain high during the experimental period of 8 weeks. Administration of 17β-estradiol to OVX (E-OVX) reduced gene expression of these osteoclastic bone-resorbing enzymes 18 hours after injection. It appeared that the suppression of the osteoclastic bone-resorbing enzymes by 17β-estradiol was most effective during the first 1–2 weeks but the degree of suppression was reduced at 8 weeks after ovariectomy. In conclusion, our results suggest that estrogen prevents bone loss by reducing the mRNA levels of osteoclastic bone-resorbing enzymes in bone tissue.

Induction of osteoclast characteristics in cultured avian blood monocytes; modulation by osteoblasts and 1,25-(OH)2 vitamin D3.

Abstract: It has been established, that the osteoclast is derived from the haemopoietic stem cell, but its exact lineage is still controversial. It is sometimes suggested, that osteoclasts and monocytes/macrophages are related cells. It has also been suggested that osteoclast differentiation is regulated by osteoblasts and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). In the present paper we addressed the question whether avian monocytes can differentiate into osteoclasts in vitro, using an array of immunocytochemical, enzyme cytochemical and function markers. We have also determined the effects of osteoblasts, osteoblast conditioned medium and 1,25-(OH)2D3 on the expression of osteoclastic features on monocytes during culture. Monocytes developed tartrate resistant acid phosphatase (TRAcP) enzyme activity and antigens for all anti-osteoclast antibodies tested, during culture. However, they did not acquire the ability to resorb dentine and still showed phagocytosis of latex spheres. This indicates that the monocytes developed into cells resembling osteoclasts but lacking their function while retaining the function of macrophages. Osteoblast conditioned medium stimulated TRAcP enzyme activity and proliferation of monocytes in cultures. Addition of osteoblasts or osteoblast conditioned medium to monocyte cultures on dentine in the presence or absence of 1,25-(OH)2D3 did not result in the generation of genuine osteoclasts, nor in pit formation. 1,25-(OH)2D3 appeared to be cytotoxic to the avian monocytes in concentrations considered optimal for mouse osteoclast formation. These results suggest that avian monocytes do not readily differentiate into osteoclasts under in vitro conditions that stimulate osteoclast differentiation from bone marrow derived haemopoietic cells. Furthermore, labelling with anti-osteoclast antibodies and TRAcP as osteoclast-markers should be used only with great caution in the identification of osteoclasts formed in vitro.

Parathyroid hormone increases the number of tartrate-resistant acid phosphatase-positive cells through prostaglandin E2 synthesis in adherent cell culture of neonatal rat bones.

Abstract: Tartrate-resistant acid phosphatase (TRAP) is expressed during one of the steps of osteoclast differentiation. The involvement of prostaglandin E2 (PGE2) synthesis in PTH-induced increases in TRAP-positive cell number was studied in an improved slowly adhering cell culture system, prepared by removing preexisting osteoclasts from disaggregated bone cells obtained from 6-day-old rats. The majority of the TRAP-positive cells that appeared were mononuclear. In 1-day culture, PTH (0.025-0.25 nM) and PGE2 (1 microM) increased the number of mono- and multinucleated TRAP-positive cells. Indomethacin (50 microM) inhibited PTH-induced increase, and the inhibition was significantly abolished by the addition of PGE2. SC-19220, a specific antagonist for one class of PGE2 receptor, inhibited the increase induced by PTH and PGE2. PTH significantly stimulated the production of PGE2 in the culture. Peroxides, which are produced as byproducts of PGE2 biosynthesis, were detected in the adherent cells using dichlorofluorescene and increased the number of TRAP-positive cells. Small resorption pits were recognized on the surfaces of bone slices on which slowly adhering cells had been incubated for 3 days with PTH. These findings showed that PTH, through its effects on PGE2 synthesis and the subsequent reactions, induced the step at which TRAP is expressed, possibly during the differentiation of osteoclasts.

The number of tartrate-resistant acid phosphatase-positive osteoclasts on neonatal mouse parietal bones is decreased when prostaglandin synthesis is inhibited and increased in response to prostaglandin E2, parathyroid hormone, and 1,25 dihydroxyvitamin D3.

Abstract: The culture of parietal bones from 4-day old mice in indomethacin (Ind) for 1 day caused a large reduction in the number of tartrate-resistant acid phosphatase positive osteoclasts (TRAP+OC) relative to both control bones and to freshly isolated bones. This reduction did not occur if prostaglandin E2 (PGE2) was present. When 5-bromo-2′-deoxyuridine (BDU) was injected into 4-day old mice, newly formed TRAP+OC nuclei became labeled 1 day later; these bones were then cultured with Ind for 1 day. TRAP+OC and newly labeled TRAP+OC nuclei were commensurately decreased in number. This suggests an active down-regulation rather than merely the inhibition of new TRAP+OC formation. Incubation of bones with Ind and either PGE2, parathyroid hormone, or 1,25 dihydroxyvitamin D3 for 6 hours following a 1-day preincubation in Ind, resulted in an increase in TRAP+OC compared with Ind alone. Using BDU labeling in vitro and in vivo, we show that this increase in number of TRAP+OC is not the result of cell proliferation, but rather differentiation of postmitotic precursors.

Changes in the markers of bone metabolism following skeletal unloading.

Abstract: To elucidate the mechanism in disuse bone atrophy induced by skeletal unloading, we studied the indices of bone resorption and bone formation in the femur of tail-suspended rats. The duration of the suspension ranged from 1 to 14 days. Tartrate-resistant acid phosphatase mRNA, an index used to evaluate bone resorption, increased significantly more than the controls for the first 3 days of the tail-suspension experiments, compared those in controls. Osteocalcin and alkaline phosphatase, two common markers for bone formation, were also monitored. Osteocalcin mRNA started to decrease after 3 days of suspension. Five days later, alkaline phosphatase mRNA showed a decrease. Levels of both of these mRNAs remained low for the remaining suspension period. Sequential changes in the markers for bone metabolism indicate that the transient increase in bone resorption preceded the decrease in bone formation in the development of disuse bone atrophy induced by skeletal unloading.

Generation and characterization of putative monoclonal and polyclonal antibodies against tartrate-resistant acid phosphatase.

Abstract: The generation of monoclonal antibodies (MAbs) specific for tartrate-resistant acid phosphatase (TRAP) may aid development of a serological immunoassay for this marker of bone resorption. The lack of MAbs to TRAP largely reflects the difficulty in obtaining sufficient antigen for in vivo immunization strategies. We have circumvented this problem by using in vitro immunization, requiring a small amount of TRAP isolated from osteoclastoma tumor. Two MAbs designated 312D and 310A were generated that exhibited weak anti-TRAP activity in enzyme-linked immunosorbent assay and immunocytochemistry. A polyclonal antibody to TRAP (Ab8023) was also raised in rabbit, using synthetic peptide. Ab8023 and MAbs 310A and 312D exhibited no activity against TRAP in dot-blotting experiments. Further characterization in enzyme-liked immunosorbent assay showed that Ab8023 was remarkably specific for TRAP whereas MAb 312D cross-reacted with another metalloenzyme, human prostatic acid phosphatase.

Growth hormone and longitudinal bone growth in vivo: short-term effect of a growth hormone antiserum.

Abstract: The control of longitudinal growth is poorly understood but GH is considered to be one of the major hormones regulating postnatal growth. However, there is dispute as to whether it has a direct or indirect action. To study the role of GH we used a polyclonal antiserum to rat GH and investigated changes in cell proliferation and enzyme activities associated with bone formation and resorption during longitudinal growth. IGF-I levels were measured by two independent RIAs, DNA synthesis by bromodeoxyuridine incorporation followed by immunocytochemistry and enzyme activities were quantified in situ by microdensitometry. After 1 day the percentage of chondrocytes undergoing DNA synthesis within the proliferative zone was reduced but no other parameters were affected. By day 4 the labelling index was the same as in pair-fed animals but the number of chondrocytes synthesising DNA was reduced as was the total width of the growth plate and that of the proliferative zone. Alkaline phosphatase (associated with mineralisation) was unchanged but glucose 6-phosphate dehydrogenase activity (associated with cell proliferation) was decreased. Osteoclastic tartrate-resistant acid phosphatase activity (associated with bone resorption) was also significantly reduced. Similar changes were apparent after 10 days. At no time was the circulating level of IGF-I decreased. These data suggest that, during longitudinal growth, GH affects the number of proliferating chondrocytes but not the percentage of cells undergoing DNA synthesis, indicating that its primary role may be on the commitment of prechondrocytes to a proliferative state. Furthermore, while GH does not seem to have any effect on skeletal mineralisation it may stimulate osteoclastic resorption of the primary spongiosa.

Topography of forming and resorbing cells on endosteal surfaces of the rabbit humerus by double-staining with in situ hybridization and tartrate-resistant acid phosphatase reaction: a new model to study the bone reaction to loading

Abstract: Since the first investigations made by Wolff, it has been known that bone adapts to mechanical load. The mechanisms which guide the reaction of osteoblasts and osteoclasts to load are still insufficiently known. In situ hybridization (ISH) allows the detection of intracellular gene transcripts. Therefore, the ISH technique was further developed to allow the detection of pro-alpha 1 (I) collagen gene transcripts on undecalcified bone surfaces. Additionally, this new technique was combined with the tartrate resistant acid phosphatase technique. The combination of the two methods allows the detection of forming and resorbing cells on the same undecalcified bone surface. In addition, a new animal model was developed to study the reaction of bone to mechanical load. This model mimics the situation of bone implants (e.g. hip prosthese), which is a static situation which is dynamically loaded by the action of the patient.

Purification and characterization of multiple S6 phosphatases from the rat parotid gland

Abstract: S6 phosphatase activities, which dephosphorylate the phosphorylated S6 synthetic peptide, RRLSSLRASTSKSESSQK, were purified to near homogeneity from the membrane and cytosolic fractions of the rat parotid gland. Multiple S6 phosphatases were fractionated on Mono Q and gel filtration columns. In the cytosolic fraction, at least three forms of S6 phosphatase, termed peaks I, II, and III, were differentially resolved. The three forms had different sizes and protein compositions. The peak I enzyme, which had an approximately Mr of 68 kDa on gel filtration, appears to represent a dimeric form of the 39 kDa protein. This S6 phosphatase showed the high activity in the presence of EGTA and was completely inhibited by nanomolar concentrations of either okadaic acid or inhibitor 2. The peak II S6 phosphatase enzyme, with an Mr of 35 kDa, was activated by Mn2+. This form could be a proteolytic product of the catalytic subunit of type 1 phosphatase, due to its sensitivities to okadaic acid and inhibitor 2. The peak III enzyme, with an Mr of 55 kDa, is a Mn2+-dependent S6 phosphatase. This S6 phosphatase can be classified as a type 1 phosphatase, due to its sensitivity to okadaic acid, since the IC50 of okadaic acid is 4 nM. However, the molecular mass of this S6 phosphatase differs from that of the type 1 catalytic subunit (37 kDa) and showed less sensitivity to inhibitor 2. On the other hand, the membrane fraction contained one form of the S6 phosphatases, termed peak V (Mr 34 and 28 kDa), which could be classified as a type 1 phosphatase. This S6 phosphatase activity was greatly stimulated by Mn2+.