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Cytochemical establishment of acid phosphatase in the bovine endometrium and trophoblast during implantation.

Acid phosphatase in the endometrial surface epithelium is seen in connection with autophagy and autolysis. In the precontact and initial apposition stage, enzyme-positive Golgi vesicles, lysosomes and secretion granules all indicate autophage performance of the dark uterine epithelial cells in the sense of a histiogene embryotrophe development. At the time of progressing apposition this is joined by cell degradation with the aim of histiolytical uterine milk production. Following the completed implantation in the adhesion phase no activity with autophagy and autolysis-correlated acid phosphatase can be established. In trophoblast giant cells the localisation of acid phosphatase speaks for secretional processes. The incidence of this enzyme in the adhesion stage in "ordinary" trophoblast cells leads to the supposition of autophage processes which must be investigated in more detail. The endometrial gland epithelium shows the same acid phosphatase-dependent autophage indications in the upper third of the glands as shown in the surface epithelium prior to apposition. However, the acid phosphatase activity and the secretion deduced therefrom, thus the histiogene embryotrophe development, is conserved during the whole early gravidity of the cow, independent of the implantation process.

Acid Phosphatase↗

Mechanism of Fe(III)-Zn(II) purple acid phosphatase based on crystal structures.

Purple acid phosphatase is a widely distributed non-specific phosphomonoesterase. X-ray structures of the dimeric 111-kDa Fe(III)-Zn(II) kidney bean purple acid phosphatase (kbPAP) complexed with phosphate, the product of the reaction, and with tungstate, a strong inhibitor of the phosphatase activity, were determined at 2.7 and 3.0 angstroms resolution, respectively. Furthermore the resolution of the unligated enzyme, recently solved at 2.9 angstroms could be extended to 2.65 angstroms with completely new data. The binding of both oxoanions is not accompanied by larger conformational changes in the enzyme structure. Small movements with a maximal coordinate shift of 1 angstroms are only observed for the active site residues His295 and His296. In the inhibitor complex as well as in the product complex, the oxoanion binds in a bidentate bridging mode to the two metal ions, replacing two of the presumed solvent ligands present in the unligated enzyme form. As also proposed for the unligated structure a bridging hydroxide ion completes the coordination spheres of both metal ions to octahedral arrangements. All three structures reported herein support a mechanism of phosphate ester hydrolysis involving interaction of the substrate with Zn(II) followed by a nucleophilic attack on the phosphorus by an Fe(III)-coordinated hydroxide ion. The negative charge evolving at the pentacoordinated transition state is probably stabilized by interactions with the divalent zinc and the imidazole groups of His202, His295, and His296, the latter protonating the leaving alcohol group.

Acid Phosphatase↗

Phenotypic and quantitative relationship of red cell acid phosphatase with haemoglobin, haptoglobin, and G6PD phenotypes.

The phenotypic and quantitative relationship of red cell acid phosphatase with haemoglobin, haptoglobin, and G6PD phenotypes was investigated in three populations in the Sudan and one population in Nilgiris, India. No significant consistent association of red cell acid phosphatase phenotypes was observed with these polymorphisms. However, there was a lack of acid phosphatase AB in G6PD deficient subjects from Nilgiris. The relative quantitative expression of red cell acid phosphatase genes PA, PB, and PC was 1.0, 1.2, and 1.3, respectively. The red cell acid phosphatase activity was higher (15%) in the presence of raised haemoglobin A2 and in sickle cell anaemia (21%). Those with Hp2 had 18% higher level of acid phosphatase than those with Hp1. G6PD deficient subjects had a lower level of acid phosphatase activity (20%) than those with normal G6PD activity.

Acid Phosphatase↗

A proposed mechanism of the mitogenic action of fluoride on bone cells: inhibition of the activity of an osteoblastic acid phosphatase.

Fluoride (F) is a potent inhibitor of osteoblastic acid phosphatase activity with an apparent Ki value (10 to 100 mumol/L) that corresponds to F concentrations that increase bone cell proliferation and bone formation in vivo and in vitro. This high sensitivity of acid phosphatase to F inhibition appeared to be specific for skeletal tissues. Mitogenic concentrations of F did not increase cellular cAMP levels but significantly stimulated net protein phosphorylation in intact calvarial cells and in isolated calvarial membranes. These concentrations of F also stimulated net membrane-mediated phosphorylation of angiotensin II (which contains tyrosyl but no seryl or threonyl residues), suggesting that some of the F-stimulated protein phosphorylations could occur on tyrosyl residues. F had no apparent effect on thiophosphorylation of membrane proteins, suggesting that the F-stimulated net protein phosphorylation in bone cells was probably not mediated via activation of protein kinases. Orthovanadate or molybdate at concentrations that inhibit bone acid phosphatase activity also stimulated bone cell proliferation, supporting the idea that inhibition of bone acid phosphatase would lead to stimulation of bone cell proliferation. Mitogenic concentrations of F potentiated the mitogenic activities of insulin, EGF, and IGF-1 (ie, growth factors the receptors of which are tyrosyl kinases) to a greater extent than they potentiated the action of basic FGF (a growth factor that does not appear to stimulate tyrosyl protein phosphorylation). Based on these findings, a model is proposed for the biochemical mechanism of the osteogenic action of F in which F stimulates bone cell proliferation by a direct inhibition of an osteoblastic acid phosphatase/phosphotyrosyl protein phosphatase activity, which in turn increases overall cellular tyrosyl phosphorylation, resulting in a subsequent stimulation of bone cell proliferation.

Acid Phosphatase↗

Localisation of acid phosphatase activity on the surface of bloodstream forms of Trypanosoma congolense.

In vitro, living bloodstream forms of Trypanosoma congolense were shown to hydrolyse p-nitrophenyl phosphate, a substrate for phosphatases. This activity appears to be from an acid phosphatase because it was enhanced at low pH values, was inhibited by the acid phosphatase inhibitor sodium fluoride, and was not inhibited by the alkaline phosphatase inhibitor tetramisole. The activity did not appear to be secreted into the surrounding medium by the living parasites although phosphatase activity could be detected in the surrounding medium when dead or dying parasites were present. Studies at various temperatures indicated that at least some of this acid phosphatase activity may be associated with the surface of the parasites, rather than with endocytic or intracellular systems. This was supported by subcellular fractionation of radiolabelled parasites which showed some cosedimentation of acid phosphatase activity with radiolabelled iodine. Histochemical studies of the parasites also supported this conclusion. Electron microscopical examination of trypanosomes incubated with lead nitrate and p-nitrophenyl phosphate showed lead phosphate deposits on the surface of the parasites in addition to the expected localisation in the flagellar pocket. We conclude that Trypanosoma congolense possesses a surface-bound acid phosphatase.

Acid Phosphatase↗

A cytochemical study of the localization of acid phosphatase in Saccharomyces cerevisiae at different growth phases.

The localization of acid phosphatase in the yeast Saccharomyces cerevisiae at different growth phases had been studied. It was shown to be crucial for authentic location of acid phosphatase that the cytochemical reaction be performed on whole cells. Dimethylsulphoxide was used to alleviate the effects of fixation of the yeast cells with glutaraldehyde; the sulphoxide did not affect the distribution of acid phosphatase in the cells. It has been established that in exponentially-growing cells acid phosphatase is localized mostly in small vacuolar compartments. In mature cells, the bulk of acid phosphatase is found in the central vacuole, although a significant amount of the enzyme is detectable in the plasma membrane and the adjacent vesicles.

Acid Phosphatase↗

3-phosphoglycerate phosphatase activity in chloroplast preparations as a result of contamination by Acid phosphatase.

The presence of a nonspecific acid phosphatase which had high activity with 3-phosphoglycerate as substrate has recently been reported in Spinacia oleracea L. chloroplasts (Mulligan, Tolbert 1980 Plant Physiol 66: 1169-1173). The subcellular localization of this activity has been reinvestigated by differential centrifugation of spinach leaf homogenates. The fraction sedimenting at 1,200g comprised mostly intact chloroplasts and contained more than half the chlorophyll but only 5% of the 3-phosphoglycerate phosphatase activity present in the homogenate. The fraction of the homogenate pelleting at 5,000g contained broken chloroplasts and had considerable 3-phosphoglycerate phosphatase activity. Further purification of the 1,200g pellet fraction on a Percoll step gradient yielded greater than 95% intact chloroplasts, yet the phosphatase activity was reduced more than 15-fold on a chlorophyll basis by this purification.When the intact chloroplast and cytoplasmic fractions of mesophyll protoplasts were separated by silicone oil filtering centrifugation, the chloroplast fraction contained more than 90% of the chlorophyll but had less than 12% of the 3-phosphoglycerate phosphatase activity. By contrast, more than 60% of the 2-phosphoglycolate phosphatase was recovered in this chloroplast fraction supporting previous evidence that this phosphatase is localized in the chloroplast stroma.It is concluded that 3-phosphoglycerate phosphatase activity is not localized in the chloroplast but that the activity present in chloroplast preparations results from contamination by acid phosphatase, which either binds to the thylakoid membranes during preparation or is present as some other contaminant in the preparation. Inasmuch as the enzyme acts on a broad range of substrates its presence in chloroplast preparations, particularly when the percentage of intact chloroplasts is low, could produce artifacts in metabolic studies such as measurement of phosphorylation.

Journal Article↗

Regulation of ErbB4 phosphorylation and cleavage by a novel histidine acid phosphatase.

Signaling by a variety of ligands including epidermal growth factor, betacellulin and neuregulin is mediated by the ErbB family of receptor tyrosine kinases. Studies on the prostate have shown that ErbB2 phosphorylation and signaling can be regulated by prostatic acid phosphatase, a histidine acid phosphatase which can dephosphorylate phospho-tyrosine residues in the ErbB2 receptor. Here we report that the histidine acid phosphatase ACPT (testicular acid phosphatase), which is highly homologous to the prostatic acid phosphatase, can dephosphorylate the ErbB4 receptor, which is known to play important roles in neuronal differentiation and synaptogenesis. ACPT and ErbB4 are both expressed in the brain where they are enriched at post-synaptic sites, and furthermore they can be co-immunoprecipitated from brain. We demonstrate that ACPT can inhibit basal and neuregulin-induced tyrosine phosphorylation of ErbB4. We also show that ACPT-dependent dephosphorylation can regulate the proteolytic cleavage of ErbB4, and this process can be reversed by applying the tyrosine phosphatase inhibitor, pervanadate. Furthermore, neuregulin-dependent differentiation of PC12 cells expressing ErbB4 is prevented by co-expressing ACPT. These results indicate that ACPT acts as a tyrosine phosphatase to modulate signals mediated by ErbB4 that are important for neuronal development and synaptic plasticity.

Acid Phosphatase↗

The histochemical behaviour, electrophoretic mobility and distribution in cell fractions of acid phosphatase isozymes in prostatic cancer and benign prostate hyperplasia.

Acid phosphatase isozymes were investigated in cancerous prostatic tissue (4 cases) and benign prostatic hyperplasia (6 cases). Electron-microscopic histochemical examination of cancer tissue revealed irregular acid beta-glycerophosphatase staining in various cell organelles, including the plasma membrane, which was not seen in non-malignant tissue. Cancerous tissue homogenates also contained isozymic acid phosphatase species with high electrophoretic mobility, which was not detectable in benign tissue unless treated with detergent. Fractionation by differential centrifugation confirmed that much of the acid phosphatase activity in cancer tissue was extra-lysosomal. The detection of these isozyme properties may provide an opportunity, by means of tissue investigations, to define tumour stages earlier than on the basis of increased levels of serum acid phosphatase activity indicative of stage IV (D) prostatic cancer.

Acid Phosphatase↗

Biosynthesis and processing of prostatic and lysosomal acid phosphatases in a prostate carcinoma cell line PC-3SF12.

The biosynthesis of distinct prostatic and lysosomal acid phosphatases is demonstrated using a human prostatic carcinoma cell line, PC-3SF12. The biosynthesis and maturation of the acid phosphatases was studied by metabolic labeling with radioactive leucine, specific immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. Of the tartrate-inhibitable acid phosphatase activity in PC-3SF12 cells, 60% is lysosomal and 10% is prostatic. The lysosomal-type acid phosphatase is synthesized as precursor with a molecular weight of 68,000, some of which is converted to higher-molecular-weight precursor polypeptides (Mr 71,000 and 77,000). The multiple forms of the precursors are due to differences in the carbohydrate chains on the enzyme because biosynthesis in the presence of tunicamycin eliminates the precursor multiplicity. The initial precursor (Mr 68,000) is processed to a mature polypeptide (Mr 49,000), via intermediates with molecular weights of 62,000 and 59,000. The mature polypeptide is degraded to smaller polypeptides with molecular weights of 30,000, 28,000, and 25,000. Precursor polypeptides of the lysosomal-type enzyme are secreted in the medium. Prostatic acid phosphatase is synthesized as a precursor with a molecular weight of 110,000, which is processed via several intermediates (Mr 99,000-93,000, 77,000, and 55,000) to a mature polypeptide with a molecular weight of 49,000. Particularly during cell homogenization, or lysis, the mature polypeptide is rapidly degraded to an immunoprecipitable polypeptide with a molecular weight of 20,000. None of these polypeptides is secreted in detectable amounts into the medium. Precursors and mature and smaller polypeptides are present in human prostate extract and seminal fluid. Proteolytic degradation of prostatic acid phosphatases in cells and tissues is probably catalyzed by a plasmin-like or related trypsin-like enzyme because degradation of the mature prostatic phosphatase polypeptide is completely prevented by addition of the plasmin inhibitor bovine pancreatic trypsin inhibitor. Prostatic- and lysosomal-type acid phosphatases are eventually stored at least in part in two different types of cell organelles. Testosterone does not influence the biosynthesis and secretion of either acid phosphatase in this cell line.

Acid Phosphatase↗

An acid phosphatase assay for quantifying the growth of adherent and nonadherent cells.

We describe an acid phosphatase assay for determination of cell growth based on quantification of cytosolic acid phosphatase activity. The assay is based on the hydrolysis of the p-nitrophenyl phosphate by intracellular acid phosphatases in viable cells to produce p-nitrophenol. For all cell types examined, absorbance of p-nitrophenol at 405 nm is directly proportional to the cell number in the range of 10(3)-10(5) cells. The assay can quantify as few as 1000 cells per well in 96-well microtiter plates. The acid phosphatase assay was used to count various adherent and nonadherent cells, including human tumors, L6, and HT-2 cells. We also demonstrate the utility of this assay for analysis of growth factor and cytokine bioactivity on mammalian cells in culture. In comparison to [3H]thymidine incorporation, the acid phosphatase assay has similar sensitivity but a wider linear response range. The method also shows higher sensitivity and reproducibility in comparison to cell proliferation assays based on the reduction of tetrazolium salts. Because of the ease of use, sensitivity, and low cost, the acid phosphatase method is especially suited to applications where a large number of samples are assayed.

Acid Phosphatase↗

Purification and partial characterization of an acid phosphatase from Spirodela oligorrhiza and its affinity for selected organophosphate pesticides.

An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the pure acid phosphatase resolved a single protein band that migrated to approximately 60 kDa. Nondenaturing SDS-PAGE electrophoresis revealed a single protein band around 120 kDa after staining with Coomassie Brilliant blue. Quantitative gel filtration chromatography estimated a native molecular mass of this enzyme to be 120 kDa. Thus, this acid phosphatase likely functions as a homodimer, consisting of two similar 60 kDa subunits. An electrophoretic technique using the flourogenic substrate 4-methylumbelliferyl phosphate enabled visualization of an acid phosphatase activity that corresponded to the protein band at 120 kDa on a nondenaturing PAGE gel. It was determined that the acid phosphatase had a pH optimum of 6.0 at 25 degrees C. The enzyme activity appeared to be stable over a broad range of temperatures (10-40 degrees C) and in the presence of the metals Zn2+, Mn2+, and Mg2+ as well as the chelating agents ethylenedinitrilotetraacetic acid and ethylene glycol tetraacetic acid. It was shown that this acid phosphatase could hydrolyze a variety of physiological organophosphate compounds including beta-glycerophosphate, phosphoserine, adenosine triphosphate, adenosine diphosphate, adenosine monphosphate, and pyrophosphate. Furthermore, analysis using capillary electrophoresis demonstrated that this hydrolytic enzyme could transform a wide array of organophosphate pesticides including S-2-ethylthioethyl O,O-dimethylphosphorothioate (demeton-S-methyl); S-1,2-bis(ethoxycarbonyl)ethyl O,O-dimethylphosphorodithioate (malathion); O,O-dimethyl O-4-nitrophenyl (paraoxon); O,O,O,O-tetraethyldithiopyrophosphate (sulfatep); O-2-chloro-4-nitrophenyl O,O-dimethylphosphorothioate (dicapthon); and 2,2-dichlorovinyl dimethylphosphate (dichlorvos).

Acid Phosphatase↗

Evaluation of acid phosphatase as a confirmation test for Clostridium perfringens isolated from water.

AIMS: To evaluate testing for acid phosphatase as an alternative method for the confirmation of Clostridium perfringens isolated from water. METHODS AND RESULTS: Sixty-two reference strains of Clostridium were tested for their ability to produce acid phosphatase, as well as reduction of sulfite on tryptose sulfite cycloserine agar (TSC) and production of fluorescence in TSC supplemented with 4-methylumbelliferylphosphate (MUP). Additionally 155 environmental presumptive C. perfringens isolates from TSC incubated at 44 degrees C were identified and tested for acid phosphatase production and by the conventional MNLG (testing for motility, nitrate reduction, lactose fermentation and gelatin liquefaction) confirmation procedure. Twenty-seven strains from 15 species of Clostridium-reduced sulfite to some extent on TSC incubated at 44 degrees C, with a significant number of species being able to grow well at this temperature, indicating that a confirmation step is needed for the enumeration of C. perfringens on this medium. All 10 strains of C. perfringens tested, together with one strain each of Clostridium baratii and Clostridium rectum produced acid phosphatase. These also produced fluorescence on MUP supplemented TSC, as did 13 strains of acid phosphatase negative, sulfite-reducing clostridia, representing nine species. Of the environmental isolates, 114 were identified as C. perfringens of which 108 (94.7%) were confirmed by the acid phosphatase test compared with 104 (91.2%) by the MNLG tests. CONCLUSIONS: Testing for acid phosphatase production is at least as reliable, and much simpler to perform, than the current standard confirmation MNLG procedure. Incorporation of MUP into TSC does not reliably improve the identification of presumptive C. perfringens. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of testing for acid phosphatase as a confirmation test for C. perfringens would substantially simplify the analysis for this bacterium from water samples, and reduce the analysis time to confirmed counts.

Acid Phosphatase↗

Reduction of low-molecular-weight acid phosphatase activity in Alzheimer brains.

Recent studies in Alzheimer brains have shown aberrant protein phosphorylation, suggesting an alteration in protein kinases and/or phosphoprotein phosphatases. In the present study, the activity of acid phosphatase was investigated in samples prepared from postmortem normal human and Alzheimer brains. p-Nitrophenyl phosphate, a nonprotein phosphoester, was used as a substrate for acid phosphatase. The separation profile on Sephadex G-100 gel filtration chromatography revealed that two major forms of high-molecular-weight and low-molecular-weight acid phosphatase were present in the crude extracts of both rat and human brains. Another class of zinc ion (Zn2+)-dependent acid p-nitrophenyl phosphatase was also detected in rat and human brains. In Alzheimer brains, the low-molecular-weight acid phosphatase activity was significantly decreased compared to that in control brains; however, the high-molecular-weight and Zn(2+)-dependent acid phosphatase activity in control and Alzheimer brains was not different. These results suggest that reduced activity of the low-molecular-weight acid phosphatase, which possesses phosphotyrosine protein phosphatase activity, might be linked to aberrant protein tyrosine phosphorylation found in Alzheimer brains.

Acid Phosphatase↗

A solid-phase immunoadsorbent assay for serum prostatic acid phosphatase.

A solid-phase immunoadsorbent assay for serum prostatic acid phosphatase (PAP) measurement has been developed as modified from our previously reported immunofluoroassay, utilizing the specific anti-PAP antibodies conjugated to CNBr-activated Sepharose 4B. The serum prostatic acid phosphatase was bound, and separated from other acid phosphatases and serum proteins, by the solid-phase anti-PAP IgG Sepharose 4B. The enzyme activity was quantitated by measuring the enzyme hydrolytic product, alpha-naphthol, from a primary standard solution. The entire procedure could be performed within four hours. The sensitivity of this method was 0.22 I.U./l of enzyme activity or 0.88 ng of prostatic acic phosphatase protein per ml of serum. Normal range of serum prostatic acid phosphatase as determined by this assay was found to be 0.4--2.4 I.U./l of enzyme activity (or 1.60--9.60 ng of enzyme protein per ml of serum). Initial clinical evaluation showed that 19 of 25 patients with early stages of prostatic cancer and 12 of 14 patients with metastatic prostatic cancer exhibited an elevated enzyme level (overall 79%), as compared with only six and eight patients, respectively (overall 36%), by a conventional chemical method.

Acid Phosphatase↗

A study of the acid phosphatase in the cells of bone and soft parts tumors, and of other tumorous conditions.

The acid phosphatase in the cells of bone and soft parts tumors, and of other tumorous conditions in our Department of Orthopedic Surgery in Kumamoto University Medical School from mid-1979 through mid-1983 were analysed by light microscopic and electron microscopic histochemical studies and their inhibition studies. The histochemical and their inhibiting studies of acid phosphatase by azo dye method in the cells of bone and soft parts tumors and of other tumorous conditions were undertaken in order to characterize them with a view to providing helpful diagnostic features. The acid phosphatase in some giant cells and tumor cells of several kinds of tumors, whose reaction against inhibitors was different from that of lysosomal acid phosphatase, was observed. In the giant cells of giant cell tumor of bone, acid-para-nitrophenyl phosphatase was demonstrated by the method of Miyayama , et al. using sodium-para-nitrophenyl phosphate as a substrate. In addition, the fine structural localization of acid phosphatase in giant cell tumor of bone was studied by Gomori's method and by the method of Miyayama , et al. By Gomori's method, acid phosphatase activity was demonstrated in lysosome, secondary lysosome-like organelles and the digestive vacuoles in the giant cells. In the stromal cells, that activity was demonstrated in lysosomes. By the method of Miyayama , et al., acid para-nitrophenyl phosphatase was demonstrated in the Golgi complex and the cisternae of the rough endoplasmic reticulum in the giant cell. Therefore, in the giant cells and the tumor cells of some kinds of tumors, non-lysosomal acid phosphatase besides lysosomal acid phosphatase was recognized. The demonstration of non-lysosomal acid phosphatase was a useful tool for the differential diagnosis of tumors and tumorous conditions in bone and soft parts.

Acid Phosphatase↗

Structure and function of yeast acid phosphatase.

A pronounced heterogeneity of purified acid phosphatase has been observed. The size heterogeneity which was demonstrated by electrophoretic methods is in agreement with the finding that the enzyme preparation contains families of molecules differing in the carbohydrate content (38% - 70%). The charge heterogeneity was shown by isoelectric focusing indicating the existence of several slightly different protein chains in the enzyme preparation. It was found that the enzyme is a dimer with a mean molecular weight of 252000 Daltons. There are 16 N-glycosidically linked carbohydrate chains, differing in size from 15-150 mannose units, and a very small amount of O-glycosidically linked mannose. Properties of acid phosphatase deglycosilated by treatment with beta-endo-N-acetylglucosaminidase, were compared with the native enzyme and it was found that the carbohydrate chains do not play a direct role in the catalytic activity of the enzyme, but stabilize the tridimensional structure of the molecule. A drastic increase of sensitivity of the deglycosilated enzyme against proteolysis was found.

Acid Phosphatase↗