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At least 343 records · Page 19Linked to original sources
PubMed1978

Serum prostate-specific acid phosphatase: development and validation of a specific radioimmunoassay.

We describe radioimmunoassay for human prostatic acid phosphatase [orthophosphoric-monoester phospho-hydrolase (acid optimum), EC 3.1.3.2] in serum, with use of monospecific antisera raised in rabbits against highly purified acid phosphatase from human prostates. The antiserum did not cross react with partly purified acid phosphatases from human spleen, erythrocytes, or synovial tissues. 125I-labeled acid phosphatase was prepared by a Chloramine T method, and the bound and free antigen was separated in the assay by use of anti-rabbit gamma-globulin raised in sheep. Uniform low nonspecific binding of the [125I]acid phosphatase was achieved by using acid-phosphatase-free serum to prepare standard curves and diluted samples of serum with high acid phosphatase activities. Concentrations of immunoreactive acid phosphatase in the serum of healthy men ranged from less than 1 to 10 microgram/liter and for 12 patients with advanced prostatic carcinoma between 100 and 500 microgram/liter. The concentrations of the enzyme in sera of patients with benign prostatic hyperplasia were very similar to those in sera of the reference group.

Acid Phosphatase↗
PubMed1981

Modulation of a cell surface glycoprotein in yeast: acid phosphatase.

Upon inorganic phosphate starvation the cell wall glycoprotein acid phosphatase of yeast Saccharomyces cerevisiae is derepressed. Purified acid phosphatase isolated from early log phase cells differs in reactivity and stability from acid phosphatase from late log phase cells indicating that the two enzymes are structurally different. This demonstrates that the yeast cell has not only the capacity to regulate the amount of acid phosphatase but also the ability to vary (modulate) the structure of the secreted enzyme. Modulation of acid phosphatase may be a mechanism which is involved in morphogenetic and behavioral differentiation of the yeast cell.

Acid Phosphatase↗
PubMed1976

Acid phosphatase heterogeneity in horse neutrophil and eosinophil leukocytes.

Two distinct groups of acid phosphatase containing granules were characterized in neutrophils, each group displaying different multiple forms of the enzyme. The heavy granule acid phosphatase showed a lysosomal location. A second lighter group of particles contained a thermolabile, thiol-dependent acid p-nitrophenyl and alpha-naphtylphosphatase, an enzyme clearly different from lysosomal acid phosphatase. Acid phosphatase activity from eosinophil leukocytes appeared to be totally associated with the typical eosinophil granules. On mechanical disruption of these particles, an acid phosphatase was released which differed in substrate and inhibitor specificity, in electrophoretic pattern, and in thermosensitivity, from the remaining matrix-bound enzyme.

Acid Phosphatase↗
PubMed1976

Distribution of acid phosphatase in chick Hensen's node.

Acid phosphatase distribution and yolk drop ultrastructure in Hensen's node of chick blastoderm at Hamburger-Hamilton stages 3 and 4 are described. At stage 3 large deposits of the reaction product are localised in type-A yolk drops, where signs of intensive degradation are seen. It seems that during this degradation lipid droplets are being formed in the degrading yolk drops. Partly digested yolk drops with a vesicular appearance are extruded from the cells, especially in deeper layers of the node. The phosphatase reaction product is also distributed into intercellular spaces. At stage 4 the cells of the node are more vacuolated and contain acid phosphatase and electron-dense yolk drops in lesser amounts. The possible physiological role of acid phosphatase in Hensen's node during gastrulation is discussed.

Acid Phosphatase↗
PubMed2003

Partial purification and kinetic characterization of acid phosphatase from garlic seedling.

The objective of this study was to obtain purer acid phosphatases than produced by prior art by operating under conditions that improve the final product. The study features are the use of a mild nonionic detergent, 40-80% saturation with (NH4)2SOm4, maintained at low temperature to remove impurity, and the use of chromatografic columns to concentrate the acid phosphatase and remove non-acid phosphatase proteins with lower or higher molecular weights. Acid phosphatase was isolated and purified from garlic seedlings by a streamline method without the use of proteolytic and lipolytic enzymes, butanol, or other organic solvents. Grown garlic seedlings of 10- 15 cm height were homogenized with 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. After homogenization, the supernatant was filtered with paper filters. Filtrated supernatant was cooled to 4 degrees C, followed by a threestep fractionation of the proteins with ammonium sulfate. The crude enzyme was isolated as a green precipitate that was dissolved in a small amount of 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. Garlic seedling acid phosphatase was purified with ion-exchange chromatography (DEAE cellulose). The column was equilibrated with 0.1 M acetate buffer. Acid phosphatase was purified 40-fold from the starting material. The specific activity of the pure enzyme was 168 U/mg. A variety of stability and activity profiles were determined for the purified garlic seedling acid phosphatase: optimum pH, optimum temperature, pH stability, temperature stability, thermal inactivation, substrate specificity, effect of enzyme concentration, effect of substrate concentration, activation energy, and effect of inhibitor and activator. The molecular mass of acid phosphatase was estimated to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH was 5.7 and the optimum temperature was 50 degrees C. The enzyme was stable at pH 4.0-10.0 and 40-60 degrees C. Activation energy was between 10 and 20 kcal, and as Michaelis Menten coefficients, Vm values were 100 and 20 mM/s and Km values were 21.27 and 8.33 mM for paranitrophenylphosphate and paranitrophenyl, respectively. Studies of the effect of metal ions on enzyme activity showed both an activating and a deactivating effect. While Cu, Mo, and Mn showed strong inhibitory effects, Na, Ca, and K were the significant activators of acid phosphatase.

Acid Phosphatase↗
PubMed1998

The glycosylphosphatidylinositol-anchored phosphatase from Spirodela oligorrhiza is a purple acid phosphatase.

We recently presented clear evidence that the major low-phosphate-inducible phosphatase of the duckweed Spirodela oligorrhiza is a glycosylphosphatidylinositol (GPI)-anchored protein, and, to our knowledge, is the first described from higher plants (N. Morita, H. Nakazato, H. Okuyama, Y. Kim, G.A. Thompson, Jr. [1996] Biochim Biophys Acta 1290: 53-62). In this report the purified 57-kD phosphatase is shown to be a purple metalloenzyme containing Fe and Mn atoms and having an absorption maximum at 556 nm. The phosphatase activity was only slightly inhibited by tartrate, as expected for a purple acid phosphatase (PAP). Furthermore, the protein cross-reacted with an anti-Arabidopsis PAP antibody on immunoblots. The N-terminal amino acid sequence of the phosphatase was very similar to those of Arabidopsis, red kidney bean (Phaseolus vulgaris), and soybean (Glycine max) PAP. Extracts of S. oligorrhiza plants incubated with the GPI-specific precursor [3H]ethanolamine were treated with antibodies raised against the purified S. oligorrhiza phosphatase. Radioactivity from the resulting immunoprecipitates was specifically associated with a 57-kD band on sodium dodecyl sulfate-polyacrylamide gels. These results, together with previous findings, strongly indicate that the GPI-anchored phosphatase of S. oligorrhiza is a PAP.

Acid Phosphatase↗
PubMed1975

A solid-phase radioimmunoassay for human prostatic acid phosphatase.

A solid-phase technique for radioimmunoassay of human prostatic acid phosphatase (EC 3.1.3.2) is described. Human prostatic acid phosphatase was purified from prostatic fluid. Monospecific antisera to the purified acid phosphatase were produced in rabbits. Disposable polypropylene tubes were coated with antiserum and used for radioimmunoassay with 125I-acid phosphatase. The nonspecific binding was minimized by saturating the binding sites of the tubes with bovine serum albumin. The working range of the technique was 1 to 30 ng of antigen. The solid-phase radioimmunoassay is rapid, sensitive, and efficient. In preliminary clinical trials it was shown that (a) patients with advanced prostatic cancer had elevated prostatic acid phosphatase levels by both enzymatic assay and radioimmunoassay assays, and (b) patients with other cancers were in the normal range for prostatic acid phosphatase.

Acid Phosphatase↗
PubMed1978

[Activity of acid phosphatase in saliva of industrial workers exposed to fluorine compounds].

Assuming that fluorine is a strong inhibitor of phosphatases, the activity of acid phosphatase in the saliva of 42 phosphorus fertilizers plant workers was studied. The workers were chronically exposed to fluorine compounds. Saliva had been collected for studies before the workers entered the productive plant and after the 1st and 2nd working hour. Enzyme activity was determined by Bessey and Lowry's method and was related to both the volume of the saliva and total protein level. The studies revealed a significant decrease in the activity of acid phosphatase after the 1st and 2nd working hour in those working in the division with a higher exposure to fluorine compounds. The results of these studies were confirmed by in vitro studies carried out additionally. Taking into account biological activity of acid phosphatase it seems that the observed phenomenon of inhibiting enzymatic activity may be connected with the frequency of parodontopathy in the studied group of workers. The results of these studies may be used for working out a biological test of workers' exposure.

Acid Phosphatase↗
PubMed1991

A new oxygen-regulated operon in Escherichia coli comprises the genes for a putative third cytochrome oxidase and for pH 2.5 acid phosphatase (appA)

The Escherichia coli acid phosphatase gene appA is expressed in response to oxygen deprivation and is positively controlled by the product of appR (katF) which encodes a putative new sigma transcription-initiation factor. However, transcription of appA from its nearest promoter (P1) did not account for total pH 2.5 acid phosphatase expression and was not subject to regulation. The cloned region upstream of appA was extended and analyzed by insertions of transposon TnphoA and by fusions with lacZ. It contains two new genes, appC and appB, which both encode extracytoplasmic proteins. appC and appB are expressed from a promoter (P2) lying just upstream of appC. Both genes are regulated by oxygen, as is appA, and by appR gene product exactly as previously shown for appA. Analysis of the nucleotide sequence and of the origins of transcription have confirmed that the P2-appC-appB- (ORFX)-P1-appA region is organized on the chromosome as an operon transcribed clockwise from P2 and that P1 is a minor promoter for appA alone. Genes appC and appB encode proteins of Mr 58,133 and 42,377, respectively, which have the characteristics of integral membrane proteins. The deduced amino acid sequences of appC and appB show 60% and 57% homology, respectively, with subunits I and II of the E. coli cytochrome d oxidase (encoded by genes cydA and cydB). The notion that the AppC and AppB proteins constitute a new cytochrome oxidase or a new oxygen-detoxifying system is supported by the observation of enhanced sensitivity to oxygen of mutants lacking all three genes, cyo (cytochrome o oxidase), cyd (cytochrome d oxidase) and appB, compared to that of cyo cyd double mutants.

Acid Phosphatase↗
PubMed1981

Purification, enzymatic properties, and active site environment of a novel manganese(III)-containing acid phosphatase.

A new manganese-containing acid phosphatase has been isolated and crystallized from sweet potato tubers. The pure enzyme contains one atom of manganese per Mr = 110,000 polypeptide and shows phosphatase activity toward various phosphate substrates. The pH optimum of the enzyme was 5.8 and the enzyme activity was inhibited by Cu2+, Zn2+, Hg2+, AsO43-, and MoO42-. This stable metalloenzyme is red-violet in color with an intense absorption band at 515 nm (epsilon - 2460). Our electronic, circular dichroism, and electron spin resonance findings strongly indicate that the Mn-valence state of the native enzyme is trivalent. When the Mn-enzyme is excited by the 5145 A line of Ar+ laser, prominent Raman lines at 1230, 1298, 1508, and 1620 cm-1 were detected. This Raman spectrum can probably be interpreted in terms of internal vibration of a coordinated tyrosine phenolate anion. The tryptophan-modified enzyme showed a positive Raman band at 370 cm-1, which is preferentially assigned to a Mn(III)-S streching mode. The modification of the Mn-enzyme by N-bromosuccinimide led to a large decrease in the fluorescence intensity of 335 nm which was dominated by its tryptophan residues within a considerable hydrophobic environment. The acid phosphatase activity was significantly decreased by the tryptophan modification. With respect to the active site donor sets, the Mn(III)-containing acid phosphatase is distinctly different from the Zn(II)-containing alkaline phosphatase. Of interest is also the appreciable similarity of some enzymatic and spectroscopic properties between the present enzyme and uteroferrin.

Acid Phosphatase↗
PubMed1995

Hairy cell identification by immunohistochemistry of tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAcP) has been an indispensible marker for hairy cell leukemia (HCL) for over two decades. However, the traditional TRAcP cytochemical stain cannot be performed effectively on sections of paraffin-embedded tissues that are important resources for histopathologic evaluation in diagnosis and treatment of HCL. Wide variation in expression of TRAcP activity by hairy cells (HCs) within and among patients is an interesting biologic phenomenon that has not been explained and can cause some diagnostic uncertainty as well. To solve the problem of staining TRAcP in paraffin sections and to begin to address the questions of variable TRAcP expression in HCL, we developed a monoclonal antibody to TRAcP, 9C5, for immunohistochemical identification of HCs. In smears of blood and bone marrow, immunocytochemistry of TRAcP using 9C5 was as specific but slightly less sensitive than direct cytochemical staining of enzymatic activity. In paraffin sections of spleen and bone marrow from HCL patients, immunohistochemistry with 9C5 stained the HCs with high sensitivity and specificity and clearly showed the characteristic diffuse infiltration by HCs. Other cells noted to stain strongly with 9C5 were occasional macrophages in bone marrow smears and osteoclasts and occasional tissue macrophages in paraffin sections. These are cells known to express abundant TRAcP activity. Immunohistochemistry with anti-TRAcP monoclonal antibody 9C5 may have utility as an added option in the diagnosis of HCL, as a means to evaluate residual disease in HCL patients undergoing new treatments, and as a way to address questions regarding variable expression of TRAcP activity by HCs within and among patients with HCL. Also, 9C5 has potential as a reagent for the immunoassay of bone-derived serum TRAcP in patients with certain bone diseases and cancers with bone metastasis.

Acid Phosphatase↗
PubMed2003

Flow cytoenzymology of intracellular tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRACP) is a cytochemical marker for hairy cell leukemia, macrophages, dendritic cells, and osteoclasts. Our purpose was to develop multicolor cytofluorometric methods to evaluate intracellular TRACP enzymic activity using a fluorogenic cytochemical reaction in combination with immunochemical stains for distinct surface membrane antigens. Monocyte-derived dendritic cells (DCs) were the model TRACP-expressing cells studied. Intracellular TRACP activity was disclosed using naphthol-ASBI phosphate as substrate with fast red-violet LB salt as coupler for the reaction product. Before the TRACP enzymic reaction, surface antigens, CD86 and CD11c of DCs, were bound with specific fluorescent antibodies to test compatibility of surface labeling and intracellular staining. TRACP activity varied in DCs from donor to donor but was reproducible on repeated examinations of each sample. Samples could be stained for simultaneous analysis of surface antigens and intracellular TRACP activity, provided certain technical details were observed. The TRACP reaction time should not exceed 9 min and the cell number should not exceed 2 x 10(5)/100 micro l test. Fluorescent surface labels did not affect the intensity of the TRACP stain, but the intensity of some surface labels may be diminished by elution of low-affinity antibodies during the TRACP reaction. Readjustment of the threshold settings in triple-labeled cells is needed to compensate for this phenomenon. Intracellular TRACP activity can be quantitated in subpopulations of cells within mixed cell populations by flow cytofluorometry using simple cytochemical methods in combination with fluorescent antibodies to cell-surface and other differentiation antigens. The cytochemical method should be useful for basic investigations of differentiation, maturation, and function of macrophages, DCs, and osteoclasts, and for diagnosis and management of hairy cell leukemia.

Acid Phosphatase↗
PubMed1999

Intracellular fragmentation of bone resorption products by reactive oxygen species generated by osteoclastic tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAP) is highly expressed in bone-resorbing osteoclasts and activated macrophages. It has been suggested that a redox-active iron in the binuclear iron center of TRAP could have the capacity to react with hydrogen peroxide to produce highly destructive reactive oxygen species (ROS). Here we show that TRAP can generate ROS in vitro and that cells over-expressing TRAP produce higher amounts of intracellular ROS than their parent cells. We further demonstrate that these ROS can be targeted to destroy collagen and other proteins. In resorbing osteoclasts, TRAP was found in transcytotic vesicles transporting matrix degradation products through the cell, suggesting that TRAP-facilitated fragmentation of endocytosed material takes place in a specific cellular compartment. These results suggest that bone matrix degradation occurs not only extracellularly in the resorption lacunae but also intracellularly in the transcytotic vesicles. We propose that proteins containing redox-active iron could represent a novel mechanism of physiological fragmentation of organic molecules. This mechanism could be important in tissue remodeling and as a defense mechanism of phagocytosing cells.

Acid Phosphatase↗
PubMed1981

Clinical applicability of acid phosphatase isoenzyme assay.

We compared electrophoretic evaluation of acid phosphatase isoenzymes with spectrophotometric determination of prostatic acid phosphatase in terms of clinical utility. In all of 33 cases of prostatic carcinoma, an increased prostatic fraction was detected; in nine prostatectomized patients, this fraction returned to normal as measured by either technique. Abnormal spectrophotometric results were also seen in 10 cases of benign prostatic hypertrophy and seven cases of non-prostatic disorders, but only two benign prostatic hypertrophy and one non-prostatic case showed a prostatic band (band 2) in an electrophoretogram. Band 2 was not demonstrated in 463 patients affected by a great variety of diseases but without prostatic disorders. A weak band 5 was seen in patterns for most patients, except for cases with metastatic bone tumor and Gaucher's disease, whose serum showed a strong band 5. The specificity of bands 2 and 5 seems to be confirmed by this large series of patients. Measurement of acid phosphatase isoenzymes is recommended as a routine screening test for patients whose serum acid phosphatase is abnormally high, because the isoenzyme study not only indicates the presence or absence of prostatic cancer but also whether or not there is bony metastasis. Other disorders such as Gaucher's disease, different kinds of leukemias, and thrombocythemia may also be detected and distinguished by this screening technique.

Acid Phosphatase↗
PubMed1998

Species specificity of monoclonal antibodies to human tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAP) is expressed abundantly by osteoclasts and is required for bone resorption. This enzyme is emerging as an important biomarker in bone pathology, both for histochemical identification of osteoclasts and as a serum marker of osteoclast activity and increased bone turnover. Rat and mouse models are becoming popular systems for studying osteoclast development, bone physiology and morphogenesis, and bone diseases such as osteoporosis. We have developed two unique antibodies to human TRAP purified from hairy cell leukemia spleen. Both antibodies (9C5 and 14G6) are suitable for immunohistochemistry of osteoclasts and macrophages. Only one (14G6) is capable of immunoprecipitating active TRAP from human cell lysates. Antibody 9C5 reacts with a denatured epitope of TRAP while antibody 14G6 probably reacts with a native, conformational determinant. The high degree of homology among TRAPs of various species predicts that these antibodies should be suitable for work in experimental animals as well as humans. Immunohistochemical staining, electrophoretic analyses, immunoprecipitation and immunoblotting assays of human rat and mouse TRAP were carried out to test the validity of these antibodies as cell markers in rodents. Both antibodies were suitable for immunohistochemistry in all species. Antibody 9C5 was suitable for immunoblotting of denatured TRAP of all species tested. Antibody 14G6 reacted with the native TRAP of humans only and failed to immunoprecipitate mouse or rat TRAP activity. Although TRAP is a phylogenetically conserved protein, subtle, species-specific determinants exist. Care should be exercised when anti-TRAP antibodies are used for immunoassay in experimental animals.

Acid Phosphatase↗
PubMed1989

Leishmania donovani: immunochemical localization and secretory mechanism of soluble acid phosphatase.

Monoclonal antibodies specific for the soluble, secreted acid phosphatase (EC 3.1.3.2) of Leishmania donovani were used to investigate the localization of this enzyme in extracellular promastigotes and intracellular amastigotes. Indirect immunofluorescence showed a weak general staining in the promastigote cytoplasm, together with strong fluorescence in the flagellar reservoir. Immunofluorescence studies on U937 cells infected in vitro with L. donovani showed that the pathogenic amastigote stage also produced soluble acid phosphatase. Metabolic labeling experiments using promastigotes indicated that the intracellular enzyme was soluble prior to secretion and no evidence was found for the association of secretory acid phosphatase with cell membranes after protein synthesis. The rapid release of acid phosphatase from the flagellar reservoir was energy dependent and may be coupled to beating of the flagellum. The results demonstrated that acid phosphatase was secreted into the flagellar reservoir by Leishmania promastigotes using a conventional constitutive secretory mechanism, and subsequently released from the reservoir into the extracellular medium.

Acid Phosphatase↗
PubMed1976

Histochemical and biochemical study on acid phosphatase activity in differentiating liver of chick.

1. Acid phosphatase activity at different ontogenetic stages of liver in chick has been studied both by histochemical and quantitative biochemical methods. 2. It has been observed that the activity of acid phosphatase does not remain constant, but undergoes changes at different phases of epigenesis. The activity rhythmically becomes higher and lower throughout the whole period of epigenesis. Comparatively higher activities have been found up to 8 days of incubation, and then the activities become lower up to 14 days and again increased at later periods of development. 3. Higher acid phosphatase activity has been observed in the perivascular connective tissue than the parenchymal cells from the 8th to the 20th day of incubation. 4. The acid phosphatase activity is high in the nuclei of early developing red blood corpuscles, but in advanced stage of R.B.C. the phosphatase activity is very high in the cytoplasm, and it is practically absent in the nuclei. 5. The activity of acid phosphatase may have some relation with the cell death and catabolic processes during chick liver morphogenesis. The relation of the phosphatase activity with the growth and differentiation of the liver has been discussed.

Acid Phosphatase↗
PubMed1986

Biochemical and immunological relationships of prostatic and leukocytic acid phosphatases and their subcellular localization.

Human acid phosphatases from various sources were purified by a new rapid procedure including chromatofocusing and gel permeation chromatography on an FPLC system. Isoenzymes 2a and 4 (Mr 105,000 and 76,000, respectively) are the prevalent acid phosphatases in prostate, leukocytes and bone marrow. Additional forms are proteolytic products formed during isolation procedure. The source of elevated acid phosphatase in blood serum in metastasizing prostatic carcinoma can be either prostate itself, the tumour or, more likely, bone marrow cells destroyed during the invasion by the tumour cells. Distribution of isoenzymes is not organ specific. It was found using an antiserum against isoenzyme 4 that immunoreactivity is confined to the specific secretory granules both of prostatic epithelium and leukocytes.

Acid Phosphatase↗