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Acid phosphatase isoenzyme mapping in Leishmania.

The profiles of acid phosphatase isoenzymes of several well defined species of the genus Leishmania were compared. The profiles were generated after isoelectric focusing of parasite extracts in polyacrylamide and incubation of the gels with an appropriate substrate coupled to an azo dye. Analysis of the zymograms showed that there is species-specificity of the acid phosphatase isoenzyme maps in Leishmania. It was also demonstrated that different strains of the same species present identical pattern of enzyme activity. The method even enabled the differentiation of closely related species which were previously difficult to identify. Some technical aspects of the isoelectric focusing procedure are discussed. The method described here can be used as an aid for species identification of Leishmania.

Acid Phosphatase↗

Naphthol-ASBI phosphate as a preferred substrate for tartrate-resistant acid phosphatase isoform 5b.

Tartrate-resistant acid phosphatase (TRAP) isoform 5b is a potential serum marker for osteoclastic activity. Biochemical assays for serum TRAP activity with para-nitrophenylphosphate (pNPP) have low specificity for bone because of hydrolysis by unrelated nontype 5 TRAPs of blood cells and by related isoform 5a. Our purpose was to increase the specificity of TRAP assay for osteoclastic activity by using naphthol-ASBI phosphate (N-ASBI-P) as a substrate for serum type 5 TRAP activity and heparin as an inhibitor of isoform 5a. TRAP activity in individual and pooled sera of normal subjects and patients with endstage renal disease (ESRD) and rheumatologic diseases was quantitated using pNPP and N-ASBI-P as substrate at pH 5.5 and 6.1. For some experiments, heparin (23U/ml) was added as a specific inhibitor of isoform 5a activity. Isoforms 5a and 5b were separated from serum pools by cation exchange chromatography and identified by nondenaturing polyacrylamide gel electrophoresis (PAGE). N-ASBI-P was selectively hydrolyzed by TRAP isoform 5b. TRAP assays with pNPP and N-ASBI-P correlated only in ESRD sera, which contained primarily isoform 5b. The two assays did not correlate in normal or rheumatic sera with significant amounts of 5a. Heparin inhibited isoform 5a activity approximately 50% but had little effect on isoform 5b activity. Biochemical assay of serum TRAP activity can be made specific for isoform 5b by using N-ASBI-P and heparin. This method can be adapted to simple microplate biochemical or immunochemical assays. This simplified method for assessment of osteoclastic TRAP 5b activity warrants a detailed investigation in diseases of bone metabolism.

Acid Phosphatase↗

Characterization of the 5'-flanking region of the human tartrate-resistant acid phosphatase (TRAP) gene.

Tartrate-resistant acid phosphatase (TRAP) is expressed at high levels in osteoclasts and may play an important role in the bone resorptive process. However, factors regulating human TRAP gene expression have not been clearly defined. Therefore, we isolated a genomic clone (CL-9) for TRAP containing a 14-kb insert. A restriction map was generated for this insert, and a 2.6-kb ApaI fragment containing the 5'-flanking region was subcloned. Sequence analysis of this fragment revealed the presence of candidate transcription factor-binding sequences for H-APF-1, SP1, GATA2, and the c-Myc proto-oncogene. PCR analysis of RNA isolated from human osteoclastomas and pagetic bone revealed a 276-bp intron at -1 bp to -276 bp relative to the ATG and a transcript originating from this intron. Rapid amplification of the 5' end of the human TRAP mRNA by PCR indicated the presence of a 93-bp untranslated region 5' from the intron. Promoter activity was detected in the DNA fragment from +1 bp to -1903 bp relative to the ATG initiation codon, which drove the transient expression of a luciferase reporter gene when transfected into HRE H9 rabbit endometrial cells. Comparison of the human TRAP 5'-flanking region with mouse TRAP and uteroferrin revealed 41% and 47% homology, respectively. This suggests that regulation of human TRAP gene expression may differ from that for the murine TRAP gene.

Acid Phosphatase↗

Purification and characterization of a low-molecular-weight bovine kidney acid phosphatase.

A relative low molecular mass bovine kidney acid phosphatase was purified 1,640-fold to homogeneity, with 7% recovery. The purified enzyme (specific activity 100 mumol min-1 mg-1) was electrophoretically homogeneous with a relative molecular mass of 17.8 kDa, as determined by SDS-polyacrylamide gel electrophoresis. A broad pH optimum of 4.0-5.5 and a maximal enzyme activity at 60 degrees C were determined for the p-nitrophenyl phosphate hydrolysis. Apparent Km values of 0.14 mM, 0.4 mM, 0.3 mM and 7.9 mM were obtained, at 37 degrees C and pH 5.0, for the best substrates p-nitrophenyl phosphate, beta-naphtyl-phosphate, flavin mononucleotide and tyrosine-phosphate, respectively. The enzyme activity was enhanced by guanosine but inhibited by ZnCl2 and CuSO4, p-cloromercuribenzoate and ammonium molybdate. Vanadate (Ki 0.47 microM), pyridoxal 5'-phosphate (Ki 2.2 microM), inorganic phosphate (Ki 0.77 microM) are competitive inhibitors. Both glycerol and methanol increased significantly the acid phosphatase activity, acting as good phosphate acceptors in the transphosphorylation reaction.

Acid Phosphatase↗

Distribution pattern of acid phosphatase activity in human peripheral blood leukocytes: a cytochemical scanning electron microscopy study.

The distribution patterns of acid phosphatase hydrolytic activity were studied in human peripheral blood cells with enzymocytochemical techniques together with light and scanning electron microscopy in the secondary and backscattered electron imaging modes. The acid phosphatase reaction product was seen in three different patterns of distribution: focal, granular and diffuse. These patterns were correlated with similar findings obtained with light microscopy. Acid phosphatase distribution patterns seen with SEM in the BEI mode were also correlated with the surface morphology of peripheral blood cells seen in the SEI mode. Cells exhibiting the focal pattern were smooth-surfaced with few microvilli; cells showing a granular pattern presented microvilli and microridges; ruffles were characteristic of cells with a diffuse pattern of activity. No reaction product was seen in cells bearing microvilli or ridges. Our findings demonstrate the correlation between acid phosphatase activity patterns and surface features in different subpopulations of peripheral blood cells.

Acid Phosphatase↗

Resolution of the low-molecular-weight acid phosphatase in avian pectoral muscle into two distinct enzyme forms.

Three distinct acid phosphatases were recently reported in avian breast muscle [J. H. Baxter and C. H. Suelter (1984) Arch. Biochem. Biophys. 228, 397-406]. Of the increased acid phosphatase activity in dystrophic muscle compared to normal muscle, 84% can be accounted for as a low-molecular-weight, cytosolic form. This low-molecular-weight form has now been purified and resolved into two distinct forms, A and B, differing in isoelectric point, apparent molecular weight, substrate specificity, and activation by guanosine. One of the two enzymes exhibits substrate inhibition with 4-methylumbelliferyl phosphate, indicating a further difference. The evidence suggests that both enzymes are Class IV acid phosphatases. Their concentrations are highest in tissues with a high catabolic activity.

Acid Phosphatase↗

[Acid phosphatase in seminal fluid--method of estimation and diagnostic significance].

A new method for the estimation of acid phosphatase in seminal fluid based on the use of p-nitro-phenylphosphate is described as simple in practicability and appropriate for routine in clinics. The frequency distribution of acid phosphatase activity is specified with 66 samples showing a normal spermiogram. The values spread logarithmically with an average of 400 U/ml and a standard deviation ranging from 200 U/ml to 880 U/ml. Correlations between acid phosphatase content and general, cytological and biochemical spermiogram-parameters are investigated. A plain negative correlation with the pH (r = 0,4615, n = 250) and a very strong positive correlation with citrate (r = +0,9029, n = 200) turned out. The significance of these correlations is discussed. Among the biochemical parameters in spermiogram both citrate and acid phosphatase--each representing prostatic function--are equivalent in diagnostical significance.

Acid Phosphatase↗

Isoenzymatic study of leucocytic acid phosphatase in haematologic diagnosis.

Isoenzymatic study of leucocytic acid phosphatase under normal conditions identifies 3 isoenzymatic bands, which exhibit a noticeable cell specificity. Band 2 is granulocytic, band 3 lymphocytic and band 4 monocytic in origin. Pathologic deviations in the isoenzymatic pattern are both qualitative and quantitative. For some diseases such as chronic lymphocytic leukaemia there is a well-defined, differential pattern according to immunological B- or T-cell origin. The more significant qualitative aspects are related to the appearance of abnormal bands, especially band 3b, indicating blastic cellularity, and 5, corresponding to hairy cells. The isoenzymatic analysis of acid phosphatase activity is a simple haematologic complementary test, particularly useful in the differential diagnosis of lymphoproliferative disorders with peripheral blood manifestations.

Acid Phosphatase↗

Characterization of Enterobacter cloacae and E. sakazakii by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases.

Acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases of 34 strains of Enterobacter cloacae and 22 strains of Enterobacter sakazakii were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. The two species could be separated on the basis of distinct electrophoretic patterns of all enzymes analysed. Glutamate dehydrogenase and acid phosphatase were detected exclusively in E. cloacae, whereas esterase bands were more intensively stained in E. sakazakii. For each species, two zymotypes could be distinguished, on the basis of electrophoretic mobilities of malate dehydrogenase and banding patterns of esterase for E. cloacae, and by both isoelectric point and electrophoretic mobilities of an esterase and of lactate and malate dehydrogenases for E. sakazakii. The high degree of enzyme polymorphism within the two species permitted precise identification of strains. The variations in electrophoretic patterns might therefore provide useful epidemiological markers.

Acid Phosphatase↗

Acid phosphatase and lipid peroxidation in human cataractous lens epithelium.

The anterior lens epithelial cells undergo a variety of degenerative and proliferative changes during cataract formation. Acid phosphatase is primarily responsible for tissue regeneration and tissue repair. The lipid hydroperoxides that are obtained by lipid peroxidation of polysaturated or unsaturated fatty acids bring about deterioration of biological membranes at cellular and tissue levels. Acid phosphatase and lipid peroxidation activities were studied on the lens epithelial cells of nuclear cataract, posterior subcapsular cataract, mature cataract, and mixed cataract. Of these, mature cataractous lens epithelium showed maximum activity for acid phosphatase (516.83 moles of p-nitrophenol released/g lens epithelium) and maximum levels of lipid peroxidation (86.29 O.D./min/g lens epithelium). In contrast, mixed cataractous lens epithelium showed minimum activity of acid phosphatase (222.61 moles of p-nitrophenol released/g lens epithelium) and minimum levels of lipid peroxidation (54.23 O.D./min/g lens epithelium). From our study, we correlated the maximum activity of acid phosphatase in mature cataractous lens epithelium with the increased areas of superimposed cells associated with the formation of mature cataract. Likewise, the maximum levels of lipid peroxidation in mature cataractous lens epithelium was correlated with increased permeability of the plasma membrane. Conversely, the minimum levels of lipid peroxidation in mixed cataractous lens epithelium makes us presume that factors other than lipid peroxidation may also account for the formation of mixed type of cataract.

Acid Phosphatase↗

Immunohistochemical localization of prostate-specific acid phosphatase and prostate-specific antigen in stage A2 adenocarcinoma of the prostate: prognostic implications.

Previous immunohistochemical studies with prostate-specific acid phosphatase and prostate-specific antigen documented the diagnostic value of these antigens in the identification of metastatic prostatic adenocarcinomas and in the differentiation of primary prostatic adenocarcinomas from poorly differentiated transitional cell carcinomas. Although attempts have been made to correlate immunostaining with degree of tumor differentiation, no study has directly assessed the relation of either prostate-specific acid phosphatase or prostate-specific antigen immunoreactivity of tumors with their biologic behavior. Nineteen patients with predominantly intermediate Gleason grade untreated stage A2 carcinomas of the prostate were studied by the unlabeled antibody immunoperoxidase technique for prostate-specific acid phosphatase and prostate-specific antigen in an attempt to identify those in whom the disease would progress without further therapeutic intervention. Of the 12 carcinomas with areas of either weak or negative prostate-specific acid phosphatase staining, nine progressed. Two of the seven carcinomas that did not have these foci of poor immunostaining also progressed. Although there was a trend for foci of poor immunoreactivity to predict tumor progression, the correlation was not significant. All seven patients who had tumor foci with weak or negative prostate-specific antigen immunostaining experienced progression of the disease. Of the 12 patients with only moderate or intense staining, the tumors did not progress in eight. This correlation between foci of poor immunoreactivity and progression of disease was statistically significant. When the study was repeated by varying the technique without awareness of previous grading results, the same predictive results were obtained. When results of prostate-specific antigen immunostaining were compared with those achieved with prostate-specific acid phosphatase, the superiority of the prostate-specific antigen antisera for labeling prostatic tissue was evident. This study suggests that prostatic cancers consist of subpopulations of cells with differing immunoreactive properties and that the presence of cells that lack sufficient differentiation to express normally present immunologically recognizable antigens is an indication of potentially more aggressive neoplasms.

Acid Phosphatase↗

A quantitative method for measurement of lysosomal acid phosphatase latency in cultured rat heart cells with 210Pb.

A method is described for measuring the latency of lysosomal acid phosphatase in cultured rat heart endotheloid cells. 210Pb was added to a medium used to demonstrate acid phosphatase activity by the Gomori lead method, and the amount of lead deposited was measured with a liquid scintillation counter. Deposition rates were measured after enzyme activation pretreatments with acetate buffer (pH 5.0) at various osmolalities, and after formaldehyde fixation. Formaldehyde, alloxan, or fluoride in the Gomori medium were evaluated for their differential effects on lysosomal and non-lysosomal acid phosphatase. The method was found to provide a sensitive, rapid and quantitative evaluation of acid phosphatase latency and should be useful for studying the integrity of lysosomes within cells.

Acetates↗

Effect of melatonin-free pineal extract on acid phosphatase activity in the testosterone-treated, castrated rat.

The effect of a melatonin-free pineal extract on acid phosphatase activity in the prostate of castrated and testosterone-treated rat was studied. Prostatic acid phosphatases in the mature rat are lysosomal and secretory, the former being inhibited by tartrate. Castration induces a decrease in the secretory phosphatases and an increase in the lysosomal ones with a subsequent increase in the tartrate-induced inhibition index. The specific activity of total acid phosphatase also rises. Administration of the pineal extract in the first 4 post-surgical days did not have a significant effect, but when it was prolonged to 7 days, it partially inhibited the effect of castration on prostatic acid phosphatase. Administration of testosterone to the castrated rat in the first 4 post-castration days, partially compensated the prostatic alterations, owing to the fact that the prostate is under the control of androgenic hormones. Administration of both pineal extract and testosterone in the first 4 post-castration days partially inhibited the effect of testosterone. The equillibrating role of the melatonin-free pineal extract and its effect in relation to the amount of time lapsed from surgery is discussed.

Acid Phosphatase↗

Elevation of serum acid phosphatase in cancers with bone metastasis.

In patients with nonprostatic cancer serum acid phosphatase activity is usually elevated when bone metastasis is present but not when bone metastasis is absent. The fraction responsible for serum enzyme elevation is a normal component of serum; it appears in gel electrophoresis as band 5; and is tartrate-resistant. It is suggested that the origin of acid phosphatase elevation is bone osteoclasts rather than cancer tissue, as is the case with prostatic carcinoma. Determination of serum acid phosphatase activity may be useful in the detection of bone metastasis.

Acid Phosphatase↗

A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings.

Phytic acid (myo-inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds. During germination, stored reserves are used as a source of nutrients by the plant seedling. Phytic acid is degraded by the activity of phytases to yield inositol and free phosphate. Due to the lack of phytases in the non-ruminant digestive tract, monogastric animals cannot utilize dietary phytic acid and it is excreted into manure. High phytic acid content in manure results in elevated phosphorus levels in soil and water and accompanying environmental concerns. The use of phytases to degrade seed phytic acid has potential for reducing the negative environmental impact of livestock production. A phytase was purified to electrophoretic homogeneity from cotyledons of germinated soybeans (Glycine max L. Merr.). Peptide sequence data generated from the purified enzyme facilitated the cloning of the phytase sequence (GmPhy) employing a polymerase chain reaction strategy. The introduction of GmPhy into soybean tissue culture resulted in increased phytase activity in transformed cells, which confirmed the identity of the phytase gene. It is surprising that the soybean phytase was unrelated to previously characterized microbial or maize (Zea mays) phytases, which were classified as histidine acid phosphatases. The soybean phytase sequence exhibited a high degree of similarity to purple acid phosphatases, a class of metallophosphoesterases.

6-Phytase↗

Comparison of the acid-phosphatase staining and polymerase chain reaction for detection of Dirofilaria repens infection in dogs in Korea.

This study was performed to compare acid-phosphatase staining with polymerase chain reaction (PCR) analysis for the diagnosis of Dirofilaria repens infection. The infection of D. repens was confirmed in Korean reared German shepherd dogs. Knott's tests were carried out for the detection of microfilaria in 543 Korean reared German shepherd dogs (255 females and 288 males). Eighty four of the 543 dogs (15.5%) showed microfilaria-positive reactions with the modified Knott's test, and the test-positive microfilariae were then examined by both acid phosphatase staining and PCR analysis. Six (7.1%) and 17 (20.2%) of the 84 microfilaria-positive samples, by the Knott's tests were positive to D. repens by acid-phosphatase staining and in D. repens-specific PCR analysis, respectively. All samples found to be positive by the acid-phosphatase staining were also found to be positive by PCR analysis. Therefore, we conclude that PCR analysis (20.2%) is more valuable for the diagnosis of D. repens infection than acid-phosphatase staining (7.1%) (p<0.001).

Acid Phosphatase↗

Theoretical investigations of prostatic acid phosphatase.

The phosphotyrosyl protein phosphatase activity of prostatic acid phosphatase (PAP) has been well established. It has also been suggested that PAP partly regulates the activity of growth factor receptors by dephosphorylating the autophosphorylysable tyrosines in them. We studied the binding of the peptides from epidermal growth factor receptor (EGFR) and its homolog (ErbB-2), corresponding to their autophosphorylation sites, to PAP using theoretical modeling and molecular dynamics (MD) simulation methods. Nine different peptides, each with a phosphotyrosine residue, were docked on human PAP. The binding energies of these peptide-PAP complexes were calculated theoretically and compared to experimentally obtained affinities. The peptide Ace--DNLpYYWD--NH2 from ErbB-2(1197-1203) showed the most favorable free energy of binding when estimated theoretically. The results demonstrate that the presence of another tyrosine residue proximate to C-terminal of autophosphorylysable Tyr enhances the binding affinity considerably. The presence of a bulky group instead prevents the binding, as is observed in case of peptide Ace--NLYpYWDQ--NH2 which failed to bind, both in theoretical calculations and experiments. Thus we demonstarted that PAP could potentially bind to EGFR and Erbb-2 and dephosphorylate them. Thus it could be involved in the regulation of the function of such receptors. In addition, complexes of a peptide from AngiotensinII and phosphotyrosine(pY) with human PAP were also modeled. The effects of different protonation states of the titratable active site residues on ligand (pY) binding have also been investigated. For a favorable binding His12 and Asp258 should be neutral, His257 should be positively charged and the phosphate group of the ligand should be in PO(4) (3-) state. Furthermore, the analysis of protein motion as observed during simulations suggests the loop-loop contact in the PAP dimer to be of importance in cooperativity.

Acid Phosphatase↗

Acid phosphatase changes associated with development of male sterile and fertile maize (Zea mays L.).

Free and bound acid phosphatase were investigated biochemically and electrophoretically in male sterile and fertile plants. Scutella at the 72-hr seedling stage and anthers with enclosed reproductive tissue at the premeiosis, meiosis, and mature developmental stages were tested. Biochemical data show that while the total amount of free acid phosphatase behaved similarly in fertile and sterile plants, specific activities decreased in fertile plants and remained unchanged or increased in sterile plants. Total amounts of bound acid phosphatase increased significantly in fertile plants while specific activities decreased. In sterile anthers both specific activity and amount of bound acid phosphatase decreased significantly (except cms-S). Electrophoretic results indicated that the basic form of the enzyme was very similar in each location.

Acid Phosphatase↗