Inhibition Of Acid Phosphatase Research Articles

Specific enzymatic dephosphorylation of the retinoblastoma protein.

The retinoblastoma gene product (RB) undergoes cell cycle-dependent phosphorylation and dephosphorylation. Pulse-chase experiments revealed that the change in RB gel electrophoretic migration which occurs near mitosis is due to enzymatic dephosphorylation (J. W. Ludlow, J. Shon, J. M. Pipas, D. M. Livingston, and J. A. DeCaprio, Cell 60:387-396, 1990). To determine the precise timing of RB dephosphorylation and whether a specific phosphatase is active in this process, we have utilized a nocodazole block and release protocol which allows a large population of cells to progress synchronously through mitosis. In such experiments, RB dephosphorylation began during anaphase and continued until complete dephosphorylation was apparent in the ensuing G1 period. In addition, late mitotic cell extracts were capable of dephosphorylating RB in vitro. This RB-specific mitotic phosphatase activity was more active in anaphase extracts than in pro- or metaphase extracts, which is consistent with the results obtained in vivo. Okadaic acid and protein phosphatase inhibitors 1 and 2 inhibited this specific RB phosphatase activity. These results suggest a role for serine and threonine phosphoprotein phosphatase type 1 in the late mitotic dephosphorylation of RB.

Differential therapeutic responses of thiol compounds in the reversal of methylmercury inhibited acid phosphatase and cathepsin E in the central nervous system of rat.

Though considerable headway has been made in elucidating the effect of methylmercury on the biochemical machinery of nervous system, the studies on the alterations in the levels of acid hydrolases received less attention. Being a lysosomal marker, acid phosphatase is one of the most extensively studies enzymes amongst the acid hydrolases. Its significance in various key physiological as well as pathological processes is well preserved in literature. Cathepsin E, an aspartic proteinase, has been demonstrated in a number of cells and tissues within the human body, rat, E. coli where its role is implicated in a number of important metabolic processes. In the present paper, we report the results of the differential levels of inhibition of these enzymes with methylmercury as well as their differential recoveries with two thiols (N-acetyl-DL-homocysteine thiolactone and glutathione) in various neuroanatomical areas (olfactory bulbs, cerebral hemispheres, cerebellum, medulla oblongata and spinal cord) of rat. 22 refs., 5 figs.

Multiple binding sites for tetrahedral oxyanion inhibitors of bovine spleen purple acid phosphatase

The theory of multiple inhibition kinetics has been extended to enzymes for which one inhibitor is noncompetitive and the other exhibits mixed inhibition. Plots of reciprocal velocity versus the concentration of either inhibitor at various fixed concentrations of the second inhibitor are predicted to give parallel lines if binding of the inhibitors is mutually exclusive and intersecting lines if the inhibitors interact at different sites on the enzyme. Application of this analysis to the purple acid phosphatase from bovine spleen in the presence of molybdate (a noncompetitive inhibitor) and phosphate (which exhibits mixed inhibition) results in parallel lines in the reciprocal velocity plots, indicating that phosphate and molybdate compete for a common site; since molybdate is a noncompetitive inhibitor, this site is inferred to be distinct from the site at which substrate binds and is hydrolyzed. Extension of these ideas suggests that phosphate ester substrates should be capable of binding to the molybdate-binding site as well as to the active site, and evidence for substrate inhibition at high substrate concentrations has been obtained. The implications of these findings for interpretation of previous spectroscopic studies of purple acid phosphatase complexes with tetrahedral oxyanions are discussed.

137. Differentiation control in human breast tumor cells by okadaic acid and related phosphatase inhibitors

137. Differentiation control in human breast tumor cells by okadaic acid and related phosphatase inhibitors

Effect of ultrasound guided core biopsy of prostate on serum concentration of prostate specific antigen and acid phosphatase activity.

Forty-three patients had their serum concentrations of prostate specific antigen and activity of tartrate inhibited acid phosphatase measured before and after digital rectal examination, transrectal ultrasonography and transrectal core biopsy. Transrectal core biopsy significantly increased the values for both tumor markers but rectal examination and ultrasonography without biopsy had no such effect. The measurements returned to normal within one week of biopsy in all but four patients who still had slightly increased concentrations of prostate specific antigen. We recommend that the concentration of prostate specific antigen and activity of tartrate inhibited acid phosphatase are checked before biopsy of the prostate is carried on.

1. G.proteins and signalling mechanisms in osteoblasts

Purple acid phosphatases are metal-containing hydrolases. While their precise biological role(s) is unknown, the mammalian enzyme has been linked in a variety of biological circumstances (e.g., osteoporosis) with increased bone resorption. Inhibition of the human enzyme is a possible strategy for the treatment of bone-resorptive diseases such as osteoporosis. Previously, we determined the crystal structure of pig purple acid phosphatase to 1.55 Å and we showed that it is a good model for the human enzyme. Here, a study of the pH dependence of its kinetic parameters showed that the pig enzyme is most efficient at pH values similar to those encountered in the osteoclast resorptive space. Based on the observation that phosphotyrosine-containing peptides are good substrates for pig purple acid phosphatase, peptides containing a range of phosphotyrosine mimetics were synthesized. Kinetic analysis showed that they act as potent inhibitors of mammalian and plant purple acid phosphatases, with the best inhibitors exhibiting low micromolar inhibition constants at pH 3–5. These compounds are thus the most potent organic inhibitors yet reported for the purple acid phosphatases.

Comparison of assays for prostatic and total acid phosphatase

Total and tartrate inhibited acid phosphatase was determined on the Technicon Chem 1 and evaluated against a Cobas-Bio centrifugal analysis procedure and an immunochemical method. Precision and reference range studies were performed for the Chem 1 acid phosphatase procedure and correlation was established with the other methods. The Chem 1 method for measuring total and prostatic acid phosphatase is a sensitive method with good correlation to the centrifugal analysis and the immunochemical method. The assay is fully automated and requires no manual off-line sample preparation.

Ultrastructural and histochemical study of β‐tricalcium phosphate resorbing cells in periodontium of dogs

beta-TCP was implanted in surgically prepared alveolar bone defects on the mesial side of the upper canine. The dogs that we used were sacrificed after 5 weeks, fixed by perfusion, and the beta-TCP resorbing cells were examined ultrastructurally and histochemically, with the following results: (1) beta-TCP was resorbed by macrophages and multinucleated giant cells. (2) Mitochondria, vacuoles and Golgi apparatus were abundant in beta-TCP-resorbing multinucleated giant cells that possessed neither ruffled borders nor clear zones. (3) The addition of tartric acid inhibited acid phosphatase activity in the cytoplasm of the multinucleated giant cells and macrophages.

Role of protein kinase C and phosphorylation in bombesin-evoked gastrin release from isolated perfused rat stomach

We have recently reported that bombesin (BBS)-stimulated gastrin release is principally dependent on a Ca 2+/calmodulin intracellular pathway, and that it is independent of the cyclic AMP-mediated pathway. Recently it was demonstrated that stimulation of protein kinase C (PK-C) resulted in increased gastrin release from the isolated canine G-cells in cultures. The role of PK-C in the BBS-evoked gastrin release, however, remains unexamined. In this study we examined a possible role of PK-C in the secretion of BBS-stimulated gastrin from isolated perfused rat stomach. The effect of phosphorylation on gastrin release, in response to BBS, was also determined. Administration of phorbol ester (PMA 10–100 nM, a PK-C activator) alone significantly provoked gastrin release, but markedly inhibited the BBS (1 nM) stimulated gastrin secretion in a dose-dependent manner. Molybdic acid (phosphatase inhibitor), caused an enhancement of BBS-evoked gastrin response at doses of 5 or > 5 mM. These results suggest that: (1) diacylglycerol/PK-C pathway may exert a negative feedback control over BBS-induced gastrin release; (2) phosphorylation step is required for gastrin secretion in response to BBS.

A New Zn2+-stimulated Sphingomyelinase in Fetal Bovine Serum

Fetal bovine serum contains a Zn2+-dependent sphingomyelinase with optimal activity at pH 5.5 in vitro. Activity could be demonstrated with a liposomal sphingomyelin substrate suspension but was stimulated up to 15-fold by Triton X-100. Under a variety of conditions tested, phosphatidylcholine, lysophosphatidylcholine, glycerophosphocholine, and p-nitrophenyl phosphate were not substrates for this activity. Several inhibitors of serum alkaline and acid phosphatases had no effect on the activity. The enzyme resembles the acid lysosomal sphingomyelinase in pH optimum and inhibition by AMP but differs in inhibition by EDTA, stimulation by Zn2+, and heat lability at 55 degrees C. It resembles the neutral, Mg2+-stimulated enzyme in inhibition by EDTA and heat lability but differs in metal ion requirement and pH optima. Of the sera tested, activity was highest in fetal bovine serum, with fetal bovine greater than newborn bovine greater than horse greater than human; more than 95% of the activity is in the lipoprotein-free infranatant of serum (d greater than 1.21). This activity appears to be a hitherto undescribed sphingomyelinase. Its biological functions are not known but may subserve a special role in sphingomyelin catabolism in the circulation, in blood vessel walls, or in the tissue(s) of origin.

Histochemical and cytochemical studies of neutral pH p-NPPase on lysosomal membrane in rat liver.

The neutral pH p-nitrophenyl phosphatase activity (p-NPPase) on the lysosomal membrane in rat liver cells was demonstrated histo- and cytochemically. The enzyme activity was observed mainly on the lysosomal membrane surfaces and its matrices by using the original incubation medium consisting of 5 mM Mg salt p-NPP, 50 mM KCl, 2.5 mM levamisole, 25% DMSO and 2 mM lead citrate in 25 mM Tricine-KOH buffer, pH 7.3. When an inhibitor of acid phosphatase (5-10 mM, NaF) was added to the incubation medium, the enzyme activity on lysosomal matrices was inhibited but the activity on membranes was not. Either 4, 4-diisothiocyanatostilbene-2, 2′-disulphonic acid (10 mM DIDS) or N-ethylmaleimide (10 mM NEM) in the incubation medium could inhibit the enzyme activity on the lysosomal membrane but not in the matrices. The enzyme activity of both lysosomal membranes and its matrices was inhibited when the medium contained NaF and DIDS, or NaF and NEM at the same time. Furthermore, if the Mg salt p-NPP was replaced by Na salt p-NPP in the NaF containing standard incubation medium, the enzyme activity was not observed at all on lysosomes. These results suggest that the lysosomal membrane p-NPPase activity at neutral pH may represent H+-ATPase activity.

Phosphorylation status of nuclear and cytosolic androgen receptors in the rat ventral prostate

We demonstrate that endogenous phosphatases are active in cytosolic and nuclear androgen receptor fractions from the rat ventral prostate. Under our androgen binding assay conditions, the effect of acid phosphatase inhibitors (sodium fluoride, tartaric acid, sodium orthovanadate) on the endogenous phosphatases could be correlated with an increase in dihydrotestosterone (DHT) binding to fractions of partially purified cytosolic androgen receptor. In contrast, tetramisole, an alkaline phosphatase inhibitor, did not alter the binding of DHT to the same receptor fraction. Immunoprecipitation of androgen receptor fractions with polyclonal anti-phosphotyrosine antibody resulted in the recovery of [3H]-DHT binding activity from nuclear receptor fractions and partially purified cytosolic receptor fractions prepared from 20- to 24-hr castrated rats. In control fractions depleted of androgen receptor, negligible levels of binding activity were recovered following immunoprecipitation with the antibody. Therefore, acid phosphatases may be acting on phosphotyrosyl residues of the androgen receptor, thus playing a role in the dephosphorylation and inactivation of the androgen receptor.

Aspergillus ficuum phytase: partial primary structure, substrate selectivity, and kinetic characterization.

Purified Aspergillus ficuum phytase's partial primary structure and amino acid and sugar composition were elucidated. Determination of kinetic parameters of the enzyme at different pH values and temperatures indicated no significant alteration of the Km for phytate while the Kcat was affected. The enzyme was able to release more than 51% of the total available Pi from phytate in a 3.0 hr assay at 58 degrees C, but the Kcat dropped to 15% of the initial rate. Substrate selectivity studies revealed phytate to be the preferred substrate. The pH optima of phytase was 5.0, 4.0, and 3.0 for phytate, ATP, and polyphosphate, respectively. The enzyme had varied sensitivity towards cations. While Ca++ and Fe++ produced no effect on the catalytic rate of the enzyme, Cu+, Cu++, Zn++, and Fe were found to be inhibitory. Mn++ was observed to enhance enzyme activity by 33% at 50 microM. Known inhibitors of acid phosphatases e.g. L (+)-tartrate, phosphomycin, and sodium fluoride had no effect on enzyme activity.

Effect of two gold compounds on lysosomes.

The effect of two gold(I) compounds on stability of lysosomes in vitro was studied. Lysosomes from homogenates of rat kidney cortex were isolated by differential centrifugation. These lysosomes were incubated at 37 degrees C with widely varied concentrations of sodium aurothiomalate and sodium aurothiosulphate for 5, 35, and 65 minutes. Acid phosphatase activities were measured and used as an indication of lysosomal membrane stability in the presence and absence of drugs. The enhanced release of acid phosphatase from lysosomes by aurothiomalate and aurothiosulphate was related to dose, but the drugs differed substantially in their potencies. The disruptive effect on lysosomes was more marked for aurothiosulphate than for aurothiomalate. In addition, both drugs inhibited acid phosphatase activities at relatively high gold concentrations. Aurothiomalate had a moderate and aurothiosulphate a weaker inhibitory effect on the enzyme. Our results indicate that aurothiomalate and aurothiosulphate exert their beneficial effect in the treatment of rheumatoid arthritis through mechanism(s) other than lysosomal membrane stabilisation.

Phosphatase activity in corn and soybean roots: Conditions for assay and effects of metals

Studies with sterile root materials showed that the optimum pH values of phosphatase activity in three varieties of each of corn (Zea mays L.) and soybean (Glycine max. L.) were 4 and 5, respectively. The activity on either side of the optimum pH fell sharply, and there was no activity at pH 9. Thus, these roots contain acid but no alkaline phosphatase activity. Acid phosphatase activity was not uniformly distributed in roots and root hairs. Studies with 20 metals showed that their effectiveness in inhibiting acid phosphatase activity of roots varied with the type of plant used. When the metals were compared at 250 μM (1.25 μmole. 5 mg−1 of homogenized roots), the inhibition of acid phosphatase of corn and soybean roots showed that Ag(I), Fe(III), Se(IV), V(IV), As(V) and Mo(VI) were the most effective inhibitors of this enzyme in corn roots, with percentage inhibition ≥30%. In addition to these metals, Sn(II), Hg(II), and W(VI) inhibited acid phosphatase in soybean roots by >30%. Other metals and one non-metallic element that inhibited acid phosphatase activity in corn and soybean roots were: Cu(I), Cu(II), Cd(II), Ni(II), Fe(II), Pb(II), Ba(II), Co(II), Mn(II), Zn(II), B(III), As(III), Cr(III), and Al(III); their degrees of effectiveness varied with type of roots used. Generally, the inhibitory effect of the metals was much less when their concentration was decreased by 10-fold. In addition to the effect of these elements, phosphate ion inhibited acid phosphatase activity of corn and soybean roots. Related anions such as NO 2 − , NO 3 − , Cl−, and SO 4 2− were not inhibitory.

Androgen metabolism and regulation of rat ventral prostate growth and acid phosphatase during sexual maturation.

Androgen metabolism and the regulation of rat ventral prostate cell proliferation and secretory function were examined during sexual maturation. Changes in acid phosphatase (AP) characteristics were measured as a marker of androgen-dependent prostatic secretory function. In immature (21-day-old) rats, total AP activity per cell was low (14.2 +/- 1.3 mol p-nitrophenol phosphate hydrolysed/h per mg DNA); it increased threefold as the weight, protein and DNA contents of the prostate increased to adult (65-day) levels. This corresponded with significant (P less than 0.001) increases in the staining intensities of three of the four bands of secretory AP on isoelectric focusing gels. The extent of inhibition of AP by tartrate decreased at the same time. Secretory AP is known to be relatively tartrate-resistant. The changes in AP activity occurred after prostatic 5 alpha-dihydrotestosterone (5 alpha-DHT) levels increased from 4.6 +/- 0.7 pmol/mg DNA (21 days) to reach a peak of 17.6 +/- 2.3 pmol/mg DNA at 58 days. Prostatic 5 alpha-DHT concentrations were always higher than testosterone levels. Prostatic 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-Adiol) levels were lower than 5 alpha-DHT levels except on day 58 when levels peaked dramatically at 26.2 +/- 5.5 pmol/mg DNA. Changes in prostatic 5 alpha-DHT and 3 alpha-Adiol levels corresponded with changes in 5 alpha-reductase and 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) activities. The oxidative reaction of 3 alpha-HSOR was approximately fourfold higher than the reductive reaction, indicating a preference for the formation of 5 alpha-DHT. The plasma levels of testosterone, 5 alpha-DHT and 3 alpha-Adiol cannot account for their respective prostatic levels, indicating the importance of the steroid-metabolizing enzymes in regulating intracellular androgen levels. Changes in the AP characteristics could be correlated with the androgen status of the prostate.

Mapping Three Protein Loci on a Soybean Chromosome1

Recombination frequencies between pairs of three protein loci on Linkage Group 9 in soybean [Glycine max (L.) Merr.] were determined so that they could be mapped. ‘Earlyana’, ‘Ebony’, ‘Hakucho’, ‘Jefferson’, ‘Kingwa’, and ‘Wilson’ were intercrossed and the F2 segregation frequencies of the markers were used to calculate map distance among the acid phosphatase (Ap), leucine aminopeptidase (Lapl), and trypsin inhibitor (Ti) loci. The recombination frequency between Ap and Lapl was estimated as 23.6 ± 1.0%. The recombination frequency between Ap and Ti was estimated at 6.6 ± 0.5%, which is significantly different from 16.2 ± 1.5% reported previously. The map distance between the Lapl and Ti loci was calculated to be 16.3 ± 1.0 map units. The three loci in Linkage Group 9 were mapped on the basis of the recombination values obtained in this study. The order of the three loci is Ap‐Ti‐Lapl.

Leishmania donovani: Surface membrane acid phosphatase blocks neutrophil oxidative metabolite production

We show that a purified preparation of the prominent tartrate-resistant acid phosphatase (E.C.3.1.3.2), isolated from the external surface of the intracellular parasite Leishmania donovani (promastigote form), inhibits toxic oxidative metabolite production of neutrophils. Preincubation of a neutrophil suspension (2.5 × 10 6 cells/ml) for 15 min at 37 C with 250 units (1 unit equals 1 nmole of 4-methylumbelliferyl phosphate cleaved per hr at pH 5.5) of the acid phosphatase in Krebs-Ringer phosphate buffer (pH 7.4) decreased O 2 consumption, O 2 − production, and H 2O 2 production of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils to 15–25% of control values. The acid phosphatase also affected concanavalin A-stimulated O 2-production by neutrophils, but had no effect on the rate of phorbol myristic acetate-stimulated O 2 − production, chemotactic peptide binding, degranulation, or membrane depolarization. Addition of an acid phosphatase inhibitor (Complex E; (NH 4) 6[P 2Mo 18O 62]·9H 2O) to suspensions of opsonized promastigotes and neutrophils resulted in a threefold or greater enhancement of O 2 − production. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.

Cytochemical localization of acid phosphatase in the rabbit ovarian follicle.

The different cell layers in the follicle wall in rabbit ovaries were examined for acid phosphatase activity during the ovulatory process. The tissue was taken before the process began and at 5, 7, 9, 10 and 11hr after it was induced by human chorionic gonadotropin. The ovaries were perfused with a glutaraldehyde fixative, cut with a microslicer, incubated for 60min at 37°C in Gomori's acid phosphatase medium, and then routinely prepared for light and electron microscopy. Control tissue, which was incubated with the acid phosphatase inhibitor, sodium fluoride, did not reveal any enzyme activity. The results show acid phosphatase reaction product in all the major cell types in the follicle wall. The enzyme is located mainly in lysosomes and Golgi apparatus. It is not present in the electron-dense granules which are characteristic of the cytoplasm of surface epithelial cells. The acid phosphatase in the thecal layers of the follicle appears to be localized, not only in lysosomes and Golgi apparatus, but also in the multivesicular structures which are present in the thecal fibroblasts of this tissue. Collectively, the data indicate that acid phosphatase and other lysosomal enzymes may contribute, at least in part, to the hydrolytic degradation of follicular connective tissue during ovulation.

The [formula omitted] phosphatase activity of ubiquitin from bovine erythrocytes

1.1. Ubiquitin, a unique protein with esterase and carbonic anhydrase activity, has been found to have also a p-nitrophenyl phosphatase activity.2.2. This phosphomonoesterase activity of ubiquitin has an acidic pH optimum; its true substrate appears to be the phosphomonoanion, with a Km of 1.8 × 10−3M.3.3. It is competitively inhibited by the typical acid phosphatase inhibitors, arsenate (Ki = 1.3 × 10−3M), molybdate (Ki = 1.2 × 10−6M), and phosphate (Ki = 1.4 × 10−3M).4.4. These inhibitors have no effect on the CO2 hydration and p-nitrophenyl acetate esterase activities of the ubiquitin.5.5. Acetazolamide slightly inhibited the p-nitrophenyl phosphatase activity.