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PubMed1994

Human liver plasma membranes contain an enzyme activity that removes membrane anchor from alkaline phosphatase and converts it to a plasma-like form.

Treatment of liver plasma membranes with Triton X-100 allowed an endogenous alkaline phosphatase-converting activity to convert amphiphilic alkaline phosphatase (membrane anchor covalently attached) to hydrophilic dimers that resemble the enzyme found in normal plasma. The Triton-solubilized activity was unaffected by protease inhibitors. Amphiphilic alkaline phosphatase purified from human liver and placenta were both substrates. The Triton-solubilized enzyme would not hydrolyze L-3-phosphatidyl(2-3H)-inositol or p-nitrophenylphosphoryl choline, nor would it cleave endogenous alkaline phosphatase from intact plasma membranes. These observations and the analysis of the protein product of the hydrolysis of placental alkaline phosphatase, following treatment with the converting activity, indicated that the enzyme has the specificity of a glycosyl-phosphatidylinositol phospholipase D. Further characterization of the enzyme activity suggests additional similarities with the glycosyl-phosphatidylinositol phospholipase D found in mammalian plasma. Alkaline phosphatase-converting activity in plasma membranes represented the same percent of total protein as it did in whole liver, whereas serum contained 3- to 10-times this amount. Endogenous converting activity in plasma membranes was not solubilized by salt washes, sonication, or repeated freeze-thaw treatments. We believe it is unlikely that the alkaline phosphatase-converting activity in liver plasma membranes resulted from adsorption of the enzyme present in plasma.

Alkaline Phosphatase↗
PubMed1990

A difference in the regulation of mRNA expression between the phenotypic and the embryonic alkaline phosphatase genes in human cancer cells.

The steady-state levels of mRNAs encoding alkaline phosphatase isoenzymes were examined in two human breast carcinoma cell lines. MDA-MB-157 cells expressed the phenotypic breast alkaline phosphatase and BT20 cells expressed the nonphenotypic placental alkaline phosphatase isoenzyme, frequently reexpressed in neoplasms. Dexamethasone (DEX), which elicits a general effect on phosphatase expression, and 1,25-dihydroxy vitamin D3 (1,25(OH)2D3), a promoter of cell differentiation that correspondingly effects embryonic phosphatase expression, were chosen as perturbing agents for these experiments. RNA blot analysis showed a single RNA species of approximately 2.6 kb under all treatment conditions in BT20 cells and a single RNA species of 2.6 kb under each condition in MDA-MB-157 cells. The results showed that the expression of both the AP isoenzyme mRNA phenotypic of breast produced by MDA-MB-157 cells and the embryonic alkaline phosphatase isoenzyme (PLAP) mRNA produced by BT20 cells was increased by treatment with DEX. By comparison 1,25(OH)2D3 caused an increase in the tissue-unspecific AP mRNA in the MDA-MB-157 cells, but caused a decrease in PLAP mRNA levels in BT20 cells. The level of each isoenzyme mRNA species is altered by either hormone in a dose- and time-dependent manner in both cell lines. In BT20 cells, treatment with cycloheximide showed that ongoing protein synthesis is not required to potentiate the PLAP mRNA response to DEX, but is required for the action of 1,25(OH)2D3. However, protein synthesis is required for the action of both hormones in the MDA-MB-157 cells which make the breast phenotypic AP. These data demonstrate that the DEX- and 1,25(OH)2D3-regulated expression of both of these alkaline phosphatase isoenzymes occurs via a complex mechanism involving control of mRNA abundance, not translational control of constant message levels.

Alkaline Phosphatase↗
PubMed1988

Intestinal alkaline phosphatase in patients with chronic renal failure.

Because of the suggestion that intestinal alkaline phosphatase was elevated in the serum of patients with chronic renal failure, we studied the serum of 42 patients undergoing hemodialysis with elevated enzyme activity. Using a sensitive and specific electroimmunoassay for the intestinal isoenzyme, 26 of 42 serum samples were positive, compared with 3 of 25 samples obtained from hospitalized patients with elevated phosphatase activity. The fractional amount of this isoenzyme was also higher, ranging from 1.5% to 41% of the total serum phosphatase, compared with 0.1%-1.2% in control sera. Kidneys removed during transplantation or postmortem contained a membranous phosphatase with immunologic activity identical to the intestinal isoenzyme in 5 of 6 patients. This enzyme accounted for 8%-21% of the total kidney phosphatase activity. By morphology the immunoreaction was localized to the apical membranes of the collecting tubules. Thus, the kidney is the likely source of the observed increase in serum intestinal-type phosphatase activity noted in patients with chronic renal failure. An elevation in the intestinal isoenzyme rather than the presence of early metabolic bone disease or hepatic disease should be considered in renal failure patients with mildly elevated (up to 50% over normal) total serum alkaline phosphatase.

Alkaline Phosphatase↗
PubMed1989

Decrease of acid phosphatase activity in murine peritoneal macrophages infected with Leishmania donovani.

The effect of infection with Leishmania donovani on the activity and isoenzyme composition of acid phosphatase within individual murine peritoneal macrophages maintained in vitro was studied. Concentrations of acid phosphatase activity and number of intracellular parasites were quantitated using a computer-assisted cytospectrophotometry system. Changes in the isoenzyme composition of macrophages during infection with L. donovani were detected by comparing the patterns of acid phosphatase levels between macrophages treated in the absence and presence of an enzyme inhibitor. It was observed that the concentration levels of acid phosphatase activity in macrophages were decreased significantly by infection with L. donovani. An inverse relation existed between concentration of acid phosphatase activity and the number of intracellular L. donovani. Reduced concentrations of acid phosphatase activity were also observed in macrophages uninfected but exposed to L. donovani. The isoenzyme composition in macrophages did not change during the course of infection with L. donovani. These results demonstrate that L. donovani reduces the acid phosphatase activity of macrophages.

Acid Phosphatase↗
PubMed1990

Change in the isoenzyme composition of acid phosphatase in murine peritoneal macrophages exposed to or infected with Trypanosoma cruzi.

The effect of infection with Trypanosoma cruzi on the activity and isoenzyme composition of acid phosphatase within individual murine peritoneal macrophages maintained in vitro was studied. Concentrations of acid phosphatase activity and number of intracellular parasites were quantitated by using a computer-assisted cytospectrophotometry system. Changes in the isoenzyme composition of macrophages during infection with T. cruzi were detected by comparing the patterns of acid phosphatase levels between macrophages treated in the absence and presence of an enzyme inhibitor. It was observed that the concentration levels of acid phosphatase activity in macrophages did not change significantly by infection with T. cruzi. Also, the concentration levels of acid phosphatase activity did not change in macrophages uninfected but exposed to T. cruzi. On the other hand, the isoenzyme composition of acid phosphatase did change in macrophages exposed to or infected with T. cruzi. These results demonstrate that Trypanosoma cruzi affects the acid phosphatase composition of macrophages.

Acid Phosphatase↗
PubMed1984

Cellular enzyme-linked immunospecific assay (CELISA). IV. Inhibition of endogenous cellular alkaline phosphatase activity.

The cellular enzyme-linked immunospecific assay (CELISA) uses ligands conjugated to calf intestinal alkaline phosphatase to quantitate antibody bound to antigens on the surfaces of whole cells. Since the endogenous alkaline phosphatase present in many cell types contributes unwanted background noise to the assay, ways of inhibiting endogenous cellular alkaline phosphatase were investigated. We found that the endogenous alkaline phosphatase in human peripheral blood mononuclear cells was incompletely inhibited by EDTA or L-cysteine. Levamisole, however, inhibited endogenous cellular alkaline phosphatase completely without impairing the sensitivity of the CELISA. The use of levamisole is recommended for assays that use alkaline phosphatase conjugates to detect molecules on the surfaces of cells that also contain endogenous alkaline phosphatase.

Alkaline Phosphatase↗
PubMed1985

Characterization of elevated plasma alkaline phosphatase activity in genetically obese mice.

Alkaline phosphatase activity in obese mice (C57BL/6J ob/ob) was significantly increased from 18 to 63 weeks of age when compared to that of their lean controls (C57BL/6J +/?). In 5 week old animals, the earliest age examined, the circulating activity of alkaline phosphatase was similar in both obese mice and their lean counterparts. To characterize the circulating alkaline phosphatase activity in the obese mouse and its lean counterpart, the response of the enzyme to fasting, various inhibitors, heat inactivation, and urea denaturation was examined and compared. L-homoarginine and L-p-bromotetramisole inhibited to a large extent the circulating activity of alkaline phosphatase in both obese mice and their lean controls in the fed state, while L-phenylalanine had essentially no effect. Even though the response of alkaline phosphatase in plasma to several inhibitors was similar, the rate of denaturation by urea of enzyme activity in plasma was significantly slower in obese mice than in their lean controls in the fed state. While the rate of inactivation of alkaline phosphatase activity in plasma for the initial two minutes at 56 degrees C was similar in obese mice and their lean counterparts, the subsequent rate of heat inactivation was significantly slower in the plasma from obese mice. Thus, both obese and lean mice in the fed state have a circulating activity of alkaline phosphatase in plasma with a greater contribution from a skeletal isoenzyme and a lesser one of intestinal origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1982

The in vitro synthesis and secretion of alkaline phosphatase from first trimester human decidua.

We studied the in vitro synthesis and secretion of alkaline phosphatase by human first trimester decidual tissue incubated in organ culture. Decidua synthesizes two different alkaline phosphatase isoenzymes; heat stable and heat labile. decidual tissue maintains a constant pool of alkaline phosphatases. The synthesis of alkaline phosphatase may be the driving force for its secretion. The de novo synthesis and secretion rate of alkaline phosphatases were also investigated. The higher specific radioactivities of the secreted enzymes than those found in the tissue may suggest that newly synthesized alkaline phosphatase is preferentially released. The intracellular distribution of the alkaline phosphatase isoenzymes was compared as well. The characteristics of the two isoenzymes are different in human decidua of maternal origin from those previously reported in the human placenta originating from embryonic stem cells.

Alkaline Phosphatase↗
PubMed1996

Protein phosphotyrosine phosphatases in Ascaris suum muscle.

Two forms of protein tyrosine phosphatases were partially purified from the musculo-cutaneous layer of Ascaris suum. A 50-55-kDa soluble form of the phosphatase cross-reacted with antisera raised against human PTP-1B and TC-PTP. Like the enzyme of human origin the phosphatase from Ascaris exhibited a preference for anionic substrates (tyrosine-phosphorylated carboxymethylated and maleylated lysozyme) and was inhibited by micromolar concentrations of vanadate, molybdate, Zn2+, heparin, and poly(Glu4Tyr). As revealed by immuno-cytochemistry, the phosphatase was mainly localized and appeared equally distributed in the cytoplasm, apart from the myofibrils, possibly in loose association with cytoskeletal elements. A second tyrosine phosphatase of 180 kDa molecular mass was mainly found in detergent extracts from a microsomal fraction. It showed no cross-reactivity with antisera raised against soluble mammalian phosphatases and dephosphorylated a basic substrate (Tyr-phosphorylated myelin basic protein). It was resistant to common inhibitors of mammalian tyrosine phosphatases except Zn2+ and thiol reagents.

Animals↗
PubMed1986

Regulation of alkaline phosphatase activity in rat hepatoma cells. Effects of serum proteins, cycloheximide, actinomycin D, chloroquine, dinitrophenol and potassium cyanide.

Alkaline phosphatase activity in rat hepatoma cells (R-Y121B) cultured in a monolayer at 0.5% serum was enhanced by serum, bovine serum albumin, casein and gamma-globulin, but ovalbumin, polyvinylpyrrolidone, dexamethasone, insulin and dibutyrylcyclic AMP showed little effect on alkaline phosphatase activity. In addition, cycloheximide, actinomycin D, chloroquine, dinitrophenol and potassium cyanide also increased the enzyme activity, although the incorporation of [14C]leucine into cellular proteins was almost completely inhibited in the presence of these cytotoxic substances. When R-Y121B cell homogenates were incubated at 37 degrees C, alkaline phosphatase activity increased in a pH-dependent manner: the maximal increase was observed at pH 7.1. The magnitudes of the increase differed among cell homogenates and a 4- to 10-fold increase was observed. Alkaline phosphatase in R-Y121B cells was apparently heat-stable, but that in the cells obtained from various treatments was heat labile and the latter activity decreased to less than 50% of the initial activity after 15 min of incubation at 56 degrees C. Alkaline phosphatase in the control and also in the treated cells was more sensitive to L-homoarginine than L-phenylalanine. The Lineweaver-Burk plot showed that the increases in the enzyme activity were accompanied by changes not only in V but also in Km for alkaline phosphatase reaction. Finally, it has been suggested that the increases in alkaline phosphatase activity under various conditions are due to the conversion of the molecule with a low enzyme activity to the molecule with a high enzyme activity in R-Y121B cells.

Alkaline Phosphatase↗
PubMed1975

Correlation between erythromycin and acid phosphatase in mouse liver.

1. Whole liver homogenates obtained from mice 1h after an intraperitoneal injection of erythromycin lactobionate (343 mg/kg, 1/3 LD 50) were fractionated into nuclear (7000 times g min), mitochondrial (33000 times g min) and lysosomr-rich (250 000 times g min) fractions. 2. The resulting fractions, as well as the final supernatant, were analyzed for erythromycin, acid phosphatase and protein. 3. The highest relative specific activities (per cent total protein) of erythromycin and of acid phosphatase were exhibited by the lysosome-rich fraction. 4. It was of interest, therefore, to examine the effects of erythromycin upon the free activity of acid phosphatase in soluble form and on its in vitro and in vivo release from liver lysosomes. 5. Concentrations of 1.7 - 10-4 M, 3.4 - 10-4 M, 6.8 - 10-4 M and 13. 6 - 10-4 M of erythromycin lactobionate had no significant effect upon the free activity of acid phosphatase in soluble form but retarded the release of this enzyme from the liver lysosome-rich preparation. This effect of erythromycin lactobionate was dose- and time-dependent. 6. Treatment of mice with erythromycin lactobionate (343 mg/kg, 1/3 LD 50) iwtraperitoneally for 7 days significantly decreased the unsedimentable acid phosphatase activity expressed as per cent of total activity in whole liver homogenates. This indicated an in vivo diminished release of acid phosphatase from liver lysosomes by erythromycin. 7. Since erythromycin lactobionate is ionisable it could be possible that erythromycin basis as many other cationic molecules accumulates in lysosomes. 8. The in vitro and in vivo diminished release of acid phosphatase may suggest that erythromycin decreases permeability of lysosomal membrane.

Acid Phosphatase↗
PubMed1986

NADP phosphatase as a marker in free-flow electrophoretic separations for cisternae of the Golgi apparatus midregion.

Based on cytochemical analysis, the enzyme NADP phosphatase is most concentrated in the so-called intercalary cisternae from the mid-region of the Golgi apparatus stack. Using free-flow electrophoresis to separate different Golgi regions of rat liver Golgi apparatus, the NADP phosphatase activity, based on estimation of the rate of release of inorganic phosphate from NADP under standard conditions, was similarly localized to membrane fractions from the center of electrophoretic separations. Peak specific activities for both a putative cis marker (NADH-cytochrome c reductase) and an established trans marker (galactosyltransferase) coincided with minima in NADP phosphatase activity, in agreement with the cytochemical observations. The pattern of distribution of enzyme activity for NADP phosphatase differed from that of both acid phosphatase and glucose-6-phosphatase. The pH optimum was 5.0, the Km for NADP was 0.6 mM and a corresponding production of NAD and inorganic phosphorus was shown. Taken together with other markers for free-flow electrophoresis separation, the NADP phosphatase will provide considerable utility as a specific marker to help identify intercalary cisternae of the mammalian Golgi apparatus and to monitor electrophoretic separations.

Animals↗
PubMed1984

The origin of plasma alkaline phosphatase activity in mice and rats.

Swiss albino mice displayed the highest activity of alkaline phosphatase at 4-6 weeks with a precipitous decline by 18 weeks of age to a value seen in the mature animal. Circulating activity of alkaline phosphatase was significantly higher in the rat than the mouse in the fed state. With fasting, enzyme activity declined in the rat yet increased in the mouse. The net result was significantly higher plasma alkaline phosphatase activity in the mouse than the rat after the 48 hr fast. L-Phenylalanine inhibition of plasma alkaline phosphatase was greater in plasma from the rat than the mouse in the fed state. Yet in the fed condition, L-homoarginine and L-p-bromotetramisole inhibited alkaline phosphatase activity in plasma from mice to a greater extent than in rats. Heat inactivation as well as urea denaturation of alkaline phosphatase was significantly faster with plasma of the mouse than the rat in the fed state. Thus, it appears that the alkaline phosphatase isoenzyme of skeletal origin contributes a greater proportion of the circulating activity in the fed Swiss albino mouse than occurs in the Sprague-Dawley rat in which the intestinal isoenzyme plays a greater role in the fed condition.

Aging↗
PubMed1990

Difference in sensitivity to alkaline phosphatase treatment between rat reticulocyte membranes in which beta-adrenoceptor desensitization was induced by isoproterenol, dibutyryl cAMP and phorbol ester.

The effect of alkaline phosphatase (3.1.3.1) on desensitization of beta-adrenoceptor-responsive adenylate cyclase and the role of phosphorylation in desensitization were examined. Treatment of rat reticulocytes with isoproterenol, dibutyryl cAMP and tetradecanoyl phorbol acetate (TPA) caused the desensitization of beta-adrenoceptor-coupled adenylate cyclase. When the membranes from dibutyryl cAMP- and TPA-desensitized cells were incubated with alkaline phosphatase for 60 min at 30 degrees C, pH 8.0, the desensitization of isoproterenol-stimulated adenylate cyclase was markedly attenuated in both preparations. When the membranes from isoproterenol-desensitized cells were treated with alkaline phosphatase under the same conditions, the attenuation of the desensitization of alkaline phosphatase was less than in the case of treatment with dibutyryl cAMP or TPA. In other words, isoproterenol-induced desensitization was more resistant to alkaline phosphatase treatment. Isoproterenol- and dibutyryl cAMP-induced desensitization of NaF-stimulated adenylate cyclase were also attenuated by alkaline phosphatase treatment. Although the stability of the Gs-catalytic unit complex of adenylate cyclase was reduced by isoproterenol treatment, the reduction of stability was also decreased by alkaline phosphatase treatment.

Adenylyl Cyclases↗
PubMed2004

A nonhydrolyzable analogue of phosphotyrosine, and related aryloxymethano- and aryloxyethano-phosphonic acids as motifs for inhibition of phosphatases.

Nonhydrolyzable analogues of both stereoisomers of phosphotyrosine, and a series of related aryloxy (or thio) methyl and aryloxy (or thio) ethyl phosphonic acids of the general formula RX-(CH(2))(n)-PO(3)H(2) (where X=O or S and n=1 or 2), have been tested as nonhydrolyzable mimetics of phosphatase substrates. These compounds were tested against a panel of phosphatases (two alkaline phosphatases, a protein-tyrosine phosphatase, and two serine/threonine phosphatases) with different active site motifs. The compounds exhibit competitive inhibition toward all enzymes tested, with the best inhibition expressed toward the Ser/Thr phosphatases. The stereoisomers of the phosphotyrosine analogues exhibited an unexpected difference in their inhibitory properties toward the protein-tyrosine phosphatase from Yersinia. The K(i) for the d isomer is 33-fold lower than that of the l isomer, and is more than an order of magnitude lower than the reported K(m) of the substrate l-phosphotyrosine.

Organophosphonates↗
PubMed2006

Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma.

The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5-7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K(m)=2.1x10(-3)M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase. The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis.

Acid Phosphatase↗
PubMed2002

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies.

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degrees C. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degrees C. Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme. An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells.

Alkaline Phosphatase↗
PubMed1991

An acid phosphatase as a biochemical marker for intestinal development in the nematode Caenorhabditis elegans.

We describe an acid phosphatase enzyme (EC 3.1.3.2) that is localized to the intestine of the nematode Caenorhabditis elegans and that should serve as a convenient biochemical marker for gut differentiation. In adult worms, acid phosphatase activity is located along the edge of the gut lumen in the vicinity of the intestinal brush border. All but the anterior six cells of the intestine stain for phosphatase activity; the nonstaining cells all descend from the Ea(l/r)(a/p)a cells. Acid phosphatase activity is low in oocytes and early embryos but increases substantially when embryos reach late morphogenesis stage; this increase corresponds to the appearance of a major band of acid phosphatase activity detectable on isoelectric focusing gels. We designate this band as the product of the pho-1 gene. The pattern of acid phosphatase expression in several embryonic mutants suggests that pho-1 expression in the developing intestine is lineage autonomous. We induced an isoelectric focusing variant in the pho-1 enzyme and used this to map the pho-1 locus about 1.5 map units to the left of center of chromosome II. We purified the pho-1 enzyme to homogeneity (6500-fold purification; 4% recovery of activity); the pho-1 acid phosphatase is a homodimeric glycoprotein with a subunit molecular weight of 55,000 Da. This paper establishes a new experimental system with which to investigate the molecular basis of lineage-specific gene expression during C. elegans development.

Acid Phosphatase↗