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PubMed2000

Functional characterization of osteoblasts and osteoclasts from alkaline phosphatase knockout mice.

Tissue nonspecific alkaline phosphatase (TNAP) knockout (ko) mice manifest defects in bone mineralization that mimic the phenotypic abnormalities of infantile hypophosphatasia. In this article, we have searched for phenotypic differences between calvarial osteoblasts and osteoclasts in wild-type (wt), heterozygous and homozygous TNAP null mice. In vitro release of 45Ca from calvarial bones, with and without stimulation with parathyroid hormone (PTH), revealed no functional difference between osteoclasts from the three TNAP genotypes. Studies of primary cultures of TNAP+/+, TNAP+/-, and TNAP-/- calvarial osteoblasts revealed no differences in the rate of protein synthesis or in the expression levels of messenger RNAs (mRNAs) for osteopontin (OP), osteocalcin (OC), collagen type I, core binding factor alpha1 (Cbfa 1), N-cadherin, Smad 5, and Smad 7. Release of interleukin-6 (IL-6) from calvarial osteoblasts under basal conditions and after stimulation with PTH, tumor necrosis factor alpha (TNF-alpha) or IL-1beta was similar in all genotypes. The amount of cyclic adenosine monophosphate (cAMP) accumulation also was comparable. However, although cultures of primary TNAP-/- osteoblasts were able to form cellular nodules as well as TNAP positive osteoblasts do, they lacked the ability to mineralize these nodules in vitro. Mineralization also was delayed in TNAP+/- osteoblast cultures compared with cultures of wt osteoblasts. Incubation with media supplemented with recombinant TNAP, but not with enzymatically inactive TNAP, restored mineralization in ko osteoblast cultures. Our data provide evidence that osteoblasts in TNAP null mice differentiate normally but are unable to initiate mineralization in vitro. The fact that even heterozygous osteoblasts show delayed mineralization provides a rationale for the presence of bone disease in carriers of hypophosphatasia.

Alkaline Phosphatase↗
PubMed1984

Purification and topographical location of tegumental alkaline phosphatase from adult Schistosoma mansoni.

Alkaline phosphatase, a marker for tegumental membranes of Schistosoma mansoni, was extracted using Triton X-100 from membranes purified by sucrose density gradient centrifugation. The enzyme activity was purified 6 800-fold over parasite homogenates and 118-fold over the tegumental membranes released when parasites were incubated in phosphate-buffered saline. Purification of the solubilised enzyme was achieved by binding to a Con A agarose affinity column, gel filtration of the eluted glycoproteins, and Blue affigel chromatography. The purified enzyme was shown to consist of a single glycosylated polypeptide Mr 65 000 on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Enzyme activity was associated with a possible tetramer, Mr 260 000 on gel filtration. Activity was associated with a band Mr 130 000 in sodium dodecyl sulphate-polyacrylamide gel electrophoresis run in the absence of reducing agents. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the absence of reducing agents, the size of the enzyme was shown to be similar in cercariae, schistosomula, adult schistosomes and their eggs, but it was smaller than the activity (Mr 145 000) extracted from host liver and intestinal microsomal membranes. The topography of the enzyme in the schistosome tegument was investigated by using surface radio-labelling reagents followed by its purification. The enzyme could be radio-iodinated only with difficulty in adult worms. Bolton and Hunter reagent, used at high concentration and for prolonged time periods, resulted in labelling of enzyme activity and the Mr 65 000 polypeptide subunit was also iodinated under these extreme conditions. It was concluded that the enzyme is not exposed at the schistome's surface, and is probably buried in the tegumental membrane network.

Alkaline Phosphatase↗
PubMed1997

Mammalian alkaline phosphatases are allosteric enzymes.

Mammalian alkaline phosphatases (APs) are zinc-containing metalloenzymes encoded by a multigene family and functional as dimeric molecules. Using human placental AP (PLAP) as a paradigm, we have investigated whether the monomers in a given PLAP dimer are subject to cooperativity during catalysis following an allosteric model or act via a half-of-sites model, in which at any time only one single monomer is operative. Wild type and mutant PLAP homodimers and heterodimers were produced by stably transfecting Chinese hamster ovary cells with mutagenized PLAP cDNAs followed by enzyme extraction, purification, and characterization. [Gly429]PLAP manifested negative cooperativity when partially metalated as a consequence of the reduced affinity of the incompletely metalated AP monomers for the substrate. Upon full metalation with Zn2+, however, the negative cooperativity disappeared. To distinguish between an allosteric and a half-of-sites model, a [Gly429]PLAP-[Ser84]PLAP heterodimer was produced by combining monomers displaying high and low sensitivity to the uncompetitive inhibitor L-Leu as well as a [Gly429]PLAP-[Ala92]PLAP heterodimer combining a catalytically active and inactive monomer, respectively. The L-Leu inhibition profile of the [Gly429]PLAP-[Ser84]PLAP heterodimer was intermediate to that for each homodimer as predicted by the allosteric model. Likewise, the [Gly429]PLAP-[Ala92]PLAP heterodimer was catalytically active, confirming that AP monomers act independently of each other. Although heterodimers are structurally asymmetrical, they migrate in starch gels with a smaller than expected weighted electrophoretic mobility, are more stable to heat denaturation than expected, and are more sensitive to L-Leu inhibition than predicted by a strict noncooperative model. We conclude that fully metalated mammalian APs are noncooperative allosteric enzymes but that the stability and catalytic properties of each monomer are controlled by the conformation of the second AP subunit.

Alkaline Phosphatase↗
PubMed2007

IL-1beta induces alkaline phosphatase in human phagocytes.

BACKGROUND: Alkaline phosphatase (ALPase) is found in blood plasma or serum and leukocytes and regulates intercellular processes, maintaining phosphoryl metabolites in a steady state, as well as synthesizing and hydrolyzing phosphate esters on membranes. ALPase supervises the active transport of inorganic phosphates, fats, proteins, carbohydrates and the sodium/potassium pump mechanisms. The formed elements of blood such as polymorphonuclear (PMNs) leucocytes, macrophages (MP) and some lymphocytes are high in ALPase concentrations. METHODS: In this study we have tested whether the interleukin-1 receptor antagonist (IL-lra) could influence ALPase generation in IL-1beta or lipopolysaccharide (LPS)-stimulated neutrophils and MP. Human neutrophils were isolated from heparin-anticoagulated blood drawn from healthy individuals by centrifugation in a two-step gradient, Ficoll-Hypaque. ALPase activity was assessed spectrophotometrically in test tubes containing isolated neutrophils and adherence PBMCs treated with LPS, IL-1beta and IL-1ra, alone or in combination. RESULTS: IL-lbeta or LPS enhanced ALPase in both PMNs and MP, whereas IL-1ra could not inhibit ALPase activity. We performed time course experiments at 0 min, 5 min, 1 h, 24 h, and 43 h (LPS 20 microg/mL, IL-1beta 10 ng/mL). No significant increase in ALPase activity was seen until 1 h; however, there was a rapid rise over the next few hours. In another set of experiments using IL-1ra (500 ng/mL), there was no difference between treated cells and control cells. The combination of IL-1beta plus IL-1ra did not reduce the ability of IL-1beta to induce ALPase activity. CONCLUSIONS: These data suggest that IL-1beta stimulates ALPase through other mechanisms than the release of arachidonic acid products, which are inhibited by IL-lra.

Alkaline Phosphatase↗
PubMed1994

17 beta-estradiol and parathyroid hormone potentiate each other's stimulatory effects on alkaline phosphatase activity in SaOS-2 cells in a differentiation-dependent manner.

We studied the effects of 17 beta-estradiol (E2) and PTH on alkaline phosphatase activity in SaOS-2 cells that had been passaged and grown continually in the presence (SaOS + Dex) or absence (SaOS - Dex) of 10(-8) M dexamethasone (Dex). We showed that the more differentiated SaOS + Dex cells had higher alkaline phosphatase activity and PTH-responsive adenylate cyclase than the less differentiated SaOS - Dex cells. In SaOS - Dex cells, E2 or PTH (1 x 10(-11)-1 x 10(-6) M) had no effect on alkaline phosphatase activity. On the other hand, in SaOS + Dex cells, PTH and E2 each had small but very significant stimulatory effects on alkaline phosphatase activity. The combined effects of PTH and E2 resulted in potentiation which was dose dependent for PTH and E2. 17 alpha-estradiol and tamoxifen (1 x 10(-11)-1 x 10(-6) M) had no effect on PTH-stimulated alkaline phosphatase activity in SaOS + Dex cells, but the latter inhibited the E2 effect, clearly demonstrating its specificity. In conclusion, we have shown that in more differentiated osteoblastic cells, E2 and PTH have interactive effects on alkaline phosphatase activity.

Adenylyl Cyclases↗
PubMed1997

The bone isoenzyme of alkaline phosphatase in hypercalcaemic cancer patients.

Alkaline phosphatase (AP) is the classic marker of bone formation, especially in cancer patients, but the interpretation of its measurement is complicated by the existence of various circulating isoenzymes, especially of liver origin. The introduction of a mass measurement of the bone isoenzyme of AP (BAP) by an immunoradiometric assay has markedly improved the sensitivity and the specificity of the determination. We measured BAP and other markers of bone turnover in 46 patients with tumour-induced hypercalcaemia (TIH), which is an interesting model for evaluating markers of bone formation because of the uncoupling between bone formation and bone resorption found by histomorphometric techniques. The extent of bone metastatic involvement was evaluated by planimetry on bone scintigraphy. Mean (+/- S.D.) BAP concentrations were slightly higher in patients with TIH than in healthy subjects, 15.5 +/- 8.5 versus 12.4 +/- 3.5 micrograms/L (P < 0.05). However, the scatter of the data in TIH patients was quite marked. Increased values (10/46 patients, 22%) occurred only in patients with bone metastases. Total AP, gamma GT and BGP levels, as well as markers of bone resorption, were not significantly different between patients with or without bone metastases. BAP levels were significantly correlated with AP (rs = 0.63; P < 0.01) but not with BGP levels nor with markers of bone resorption. BAP levels were also correlated with the extent of bone uptake at scintigraphy (rs = 0.54; P < 0.01), but this was not the case for total AP or BGP. In the 36 patients re-evaluated when normocalcemic after pamidronate therapy, BAP levels increased from 16.3 +/- 9.2 to 22.2 +/- 21.3 micrograms/L (P < 0.05) but there were no significant changes in AP or BGP concentrations. In summary, our data confirm the existence of an uncoupling in bone turnover in TIH and indicate that cancer hypercalcaemia is another pathological condition characterised by a discordance between BAP and BGP concentrations. BAP levels appear to be a better reflection of bone metastatic involvement than total AP or BGP and their short-term increase after pamidronate therapy could reflect the recently described effects of bisphosphonates on osteoblasts.

Adult↗
PubMed1995

Induction of the blood/brain-barrier-associated enzyme alkaline phosphatase in endothelial cells from cerebral capillaries is mediated via cAMP.

The mammalian blood/brain barrier is located at the endothelial cells of the cerebral capillaries. Alkaline phosphatase is associated to a very large extent with these cells and has been established as a marker enzyme for a differentiated blood/brain barrier phenotype in vivo and in vitro. Nevertheless cultured brain capillary endothelial cells (BCEC) lose this marker enzyme because of a cessation of de novo synthesis. Since astrocytes have been shown to possess the capability to re-induce the enzymic activity of alkaline phosphatase in BCEC in vitro we were interested in the second messengers involved in the signal-transduction mechanism of this induction in BCEC. For this reason we treated cultured porcine BCEC with a water-soluble and membrane-permeable analogue of cAMP, 8-(4-chlorophenylthio)-cAMP (C1PhS-cAMP) in the absence of astrocytes. By means of enzymic activity assays we were able to show that within three days the activity of alkaline phosphatase increased up to sixfold compared with the controls. The total activity of alkaline phosphatase in C1PhS-cAMP-treated BCEC was comparable to that of freshly isolated cells. Addition of cycloheximide inhibited the alkaline phosphatase activity increase. We conclude that cAMP is one of the second messengers involved in the induction of alkaline phosphatase activity in BCEC in vitro.

Alkaline Phosphatase↗
PubMed1978

Effects of petroleum hydrocarbon solvents on alkaline phosphatase of rats.

After administration of benzene, gasoline, IOMEX or petroleum ether, liver alkaline phosphatase showed over 200% increase in activity whereas kidney alkaline phosphatase activity was depressed by 50%. The pattern of separation of isoenzymes of alkaline phosphatase in liver, kidney and serum indicated involvement of one of the isoenzymes. A simple method of detecting the incipent toxicity is described.

Alkaline Phosphatase↗
PubMedDate not supplied

[Effect of adrenalectomy on alkaline phosphatase isoenzymes in intact and pregnant mice].

The paper shows that alkaline phosphatase activity tends to decrease in the hepatic extracts, in the intestine, significantly in the blood serum and to increase in the kidneys and significantly in the bone in mice 7 days after adrenalectomy, which coincides with the changes in the amount and activity of rapidly migrating alkaline phosphatase isoforms during polyacrylamide gel electrophoresis. Pregnancy in animals enhances alkaline phosphatase activity in the extracts of organs and tissues; adrenalectomy made on day 13-14 of pregnancy does not cause so noticeable differences as in the control group.

Adrenal Glands↗
PubMed1992

Significance of a new stratification of alkaline phosphatase and extent of disease in patients with prostate carcinoma with bone metastasis.

Bone is the most frequent site of metastatic prostate cancer and the prognosis of patients with bone metastasis is poor. The authors have investigated a semiquantitative system to evaluate bone metastatic lesions in terms of cancer-specific survival. Based on the extension of disease (EOD) grade proposed by Soloway and associates, a new EOD grading system obtained from bone scintigraphy alone and EOD score obtained from bone scintigraphy and alkaline phosphatase was studied in 164 patients with prostate cancer with metastatic bone involvement. In terms of a cancer-specific survival and prostate cancer death, both the new EOD grade and the EOD score were apparently superior to eight other items studied (age, medical score, gait disturbance, histologic grade, erythrocyte sedimentation rate, prostatic acid phosphatase, and alkaline phosphatase). Multivariate analysis revealed that the EOD score was better than the new EOD grade. This improvement was due to the elimination of false-positive or nonactive metastatic bone lesions on bone scintigraphy through the alkaline phosphatase evaluation.

Aged↗
PubMedDate not supplied

A therapeutic profile of metal chelators in the detoxication of methylmercury chloride inhibited acid and alkaline phosphatases in different areas of the central nervous system of rats.

Acid and alkaline phosphatase changes in various parts of the central nervous system (olfactory bulbs, cerebral hemispheres, cerebellum, medulla oblongata, and spinal cord) were analyzed during methylmercury chloride (MMC) treatment with a dose of 5 mg/kg/day body weight. The drug was subcutaneously introduced into the animals and the enzymes were analyzed after 2, 7, and 15 days' treatment. One group of animals was treated for seven days and kept without drug for another seven days (withdrawal group). The antagonizing capacities of four chelators, namely, N-acetyl-DL-homocysteine thiolactone (NAHT), D-penicillamine (DPA), glutathione (GSH), and sodium selenite (SEL), were also analyzed in relation to the restoration of enzymes. Study results show a linear inhibition of acid and alkaline phosphatases with increasing duration of MMC treatment. However, the magnitude of enzymatic inhibition is different in different brain areas. After 15 days' treatment, maximum inhibition of acid and alkaline phosphatases was recorded in the spinal cord and cerebellum, respectively. Chelators also exhibited differential recovery of the enzymes in various animal groups, as well as in discrete brain areas. No uniformity in the recovery of the enzymes with chelators was observed. However, study results show that biochemical parameters are good indicators of early recognition of neurotoxicity.

Acid Phosphatase↗
PubMed2002

Maternal serum urea resistant alkaline phosphatase in Down syndrome pregnancy.

BACKGROUND: The normal levels of alkaline phosphatase (AP) activity in maternal serum are virtually the same as those observed in Down syndrome (DS) pregnancies at 14-20 weeks' gestation. Using urea inhibition of AP, we observed an atypical AP isoenzyme in the neutrophils of mothers with trisomy 21 fetuses. AIM: To assess the use of urea as a selective inhibitor of serum AP in order to seek a possible diagnostic difference between normal and DS pregnancies. STUDY DESIGN AND SUBJECTS: Serum AP samples from 24 DS pregnancies and 204 control cases were examined at 12-22 weeks' gestation with and without 2.5 M urea AP inhibition at 18 degrees C for 2 h. The levels of AP activity obtained without urea and the percentage urea AP inhibition were analyzed in the two groups. RESULTS: Without urea treatment, no significant difference of total alkaline phosphatase activity levels was detected between the 204 normal controls and the 24 DS samples. Using 2.5 M urea AP inhibition, after 120 min of exposure at 18 degrees C, the residual activity, as a percentage of initial values of AP, showed significantly higher resistance in the DS samples (> or = 50 IU/l of total AP activity) at 15-22 weeks' gestation. However, at 12-14 weeks (< or = 45 IU/l of total AP activity), no significant difference was found between the DS and control cases. CONCLUSION: Serum urea resistant alkaline phosphatase in DS pregnancies showed a significant difference only at 15-22 weeks' gestation, compared with normal controls.

Adult↗
PubMed1999

Elevated serum alkaline phosphatase may enable early diagnosis of ovarian cancer.

A case of endometrioid ovarian carcinoma associated with elevated levels of serum placental-like alkaline phosphatase (PLAP) is presented. Two and a half years before a final diagnosis was made following explorative laparotomy, an incidental blood test revealed elevated alkaline phosphatase in the patient's serum. A thorough investigation for the source of this elevation was negative. Postoperative immunohistochemical staining of the tumor, showed diffuse stain with PLAP, along with gradual decline to normal values of serum total alkaline phosphatase. It is suggested, that whenever serum alkaline phosphatase is elevated due to unknown reason, an investigation including alkaline phosphatase isoenzymes, serum Ca-125, trans-vaginal pelvic sonogram and even diagnostic laparoscopy, should be considered in a search for early preclinical ovarian cancer.

Adenocarcinoma↗
PubMed1981

Lack of relationship between activity of intestinal alkaline phosphatase and calcium or phosphate absorption.

The effects of vitamin D3 and the aqueous extract of Solanum malacoxylon on intestinal alkaline phosphatase and tissue phosphate content were studied on rachitic chicks treated with large doses of ethane-1-hydroxy-1,1 diphosphonate (EHDP). The EHDP treatment blocks the increase of intestinal calcium or phosphate absorption induced by the vitamin D3, while it has no effects on the rise of intestinal alkaline phosphatase activity or the increment in tissue phosphate content. The lack of correlation between the increment of alkaline phosphatase and that of Ca or phosphate absorption in vitamin D3 plus EHDP treated chicks excludes a participation of the alkaline phosphatase in the mechanism of Ca or P intestinal absorption. The Ca or phosphorus absorption are elicited specifically by 1,25-(OH)2-D3, while alkaline phosphatase activity and phosphate tissue concentration respond to a broader spectrum of stimuli.

Alkaline Phosphatase↗
PubMed2005

Phosphodiesterase activity of alkaline phosphatase in ATP-initiated Ca(2+) and phosphate deposition in isolated chicken matrix vesicles.

Inorganic pyrophosphate is a potent inhibitor of bone mineralization by preventing the seeding of calcium-phosphate complexes. Plasma cell membrane glycoprotein-1 and tissue nonspecific alkaline phosphatase were reported to be antagonistic regulators of mineralization toward inorganic pyrophosphate formation (by plasma cell membrane glycoprotein-1) and degradation (by tissue nonspecific alkaline phosphatase) under physiological conditions. In addition, they possess broad overlapping enzymatic functions. Therefore, we examined the roles of tissue nonspecific alkaline phosphatase within matrix vesicles isolated from femurs of 17-day-old chick embryos, under conditions where these both antagonistic and overlapping functions could be evidenced. Addition of 25 microM ATP significantly increased duration of mineralization process mediated by matrix vesicles, while supplementation of mineralization medium with levamisole, an alkaline phosphatase inhibitor, reduces the ATP-induced retardation of mineral formation. Phosphodiesterase activity of tissue nonspecific alkaline phosphatase for bis-p-nitrophenyl phosphate was confirmed, the rate of this phosphodiesterase activity is in the same range as that of phosphomonoesterase activity for p-nitrophenyl phosphate under physiological pH. In addition, tissue nonspecific alkaline phosphatase at pH 7.4 can hydrolyze ADPR. On the basis of these observations, it can be concluded that tissue nonspecific alkaline phosphatase, acting as a phosphomonoesterase, could hydrolyze free phosphate esters such as pyrophosphate and ATP, while as phosphodiesterase could contribute, together with plasma cell membrane glycoprotein-1, in the production of pyrophosphate from ATP.

Adenosine Triphosphate↗
PubMed1978

Ultrastructural localization of alkaline phosphatases in rat incisor odontoblasts.

The localization of alkaline phosphatases in dentinogenically active rat incisor odontoblasts was studied by means of subcellular fractionation and electron microscopical histochemistry. Subcellular fractionation revealed the predominant phosphatase activity to be present in the microsome fraction and to a lesser extent in the mitochondrial fraction. Adenosine triphosphate degrading enzyme activity was determined in the presence or absence of (+/-)-6(m-bromophenyl)-5, 6-dihydroimidazo(le) (2,1-b) thiazole oxalate (R 8231). Before the histochemical study, the effects on phosphatase activities by aldehyde fixation were studied by biochemical assay. A method of fixation for optimal preservation of phosphatase activity is presented. Phosphatase electron microscopic histochemistry was performed by using ATP as a substrate and with or without addition of the inhibitor R 82319 Precipitates were seen in the membranes of vesicles present in the odontoblast process and the Golgi region. When there were signs of insufficient fixation, precipitates were also seen in the outer membranes of mitochondria. No phosphatase activity was seen in the cell membrane. ATP degrading enzyme activities mediated by nonspecific alkaline phosphatase (APase) and Ca2+ -adenosine triphosphatase thus have the same morphological localization. This close association is consistent with earlier biochemical studies.

Adenosine Triphosphatases↗
PubMed1976

The incorporation of 5-iodo-2'-deoxyuridine into the DNA of HeLa cells and the induction of alkaline phosphatase activity.

Inhibition of DNA synthesis during the period of exposure of HeLa cells to 5-iodo-2'-deoxyuridine (IUdR) inhibited the induction of alkaline phosphatase activity. This finding, taken together with previous findings that IUdR did not induce alkaline phosphatase activity in the presence of 2-fold molar excess thymidinemonstrated that IUdR incorporation into DNA is correlated with the increase in alkaline phosphatase activity. With the exception of an interim period described in the text, induction of alkaline phosphatase activity was linearly related to medium concentrations of IUdR of up to at least 3 muM. However, the extent of IUdR substitution in DNA did not appear to be related to the degree of enzyme induction. Alkaline phosphatase activity continued to increase at medium concentrations of IUdR from 1 to 3 muM, while little further substitution of DNA occurred.

Alkaline Phosphatase↗
PubMed1992

DNA hybridization assay using ATTOPHOS, a fluorescent substrate for alkaline phosphatase.

A fluorometric procedure for the detection of DNA-DNA hybrids is described. The procedure involved the detection of probe-bound alkaline phosphatase with the fluorescent substrate ATTOPHOS. This substrate is converted to ATTOFLUOR by alkaline phosphatase and fluoresces strongly at 550 nm when excited with a wavelength of 440 nm. DNA hybridization assays were performed both with dilutions of purified target plasmid DNA (pSE9 or PBR322) and whole bacterial cells. Streptavidin-alkaline phosphatase conjugates were added to react with bound probe. Fluorometric assays, as well as colorimetric assays, using 5-bromo-4-chloro-3-indolylphosphate + nitroblue tetrazolium for alkaline phosphatase activity were performed. The fluorescence of the substrate was measured at time intervals, and the slope of the regression line calculated. A slope four times greater than that of background was considered positive. One hundred femtograms or 2.2 x 10(4) molecules of homologous DNA were detected with the fluorescent assay as compared with 10,000 femtograms or 2.2 x 10(6) molecules of homologous DNA with the colorimetric assay. Similar results were obtained with whole cells. Approximately 1 x 10(3) homologous cells were detected fluorometrically and 1 x 10(5) cells were detected colorimetrically. Based on these results, we conclude that, in our hands, the DNA hybridization assay described here using ATTOPHOS as the substrate for alkaline phosphatase is a very sensitive assay for the detection of DNA-DNA hybrids.

Alkaline Phosphatase↗