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PubMed1980

Reaction rate retardation as a method for serum alkaline phosphatase isoenzyme measurement.

A method for serum alkaline phosphatase isoenzymes using an enzyme reaction rate analyser is described. The complete urea-induced degradation of enzyme activity is monitored, from which individual isoenzyme activities are obtained by calculating the constituent exponential components of the degradation curve. Activities have been measured with adequate sensitivity and selectivity for up to four isoenzyme components in normal and in pathological sera. The identity of each isoenzyme present is assigned from its characteristic degradation half-life, and by this method bone and liver alkaline phosphatase are clearly distinguished and quantitated, and a composite value for placental-intestinal alkaline phosphatase activity is obtained. The approach promises to be applicable to a wide range of isoenzymes, and in analogy with 'reaction rate' the term 'reaction rate retardation' is suggested for the procedure.

Alkaline Phosphatase↗

PubMed1991

Rat intestinal alkaline phosphatase secretion into lumen and serum is coordinately regulated.

We have reported the presence of intestinal alkaline phosphatase on particles with surfactant-like properties within enterocytes, on the luminal surface (light mucosal scrapings) and in the lumen of adult fat-fed rat intestines ((1989) J. Clin. Invest. 84, 1355). To test the physiological role of these particles, we compared the effect on particle secretion of a known inducer of luminal and serum alkaline phosphatase secretion (fat), with the effect of pharmacological stimulators (cholecystokinin and bethanecol). Fat induced a 2-3-fold increase in membrane-free phosphatase activity in serum, and in particle-bound alkaline phosphatase activity in proximal luminal washings and light mucosal scrapings, reaching a peak in both compartments 7 h after a corn oil feed. Bethanecol given subcutaneously induced a quantitatively similar increase in serum alkaline phosphatase activity and in particle-bound phosphatase activity in proximal light mucosal scrapings, reaching a peak 7.5 min after injection. Cholecystokinin also had a 2-3-fold stimulatory effect, 30 min after injection, on particle-bound phosphatase activity in proximal intestinal light mucosal scrapings and distal intestinal luminal washings. The increase in alkaline phosphatase activity in serum samples reached a peak 60 min after cholecystokinin injection. Thus, three independent stimuli increase both luminal and serum appearance of intestinal alkaline phosphatase. These data support the earlier findings that intestinal alkaline phosphatase secretion into the lumen is mediated by a secreted particle, further show that secretion into serum and lumen is coordinately regulated, and are consistent with the hypothesis that the rise in serum alkaline phosphatase activity could be related to extracellular release of the enzyme from the particles.

Alkaline Phosphatase↗

PubMed1980

[Measurement of the hepatic, intestinal and bony fractions of the serum alkaline phosphatase (author's transl)].

A trial of thermostability associated with immunological estimation of alkaline phosphatase of intestinal origin permits evaluation of the various fractions of serum alkaline phosphatase. The usual values, drawn up on the basis of data collected in 35 subjects in apparently good health and chosen among a laboratory population, indicate that on average, 9 per cent of the enzyme activity is of intestinal origin whereas the hepatic and bony fractions are respectively 25 per cent and 67 per cent. On the basis of the results presented in this paper, it appears that the method proposed is able, in the presence of raised alkaline phosphatase, to determine the tissue origin of this blood abnormality. Whereas the coefficient of correlation drawn up between the values of 5-nucleotidase and those of the hepatic fraction of serum alkaline phosphatase determined by the technic described is satisfactory, if the raised alkaline phosphatase is of hepatic origin, it becomes poor or nil, if the latter is from an extra-hepatic source. In the cases of hyperphosphatasemia of extra-hepatic origin, there is excess evaluation of the hepatic alkaline phosphatase, if one concludes from the results of 5-nucleotidase.

Alkaline Phosphatase↗

PubMed1994

Okadaic acid reverses the inhibitory effect of protein kinase C on alkaline phosphatase activity in osteoblast-like cells.

We previously reported that fetal calf serum-induced alkaline phosphatase activity is suppressed due to the activation of protein kinase C in osteoblast-like MC3T3-E1 cells (Miwa et al. (1991) Bone Miner. 14, 15-25; Kotoyori et al. (1994) Horm. Metab. Res. 26, 116-118). In the present study, we examined the effect of okadaic acid, a potent and specific inhibitor of protein phosphatase type 1 and 2A, on fetal calf serum-induced alkaline phosphatase activity in MC3T3-E1 cells. The pretreatment with okadaic acid enhanced the fetal calf serum-induced alkaline phosphatase activity in a dose-dependent manner in the range between 0.1 and 5 nM. 1-Norokadaone, a less potent analogue of okadaic acid, had little effect on the fetal calf serum-induced alkaline phosphatase activity. Okadaic acid partially reversed the suppression of fetal calf serum-induced alkaline phosphatase activity by 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C activator. The effect of okadaic acid was dose-dependent in the range between 0.1 and 5 nM. The patterns of the dose-dependency of both okadaic acid effects on fetal calf serum-induced alkaline phosphatase activity and on the suppression by 12-O-tetradecanoylphorbol-13-acetate were similar. These results strongly suggest that protein phosphatase type 1 and/or 2A act as a regulator of alkaline phosphatase activity at a point downstream from protein kinase C in osteoblast-like cells.

Alkaline Phosphatase↗

PubMed1977

Alkaline phosphatase activity in human bladder tumor cell lines.

The cellular localization and isoenzyme pattern of alkaline phosphatase in five cell lines derived from human bladder carcinomas (T24, RT4, RT112, J82, EJ) shown not to be HeLa cells has been established. RT112 cells had a high level of alkaline phosphatase. RT4 had a moderate amount of alkaline phosphatase but in the other three lines, levels were extremely low. Prednisolone caused a small (2 to 3-fold) increase in total alkaline phosphatase in T24 and RT112 lines only. Electrophoretic separation of isoenzymes showed that RT112 and RT4 cells (derived from more highly differentiated tumor types) had three heat stable bands equivalent to placental alkaline phosphatase and three slower bands of a modified placental type. Prednisolone increased only the former. In T24 cells the enzyme resembled the liver-type alkaline phosphatase in electrophoretic mobility and sensitivity to heat denaturation. Cytochemical studies confirmed the presence of cell surface-associated extramembraneous placental type enzyme in RT112 cells. All five cell lines had small deposits of intramembraneous alkaline phosphatase in the plasma membrane and deposits associated tith the mitochondrial membranes and the endoplasmic reticulum that were not completely inhibited by phenylalanine or Levamisole.

Alkaline 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

Purification and partial characterization of alkaline phosphatase of matrix vesicles from fetal bovine epiphyseal cartilage. Purification by monoclonal antibody affinity chromatography.

Alkaline phosphatase of matrix vesicles isolated from fetal bovine epiphyseal cartilage was purified to apparent homogeneity using monoclonal antibody affinity chromatography. The enzyme from the butanol extract of matrix vesicles bound specifically to the immobilized antibody-Sepharose in the presence of 2% Tween 20 whereas the major portion of nonspecific protein was removed by this single step. Of various agents tested, 0.6 M 2-amino-2-methyl-1-propanol, pH 10.2, was the most effective in eluting 80-100% of the enzyme initially applied. Both Tween 20 and 2-amino-2-methyl-1-propanol associated with the eluted enzyme were effectively removed by the sequential application of DEAE-cellulose and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of a dominant band (using silver staining) corresponding to a molecular weight of 81,000. This molecular weight was nearer reported values for rat liver (Ohkubo, A., Langerman, N., and Kaplan, M. M. (1974) J. Biol Chem. 249, 7174-7180) and porcine kidney (Cathala, G., Brunel, C., Chapplet-Tordo, D., and Lazdunski, M. (1975) J. Biol. Chem. 250, 6040-6045) alkaline phosphatase, than to previously reported values for chicken (Cyboron, G. W., and Wuthier, R. E. (1981) J. Biol. Chem. 256, 7262-7268) and fetal calf (Fortuna, R., Anderson, H. C., Carty, R. P., and Sajdera, S. W. (1980) Calcif. Tissue Int. 30, 217-225) cartilage matrix vesicle alkaline phosphatase. The purified alkaline phosphatase was activated by micromolar Mg2+. The amino acid composition of cartilage alkaline phosphatase was found to be similar to that previously described for porcine kidney (Wachsmuth, E. D., and Hiwada, K. (1974) Biochem. J. 141, 273-282). Double immunoprecipitation data indicated that monoclonal antibody against cartilage alkaline phosphatase cross-reacted with fetal bovine liver or kidney enzyme but failed to react with calf intestinal or rat cartilage enzyme. Thus these observations suggest that alkaline phosphatase of matrix vesicles from calcifying epiphyseal cartilage is a liver-kidney-bone isozyme.

Alkaline Phosphatase↗

PubMed1978

Purification and characterization of alkaline phosphatase from cultured rat ascites hepatoma cells.

Alkaline phosphatase of cultured rat ascites hepatoma cells has been purified by butanol extraction, DEAE-cellulose column chromatography, gel filtration through Sephadex G-200, concanavalin A-Sepharose affinity chromatography, and polyacrylamide gel electrophoresis. Affinity chromatography confirmed the glycoprotein nature of alkaline phosphatase from cultured rat ascites hepatoma cells. Electrophoresis on polyacrylamide gels of various concentrations indicated a molecular weight of 290,000. The molecular weight of the subunit was estimated to be 72,000 by SDS-polyacrylamide gel electrophoresis. These findings suggest that alkaline phosphatase of cultured rat ascites hepatoma cells is a tetramer with a subunit molecular weight of 72,000.

Alkaline Phosphatase↗

PubMed1992

Cloning and expression in Escherichia coli of an alkaline phosphatase (phoA) gene from Zymomonas mobilis.

An alkaline phosphatase (phoA) gene from Zymomonas mobilis was isolated in Escherichia coli CC118 by use of the plasmid Bluescript KS+. The origin of the 6.4-kb DNA fragment in pZAP1 from the chromosome of Z. mobilis was confirmed by Southern blotting and hybridization studies. The Z. mobilis phoA gene was localized at one end of the chromosomal insert on plasmid pZAP1. The Z. mobilis phoA gene was expressed from its own promoter in E. coli, and the enzyme was localized to the periplasmic space. Z. mobilis alkaline phosphatase activity in E. coli was repressed in high-phosphate media and derepressed under a phosphate-limited growth condition. These results suggest that Z. mobilis alkaline phosphatase is subjected to normal regulation in E. coli.

Alkaline Phosphatase↗

PubMed1998

Assessment of serum total and bone alkaline phosphatase measurement in clinical practice.

The aim of the study was to measure serum levels of the bone-specific isoenzyme of alkaline phosphatase in normal subjects and patients with metabolic bone disease by using an immunoadsorption assay. We studied 140 healthy adults, 122 patients affected by metabolic bone disease and 15 patients with cholestatic liver disease. Mean values of the bone-specific isoenzyme of alkaline phosphatase in healthy men were significantly higher than those found in premenopausal women (17.8 +/- 4.2 U/l vs 15.6 +/- 4.6 U/l, p < 0.02); postmenopausal women had significantly higher levels of bone-specific isoenzyme of alkaline phosphatase (22.6 +/- 6.4 U/l) than premenopausal women (p < 0.0001). After the menopause total alkaline phosphatase increased by 46%, while the increase in bone-specific isoenzyme of alkaline phosphatase was 39%. No significant correlations were found between bone-specific isoenzyme of alkaline phosphatase and either age or years since menopause, in postmenopausal subjects. In patients with bone-specific isoenzyme of alkaline phosphatase above the upper limit of normal, the assay had a sensitivity of 100% only in patients with Paget's disease of bone. In patients with cholestatic liver disease we found no correlation between bone-specific isoenzyme of alkaline phosphatase and either total alkaline phosphatase and gamma-glutamyl transpeptidase, while a positive correlation was found between total alkaline phosphatase and gamma-glutamyl transpeptidase. Our results confirm the role of bone-specific isoenzyme of alkaline phosphatase assay in clinical research; however, its usefulness in clinical practice is unclear once liver involvement has been excluded.

Adult↗

PubMed1991

Yeast fructose-2,6-bisphosphate 6-phosphatase is encoded by PHO8, the gene for nonspecific repressible alkaline phosphatase.

Yeast fructose-2,6-bisphosphate 6-phosphatase has been purified 7000-fold by heat treatment, poly(ethylene glycol) precipitation, ion-exchange chromatography with Q-Sepharose Fast Flow and Mono Q followed by affinity chromatography with concanavalin-A-Sepharose and gel filtration with Superose 12. The purified dimeric enzyme contains 1.5 mol zinc and 1.3 mol copper/mol subunit. It reacts with fructose 2,6-bisphosphate [Fru(2,6)P2] as well as with p-nitrophenyl phosphate (NpP) showing a pH optimum at pH 6-6.5 with Fru(2,6)P2 [Plankert, U., Purwin, C. & Holzer, H. (1988) FEBS Lett. 239, 69-72] and above pH 9.0 with NpP. The following observations suggest that activity with both substrates depends on the same protein. (a) During 7000-fold purification, the ratio of activity with NpP to that with Fru(2,6)P2 remained constant. (b) The time course of inactivation of enzyme activity in dilute solution at 30 degrees C is similar for both substrates. (c) At increasing temperatures, inactivation of enzyme activity measured with both substrates proceeds at nearly identical rates. (d) Activity with both substrates is found preferentially in the vacuoles. (e) Mutants defective in the nonspecific alkaline phosphatase coded by the PHO8 gene are also defective in Fru(2,6)P2 6-phosphatase activity. (f) A proteinase A mutant, defective in processing and activation of nonspecific alkaline phosphatase coded by the PHO8 gene, also fails to activate Fru(2,6)P2 6-phosphatase.

Alkaline Phosphatase↗

PubMed1989

Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver: lack of sensitivity to alkaline phosphatase when compared with that of glucocorticoid receptor.

Rat hepatic cytosol was treated with alkaline phosphatase in order to determine if dephosphorylation altered the ability of Ah receptor to bind 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD). Glucocorticoid receptor was studied for comparison. As previously had been shown in other laboratories, treatment of cytosol with purified alkaline phosphatase dramatically reduced the subsequent ability of glucocorticoid receptor to bind hormone. However, alkaline phosphatase had no effect on the ability of Ah receptor to bind [3H]TCDD. If either glucocorticoid receptor or Ah receptor was occupied by its ligand prior to exposure to alkaline phosphatase there was no loss in ligand binding capacity. Crude alkaline phosphatase (containing some protease activity) substantially reduced the ability of glucocorticoid receptor to bind hormone and shifted the sedimentation position of the glucocorticoid receptor from approximately 8 S to approximately 2 S. Crude alkaline phosphatase did not reduce the ability of Ah receptor to bind [3H]TCDD and did not alter sedimentation of the 9 S [3H]TCDD. Ah receptor complex. Although the Ah receptor appears to be a member of the steroid receptor superfamily, the lack of effect of alkaline phosphatase on Ah receptor (compared to the sensitivity of glucocorticoid receptor) highlights another significant difference in molecular characteristics between the Ah receptor and the receptors for steroid hormones.

Alkaline Phosphatase↗

PubMed1994

The secretion of intestinal alkaline phosphatase (IAP) from the enterocyte.

Rat intestinal alkaline phosphatase exists in two separate forms, differing markedly in the carboxyterminal coding region, as well as in the 3' untranslated regions. It is secreted bound to a phospholipid-rich particle (surfactant-like particle) which appears to have unique properties, but whose role is still uncertain. Evidence is presented to suggest that intestinal alkaline phosphatase secretion is mediated by this particle. The challenge for the future is to study the biology of the surfactant-like particle, to ascertain its role in gut physiology, and to determine how the dual alkaline phosphatases are involved in this specialized secretory apparatus.

Alkaline Phosphatase↗

PubMed1993

Characterization of the alkaline phosphatase expressed on the surface of a Hodgkin's lymphoma cell line.

Alkaline phosphatase solubilized from a human Hodgkin's lymphoma cell line (L428) was compared with purified amphiphilic and hydrophilic forms of the enzyme from human liver, and with the enzyme solubilized from a cultured osteosarcoma cell line (Saos-2). Purified hydrophilic alkaline phosphatases from human placenta and intestine were also compared in some experiments. Alkaline phosphatase was released from the plasma membrane of intact lymphocytes by phosphatidylinositol phospholipase C and thus is anchored to the outside of the plasma membrane by covalently attached phosphatidylinositol. Enzyme released in this way was hydrophilic and that solubilized with Triton X-100 was amphiphilic, as assessed by adsorption to octyl-Sepharose. Lymphocyte alkaline phosphatase, when released from the membrane by phosphatidylinositol phospholipase C or solubilized by Triton X-100, had apparent M(r) values on gradient gel electrophoresis of 227 and 494 kDa, respectively. These values were consistently higher than equivalent ones obtained with enzymes purified from human liver, but were similar to those of cultured osteosarcoma cells. Isoenzyme-specific inhibitors of alkaline phosphatase showed similar patterns of inhibition between the enzyme from L428 cells and the tissue-nonspecific (liver/kidney/bone) isoenzyme from human liver. Heat stabilities were similar for the enzymes from L428 and Saos-2 (bone isoform) cell lines, but differed significantly from those of liver, intestine and placenta. We conclude that the alkaline phosphatase expressed in this lymphoma cell line (L428) has properties that most closely resemble those of the tissue-nonspecific isoenzyme found normally in osteoblasts of bone (bone isoform).

Alkaline Phosphatase↗

PubMedDate not supplied

Placental and germ cell alkaline phosphatase RFLPs and haplotypes associated with spontaneous abortion.

Placental alkaline phosphatase (PLAP) and germ cell alkaline phosphatase (GCAP) are controlled by closely linked genes on chromosome 2q34-q37. In previous investigations, associations have been found between PLAP types and spontaneous abortion. In this study, PLAP and GCAP RFLPs and haplotypes were found to show highly significant associations with spontaneous abortions in the Finnish and Swedish populations. However, different associations were found in the Finnish and Swedish populations. The Finnish abortions were associated with the GCAP allele PstI(b) 2 and the Swedish abortions with the PLAP allele PstI(a) 2. A possible mechanism behind the associations may therefore be linkage disequilibria with deleterious alleles within or close to the alkaline phosphatase gene complex.

Abortion, Spontaneous↗

PubMedDate not supplied

Alkaline phosphatase as tumor marker.

Applications and limitations of alkaline phosphatase as a tumor marker are discussed. This review focuses on three characteristic isoenzymes that have been found in the serum of cancer patients: (1) the Regan isoenzyme in bronchogenic carcinoma; (2) the Nagao isoenzyme in pleuritis carcinomatosa; and (3) the hepatoma alkaline phosphatase. Tumor variants of alkaline phosphatase are classified based on their heat stability properties resembling or differing from the placental isoenzyme. They can also be differentiated in their behavior to specific inhibitors. The presence of electrophoretic variants and enzyme-immunoglobulin complexes in other cancers are also described.

Alkaline Phosphatase↗

PubMed1985

Different genes code for alkaline phosphatases from human fetal and adult intestine.

Alkaline phosphatases from human adult intestine and fetal intestine (meconium) were purified and compared. Electrophoresis in SDS showed one band of protein in the former. There were three bands of protein in the latter, all with essentially the same peptide map. Thus, two of the bands probably arose by proteolysis of the third, which was largest (Mr 73000). In gradient gels of polyacrylamide the alkaline phosphatase from fetal intestine showed only one band of protein coincident with the band of activity (Mr 151000). Radiolabeled mapping showed that the tryptic peptides of the alkaline phosphatase from fetal intestine were distinctly different from those of adult intestine and human liver, and placenta, indicating a gene distinct from the three that code for the enzyme in liver/kidney/bone, placenta, and adult intestine.

Adult↗

PubMed2005

Alkaline phosphatase is involved in the control of adipogenesis in the murine preadipocyte cell line, 3T3-L1.

OBJECTIVE: As alkaline phosphatase may play a role in cell differentiation, our aim was to study the possible role of this enzyme in the differentiation of preadipocytes (3T3-L1 cells) into adipocytes. RESEARCH METHODS AND PROCEDURES: 3T3-L1 cells were grown in medium containing insulin, dexamethasone and IBMX to induce adipogenesis. Adipogenesis was measured using the triglyceride-specific dye, oil red O at 0, 3, 7 and 11 days after initiation of adipogenesis in the presence or absence of the alkaline phosphatase inhibitors, levamisole, histidine and Phe-Gly-Gly. Intracellular localisation of the enzyme was detected using ELF-phosphatase, a fluorescent substrate and alkaline phosphatase gene expression was assessed using RT-PCR. RESULTS: Alkaline phosphatase activity was detected in untransformed cells (1.91+/-0.62 mU/mg protein) and activity increased 11.5+/-1.4-fold after 11 days treatment with transformation medium and 5.3+/-0.3-fold in transformation medium containing levamisole (p<0.05). Triglyceride content of cells increased 3.1+/-0.2-fold after 11 days treatment with transformation medium and 2.1+/-0.3-fold in the presence of levamisole (p<0.005). Histidine inhibited adipogenesis and alkaline phosphatase to a greater extent than did levamisole, but Phe-Gly-Gly had no effect on these variables. Alkaline phosphatase was localised around the lipid droplets of the cells. Gene expression of alkaline phosphatase increased during adipogenesis. DISCUSSION: This study demonstrates that tissue-nonspecific alkaline phosphatase is present in 3T3-L1 cells and that it may play a role in the control of adipogenesis.

3T3-L1 Cells↗