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At least 1,117 records · Page 62Linked to original sources
PubMed1999

pSKAP/S: An expression vector for the production of single-chain Fv alkaline phosphatase fusion proteins.

The vector pSKAP/S was constructed to enable overexpression of single-chain variable fragment antibody (scFv)-alkaline phosphatase fusion proteins. In pSKAP/S, the scFv were genetically fused to the mutated Escherichia coli PhoA/S gene that encodes an alkaline phosphatase with increased specific activity. The restriction sites incorporated into pSKAP/S allowed the scFv genes to be easily transferred from pUC119-derived phagemid vectors that are used frequently in phage display antibody library technology. Strong transcriptional control of expression was achieved using the tetracycline promoter, and induction of different individual clones with anhydrotetracycline resulted in secretion of most of the scFv-alkaline phosphatase fusion proteins into the culture medium. Although some of the clones secreted fusion proteins that were retained in the periplasm, these proteins could be isolated with a simple extraction procedure. Increased amounts of a scFv-alkaline phosphatase fusion protein were obtained when expressed in the pSKAP/S vector compared with expression in a vector incorporating the lac promoter. Testing for binding of the scFv-alkaline phosphatase fusion proteins to antigen was possible in an ELISA without the need for additional enzyme-conjugated antibodies. The pSKAP/S vector was successfully used to obtain scFv fragments from a preparation of phage-antibody clones after subcloning and expression of individual clones as scFv-alkaline phosphatase fusions, whereas fewer clones (and clones with different properties) were obtained from the same phage-antibody preparations when expressed as soluble scFv fragments. Therefore, the pSKAP/S vector was shown to be useful in extending the range of scFv obtained from phage display libraries.

Alkaline Phosphatase↗
PubMed1993

Characterization of immobilized Escherichia coli alkaline phosphatase reactors in flow injection analysis.

The characterization of immobilized Escherichia coli alkaline phosphatase reactors used in flow injection analysis is reported for factors such as optimum pH, activity, ionic strength, product inhibition, and substrate specificity. The kinetics of the immobilized enzyme was studied, and mathematical descriptions were developed for the use of an immobilized enzyme packed-bed reactor to evaluate the kinetic parameters and the number of active sites on the immobilized enzyme. Suppression of phosphatase activity by orthophosphate was found to be significantly reduced, and the Michaelis-Menten constant increased when the enzyme was immobilized and packed in a reactor. Immobilized E. coli alkaline phosphatase exhibited similar activity at pH 8 in Tris-HCl, NaHCO3 and borate-HCl buffers but slightly lower activity in NH3H2O-NH4Ac buffer. The performance of the immobilized enzyme reactor was not affected by the presence of up to 10 M Mg(II), Ni(II), Cd(II), Co(II), Mn(II), Cu(II), or urea, 1 M Fe(II), or 0.1 M Fe(III) in the substrate stream. The chelating agent EDTA, however, gradually deactivated the immobilized enzyme. The periodic restoration of enzyme activity was achieved following the removal and addition of zinc ions. The immobilized E. coli alkaline phosphatase packed-bed reactor was used to measure the alkaline phosphatase available phosphorus content of a number of model organophosphorus compounds. p-Nitrophenyl phosphate showed a linear response in the range of 1.6 x 10(-7)-1.6 x 10(-4) M. This study forms part of a larger program to develop enzymatic systems for water quality measurement.

Alkaline Phosphatase↗
PubMed1985

Alkaline phosphatase and 5'-nucleotide phosphodiesterase from bovine intestine are cross-reactive.

Polyclonal antibodies to native alkaline phosphatase and to native 5'-nucleotide phosphodiesterase were found to strongly cross-react with both enzymes. The antibodies also cross-react with both denatured enzymes, with glycopeptides from 5'-nucleotide phosphodiesterase, and with the oligosaccharides remaining after Pronase E digestion of the phosphodiesterase. They do not cross-react with either enzyme after their oligosaccharides have been modified or removed by periodate or trifluoromethanesulfonic acid treatment. Antibodies to denatured 5'-nucleotide phosphodiesterase do not bind to the native phosphodiesterase or alkaline phosphatase but do cross-react with denatured alkaline phosphatase even after removal or modification of the carbohydrate moieties. These results suggest that antibodies to denatured 5'-nucleotide phosphodiesterase may recognize amino acid sequence homology between alkaline phosphatase and 5'-nucleotide phosphodiesterase. However, antibodies to native enzymes apparently recognize cross-reactive determinants of the native enzymes which are carbohydrate in nature. This is the first report of antimammalian alkaline phosphatase antibodies which recognize the carbohydrate moieties of the enzyme.

Alkaline Phosphatase↗
PubMed1994

[Excessive rise in plasma alkaline phosphatase in the last trimester of pregnancy].

This article is concerned with the increase of alkaline phosphatase of a 32-year old primipara, five to six times higher than the upper basal values, during the last trimenon of pregnancy. The physiological increase of serum total alkaline phosphatase levels during pregnancy were two to three times higher than the basal values (4, 11). Hepatopathies, osteopathies, endocrinological disorders, infections as well as other medical causes were excluded by anamnesis due to close-meshed laboratory chemical and clinical controls relating to differential diagnosis. Thus, only pregnancy could be considered as a trigger mechanism. Differentiating the alkaline phosphatase by laboratory chemical means in the course of this pregnancy, an isolated increase of the alkaline phosphatase placenta isoenzyme (PLAP) was seen. Besides premature labour pains, which led to the admission of the patient to the maternity ward, around a calculated 32nd week of pregnancy, a discreet foetal growth retardation was recognised, which continued to increase constantly up to a minus discrepancy of three weeks. The delivery of a dystrophic girl took place at the beginning of the 37th week of pregnancy. Post partum alkaline phosphatase levels normalised to basal values. The correlation: the increase in PLAP and intra-uterine foetal growth-retardation was evident in the case presented and was also discussed in connection with previous observations by other authors of similar studies.

Adult↗
PubMed1977

The change of alkaline phosphatase binding conditions with trophoblast membrane at different stages of human placenta.

Alkaline phosphatase was brought into solution from microsomal fractions of placentas of varying gestational age by using gradually increasing concentrations of proteinase papain. When the activity of the soluble alkaline phosphatase (S) was compared with that of the non-soluble residue (R), the S/R ratio rose as pregnancy progressed. The electrophoretic pattern showed that in serum from pregnant women the papain-soluble alkaline phosphatase corresponded to the heat-stable one. These results indicate that the cytoplasmic membrane of the trophoblast changes with the growth of the placenta so that this enzyme is easily dissolved by papain. It is probable that alkaline phosphatase molecules easily enter the maternal blood stream in late pregnancy.

Alkaline Phosphatase↗
PubMed1987

Triton X-100 solubilized bone matrix-induced alkaline phosphatase.

1. Solubilized and membrane-bound alkaline phosphatase showed Michaelis-Menten behavior in a wide range of different substrate concentrations. 2. Membrane-bound alkaline phosphatase has a molecular weight of 130,000 and its minimum active configuration comprises two identical subunits of about 65,000. 3. The two forms of the enzyme behave similarly with respect to NaCl, urea and guanidine HCl. 4. Catalytic groups have pK values of about 8.5 and 9.7 for both membrane-bound and solubilized enzyme.

Alkaline Phosphatase↗
PubMed1978

Ultrastructural localization of alkaline phosphatase in rat eosinophil leucocytes.

The ultrastructural localization of alkaline phosphatase in eosinophil leucocytes, obtained from experimentally-induced peritoneal exudates in rats, has been studied using an osmiophilic technique with 2-naphthylthiolphosphoryl dichloride as substrate, fast Blue BBN as diazonium salt and postosmication with 1% aqueous osmium tetroxide. With this method identical incubation procedures could be used for both light and electron microscope examination. Eosinophils were the only cells which contained alkaline phosphatase. The enzyme was predominantly associated with the outer surface of the plasma membrane, being present in much lower concentrations in cytoplasmic cisternae. Eosinophil granules only rarely showed reaction product. The plasma membrane location of alkaline phosphatase in eosinophil leucocytes is identical to that recently demonstrated in the human neutrophil.

Alkaline Phosphatase↗
PubMed1976

The differentiation of proximal and distal tubules in the male rat kidney: the appearance of aldolase isozymes, aminopeptidase and alkaline phosphatase during ontogeny.

1. The specific activities of aminopeptidase, alkaline phosphatase and aldolase isozymes were measured in homogenates of kidneys taken at different stages of ontogeny. The cellular localization of these enzymes was studied in cryostat tissue sections using substrate linked assays for aminopeptidase and alkaline phosphatase and the mixed aggregation immuno-cytochemical technique for aldolase isozymes; local enzyme concentrations were estimated photometrically. 2. The presence of both aldolase-A and aldolase-B was demonstrated in all metanephrogenic cells (and at still higher concentrations in collecting tubule cells) of the rat fetus 16 days after conception and in the undifferentiated cells of the neogenetic zone of kidney up to 8 days after birth; no aminopeptidase or alkaline phosphatase could be found in these cells. 3. Measurements made on stained tissue sections show that the shift towards aldolase-B, seen in homogenate analyses, is due to a change in the relative amounts of proximal tubules. No evidence was seen for repression in the synthesis of aldolase-A or aldolase-B monomers in the different kidney cells during ontogeny. 4. Two transitions in the mode of nephron differentiation were observed: one was shortly after birth, the other followed weaning. Before the first transition the concentrations of the enzymes increased to different degrees, such that the enzymes reached concentrations comparable with those as in the cells of adult rats by 2 to 4 days post partum. After the second transition proximal tubule size and specific activity of brush border membrane enzymes increased 3 fold. In contrast, the distal tubules did not increase significantly in size, but their aldolase-A concentration increased 3 fold. 5. Evidence based on enzyme quantification and morphometry in kidney sections is presented to demonstrate that the proximal tubule cells show functional adaptation by two independent mechanisms: specific amplification of gene expression and hypertrophy. In contrast, the distal tubule shows functional adaptation only by specific amplification of gene expression.

Alkaline Phosphatase↗
PubMed1990

Induction of alkaline phosphatase in mouse L cells by overexpression of the catalytic subunit of cAMP-dependent protein kinase.

Mouse L929 cells were used to study the mechanism of cAMP induction of alkaline phosphatase (AP) activity. Following treatment with 200 microM 8-chlorophenylthio-cAMP (CPT-cAMP), alkaline phosphatase enzyme activity was observed to increase 80-fold after 24 h. The CPT-cAMP dose response of the alkaline phosphatase enzyme activity correlated well with the CPT-cAMP activation of cAMP-dependent protein kinase in L cells. A cDNA clone for the alkaline phosphatase was isolated and used to demonstrate a 10-fold increase in alkaline phosphatase mRNA levels after a 24-h treatment of L cells with CPT-cAMP. Increased mRNA levels were first detected 4-6 h, after CPT-cAMP treatment, and the level of alkaline phosphatase mRNA decreased rapidly after removal of CPT-cAMP. In vitro nuclear transcription studies showed that a 3-fold increase in alkaline phosphatase gene transcription was detectable 6 h after CPT treatment, and this increase was blocked by cycloheximide. In order to determine if the catalytic (C) subunit of cAMP-dependent protein kinase was able to mediate the induction of AP, L cells were transfected with expression vectors containing the metallothionein promoter and coding for the C alpha isoform of the catalytic subunit of cAMP-dependent protein kinase or for a catalytic subunit in which lysine 72 had been mutated to methionine (C alpha K72M). Zinc treatment of stably transfected cells expressing the wild-type C subunit showed an increase in protein kinase activity and an increase in AP activity. Zinc treatment of cells containing the mutant C subunit expression vector produced an increase in the amount of a protein which was recognized by C subunit antibodies on Western blots, but these cells showed no increase in protein kinase activity or in AP activity. We conclude that the C subunit is sufficient for transcriptional induction of the AP gene and that the phosphotransferase activity of the C subunit is required for this induction.

Alkaline Phosphatase↗
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↗
PubMed2000

Mutation of Arg-166 of alkaline phosphatase alters the thio effect but not the transition state for phosphoryl transfer. Implications for the interpretation of thio effects in reactions of phosphatases.

It has been suggested that the mechanism of alkaline phosphatase (AP) is associative, or triester-like, because phosphorothioate monoesters are hydrolyzed by AP approximately 10(2)-fold slower than phosphate monoesters. This "thio effect" is similar to that observed for the nonenzymatic hydrolysis of phosphate triesters, and is the inverse of that observed for the nonenzymatic hydrolysis of phosphate monoesters. The latter reactions proceed by loose, dissociative transition states, in contrast to reactions of triesters, which have tight, associative transition states. Wild-type alkaline phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate approximately 70 times faster than p-nitrophenyl phosphorothioate. In contrast, the R166A mutant alkaline phosphatase enzyme, in which the active site arginine at position 166 is replaced with an alanine, hydrolyzes p-nitrophenyl phosphate only about 3 times faster than p-nitrophenyl phosphorothioate. Despite this approximately 23-fold change in the magnitude of the thio effects, the magnitudes of Bronsted beta(lg) for the native AP (-0.77 +/- 0.09) and the R166A mutant (-0.78 +/- 0. 06) are the same. The identical values for the beta(lg) indicate that the transition states are similar for the reactions catalyzed by the wild-type and the R166A mutant enzymes. The fact that a significant change in the thio effect is not accompanied by a change in the beta(lg) indicates that the thio effect is not a reliable reporter for the transition state of the enzymatic phosphoryl transfer reaction. This result has important implications for the interpretation of thio effects in enzymatic reactions.

Alanine↗
PubMed1990

Establishment of hybridomas secreting monoclonal antibodies to placental alkaline phosphatase and development of an enzyme immunoassay for its determination.

We established seven hybridomas secreting murine IgG monoclonal antibodies (MoAbs) to placental alkaline phosphatase (PLAP). The seven hybridomas were designated (1) 7C6, (2) 6G10, (3) 5B9, (4) 6D5, (5) 6B5, (6) 11G6 and (7) 3E10, respectively. The characteristics of these hybridomas were evaluated by radioimmunoassay (RIA) with 125I-PLAP. Their reactivity with the intestinal alkaline phosphatase, one of the alkaline phosphatase isozymes, was (1) 0.04, (2) 0.2, (3) 1.4, (4) 1.8, (5) 0, (6) 4.0 and (7) 6.2(%), respectively. None of them showed signs of cross-reactivity with the liver-type alkaline phosphatase, also one of the alkaline phosphatase isozymes, within a PLAP concentration of 2,000 IU/l. The subtype of 5B9 was IgG1, and that of the others was IgG2a. We then used 7C6, to develop a sensitive, specific and convenient enzyme immunoassay (EIA) for the determination of PLAP, and assayed sera from patients with various gynecologic diseases. The incidence of increased PLAP was 6.4% in patients with benign diseases, 21.5% in cervical cancer, 36.4% in endometrial carcinoma, and 39.5% in malignant ovarian tumors. The specificity for malignant diseases seemed to be higher than that of CA125. Among endometrial carcinomas, well-differentiated adenocarcinoma had the highest incidence of an increased concentration. Among malignant ovarian tumors, serous cystadenocarcinoma, endometrioid carcinoma, dysgerminoma and Krukenberg's tumor showed a higher incidence than the other types.

Alkaline Phosphatase↗
PubMed1984

Hepatic clearance of rat plasma intestinal alkaline phosphatase.

The mechanism and route of clearance of intestinal alkaline phosphatase from plasma have been studied in rats to define the magnitude of hepatic extraction and biliary excretion of the enzyme. Plasma clearance, tissue distribution, and biliary excretion of enzyme were followed after intravenous administration of physiological amounts of 125I-labeled rat intestinal alkaline phosphatase. The plasma disappearance curve was biphasic; the initial phase was rapid, during which 50% of injected enzyme was selectively extracted by the liver over 5 min. Less than 4% of total hepatic radioactivity was excreted into bile over 80 min; this was shown by chromatographic analysis to be degraded enzyme only. Rapid clearance of enzyme could be significantly slowed by injection of large amounts of mannan or N-acetylglucosamine-bovine serum albumin, but not by desialylated fetuin, demonstrating that clearance was probably mediated by mannose/N-acetylglucosamine-specific receptors. It is concluded that, under physiological conditions, rat plasma intestinal alkaline phosphatase is rapidly cleared from the circulation by the liver. However, biliary excretion of undergraded enzyme is negligible, and a physiologically significant enterohepatic circulation seems most unlikely.

Alkaline Phosphatase↗
PubMed1990

Differential effects of sodium butyrate and hyperosmolality on the modulation of alkaline phosphatases of LoVo cells.

LoVo cells produce term-placental and intestinal alkaline phosphatases. Hyperosmolality and sodium butyrate increase the levels of both, but the effect of sodium butyrate is more pronounced on the intestinal enzyme. When applied together, induction of term-placental alkaline phosphatase is additive and that of the intestinal enzyme is synergistic. Induction by either stimulus or by their mixture is independent of cell density. However, whereas the effect of hyperosmolality is readily reversible, induction by sodium butyrate is not. No synergistic increase in intestinal alkaline phosphatase activity occurs when cells are sequentially treated with hyperosmolality and sodium butyrate or vice versa. This indicates that only when applied concurrently does one inducer amplify the effect of the other. Since the normal colonic mucosa produces intestinal alkaline phosphatase, its predominant induction by sodium butyrate in LoVo cells may reflect a more differentiated state.

Alkaline Phosphatase↗
PubMed1999

A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity.

We describe a high-resolution, fluorescence-based method for localizing endogenous alkaline phosphatase in tissues and cultured cells. This method utilizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. We compared zebrafish intestine, ovary, and kidney cryosections stained for endogenous alkaline phosphatase using four histochemical techniques: ELF-97 phosphate, Gomori method, BCIP/NBT, and naphthol AS-MX phosphate coupled with Fast Blue BB (colored) and Fast Red TR (fluorescent) diazonium salts. Each method localized endogenous alkaline phosphatase to the same specific sample regions. However, we found that sections labeled using ELF-97 phosphate exhibited significantly better resolution than the other samples. The enzymatic product remained highly localized to the site of enzymatic activity, whereas signals generated using the other methods diffused. We found that the ELF-97 precipitate was more photostable than the Fast Red TR azo dye adduct. Using ELF-97 phosphate in cultured cells, we detected an intracellular activity that was only weakly labeled with the other methods, but co-localized with an antibody against alkaline phosphatase, suggesting that the ELF-97 phosphate provided greater sensitivity. Finally, we found that detecting endogenous alkaline phosphatase with ELF-97 phosphate was compatible with the use of antibodies and lectins. (J Histochem Cytochem 47:1443-1455, 1999)

Alkaline Phosphatase↗
PubMed1983

Studies of the Regan alkaline phosphatase isoenzyme in plasma from a lung-carcinoma patient.

We compared an alkaline phosphatase (EC 3.1.3.1) isoenzyme from a plasma sample of a patient with carcinoma of the lung, who was suspected of having the Regan-type isoenzyme, with placental serum alkaline phosphatase and with tissue preparations from placenta, liver, bone, and intestine. The placental and Regan phosphatases responded similarly to heat treatment and modification by various inhibitors, except that the Regan isoenzyme was more resistant to EDTA inactivation than was the placental serum alkaline phosphatase, but more sensitive than placental tissue alkaline phosphatase. Isoelectric focusing and Cellogel electrophoresis showed the Regan isoenzyme to have a mobility similar to that of the placental type. We conclude that the isoenzyme was of the Regan type and support the contention that the placental and Regan isoenzymes are products of the same structural gene, with small differences due to post-genetic modification.

Alkaline Phosphatase↗
PubMed1984

Alkaline phosphatase activity and tetracycline incorporation during initial orthodontic tooth movement in rats.

The activity of alkaline phosphatase and the incorporation of tetracycline as signs of bone formation were studied after orthodontic tooth movement for 10 h to 6 days in rats. Defined low or high forces were used. A moderate activity of non-specific alkaline phosphatase was found in the periodontal membrane (PDM) in untreated rats and in rats treated with low forces. In addition, all bone surfaces were outlined with a narrow band of intense non-specific alkaline phosphatase activity that was vanadate- and levamisole-resistant. Likewise, tetracycline was incorporated on all bone surfaces. The bone formation rate was low and uniform within the alveolus, indicating that no intra-alveolar drift of the molar occurred in the untreated rats. Orthodontic forces gradually inhibited vanadate- and levamisole-resistant alkaline phosphatases and tetracycline incorporation on the bone surfaces in the pressure zones of the PDM, depending on the magnitude of the force. It was suggested that the disappearance of these isoenzymes, in a limited area, as seen in the pressure zones, was associated with inhibited bone formation and subsequent initiation of bone resorption. On the tension side a slight reduction and redistribution of vanadate- and levamisole-resistant alkaline phosphatase activity could be noted irrespective of the magnitude of the applied force.

Alkaline Phosphatase↗
PubMed1992

Transcriptional regulation of the human placental-like alkaline phosphatase gene and mechanisms involved in its induction by sodium butyrate.

The human alkaline phosphatases constitute a multigene family with at least four members. Placental-like alkaline phosphatase (PLAP) is of particular interest because it is frequently present in tumors, where it serves as a marker of malignant transformation. Moreover, its expression is highly inducible by differentiating agents such as sodium butyrate. In the present study we have examined the PLAP gene promoter in order to better understand the mechanisms involved in its expression and induction. The PLAP promoters from four colon cancer cell lines with widely varied butyrate-inducible alkaline phosphatase activity were thermally amplified and sequenced. The overall sequence similarity of this region was found to be 99% between cell lines; thus, sequence variation of the promoter does not appear to account for the differential expression of this marker. We therefore analyzed the activity of the LS174T cell PLAP promoter using transient transfection experiments. Here, the 5'-flanking region of the gene was found to have positive regulatory elements in nucleotides -1 to -170 and -363 to -512 (relative to the start of transcription). A negative control element was also found to be present in the region between nucleotides -170 and -363. Mobility shift electrophoresis indicated that a nuclear factor bound to the promoter between bases -182 and -341. Furthermore, the activity of the PLAP promoter was found to be inducible by sodium butyrate. In contrast, the closely related placental alkaline phosphatase gene promoter exhibited almost no response to this agent. These results confirm that the activity of the PLAP promoter is stimulated by sodium butyrate and delineate regions that control this induction process.

Alkaline Phosphatase↗