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PubMed1992

Phosphotransferase activity associated with rat osseous plate alkaline phosphatase: a possible role in biomineralization.

1. Alkaline phosphatase from rat osseous plate catalyzed the transfer of phosphate from p-nitrophenylphosphate to glycerol, ethanolamines, Tris, glucose and 1-amino-1-methyl-2-propanol, in a wide range of pH. Serine did not stimulate phosphotransferase activity of the enzyme. 2. The best phosphotransferase acceptors were diethanolamine and glycerol while glucose was the poorest phosphotransferase acceptor used. 3. Diethanolamine and glycerol affected both VM and KM of p-nitrophenylphosphate hydrolysis with activation constants (KA) of 0.25 and 0.85 M, respectively. 4. A kinetic model was proposed for the phosphotransferase reaction observed with alkaline phosphatase from rat osseous plates.

Alkaline Phosphatase↗

PubMed2002

Acid and alkaline phosphatase activities in the clam Scrobicularia plana: kinetic characteristics and effects of heavy metals.

The acid and alkaline phosphatase activities of the clam Scrobicularia plana have been partially characterised in different organs and tissues (digestive gland, gills, foot, siphon and mantle) and the 'in vitro' effect of heavy metals on both types of enzymatic activity have been analysed. The optimal pH ranged between 4.0 and 5.5 for acid phosphatase activity and 8.5 and 9.5 for alkaline phosphatase activity. The apparent optimum temperature was in the 30-60 degrees range for acid phosphatase activity and in the 30-40 degrees C range for alkaline phosphatase activity. The effect of substrate concentration on enzymatic activities in the tissues showed a good fit to the Michaelis-Menten model. For both types of enzymatic activity, the highest values were found in the digestive gland. The effect of heavy metals was dependent on the tissue analysed. Mercury showed the highest inhibition in the organs/tissues and the parameters Km and Vmax were modified when the inhibitor concentration increased, thus indicating a mixed type of inhibition.

Acid Phosphatase↗

PubMed1999

The integration host factor (IHF) affects the expression of the phosphate-binding protein and of alkaline phosphatase in Escherichia coli.

The genes encoding alkaline phosphatase (phoA) and the inducible inorganic phosphate transport system Pst (pstS,C,A,B,U) belong to the PHO regulon. Mutants of Escherichia coli lacking the global regulatory protein integration host factor (IHF) show an increased level of alkaline phosphatase and a decreased level of Pst. IHF binds weakly but specifically to a DNA fragment containing the promoter region of the pst operon but does not bind to a fragment that includes the promoter region of phoA. It is proposed that IHF is a positive regulator of the pst operon and as such controls indirectly the expression of phoA.

Alkaline Phosphatase↗

PubMed1977

Expression of liver and placental alkaline phosphatases in Chang liver cells.

This paper presents evidence that a protein characteristic of differentiated liver cells, liver alkaline phosphatase is synthesized by the Chang liver cell line. Liver alkaline phosphatase was demonstrated by immunochemical assay, 32P-labeling and polyacrylamide gel electrophoresis, immunofluorescence microscopy, and the fluorescence-activated cell sorter. The synthesis of the liver enzyme by the Chang liver cells is interpreted to indicate fidelity of the Chang cells to their origin from human liver tissue. Chang liver cells also synthesize a phosphatase which is similar if not indentical to the placental alkaline phosphatase. Since a placental-type alkaline phosphatase has been observed in a number of non-trophoblastic cell lines and also in some neoplasms, it does not seem reliable as an index of the origins of the cell line. Because of the claims that Chang liver cells are actually HeLa cells, HeLa cells were studied in tandem with the Chang cells. The results showed that the HeLa cells do not make the liver type phosphatase. The data are discussed in relation to the question of HeLa cell contamination of the Chang cell line and the validity of criteria normally used to identify cell lines.

Alkaline Phosphatase↗

PubMed1985

Immunoelectron microscopic double labeling of alkaline phosphatase and penicillinase with colloidal gold in frozen thin sections of Bacillus licheniformis 749/C.

The subcellular distribution of alkaline phosphatase and penicillinase was determined by double labeling frozen thin sections of Bacillus licheniformis 749/C with colloidal gold-immunoglobulin G (IgG). Antipenicillinase and anti-alkaline phosphatase antibodies were used to prepare complexes with 5- and 15-nm colloidal gold particles, respectively. The character of the labeling of membrane-bound alkaline phosphatase and penicillinase was different: the immunolabels for alkaline phosphatase (15-nm particles) were bound to a few sites at the inner surface of the plasma membrane, and the gold particles formed clusters of various sizes at the binding sites; the immunolabels for penicillinase (5-nm particles), on the other hand, were bound to the plasma membrane in a dispersed and random fashion. In the cytoplasm, immunolabels for both proteins were distributed randomly, and the character of their binding was similar. The labeling was specific: pretreating the frozen thin sections with different concentrations of anti-alkaline phosphatase or penicillinase blocked the binding of the immunolabel prepared with the same antibody. Binding could be fully blocked by pretreatment with 800 micrograms of either antibody per ml.

Alkaline Phosphatase↗

PubMed1994

Multicomponent analysis for alkaline phosphatase isoenzyme determination by multiple linear regression.

Alkaline phosphatase (EC 3.1.3.1) isoenzymes in serum may be determined by multicomponent analysis of the enzyme activities in the presence of multiple inhibitors. To determine inhibition coefficients of the isoenzymes, we used multiple linear regression analysis to compare alkaline phosphatase activities in the presence of known inhibitors with electrophoretically determined isoenzyme activities in plasma and serum samples. All possible combinations of exactly determined and overdetermined linear systems of inhibitors were ranked according to their prediction error to select an optimum set. The best multicomponent system for prediction included the use of levamisole, phenylalanine, and heat inhibition at 56 degrees C and 65 degrees C to determine bone, hepatic, intestinal, and placental isoenzymes. Consideration of the hepatic isoenzyme as liver and macromolecular fractions resulted in significantly worse predictions. Error analysis involving repeat determinations and a simplex optimization of the inhibition coefficients indicated that the inaccuracy of the comparison electrophoretic method may have been a major factor affecting poor isoenzyme prediction in some samples.

Alkaline Phosphatase↗

PubMed1987

Activation of alkaline phosphatase with Mg2+ and Zn2+ in rat hepatoma cells. Accumulation of apoenzyme.

In Reuber rat hepatoma cells (R-Y121B), alkaline phosphatase activity increased without de novo enzyme synthesis (Sorimachi, K., and Yasumura, Y. (1986) Biochim. Biophys. Acta 885, 272-281). The enzyme was partially purified by butanol extraction from the particulate fractions. The incubation of the extracted alkaline phosphatase with the cytosol fraction induced a large increase in enzyme activity (5-10-fold of control). The dialyzed cytosol was more effective than the undialyzed cytosol during an early period of incubation at 37 degrees C. This difference between the dialyzed and the undialyzed cytosol fractions was due to endogenous Na+. For maximal activation of the enzyme, both Mg2+ above 1 mM and Zn2+ at low concentrations (below 0.01 mM) were needed, although Zn2+ at high concentrations (above 0.1 mM) showed an inhibitory effect. Zn2+ and Mg2+ alone slightly increased alkaline phosphatase activity. This activation of the enzyme was temperature dependent and was not observed at 0 or 4 degrees C. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the increase in alkaline phosphatase activity did not involve the fragmentation of the enzyme and that 65Zn2+ bound to it during enzyme activation with 65Zn2+ and Mg2+. The cytosol fraction not only supplied Zn2+ to the nascent enzyme but also increased the maximal enzyme activity more than did direct addition of metal ions. Ferritin and metallothionein contributed to the activation of alkaline phosphatase with the metal ions. Since the binding of Zn2+ and Mg2+ to the nascent alkaline phosphatase is disturbed in Reuber rat hepatoma cells (R-Y121B), the apoenzyme is accumulated inside the cells. The binding of Zn2+ and Mg2+ to the apoenzyme readily takes place in the cell homogenates accompanied by an increase in catalytic activity without new enzyme synthesis.

Alkaline Phosphatase↗

PubMed1996

The flare in serum alkaline phosphatase activity after orchiectomy: a valuable negative prognostic index for progression-free survival in prostatic carcinoma.

PURPOSE: We determined whether an early flare in serum alkaline phosphatase activity after orchiectomy was of prognostic value for progression-free survival in patients with advanced prostatic carcinoma. MATERIALS AND METHODS: A retrospective analysis of a data base from a Dutch multicenter study on prostatic carcinoma was done to determine the prognostic value of a flare in alkaline phosphatase activity after orchiectomy in 112 patients with metastatic (75%) or locally advanced (25%) disease. Cox's proportional hazards models and Kaplan-Meier survival curves were used. RESULTS: Of the patients 50% had initially increased alkaline phosphatase levels and a flare in activity was demonstrated in 87% 2 to 4 weeks after orchiectomy. The prostate specific antigen nadir (cutoff 4 ng./ml.) 6 months after orchiectomy was of significant prognostic value for progression-free survival. A flare in alkaline phosphatase activity after orchiectomy demonstrated an early significant prognostic value for progression-free survival, independent of the serum alkaline phosphatase activity. CONCLUSIONS: The simplicity, ready availability and cost-effectiveness of serum alkaline phosphatase activity as a prognostic index render it attractive to the clinician, particularly early in the course of prostatic carcinoma. Measuring the flare in alkaline phosphatase activity within 1 month of orchiectomy may permit early identification of patients in whom the disease is likely to progress rapidly and who would potentially benefit from aggressive treatment.

Aged↗

PubMed1997

Flow cytometry of leucocyte alkaline phosphatase in normal and pathologic leucocytes.

Leucocyte alkaline phosphatase (LAP) is an enzyme expressed on the external aspect of the neutrophilic granulocyte plasma membrane, and represents a specific marker for the fully differentiated granulocyte. In this report we characterize 1B12.1, a monoclonal antibody raised against human bone alkaline phosphatase, by its ability to recognize the LAP protein. As assessed by Western blot analysis, following electrophoresis under non-reducing conditions, the antibody specifically reacts with LAP upon forced expression of the protein in simian COS-7 fibroblasts. In addition, the 1B12.1 antibody recognizes partially purified LAP isolated from peripheral blood granulocytes. With this antibody we developed a quantitative flow-cytometry-based method for the determination of LAP. Double fluorescence flow cytometry demonstrated that the LAP protein was present in relatively high amounts in neutrophilic granulocytes, but not in monocytes, natural killer cells, or B and T lymphocytes of normal individuals. The protein was completely absent in granulocytes obtained from chronic myeloid leukaemia and paroxysmal nocturnal haemoglobinuria patients. Higher than normal levels of LAP protein were evident in neutrophilic granulocytes of patients suffering from polycythaemia vera, essential thrombocythaemia and severe aplastic anaemia. However, the highest amounts of LAP protein were present in the granulocytes of normal individuals treated with G-CSF for the isolation of peripheral blood stem cells.

Alkaline Phosphatase↗

PubMed1976

Increase in alkaline phosphatase activity in the liver of mice bearing Ehrlich ascites tumor.

In mice bearing Ehrilich ascites tumors, alkaline phosphatase activity was increased fivefold in the liver and by 50% in the kidney. In mice bearing solid tumors caused by inoculation of tumor cells into the axillary region, the activity of this enzyme in the liver was increased 11-fold, whereas the activity in the kidney did not change. Alkaline phosphatase activities in the liver and kidney were not altered by administration of adrenal steroids. Adrenalectomy, fasting, and pregnancy did not affect the activity of alkaline phosphatase in the liver and kidney. Treatment with tumor extracts or ascites fluid of normal mice increased liver alkaline phosphatase activity. These findings suggested that the elevation of liver alkaline phosphatase activity was cuased primarily by the tumor itself, and not by hormonal imbalance provoked secondarily by the presence of the tumor.

Adrenal Glands↗

PubMed1975

Disc electrophoresis of turkey serum alkaline phosphatase isoenzymes using polyacrylamide gel. 1. Procedure.

The isoenzymes of alkaline phosphatase in turkey serum were separated by disc electrophoresis using polyacrylamide gel as the supporting medium In this laboratory we have been unable to adapt polyacrylamide gel electrophoresis procedures for turkey serum that had been described by previous workers as successful in separating the isoenzymes of avian alkaline phosphatase. A rapid and repeatable procedure that yielded excellent resolution of alkaline phosphatase isoenzymes was devised. This procedure involved a step-by-step determination of the following: a suitable gel system, buffer system, staining procedure and sample preparation.

Alkaline Phosphatase↗

PubMed1989

Tryptophan phosphorescence as a monitor of the structural role of metal ions in alkaline phosphatase.

The phosphorescence properties of Trp109 in alkaline phosphatase from Escherichia coli have been utilized to probe the conformation of the polypeptide following the removal of metal ions, reconstitution with Zn2+ and Cd2+ and phosphorylation. The complete removal of metal ions induces a drastic loosening of the protein structure that extends to the inner core of the macromolecule. While binding of a single metal ion/subunit (A-site occupancy) restores the holoconformer, practically no structuring effect is observed upon B-site occupancy by the second incoming metal ion. An exception to this rule occurs at alkaline pH and when the adjacent subunit in the dimer is metal-free. Under these circumstances a conformation of the subunit more compact than that of the fully saturated dimer manifests some degree of communication across the subunit interface. The binding of more than two metal ions/monomer generally destabilizes the protein, the effect being more pronounced at acid pH. Finally, the binding of inorganic phosphate restores the native-like configuration abolishing any destabilization induced by excess metal ions and acid pH. If the negative cooperativity towards metal binding to A sites in doubly metalated forms at pH 8 is in substantial agreement with 113Cd-NMR data, the equivalence in conformation between Zn2+- and Cd2+-reconstituted alkaline phosphatase emphasizes that no serious structural changes are introduced by the metal replacement.

Alkaline Phosphatase↗

PubMed1970

Induction of rat liver alkaline phosphatase: the mechanism of the serum elevation in bile duct obstruction.

Bile duct ligation in the rat leads to a rapid increase in hepatic and serum alkaline phosphatase activity. Within 12 hr after bile duct ligation, hepatic alkaline phosphatase has increased 7-fold and serum alkaline phosphatase activity 2(1/2)-fold. The elevation in the serum activity is completely due to an increase in an isozyme that appears to originate in the liver. This serum isozyme and liver phosphatase, both partially purified by DEAE-cellulose column chromatography, have identical Michaelis constants, pH optima, and rates of heat denaturation. These isozymes migrate identically when subjected to electrophoresis on polyacrylamide gel, and their migration rates are equally slowed after neuraminidase digestion. The data suggest that the rise in hepatic alkaline phosphatase activity is dependent on de novo protein synthesis. Cycloheximide, in a dose that inhibited incorporation of leucine-(14)C into protein by 68%, inhibited the rise in liver phosphatase by 98% and that in serum by 80%. The rise in liver phosphatase activity could not be accounted for by simple retention of alkaline phosphatase that would normally appear in bile. The rise in liver activity after bile duct ligation was 240 times greater than the amount of phosphatase that normally appears in bile over a similar period of time. Cycloheximide had no effect on the bile duct ligation-induced changes in the serum and liver glutamic pyruvic transaminase.

Alanine Transaminase↗

PubMed1987

Alkaline phosphatase inactivation by mixed function oxidation systems.

Alkaline phosphatase is inactivated by mixed function oxidation systems. OH. radicals, generated via an ascorbate-modified Haber-Weiss cycle or a Fenton-type reaction, seem to be responsible for the protein oxidative damage. Experiments with hydroxyl radical scavengers, enzyme substrates, products, and metal cofactors suggest that a "site-specific" radical attack takes place at or near the active center. Vitamin E fails to protect alkaline phosphatase; uric acid, instead, is particularly effective in shielding the protein against covalent modifications.

Alkaline Phosphatase↗

PubMed1983

Adaptive increase in phytate digestibility by phosphorus-deprived rats and the relationship of intestinal phytase (EC 3.1.3.8) and alkaline phosphatase (EC 3.1.3.1) to phytate utilization.

1. The effects of phosphorus deprivation on phytate digestibility, phosphorus utilization and intestinal phytase (EC 3.1.3.8) and alkaline phosphatase (EC 3.1.3.1) in rats were investigated. 2. P deprivation was achieved by giving rats a diet containing 3 g P/kg and resulted in hypophosphataemia, hypercalcaemia, hypercalciuria, and lower levels of P absorbed and retained, and calcium retained. 3. Rats adapted to P deprivation by increasing the digestion of total dietary-P and phytate-P. 4. Levels of intestinal alkaline phosphatase and alkaline phytase were not different between the two treatment groups. 5. P deprivation in the rats given the marginal-P diet may be a result of a lower absorption of total dietary-P or increased absorption of inositol phosphates formed during the enzymatic hydrolysis of phytate which are not readily utilized by the rat. 6. These results suggest that intestinal phytase and alkaline phosphatase do not play a role in the adaptive increase in phytate digestibility by rats given marginal-P diets. The adaptation may result from enhanced phytase or alkaline phosphatase synthesis by the gastrointestinal microflora stimulated by a lower level of P in the digesta.

6-Phytase↗

PubMed1992

Clinical significance of a low serum alkaline phosphatase.

The reasons for a low serum alkaline phosphatase activity were reviewed in a 1-yr retrospective study of all the results emanating from a central, university hospital laboratory. Apart from occasional errors, hypophosphatasaemia was encountered in neonates undergoing exchange transfusions with plasma, in patients (particularly children) receiving chemotherapy and during cardio-pulmonary bypass. Low activity values were recorded in several individuals in the absence of any obvious cause. This would suggest that the definition of the lower limit of the reference range for alkaline phosphatase is arbitrary, thus limiting the use of low serum activity as a marker of disease.

Adult↗

PubMedDate not supplied

[Immobilization of enzymes on carriers with magnetic properties: the search for optimum conditions for immobilization of alkaline phosphatase from the chicken gut].

Chicken intestine alkaline phosphatase was immobilized on seven carriers by adsorption or covalent binding. The efficiency of immobilization depended on the carrier, immobilization method, and initial enzyme concentration. The heat stability of the soluble and immobilized enzyme preparations, and the dependence of the enzyme activity on pH and Zn2+ and Mg2+ ions were studied. Alkaline phosphatase immobilized on magnetic carriers displayed lower heat stability than the soluble enzyme. The temperature optima of the immobilized and soluble enzymes were 80 and 60 degrees C; the pH optima were 9.0 and 8.0, respectively. Ions Mg2+ and Zn2+ were found to play an important role in the activation of both soluble and immobilized enzymes and their stabilization at high temperatures.

Alkaline Phosphatase↗

PubMed1987

Pst I restriction fragment length polymorphism of the human placental alkaline phosphatase gene in normal placentae and tumors.

The structure of the human placental alkaline phosphatase gene from normal term placentae was studied by restriction enzyme digestion and Southern blot analysis using a cDNA probe to the gene for the placental enzyme. The DNA digests fall into three distinct patterns based on the presence and intensity of an extra 1.1-kilobase Pst I band. The extra 1.1-kilobase band is present in 9 of 27 placenta samples, and in 1 of these samples the extra band is present at double intensity. No polymorphism was revealed by digestion with restriction enzymes EcoRI, Sma I, BamHI, or Sac I. The extra Pst I-digestion site may lie in a noncoding region of the gene because no correlation was observed between the restriction fragment length polymorphism and the common placental alkaline phosphatase alleles identified by starch gel electrophoresis. In addition, because placental alkaline phosphatase is frequently re-expressed in neoplasms, we examined tissue from ovarian, testicular, and endometrial tumors and from BeWo choriocarcinoma cells in culture. The Pst I-DNA digestion patterns from these cells and tissues were identical to those seen in the normal ovary and term placentae. The consistent reproducible digestion patterns seen in DNA from normal and tumor tissue indicate that a major gene rearrangement is not the basis for the ectopic expression of placental alkaline phosphatase in neoplasia.

Alkaline Phosphatase↗