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PubMed1985

[Alkaline phosphatase activity of normal and transformed cultured human cells].

A study was made of the relationship between the activity of alkaline phosphatase and the proliferation of cultured human cells with different replicative potentials. It is shown that alkaline phosphatase plays a role as one of endogenic stimulators of cellular proliferation. The ageing of diploid cells is accompanied by a decrease in the enzyme activity. Maximum activity was observed during a period of logarithmic cell growth. Addition of placental alkaline phosphatase to the synchronized diploid cells stimulated DNA synthesis in the S-phase of the cell cycle. Heteroploid cells with a high growth rate possessed a 30-100 times higher alkaline phosphatase activity than in the diploid cells. Under certain conditions alkaline phosphatase may presumably function as a proteinkinase.

Alkaline Phosphatase↗
PubMed1985

Intralumenal alkaline phosphatase of the calf intestine.

About 80% of the total activity of the alkaline phosphatase of the calf intestine was found within the lumen and only 20% in the mucosa. The specific activity of the intralumenal enzyme fraction was about ten times higher than that of the mucosa enzyme. Differential centrifugation experiments demonstrated that the intralumenal enzyme of the proximal gut segment can be found in a fraction which exhibits sedimentation behaviour like a microsomal fraction, whilst the intralumenal enzyme of the distal gut segment was found to be soluble in the supernatant after centrifugation at 135000 X g for 60 min. Ouchterlony double diffusion analysis and antibody inhibition of alkaline phosphatase by antisera against the mucosa enzyme demonstrate that the two forms of intestinal enzyme have apparently identical antigenicity. Purified alkaline phosphatase obtained from the intralumenal fraction and from mucosa exhibits closely related pH-optima, Michaelis constants, amino acid inhibition type and inhibition constants. The results of large scale release of alkaline phosphatase bound to microvesicles are discussed with regard to results of morphological investigations in different tissues and cell cultures demonstrating the formation of intestinal membrane bodies and cell extrusion.

Alkaline Phosphatase↗
PubMed1981

Effect of cobalt on synthesis and activation of Bacillus licheniformis alkaline phosphatase.

The effect of CO2+ on the synthesis and activation of Bacillus licheniformis MC14 alkaline phosphatase has been shown by the development of a defined minimal salts medium in which this organism produces 35 times more (assayable) alkaline phosphatase than when grown in a low-phosphate complex medium or in the defined medium without cobalt. Stimulation of enzyme activity with cobalt is dependent on a low phosphate concentration in the medium (below 0.075 mM) and continued protein synthesis. Cobalt stimulation resulted in alkaline phosphate production being a major portion of total protein synthesized during late-logarithmic and early-stationary-phase culture growth. Cells cultured in the defined medium minus cobalt, or purified enzyme partially inactivated with a chelating agent, showed a 2.5-fold increase in activity when assayed in the presence of cobalt. Atomic spectral analysis indicated the presence of 3.65 +/- 0.45 g-atoms of cobalt associated with each mole of purified active alkaline phosphatase. A biochemical localization as a function of culture age in this medium showed that alkaline phosphatase was associated with the cytoplasmic membrane and was also found as a soluble enzyme in the periplasmic region and secreted into the growth medium.

Alkaline Phosphatase↗
PubMed1979

N-alkaline phosphatase: a potential disease marker for lymphoproliferative disorders.

A characteristic alkaline phosphatase (orthophosphoric monoester hydrolase, alkaline pH optimum, EC 3.1.3.1) was detected in the sera of most patients with infectious mononucleosis, acute and chronic lymphatic leukaemia, non-Hodgkin's lymphoma, Burkitt's lymphoma and nasopharyngeal carcinoma. The enzyme was also present in the sera of nine out of 26 patients with cancer of the cervix. N-APase in these cases counted 30-100% of the total alkaline phosphatase activity. N-APase was absent from the sera of healthy individuals and of patients with acute and chronic granulocytic leukaemia, breast cancer, colon cancer, rheumatoid arthritis, ulcerative colitis, systemic lupus erythematosis, hepatitis and obstructive jaundice. Only three of 22 patients with Hodgkin's disease showed n-apase activity in the serum. In infectious mononucleosis the presence of N-APase activity was well correlated with the clinical course. In 13 cases studied, the clinical improvement was associated with the decrease or disappearance of N-APase activity. N-APase activity could not be detected in white cells of acute myeloid leukaemic patients, nor in the cells of myeloid blastic crisis of chronic granulocytic leukaemia. It was present in the cells of lymphoid blastic crisis of chronic granulocytic leukaemia.

Adult↗
PubMed1978

The function of carbohydrate moiety and alteration of carbohydrate composition in human alkaline phosphatase isoenzymes.

The relationship between the structure and function of alkaline phosphatase (orthoposphoric monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) isoenzymes is under investigation in a number of laboratories. The present study deals with the effects of glycosidase digestion on the alkaline phosphatase isoenzymes. Changes in physicochemcial properties, activity, affinity for various lectins and blood group antisera, carbohydrate composition and biological half-life were investigated. The desialylated hepatic enzyme was shown to be more heat labile and more sensitive to protease digestion in the presence of 0.5% sodium dodecyl sulfate than native hepatic enzyme. Helix contents of the native and desialated hepatic enzyme were calculated to be 39.0 and 30.8%, respectively, and apparent molecular weights 175,000 and 167,000, respectively. Intestinal enzyme preparations treated with alpha-mannosidase, exo-N-acetyl-Dglucosaminidase and endo-N-acetyl-D-glucosaminidase-D displayed a decrease in enzyme activity. Among these, the alpha-mannosidase-treated enzyme activity was the most clearly reduction. The maximum activity of the alpha-mannosidase-treated intestinal enzyme was observed to change from 40 mM Mg2+ to 5--10 mM Mg2+.

Alkaline Phosphatase↗
PubMed1974

Mutationally altered rate constants in the mechanism of alkaline phosphatase.

The hydrolysis of phosphate esters by a mutationally altered alkaline phosphatase from Escherichia coli was studied by both steady-state and transient-kinetic methods. The difference between the catalytic-centre activities of the mutationally altered and the wild-type alkaline phosphatases was found to vary with pH and at optimal pH values the modified enzyme had the higher activity. Stopped-flow experiments at acidic pH values showed that transient product formation by the mutationally altered enzyme was faster than that with the wild-type enzyme whereas the rate of the steady state was slower. In the alkaline pH region, the transient was observed in the reaction of only the modified enzyme and not the wild type. These observations permit a fuller characterization of the individual steps in the catalytic mechanism of alkaline phosphatase than is possible by study of only the wild-type enzyme.

Alkaline Phosphatase↗
PubMed1975

Role of metal ions in Escherichia coli alkaline phosphatase. A study of the metal-water interaction by nuclear relaxation rate measurements on water protons.

The binding of metal to alkaline phosphatase from Escherichia coli and the binding of water and orthophosphate to the Me-2+-enzyme binary complex have been examined by water proton relaxation rate (PRR) measurements. Titration of the three paramagnetic metals, Mn2+, Cu2+, and Co2+, into apoalkaline phosphatase and the titrations of apoenzyme into metal have been carried out. Analysis of the spin-lattice relaxation rates for these titrations and of Scatchard binding curves derived from these results, as well as EPR data, show four tight manganese sites, between two and three tight copper sites, or four cobalt sites per enzyme dimer of molecular weight 80,000. The multiple sites for each metal are indistinguishable by these magnetic resonance techniques. Both the spin-lattice- and spin-spin-relaxation rates exhibit a negative temperature coefficient, showing that these processes are not exchange-limited. From a frequency dependence study of T-1 and from the T-1:T-2 ratio measured at 220 MHz, correlation times from the water-enzyme complexes have been estimated. For H20-Mn-2+-alkaline phosphatase, gamma c equals 1.55 times 10-9 s; for H20-Cu-2+ -alkaline phosphatase, gamma c equals 1.82 times 10-s; and for the cobalt complex, gamma c equals 1.0 times 10-12 s at 4 degrees. Assuming 1 water molecule bound per metal site, these correlation times correspond to the following water-metal distances: gamma (A) is 4.0 A for Mn-2+-H20, 3.4 A for Cu-2+-H20, and 2.8 A for Co-2+-H20. Thus, water is shown to bind directly to the metal atoms of alkaline phosphatase. The correlation between the length of the water-metal bond and the relative activity of the various metalloenzymes support the importance of this binding in the monophosphoesterase reaction catalyzed by alkaline phosphatase. Addition of excess orthophosphate to any of the water-metalloenzyme complexes does not displace an exchangeable water molecule from the metal site. The Mn-PO-4 distance which we have reported earlier (Zukin, R.S., Hollis, D.P., and Gray, G.A. (1973) Biochem. Biophys. Res. Commun. 53, 238) to be 7.3 A is consistent with this finding and suggests a model in which Pi binds to Mn-2+-alkaline phosphatase through a water bridge.

Alkaline Phosphatase↗
PubMed1976

Alkaline phosphatase activity of epididymal contents in boars with normal or reduced spermatogenesis.

The alkaline phosphatase activity was measured in testicular fluid and in epididymal plasma from caput and cauda epididymidis in boars with normal sperm production and in boars in which the number of spermatozoa passing from the testis to the epididymidis was reduced. The testicular fluid and the epididymal plasma from caput epididymidis contained low amounts of alkaline phosphatase in comparison with epididymal plasma from the cauda. This applies to both groups of boars e.g. boars with normal as well as with totally lacking or lowered sperm production. As no fluid resorption takes place between caput and cauda the distal part of the epididymidis must be the main production site for alkaline phosphatase. The production there is not related to the presence of spermatozoa in the duct. In the caput, on the other hand, it seems that the level of alkaline phosphatase in some way is influenced by the sperm supply to the duct.

Alkaline Phosphatase↗
PubMed1998

A superfamily of metalloenzymes unifies phosphopentomutase and cofactor-independent phosphoglycerate mutase with alkaline phosphatases and sulfatases.

Sequence analysis of the probable archaeal phosphoglycerate mutase resulted in the identification of a superfamily of metalloenzymes with similar metal-binding sites and predicted conserved structural fold. This superfamily unites alkaline phosphatase, N-acetylgalactosamine-4-sulfatase, and cerebroside sulfatase, enzymes with known three-dimensional structures, with phosphopentomutase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, phosphoglycerol transferase, phosphonate monoesterase, streptomycin-6-phosphate phosphatase, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1, and several closely related sulfatases. In addition to the metal-binding motifs, all these enzymes contain a set of conserved amino acid residues that are likely to be required for the enzymatic activity. Mutational changes in the vicinity of these residues in several sulfatases cause mucopolysaccharidosis (Hunter, Maroteaux-Lamy, Morquio, and Sanfilippo syndromes) and metachromatic leucodystrophy.

Alkaline Phosphatase↗
PubMedDate not supplied

Alkaline phosphatase histochemistry discriminates peritubular cells in primary rat testicular cell culture.

Histochemical demonstration of alkaline phosphatase activity appears to be useful in identifying rat peritubular cells in primary testicular cell culture. In both frozen sections of rat testis and Mirsky's fixed, methacrylate-embedded rat testis, the reaction product localized primarily in peritubular cells, vascular endothelium and occasionally in interstitial cells, with much smaller amounts of reaction product associated with elongating spermatids in the germinal epithelium. Occasional late-stage tubules (X-XIV) showed weak reactivity in the epithelium, associated with spermatocytes or Sertoli cells. Ultrastructurally, Gomori-method reaction product was localized to peritubular cells, lymphatics, and spermatogonia in stage VII; no staining was found consistently in Sertoli cells. In isolated cell preparations enriched for Sertoli and germ cells, 1 to 8% of the cells demonstrated alkaline phosphatase activity, while greater than 50% of the cells stained positive for alkaline phosphatase activity in peritubular-enriched fractions. The histochemical demonstration of alkaline phosphatase activity can be useful for identifying peritubular cells in primary cultures of testicular cells.

Alkaline Phosphatase↗
PubMed1988

A Technique for Automated Quantification of Canine Glucocorticoid-induced Isoenzyme of Alkaline Phosphatase.

A sensitive assay for the corticosteroid-induced alkaline phosphatase isoenzyme (CAP), adaptable to most clinical chemistry autoanalyzers, is described and validated. This assay is based on the greater than 98% inhibition of liver alkaline phosphatase isoenzyme (LAP) activity with 4.2 mM levamisole, as compared to the 42% inhibition of CAP activity. Analysis of serum with total alkaline phosphatase (AP) activity within the reference range, resulted in a reference range of 0 to 19 U/L for CAP activity. Analysis of serum from 160 clinical patients with AP activity above the reference range, revealed 73% with increased CAP activity ranging from 20 to 7,000 U/L. The diagnostic significance of this increased CAP activity is discussed.

Journal Article↗
PubMed1955

Intracellular distribution of alkaline phosphatase in rat liver cells.

1. Cytochemical studies of the intracellular distribution of alkaline phosphatase in rat liver have been made, using a fractionation procedure recently developed in this laboratory (8) and a similar but modified method not described previously. Aqueous media were used in both cases. 2. The alkaline phosphatase was found to consist of two forms, one of which is strongly activated by magnesium and one of which is not sensitive to this metal. 3. The form of the enzyme that is not activated by magnesium occurs mainly in the nuclear fraction, where it seems to be rather firmly bound. Some of this form of the enzyme is also found in the microsomes, but very little if any occurs in the soluble supernatant fraction. 4. The form of alkaline phosphatase which is activated by magnesium occurs mainly in the soluble supernatant fraction, but what is believed are significant amounts also occur in nuclei. A significant portion of this form of the enzyme can be extracted from the isolated nuclei with cold, isotonic saline solution. Some activity of this form of the enzyme is also found in the microsomal fraction. 5. Mitochondria appear to contain relatively little alkaline phosphatase of either kind. 6. The concept of a porous nuclear membrane has been invoked to explain some of the results obtained in this work. It is postulated that part at least of the form of the enzyme that is activated by magnesium is free to diffuse back and forth through pores in the nuclear membrane, whereas this is considered not to be possible for the form of the enzyme that is insensitive to magnesium as a result of the firm binding of the latter to nuclear substance.

Alkaline Phosphatase↗
PubMed1978

Alkaline phosphatase from Bacillus licheniformis. Solubility dependent on magnesium, purification and characterization.

The membrane-associated alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from Bacillus licheniformis MC14, a facultative thermophile, was purified to homogeneity in buffer containing 0.2 M Mg2+. The alkaline phosphatase purified in this manner is insoluble upon removal of the magnesium by dialysis. This insoluble alkaline phosphatase has been characterized and compared to the previously purified heat-solubilized enzyme (Hulett-Cowling, F.M. and Campbell, L.L. (1971) Biochemistry 10, 1364--1371).

Alkaline Phosphatase↗
PubMed1972

Physiological factors in the regulation of alkaline phosphatase synthesis in Escherichia coli.

Alkaline phosphatase is induced in excess phosphate media by starvation either for pyrimidines or for guanine. Induction is observed both during starvation, after a lag period, and following a period of starvation. Induction is not caused by a lowering of the internal orthophosphate pool, but is linked to alterations in the levels of the nucleotide pools. Experiments with purine-requiring mutants suggest that phosphatase is induced in wild-type strains by an adenine nucleotide. Mutations in the phoR gene can produce differential responses to the different starvation regimes.

Adenine↗
PubMed1975

Analysis of heat inactivation curves of alkaline phosphatase isoenzymes in serum.

The course of the decline in alkaline phosphatase activity during exposureof serum samples to a temperature of 56 degree C can be resolved into two phases. These represent the exponential decay of an enzyme component with an average half-inactivation time of 112 seconds and of a second component with an average half-inactivation time of 456 seconds. The more rapid fall is due to inactivation of bone alkaline phosphatase and the slower to inactivation of liver and, when present, intestinal phosphatases. The half-inactivation times of the different enzyme species show considerable variation from one serum sample to another. The implications of this variation for methods of estimating the relative proportions of alkaline phosphatase isoenzymes by selective inactivation procedures are discussed.

Alkaline Phosphatase↗
PubMed1988

5'-Nucleotide phosphodiesterase and alkaline phosphatase in tumor cells: evidence for existence of novel species in the cytosol.

Characteristics of 5'-nucleotide phosphodiesterase (phosphodiesterase I, EC 3.1.4.1) and alkaline phosphatase (EC 3.1.3.1) activities in tumor cell lines of human and murine origin were examined. Of the 15 cell lines tested, 5'-nucleotide phosphodiesterase activity in 13 cell lines and alkaline phosphatase activity in 10 cell lines were inhibited by N-ethylmaleimide and activated by dithiothreitol (N-ethylmaleimide-sensitive), and suggested to be SH-enzymes. In contrast, the two phosphohydrolases from normal tissues were inactivated by dithiothreitol, but not by N-ethylmaleimide (dithiothreitol-sensitive). There was only one tumor cell line in which both activities were dithiothreitol-sensitive. Human hepatoma PLC/PRF/5 cells appear to possess both types of 5'-nucleotide phosphodiesterase and alkaline phosphatase, and the subcellular distribution of these enzymes in this cell line was investigated. Dithiothreitol-sensitive 5'-nucleotide phosphodiesterase and alkaline phosphatase of PLC/PRF/5 cells were localized in the plasma membrane as in normal tissues, but N-ethylmaleimide-sensitive phosphohydrolases were soluble cytosolic proteins. N-Ethylmaleimide-sensitive 5'-nucleotide phosphodiesterase and alkaline phosphatase activities from other cell lines were also recovered in the cytosol. Molecular masses of cytosolic N-ethylmaleimide-sensitive phosphohydrolases were apparently smaller than their membrane-bound dithiothreitol-sensitive counterparts, as judged from gel filtration. It was concluded that many tumor cell lines lack plasma membrane 5'-nucleotide phosphodiesterase and alkaline phosphatase, but express enzymes with similar activities in the cytosol, with properties clearly distinguishable from enzymes so far characterized.

Alkaline Phosphatase↗
PubMed1974

Fluorotyrosine alkaline phosphatase from Escherichia coli: preparation, properties, and fluorine-19 nuclear magnetic resonance spectrum.

Alkaline phosphatase (EC 3.1.3.1) containing m-flurotyrosine has been prepared from E. coli grown in the presence of m-flurotyrosine. The kinetic properties of the m-fluorotyrosine enzyme measured with p-nitrophenylphosphate at pH 8.0 and dinitrophenylphosphate at pH 5.5 are essentially the same as those of normal alkaline phosphatase. However, the ability of the m-fluorotyrosine protein to refold active enzyme after acid denaturation, while unchanged at pH 5.8, was markedly decreased at pH 7.6. This result implies that the tyrosines must be in their protonated form for the protein to refold, reassociate, and take on zinc. The (19)F nuclear magnetic resonance spectrum of m-fluorotyrosine alkaline phosphatase contains resolved resonances corresponding to different chemical environments for each m-fluorotyrosine in the folded protein. This demonstrates that (19)F nuclear magnetic resonance spectroscopy of enzymes specifically labeled with (19)F, even with enzymes as large as alkaline phosphatase (molecular weight, 86,000), will provide a very valuable probe for conformational changes in proteins.

Alkaline Phosphatase↗