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PubMed2000

Effects of oxodiperoxovanadate (V) complexes on the activity of green crab (Scylla serrata) alkaline phosphatase.

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme that catalyzes the nonspecific hydrolysis of phosphate monoesters. The effects of some pollutants in seawater on the activity of the enzyme will result in the loss of the biological function of the enzyme, which will affect the exuviating crab shell and threaten the survival of the animal. In the present paper, the effects of four oxodiperoxovanadate (V) complexes on the activity of green crab alkaline phosphatase have been studied. The results show that these vanadate derivatives can lead to reversible inactivation. The equilibrium constants for binding of inhibitors with the enzyme and/or the enzyme-substrate complexes have been determined. The results show that sodium (2,2;-bipyridine)oxodiperoxovanadate, pV(bipy), and potassium oxodiperoxo-(1,10-phenanthroline)vanadate, pV(phen), are competitive inhibitors, while potassium picolinato-oxodiperoxo-vanadate, pV(pic), and oxalato-oxodiperoxovanadate, pV(ox), are mixed-type inhibitors. These results suggest that pV(bipy) is a considerably more potent competitive inhibitor than pV(phen) and that the competitive inhibition effect of pV(pic) is stronger than that of pV(ox), but the non-competitive inhibition effect of pV(ox) is stronger than that of pV(pic).

2,2'-Dipyridyl↗

PubMed1998

Extremely high levels of alkaline phosphatase in hospitalized patients.

We determined the diseases associated with extremely high levels of alkaline phosphatase in hospitalized patients. Computerized laboratory records of the Hospital of Saint Raphael identified all inpatients who had elevations of alkaline phosphatase above 1,000 U/l from April 1994 to September 1995. Thirty-seven inpatients with alkaline phosphatase levels above 1,000 U/l were identified. Six had bone involvement from malignancy or Paget's disease and were eliminated from further analysis, and 31 patients were included in the study. Levels of alkaline phosphatase ranged from 1,014 to 3,360 U/l. Ten patients had sepsis as the cause of the elevated alkaline phosphatase. These included gram-negative organisms, gram-positive organisms, and two patients with fungal sepsis. Seven of 10 patients with sepsis had an extremely high alkaline phosphatase level and a normal bilirubin, 3 of 10 patients with sepsis also had acquired immunodeficiency syndrome (AIDS). Eight patients had biliary obstruction, 7 with malignant obstruction and 1 with a common bile duct stone. Nine patients had AIDS. The cause of the elevated alkaline phosphatase in these included three with sepsis, three with mycobacterium avium intracellulare (MAI) infection, two with cytomegalovirus infection, and one with Dilantin toxicity. Three patients had diffuse liver metastases. Finally, four patients had benign intrahepatic disease, including one patient with liver hemangiomas, one patient with sarcoid hepatitis, one patient with lead toxicity, and one patient with drug-induced cholestasis. Extremely high elevations of alkaline phosphatase are most frequently seen in patients with sepsis, malignant obstruction, and AIDS. Patients with sepsis can have an extremely high alkaline phosphatase level and a normal bilirubin. A variety of other causes were also noted.

Adult↗

PubMedDate not supplied

Increased synthesis of rat liver alkaline phosphatase by bile duct ligation.

The following studies were done to determine the mechanism of the increase in rat liver alkaline phosphatase activity after bile duct ligation. Antiserum was raised in rabbits to highly purified rat liver alkaline phosphatase. In immune titration experiments, the 350% increase in rat liver alkaline phosphatase activity caused by bile duct ligation was paralleled by a similar increase in immunoprecipitated alkaline phosphatase protein. In a second set of experiments, rat liver alkaline phosphatase was labeled with L-[3H]leucine injected into portal veins. Alkaline phosphatase was purified by antibody affinity chromatography followed by immunoprecipitation. The incorporation of L-[3H]leucine into alkaline phosphatase was significantly higher in bile duct-ligated rats than in controls, 68,357 +/- 7,144 vs. 19,297 +/- 3,076 dpm per gm liver (p less than 0.001) and 349 +/- 36 vs. 104 +/- 17 dpm per gm protein (p less than 0.001). In a third set of experiments, the incorporation of L-3H-amino acid into highly purified rat liver alkaline phosphatase was measured. Rat liver alkaline phosphatase was purified by means of sequential N-butanol extraction, antibody affinity column chromatography, preparative polyacrylamide gel electrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was significantly more L-3H-amino acid incorporated into alkaline phosphatase in bile duct-ligated rats compared to sham-operated rats, 3,565 vs. 704 dpm per gm liver and 19,656 vs. 3,843 dpm/gm protein. The data suggest that bile duct ligation increases the synthesis of rat liver alkaline phosphatase.

Alkaline Phosphatase↗

PubMed1987

Control of placental alkaline phosphatase gene expression in HeLa cells: induction of synthesis by prednisolone and sodium butyrate.

HeLa S3 cells produce an alkaline phosphatase indistinguishable from the enzyme from human term placenta. The phosphatase activity in these cells was induced by both prednisolone and sodium butyrate. Both agents stimulated de novo synthesis of the enzyme. The increase in phosphatase activity paralleled the increase in immunoactivity and biosynthesis of placental alkaline phosphatase. The fully processed phosphatase monomer in control, prednisolone-treated or butyrate-treated cells was a 64.5 K polypeptide, measured by both incorporation of L-[35S]methionine into enzyme protein and active-site labeling. The 64.5 K polypeptide was formed by the incorporation of additional N-acetylneuraminic acid moieties to a precursor polypeptide of 61.5 K. However, this biosynthetic pathway was identified only in butyrate-treated cells. In prednisolone-treated cells, the processing of 61.5 K to the 64.5 K monomer was accelerated, and the presence of the 61.5 K precursor could only be detected by either neuraminidase or monensin treatment. Phosphatase mRNA which comigrated with the term placental alkaline phosphatase mRNA of 2.7 kilobases was induced in the presence of either prednisolone or butyrate. Alkaline phosphatase mRNA in untreated HeLa S3 cells migrated slightly faster than the term placental alkaline phosphatase mRNA. Butyrate also induced a second still faster migrating alkaline phosphatase mRNA. Both prednisolone and butyrate increased the steady-state levels of placental alkaline phosphatase mRNA. Our data indicate that the increase in phosphatase mRNA by prednisolone and butyrate resulted in the induction of alkaline phosphatase activity and biosynthesis in HeLa S3 cells. Furthermore, both agents induced the expression of different alkaline phosphatase gene transcripts without altering its protein product.

Alkaline Phosphatase↗

PubMed1978

Activity of alkaline phosphatase in the healing rat liver after hepatectomy.

Changes of alkaline phosphatase activity in the process of healing liver injuries of rats on which partial hepatectomy was performed were the subject of our researches. Reaction on alkaline phosphatase was performed on cryostat sections according to the method of Gomori-Takamatsu. It has been found that granulated precipitate of cobalt sulphide was localized in the cytoplasm of granulocytes, macrophages and fibroblasts. A diffusive reaction comprized clusters of newly created collagen fibres. In early stages in the centres of necrosis, the activity of alkaline phosphatase increased, in later periods a lack of the activity was observed. Steatosis of liver tissue does not correlate with the intensity of the reaction. Lack of alkaline phosphatase activity is characteristic for old cicatrices. The authors discuss the effects of the histogenetical process during healing of liver injury on the results of the histoenzymatic reactions. The authors suggest that alkaline phosphatase is involved in the mechanism of re-building collagen fibres. It is stressed in the conclusion that the localization and intensity of alkaline phosphatase in the area of healing liver lesions are variable depending on the period of healing wound of the liver and the influence of other complications.

Alkaline Phosphatase↗

PubMed1994

Effect of alkaline phosphatase on thromboxane mimetic induced platelet activation.

Recently it has been reported that alkaline phosphatase selectively inhibited thromboxane mimetic induced platelet aggregation and secretion suggesting that the phosphorylation state on the platelet surface may be important for thromboxane induced platelet activation. We report here studies attempting to elucidate the mechanism of action of alkaline phosphatase. Washed human platelet aggregation induced by the thromboxane mimetic IBOP was completely abolished when incubated with alkaline phosphatase (1 unit/ml) for 5 min. The effect was inhibited by co-incubation with 5mM phosphate. Binding studies using [125I]BOP showed that neither the affinity of IBOP for the receptor (control: 9.2 +/- 2.1 nM, alkaline phosphatase: 7.9 +/- 1.8 nM) nor the Bmax (control: 1780 +/- 320 sites/plt. alkaline phosphatase: 1920 +/- 290 sites/plt) were effected by alkaline phosphatase treatment. GTPase activity was measured in platelet membranes treated with and without alkaline phosphatase as measured by IBOP induced hydrolysis of [gamma-32P]GTP. The EC50 values for IBOP induced GTPase were similar whereas the maximum amount of released Pi in the control membranes was more than two fold greater than in alkaline phosphatase treated membranes. These studies suggest that thromboxane induced platelet activation may be dependent upon the phosphorylation state of the thromboxane receptor and/or closely associated protein.

Alkaline Phosphatase↗

PubMed1991

Bone-alkaline phosphatase as indicator of bone formation.

Bone-alkaline phosphatase was determined in patients at risk of osteoporosis due to treatment with oral corticosteroids, and in patients at risk of increased bone synthesis because of treatment with cyclosporin. Both a significant decrease of bone-alkaline phosphatase during corticosteroid treatment, and a significant increase of bone-alkaline phosphatase during cyclosporin treatment could be demonstrated. It is concluded that bone-alkaline phosphatase is a useful parameter for monitoring changes in bone formation.

Adult↗

PubMed1978

Alkaline phosphatase isoenzymes.

At the time of admission, 4 to 8% of all hospital admissions have a elevation of alkaline phosphatase. This high incidence of alkaline phosphatasemia has resulted in not only a need to identify the source of the elevated alkaline phosphatase; it has also led to a marked proliferation in laboratory procedures aimed at identifying the various isoenzymes of alkaline phosphatase. These various techniques are reviewed and their relative merits are discussed. No single technique adequately delineates all of the alkaline phosphatase isoenzymes, but the combination of heat fractionation, electrophoresis, and the measurement of an additional hepatobiliary enzyme has been shown to be able to identify the source of origin of more than 95% of all cases of alkaline phosphatasemia. The individual isoenzymes of alkaline phosphatase are discussed in detail, and the underlying pathophysiology is reviewed so that physiologic elevations of alkaline phosphatase are not confused with pathologic increases in alkaline phosphatase.

ABO Blood-Group System↗

PubMedDate not supplied

Serum alkaline phosphatase isoenzymes as markers of liver disease.

A perspective on serum alkaline phosphatase isoenzymes in liver disease is provided with a brief discussion of the location of the enzyme in liver and its presumed function. Mechanisms of entry of alkaline phosphatase into serum in liver disease are discussed. Characterization of high molecular weight alkaline phosphatase in obstructive jaundice is reviewed. The relationship between blood group O and the appearance of the intestinal enzyme in sera of such subjects with cirrhosis of liver is discussed. Properties of hepatoma alkaline phosphatase and the genesis of liver alkaline phosphatase in diseases not related to the liver are explored. Methods for detection of serum alkaline phosphatase isoenzymes in liver disease are discussed from the standpoint of the limitations of electrophoretic procedures, and the promise of procedures such as isoelectric focusing and high performance liquid chromatography that are currently non-routine.

Alkaline Phosphatase↗

PubMed1978

[A Study of the enzymes of the alkaline phosphatase in commercial sera (author's transl)].

We have examined alkaline phosphatase in five commercial sera employing three analyzers. Alkaline phosphatase in three samples is underestimated or overestimated according to the analyzers used. Therefore we have studied the isoenzymatic fractions employing the fractionation of alkaline phosphatase on celluloseacetate. Alkaline phosphatase isoenzymes have physical and biochemical marks different from human sera. In human sera on the contrary have not noticed any difference according to the analyser used.

Adult↗

PubMed1977

Disappearance rates of intravenously injected canine alkaline phosphatase isoenzymes.

The serum half-life of various canine alkaline phosphatase isoenzymes has been determined. Isoenzymes can be divided into 2 groups with regard to their half-life. Isoenzymes with a half-life of approximately 3 days include the hepatic alkaline phosphatase and the steroid-induced alkaline phosphatase. Those with a half-life of under 6 minutes include the alkaline phosphatase from placenta, kidney, intestine, and the hepatic and steroid-induced iosenzymes after removal of sialic acid. The inability to demonstrate some of the isoenzymes of alkaline phosphatase in serum of diseases animals is attributed to their short half-life. The relationship of the short half-life to the sialic acid moiety of the protein is discussed.

Alkaline Phosphatase↗

PubMed1984

Zn(II)-113Cd(II) and Zn(II)-Mg(II) hybrids of alkaline phosphatase. 31P and 113Cd NMR.

Methods have been developed for the addition of different metal ion species to the three distinct pairs of metal sites (A, B, and C) found in the dimer of apoalkaline phosphatase. This allows the preparation of hybrid alkaline phosphatases in which A and B sites of each monomer contain two different species of metal ion or the A and B sites of one monomer contain the same species of metal ion, while the adjacent monomer contains a second species. The following hybrids have been characterized in detail: (Zn(II)ACd(II)B)2 alkaline phosphatase, (Zn(II)AMg(II)B)2 alkaline phosphatase, (Cd(II)AZn(II)B)2 alkaline phosphatase, and (Zn(II)AZn(II]B)(Cd(II)ACd(II)B) alkaline phosphatase. 31P and, where appropriate, 113Cd NMR have been used to monitor the behavior of the covalent (E-P) and noncovalent (E X P) phosphointermediates and of the A and B metal ions. From the pH dependencies of the E-P in equilibrium E X P in equilibrium E + Pi equilibria, it is clear that A site metal is the dominant influence in dephosphorylation of E-P and may have a coordinated water molecule, which ionizes to ZnOH- at a low pH providing the nucleophile for dephosphorylation. A site metal also serves to coordinate phosphate in the E X P complex. B site metal has a much smaller effect on dephosphorylation rates, although it does dramatically alter the Pi dissociation rate, which is the rate-limiting step for the native enzyme at alkaline pH, and is probably important in neutralizing the charge on the phosphoseryl residue, thus potentiating the nucleophilic attack of the OH- bound at A site. Phosphate dissociation is slowed markedly by replacement of B site zinc by cadmium. There is clear evidence for long range effects of subunit-subunit interactions, since metal ion and phosphate binding at one active center alters the environments of A and B site metal ions and phosphoserine at the other active site.

Alkaline Phosphatase↗

PubMed1987

Molecular nature of three liver alkaline phosphatases detected by drug administration in vivo: differences between soluble and membranous enzymes.

1. Activities of alkaline phosphatase, liver-membranous, liver-soluble and serum-soluble, were dramatically induced in dogs by treatment with both phenobarbital and brovanexine. The treatment induced a 17-fold increase in membranous, a 155-fold increase in soluble, and a 105-fold increase in serum alkaline phosphatases. 2. There was no difference in the enzymatic behavior of the three forms of alkaline phosphatase, on heat stability, amino acid inhibition and optimum pH. 3. When the three alkaline phosphatases were treated initially with n-butanol, their apparent molecular size was identical. After treatment with phosphatidylinositol-specific phospholipase C, the liver-soluble and serum-soluble alkaline phosphatase were of the same molecular size. Liver-membranous alkaline phosphatase, however, was larger in molecular size than the other two forms, suggesting a difference between soluble and membranous alkaline phosphatase forms. 4. In terms of the sugar moiety of the three alkaline phosphatase forms, the membranous enzyme showed more of the higher affinity fraction and less of the lower affinity fraction of concanavalin A, compared with the soluble enzymes. 5. Consequently, it is possible that the membranous enzyme may be solubilized by an enzyme such as phosphatidylinositol-specific phospholipase C and modify further the sugar moiety of alkaline phosphatase molecules, resulting in serum alkaline phosphatase transfer from the soluble enzyme in liver.

Alkaline Phosphatase↗

PubMed1979

Separation and purification of the alkaline phosphatase and a phosphodiesterase from Halobacterium cutirubrum.

1. Halobacterium cutirubrum alkaline phosphatase is associated in crude extracts with a phosphodiesterase. 2. The enzymes were stabilized in buffers containing both (NH4)2SO4 and 10 mM-Mn2+. 3. Adsorption chromatography on Sepharose 6B/agarose-gel columns in the presence of 1.4M-(NH4)2SO4 gave a phosphatase-free phosphodiesterase and the alkaline phosphatase associated with some phosphodiesterase activity. 4. Further chromatography of the separated enzymes gave a good recovery of greater than 600-fold purified phosphodiesterase and greater than 3000-fold purified alkaline phosphatase. 5. The requirements of these enzymes and their relationship to each other was examined. 6. A detailed study showed that the alkaline phosphatase was adsorbed at least partially to agarose and dextran columns at all (NH4)2SO4 concentrations from 0.25 to 2M. 7. In contrast, no adsorption of the enzyme or protein standards was evident in 2.5M-KCl/l M-NaCl or 0.25 M-KCl/0.1 M-NaCl, in agreement with previous studies by Louis, Peterkin & Fitt [(1971) Biochem. J. 121, 635-641], thus confirming the validity of gel filtration in 2.5 M-KCl/1 M-NaCl as a method for determining the approximate molecular weights of extremehalophile proteins.

Alkaline Phosphatase↗

PubMed2005

An acid phosphatase from Manihot glaziovii as an alternative to alkaline Phosphatase for molecular cloning experiments.

An acid phosphatase, free of deoxyribonuclease activity, was isolated from Manihot glaziovii leaves. It had a Mr of 78 kDa and was optimally active at pH 4.3 and 52 degrees C. It was inactivated at 65 degrees C over 15 min. It had a broad substrate specificity with strongest activity towards p-nitrophenyl phosphate. The enzyme dephosphorylated linearized pUC18 DNA and preventing self-ligation under the same conditions used for calf intestine alkaline phosphatase.

Acid Phosphatase↗

PubMed1981

A particulate form of alkaline phosphatase in the yeast, Saccharomyces cerevisiae.

A new form of alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) has been identified in the yeast Saccharomyces cerevisiae. Utilizing either synthetic or natural substrates, the enzyme exhibited a broad pH activity curve with maximum activity between 8.5 and 9.0. The enzyme was nonspecific with respect to substrate, attacking a variety of compounds containing phosphomonoester linkages, but has no detectable activity against polyphosphate, pyrophosphate or phosphodiester linkages. The enzyme exhibited an apparent Km of 0.25 mM with respect to p-nitrophenyl phosphate, 0.38 mM with respect to alpha-naphthyl phosphate, and 1.0 mM with respect to 5'AMP. The enzyme is regulated in a constitutive manner and its activity does not increase during phosphate starvation or sporulation, as does the repressible alkaline phosphatase. The enzyme is tightly bound to a particulate fraction of the cell, tentatively identified as the tonoplast membrane. It is not solubilized by treatment with high concentrations of NaCl, KH2PO4 or chaotropic agents. Triton X-100 (0.1%) solubilizes 12% of the particulate activity. This enzyme is differentiated from the other alkaline phosphatases found in yeast by its chromatographic elution DEAE-cellulose, kinetic parameters, heat stability and pH stability, as well as its particulate nature. This particulate alkaline phosphatase was found in every strain examined. It has a significantly lower specific activity in the phoH mutant and a higher activity in the acid phosphatase constitutive mutant A137.

Alkaline Phosphatase↗

PubMed1986

Localization of alkaline phosphatase in Streptomyces noursei.

Alkaline phosphatase activity appeared in Streptomyces noursei strain IMET 43,716 when cultures were shifted to phosphate limitation. By using p-nitrophenylphosphate as substrate, the activity detected at pH 9.4 was cell-bound, as long as young mycelia were studied. Supernatant fluids of the cultures were only active, if partial mycelial lysis had taken place after incubation for several days under the influence of shear forces. After cytochemical staining the phosphatase reaction products were microscopically visible as grains distributed rather homogeneously within the hyphal lumen. The frequency of grains was correlated to the rate of nourseothricin production. Electron microscopy of thin sections showed the phosphatase reaction products to be only present in the cytoplasm.

Alkaline Phosphatase↗

PubMed1976

Characterization of two different alkaline phosphatases in mouse teratoma: partial purification, electrophoretic, and histochemical studies.

Alkaline phosphatase (E.C.3.1.3.1.) has been used as a marker for embryonal carcinoma cells which constitute the multipotential stem cells of the mouse teratoma. Studies by other investigators based on kinetics of thermal inactivation and L-phenylalanine inhibition have shown that the alkaline phosphatase of the teratoma differs from the mouse intestinal and liver isozymes, but resembles the isozymes of kidney and placenta. Since functional characterization of nonpurified enzymes is not the most accurate means for distinguishing different molecular forms of an enzyme, we have partially purified the enzymes from the ascitic (embryoid body) and solid tumor forms of the OTT-6050 teratoma line, and utilized the technique of electrophoresis in polyacrylamide gels to compare the teratoma enzyme with isozymes from kidney and placenta. Covalent 32PO4-labeling of the alkaline phosphatases and polyacrylamide gel electrophoresis in sodium dodecylsulfate was also used to compare the subunit molecular weights of the enzymes. The results indicate that the mouse teratoma enzyme is distinct from the kidney and placental isozymes. Since histochemical studies have localized the enzyme to the stem cell population of the teratoma, the results imply that stem cell alkaline phosphatase is a distinct isozyme. The embryoid bodies contain a second alkaline phosphatase which may correspond to the placental isozyme. This enzyme may be attributed to the outer cell layer of embryoid bodies of the ascitic tumor, since this cell type histochemically demonstrates alkaline phosphatase activity.

Alkaline Phosphatase↗