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PubMed1977

Electrophoretic studies on alkaline phosphatases in normal and zinc intoxicated rats.

Polyacrylamide gel electrophoresis was used to characterize the isoenzymes of serum alkaline phosphatase in the rat. The electrophoresis of serum from normal rats resulted in two bands of alkaline phosphatase activity. A prominent band in serum corresponded in electrophoretic mobility to the alkaline phosphatase from bone and intestinal tissue extracts and also a slower migrating liver isoenzyme. A less prominent, fast migrating band in serum had a mobility similar to a faster migrating liver tissue extract isoenzyme. This band only represented 1-2% of the total alkaline phosphatase present in the serum of normal rats but approximately 15% of the total alkaline phosphatase in the serum of rats fed excess levels of zinc. The study also revealed an alteration in the electrophoretic mobility of alkaline phosphatase in bone homogenate by the addition of deactivated serum to the homogenate. The addition of deactivated serum did not alter the electrophoretic mobility of the liver and intestinal alkaline phosphatases in rats.

Alkaline Phosphatase↗

PubMed1971

Distribution of the sites of alkaline phosphatase(s) activity in vegetative cells of Bacillus subtilis.

Sites of alkaline phosphatase activity have been located by an electron microscopic histochemical (Gomori) technique in vegetative cells of a repressible strain SB15 of Bacillus subtilis, derepressed and repressed by inorganic phosphate, and in a mutant SB1004 which forms alkaline phosphatase in a medium high in phosphate. The sites of enzyme activity were revealed as discrete, dense, and largely spherical bodies of varying sizes (20 to 150 nm). Cells of both repressible and repression-resistant strains acted on a wide variety of phosphate esters (p-nitrophenylphosphate, beta-glycerophosphate, adenosine-5'-phosphate, glucose-6-phosphate, glucose-l-phosphate, adenosine triphosphate, and sodium pyrophosphate) to produce inorganic phosphorus under conditions of alkaline phosphatase assay [0.05 m tris(hydroxymethyl)aminomethane buffer (pH 8.4) containing 2 mm MgCl(2)]. The purified alkaline phosphatase also acted on all these esters, although much less effectively on adenosine triphosphate and sodium pyrophosphate than did the cells. Comparison of the relative utilization of the various substrates by repressed and derepressed cells and purified enzyme suggested the presence of multiple enzymes in the cells. Thus, the cytochemical method of trapping the newly generated inorganic phosphorus determines the location of an alkaline phosphatase of broad substrate profile, and in addition locates the sites of other enzymes generating inorganic phosphorus under identical conditions of assay. It is intriguing that all of these enzymes usually exist in a few clusters attached to the peripheral plasma membrane. In addition to this predominant location, there were a few sites of enzyme activity in the cytoplasm unattached to any discernible structure, and also in the cell wall of the repression-resistant and of the derepressed, repressible strains.

Alkaline Phosphatase↗

PubMed1987

Properties of alkaline phosphatase isoenzymes in plasma of preterm and term neonates.

The isoenzymes of alkaline phosphatase (EC 3.1.3.1) in plasma of 37 preterm and 21 term neonates two weeks postpartum have been studied with regard to electrophoretic mobility, sialic acid content, inhibition properties, heat lability, molecular mass, and binding to lectins. Term infants generally had a single form of alkaline phosphatase present in significant amounts, identified by the above criteria as originating in bone. All the preterm neonates had two alkaline phosphatase isoenzymes in plasma, namely, bone- and fetal-type intestinal alkaline phosphatase. Liver-, placental-, and adult-type intestinal alkaline phosphatase were not detected in any of the plasma samples.

Alkaline Phosphatase↗

PubMed1981

Alkaline phosphatase activity in chronic streptozotocin-induced insulin deficiency in the rat: effect of insulin replacement.

Alterations in circulating alkaline phosphatase have been described in both man and the experimental animal with chronic insulin deficiency. We evaluated plasma and tissue alkaline phosphatase levels in freely-fed control, streptozotocin-induced diabetic and insulin-treated diabetic rats, seven weeks after the induction of diabetes. Circulating alkaline phosphatase activity was markedly elevated in the insulin deficient animal (p less than 0.001) and completely normalized following insulin administration. The elevated plasma alkaline phosphatase activity observed in the insulin deficient animals was heat-resistant and phenylalanine-sensitive, a pattern typical of the intestinal isoenzyme. Small intestinal alkaline phosphatase activity was significantly higher (p less than 0.01) in the diabetic animals, but comparable in the insulin-replaced and control rats. The intestinal isoenzyme activity was found to be strikingly insulin-sensitive; withholding insulin therapy for 36 hr prior to sacrifice resulted in an abrupt rise in both plasma and intestinal alkaline phosphatase values comparable to those observed in the insulin-deficient state. In contrast to these observations, skeletal alkaline phosphatase activity was decreased in the insulin deficient animal (p less than 0.01) and this abnormality was corrected by insulin replacement. Neither insulin deficiency nor insulin replacement resulted in any significant changes in the hepatic alkaline phosphatase isoenzyme.

Alkaline Phosphatase↗

PubMed1994

Evaluation of an immunoradiometric assay for bone alkaline phosphatase mass concentration in human sera.

The performance characteristics of an immunoradiometric assay for bone alkaline phosphatase mass concentration in human sera are reported. Within-run imprecision (n = 20) was 12.1% (mean = 7.8 micrograms/l) and 3.6% (mean = 22.8 micrograms/l), between-day imprecision (n = 8) was 10.1% (mean = 20.3 micrograms/l) and 2.8% (mean = 84.3 micrograms/l). There was a linear relationship between the concentrations of the standards employed and the counts per minute up to 120 micrograms/l. The detection limit was 0.3 micrograms/l. In 102 apparently healthy persons (51 males and 51 females; range of age: 18-56 years) the following reference intervals were established: 3.8-21.3 micrograms/l (males) and 3.4-15.0 micrograms/l (females). We compared the values obtained using the immunoassay with those obtained by precipitating of bone alkaline phosphatase with wheat-germ lectin (alkaline phosphatase activity concentration was determined at + 25 degrees C by the optimized standard method according to the Recommendations of the German Society for Clinical Chemistry). For the reference individuals the relationship between the results of the two methods is given by the following regression equation: Bone alkaline phosphatase activity concentration [U/l] = 14.81 + 3.28 x bone alkaline phosphatase mass concentration [micrograms/l] (r = + 0.783). In 89 sera from 32 patients before and after renal transplantation (range of bone alkaline phosphatase mass concentration: 2-39 micrograms/l) comparison between the two methods yielded a linear correlation coefficient of r = + 0.886.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1979

Evidence that three structural genes code for human alkaline phosphatases.

The number of structural gene loci that code for the different molecular forms of human alkaline phosphatase is unknown. Physical properties of the enzymes, immunological data, chemical inhibition and genetic studies suggest that at least three structural genes are involved: one coding for alkaline phosphatase from placenta, another for the enzyme from intestine, and one or more for the enzymes from liver, kidney and bone. Badger and Sussman have shown that alkaline phosphatases from human liver and placenta are products of different structural genes, and Greene and Sussman have shown that alkaline phosphatase from a metastasised bronchogenic carcinoma was nearly identical to the enzyme from placenta. However, other tumour-associated alkaline phosphatases and the enzymes from normal tissue other than placenta and liver have not been identified by conclusive structural criteria, and thus it is not known whether these onco-alkaline phosphatases represent ectopic production or unusual post-translational modification of the enzymes found in normal tissues. We present here, using a sensitive peptide-mapping technique, structural evidence that the enzyme forms from liver, kidney and serum from a patient with Paget's disease of bone (osteitis deformans) are products of the same structural gene and can be easily distinguished from either the intestinal or placental isoenzymes. The technqiue seems to be useful for the classification of tumour-associated alkaline phosphatases on a structural basis.

Alkaline Phosphatase↗

PubMed1994

Effect of redox environment on the in vitro and in vivo folding of RTEM-1 beta-lactamase and Escherichia coli alkaline phosphatase.

The oxidative folding mechanisms of two Escherichia coli periplasmic proteins, alkaline phosphatase and RTEM-1 beta-lactamase, have been examined in vitro and in vivo. In contrast to eukaryotic proteins, which require a relatively reducing environment for optimal folding rates, both alkaline phosphatase and beta-lactamase fold fastest under very oxidizing conditions. For example, bovine pancreatic ribonuclease exhibits an optimal folding rate in a redox buffer consisting of 1 mM GSH and 0.2 mM GSSG (Lyles, M. M., and Gilbert, H. F. (1991) Biochemistry 30, 613-619); however, both E. coli alkaline phosphatase and beta-lactamase exhibit optimal in vitro folding rates at low concentrations of GSH (< 0.4 mM) and very high concentrations of GSSG (4-8 mM). For both bacterial proteins, GSH inhibits oxidative folding. Under optimal redox conditions, the rate-limiting step for the in vitro oxidative folding of alkaline phosphatase depends on the concentration of the protein, consistent with a mechanism involving rapid oxidation followed by slow dimerization. With beta-lactamase, the oxidative folding mechanism involves a competition between disulfide bond formation and folding of the molecule into a catalytically active conformation that buries the 2 reduced cysteines in the core of the enzyme. The effects of including a thiol reductant in the growth medium on the in vivo folding of alkaline phosphatase and beta-lactamase are similar to the effects observed during in vitro folding of these enzymes. The levels of both oxidized proteins are decreased by GSH in the growth medium. However, addition of a disulfide oxidant to the growth medium does not positively affect the production of either enzyme. These observations are consistent with the idea that the oxidative folding mechanisms of E. coli periplasmic proteins and, by inference, proteins of the eukaryotic endoplasmic reticulum have evolved to accommodate constraints placed on the folding reaction by the folding environment. The consequences of differences between the folding mechanisms in eukaryotic and prokaryotic disulfide-containing proteins on the expression of eukaryotic proteins in the bacterial periplasm are discussed.

Alkaline Phosphatase↗

PubMedDate not supplied

[Localization of alkaline phosphatase in cells of Bacillus intermedius].

Alkaline phosphatase, an enzyme secreted by Bacillus intermedius S3-19 cells to the medium, was also detected in the cell wall, membrane, and cytoplasm. The relative content of alkaline phosphatase in these cell compartments depended on the culture age and cultivation medium. The vegetative growth of B. intermedius on 0.3% lactate was characterized by increased activity of extracellular and membrane-bound phosphatases. The increase in lactate concentration to 3% did not affect the activity of membrane-bound phosphatase but led to a decrease in the activity of the extracellular enzyme. Na2HPO4 at a concentration of 0.01% diminished the activity of membrane-bound and extracellular phosphatases. CoCl2 at a concentration of 0.1 mM released membrane-bound phosphatase into the medium. By the onset of sporulation, phosphatase was predominantly localized in the medium and in the cell wall. As is evident from zymograms, the multiple molecular forms of phosphatase varied depending on its cellular localization and growth phase.

Alkaline Phosphatase↗

PubMed1984

Effect of vanadate, a potent alkaline phosphatase inhibitor, on 45Ca and 32Pi uptake by matrix vesicle-enriched fractions from chicken epiphyseal cartilage.

Although alkaline phosphatase has been long associated with the mineralization process, its exact function remains to be elucidated. To clarify its possible role in matrix vesicle-mediated mineralization, we tested the effect of vanadate, a phosphate analogue and powerful competitive inhibitor of alkaline phosphatase activity, on calcium and phosphate uptakes by a matrix vesicle-enriched microsomal fraction. Vanadate was also tested in a hydroxyapatite-seeded ion uptake system to determine possible direct effects on mineral formation. The effect of vanadate on vesicle mineral ion uptake was complex; low dosages of vanadate (2-20 microM) were stimulatory to Ca2+ uptake, but were inhibitory to Pi. Higher dosages (greater than 67 microM) were inhibitory to both ions. The effect of vanadate on ion uptake was strongly influenced by the stage of vesicle loading; major effects were seen during the lag and early uptake phases, and minimal effects were seen in the terminal stages. Concentrations of vanadate highly inhibitory to vesicle ion uptake had minimal effects on ion accretion by a hydroxyapatite-seeded system. Inhibition of alkaline phosphatase activity by vanadate broadly paralleled inhibition of Pi and Ca2+ uptake; however, at low vanadate concentrations, inhibition of Pi uptake closely paralleled that of alkaline phosphatase. The data indicate that vanadate binds with high affinity to Pi-loading sites, blocking initial Pi uptake. Complexation between vanadate and Ca2+ may be responsible for the stimulation of Ca2+ uptake at early stages of vesicle ion loading with low levels of vanadate by enhancing binding of Ca2+ to the vesicles. It may also account for the selective inhibition of Ca2+ uptake during the rapid stage of vesicle ion loading with high levels of vanadate by reducing Ca2+ ion activity. The close parallelism between inhibition of early Pi uptake and of alkaline phosphatase activity supports the concept that alkaline phosphatase is involved in Pi transport during the early stages of matrix vesicle ion loading. However, the fact that only about half of the Pi uptake was affected by vanadate, despite the progressive inhibition of alkaline phosphatase activity, indicates that alkaline phosphatase is not solely responsible for Pi uptake by the matrix vesicle-enriched fraction.

Alkaline Phosphatase↗

PubMed1991

Purification and properties of two membrane alkaline phosphatases from Bacillus subtilis 168.

Two alkaline phosphatases were extracted from the membranes of Bacillus subtilis 168 stationary-phase cells and purified as homogeneous proteins by hydroxyapatite column chromatography. Alkaline phosphatases I and II differed in several properties such as subunit molecular weight, substrate specificity, thermostability, Km, pH stability, and peptide maps.

Alkaline Phosphatase↗

PubMed1995

Simultaneous demonstration of CD15 and alkaline phosphatase activity in cryostat sections of rat fetuses: a detailed technical description for the developing brain.

CD15 and alkaline phosphatase are significant markers of the developing central nervous system. CD15 is known for its interaction in cell-cell contacts, while the presence of alkaline phosphatase is related to the formation of neuronal connections. This paper describes a combined immunocytochemical and enzyme histochemical technique to demonstrate both embryonic markers in the same cryostat section. The monoclonal antibody 3B9, which was used for the detection of CD15 recognizes the immunodominant carbohydrate structure alpha-1,3-Fucosyl-N-Acetyl-Lactosamine also known as X, LewisX, FAL or SSEA-1. This antigen dissolves and is easily rinsed out of the section. The procedure starts off with the detection of CD15. The results of the immunocytochemical procedure is a reaction product which is stable at a high pH, preventing loss of the immunocytochemical reaction product during the subsequent alkaline phosphatase detection. The other embryonic marker, alkaline phosphatase, can only be demonstrated enzymehistochemically if the enzyme is still active. The immunocytochemical localization procedure decided for, does not inactivate alkaline phosphatase totally. From the colorimetrical and histochemical alkaline phosphatase determinations it was concluded that the residual alkaline phosphatase activity detected with this technique, could be intensified by adding Mg(2+)-ions to both the colorimetrical and histochemical incubation media.

Alkaline Phosphatase↗

PubMed1991

Separate promoters direct expression of phoAIII, a member of the Bacillus subtilis alkaline phosphatase multigene family, during phosphate starvation and sporulation.

Alkaline phosphatase (APase) expression can be induced in Bacillus subtilis by phosphate starvation or by sporulation. We have recently shown that there are multiple APase structural genes contributing to the total alkaline phosphatase expression in B. subtilis. The expression of the alkaline phosphatase III gene (phoAIII) was analysed under both phosphate-starvation induction and sporulation induction conditions. phoAII is transcribed from two promoter regions, PV and PS. The PV promoter initiated transcription 37 bp before the translation initiation codon and was used to transcribe phoAIII during phosphate-starvation induction in vegetative cells. The PS promoter initiated transcription 119 bp before the translation initiation codon and was used during sporulation induction. Genes which have previously been shown to affect total vegatative APase, pho regulon genes phoP, phoR and phoS, affected expression of phoAIII during phosphate starvation. Genes known to affect expression of total sporulation APase, i.e. spoIIA, spoIIG and spoIIE, affected phoAIII expression during sporulation induction. Our data show that one member of the APase multigene family, phoAIII, contributes to the total APase expression both during phosphate-starvation induction and sporulation induction, and that the mechanism of regulation includes two promoters, each requiring different regulatory genes.

Alkaline Phosphatase↗

PubMed1986

Conversion of human placental alkaline phosphatase from a high Mr form to a low Mr form during butanol extraction. An investigation of the role of endogenous phosphoinositide-specific phospholipases.

Alkaline phosphatase in a wide range of tissues has been shown to be anchored in the membrane by a specific interaction with the polar head group of phosphatidylinositol. It has previously been suggested that the production of low Mr alkaline phosphatase during the commonly used butanol extraction procedure may result from the activation of an endogenous phosphoinositide-specific phospholipase C which removes the 1,2-diacylglycerol responsible for membrane anchoring. This conversion process was investigated in greater detail with human placenta used as the source of alkaline phosphatase. Mr and hydrophobicity of the alkaline phosphatase were determined by gel filtration on TSK-250 and partitioning in Triton X-114, respectively. Alkaline phosphatase extracted from human placental particulate fraction with butanol at pH 5.4 or released by incubation with Staphylococcus aureus phosphatidylinositol-specific phospholipase C produced a form of alkaline phosphatase of Mr approx. 170,000 and relatively low hydrophobicity. By contrast, the butanol extract prepared at pH 8.3 was an aggregated form of Mr approx. 600,000 and was relatively hydrophobic. The effect of a variety of inhibitors and activators on the amount of low Mr alkaline phosphatase produced during butanol extraction revealed that it was a Ca2+- and thiol-dependent process. Proteinase inhibitors had no effect. [3H]Phosphatidylinositol hydrolysis by the particulate fraction, unlike low Mr alkaline phosphatase production, was relatively sensitive to heat inactivation, indicating that the phosphoinositide-specific phospholipases C from cytosol and lysosomes were unlikely to be responsible for conversion. A butanol-stimulated activity which removed the [3H]myristic acid from the variant surface glycoprotein ( [3H]mfVSG) of Trypanosoma brucei was detectable in the human placental particulate fraction. Since this activity was acid active, Ca2+- and thiol-dependent and relatively heat stable, it may be the same as that responsible for production of low Mr alkaline phosphatase. The only 3H-labelled product identified was phosphatidic acid, suggesting that the [3H]mfVSG-cleaving activity is a phospholipase D. These data strongly support the proposal that production of low Mr alkaline phosphatase during butanol extraction is an autolytic process occurring as the result of an endogenous phospholipase. However, they also suggest that the lysosomal and cytosolic phosphoinositide-specific phospholipases C that have previously been described in many mammalian tissues are not responsible for this process.

1-Butanol↗

PubMed1979

Occurrence of heat-labile Regan type of alkaline phosphatase in hematopoietic tumors.

The physiocochemical and immunological properties of alkaline phosphatase extracted from Hodgkin's nodes, non-Hodgkin's lymphoma nodes and leukemic leukocytes have been studied. The alkaline phosphatase from these three tumor types possesses the same biophysical and biochemical properties and immunological determinants as the placental alkaline phosphatase. However, it is more heat-labile than the placental isoenzyme. Immunological experiments indicate that, of these tumor types, Hodgkin's tumor contains the largest amounts of heat-labile Regan type of alkaline phosphatase.

Alkaline Phosphatase↗

PubMed1992

Allelic amino acid substitutions affect the conformation and immunoreactivity of germ-cell alkaline phosphatase phenotypes.

The gene encoding placental alkaline phosphatase (PLAP) displays a well-documented allelic polymorphism. Likewise, different phenotypes exist for the PLAP-related germ-cell alkaline phosphatase (GCAP). We investigated the extent to which various allelic GCAP positions are critical in determining the enzymatic, structural, and immunological properties of GCAP phenotypes. Three homozygous GCAP phenotypes [JEG3, BeWo, and wild-type (wt) GCAP] were analyzed and compared with a "core" GCAP mutant that contains the seven amino acid substitutions that are consistently different between PLAP and GCAP but are common to the three known allelic GCAP genotypes. Although some substitutions could influence the electrophoretic behavior of the phenotypes, the allelic differences did not affect the kinetic properties of GCAP. However, they did affect the immunoreactivity and conformation of the variants as detected with a panel of 18 epitope-mapped monoclonal antibodies (MAbs) to PLAP. The selective immunoreactivity of the PLAP/GCAP-discriminating MAb C2 was critically dependent on the nature of the allelic residues 133 and 361 in GCAP. Residue 133 was also important for the general stability of the molecule because BeWo and wt GCAP, which have Asn133 and Val133, respectively, instead of Met133, showed a consistently reduced heat stability compared to core GCAP and JEG3. Because the core GCAP mutant consistently shows the characteristics of wt GCAP, its use as an antigen should allow the generation of monoclonal antibodies to GCAP that will not cross-react with PLAP and whose immunoreactivity will only marginally be influenced by allelic GCAP variation.

Alkaline Phosphatase↗

PubMed1999

[The effect of immobilized salivary alkaline phosphatase on remineralization processes].

Relationship between alkaline phosphatase immobilized on apatite and remineralization processes has been studied. The enzyme notably accelerated the remineralization. The course of enzymatic remineralization depended on the source of phosphorus (inorganic phosphorus or beta-glycerophosphate). Calcium incorporation in remineralizing surface was the most rapid in the presence of beta-glycerophosphate. This may be explained by a higher rate of formation of apatite under the effect of immobilized alkaline phosphatase, than of brushite or vitlokite.

Alkaline Phosphatase↗

PubMed1977

Induction of placental alkaline phosphatase in choriocarcinoma cells by 5-bromo-2'-deoxyuridine.

Growth of choriocarcinoma cells in the presence of 5-bromo-2'-deoxyuridine (BrdUrd) results in a 30- to 40-fold increase in alkaline phosphatase activity. The effects of BrdUrd is specific for phosphatase with an alkaline pH optimum. The induction by BrdUrd is probably not due to the production of an altered enzyme, since the induced enzyme resembles the basal enzyme in thermal denaturation and kinetic properties. Enzyme induction can be prevented by thymidine but not by deoxycytidine or deoxyuridine. The induction of alkaline phosphatase appears to require incorporation of the BrdUrd into cellular DNA. The presence of BrdUrd in the growth medium is not necessary for alkaline phosphatase induction in proliferating cells containing BrdUrd-substituted genomes. However, enzyme induction and maintenance of the induced levels of alkaline phosphatase in nonproliferating cells containing BrdUrd-substituted DNA requires the presence of the analogues in the medium. The induction of alkaline phosphatase by BrdUrd in probably an indirect process.

Alkaline Phosphatase↗

PubMedDate not supplied

Determination of serum acid and alkaline phosphatase using 4-methylumbelliferyl phosphate.

The present report compares the performance of several colorimetric substrates presently employed in clinical laboratories for the determination of serum acid and alkaline phosphatase with that of the fluorogenic phosphatase reagent 4-methylumbelliferyl phosphate. Acid and alkaline phosphatase assays were performed on reference hospital populations in Nigeria and the United States. The results of both acid and alkaline phosphatase determinations indicate that the coefficient of variation is smaller for the fluorometric assay than for the colorimetric methods that employ either phenyl phosphate or p-nitrophenyl phosphate as the substrate. Furthermore, serum acid phosphatase determinations using 4-methylumbelliferyl phosphate identified increased serum enzyme levels in 6/6 cases of prostatic carcinoma and 9/9 cases of Gaucher's disease. The occurrence of elevated serum alkaline phosphatase levels in 24/24 cases involving liver and bone disease was also confirmed by the fluorometric alkaline phosphatase assay. In addition, sera from eight patients with the connective tissue disease, osteogenesis imperfecta, were found to contain normal levels of acid and alkaline phosphatase by the fluorometric assays. From these results it appears that the sensitive and rapid fluorometric assay procedures can be readily employed in the clinical pathology laboratory for the determination of serum acid and alkaline phosphatase levels.

Acid Phosphatase↗