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Results for “ALKALINE PHOSPHATASE”

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PubMed1998

The effects of N-thiophosphoryl amino acids on the activity of green crab (Scylla Serrata) alkaline phosphatase.

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme which catalyzed the nonspecific hydrolysis of phosphate monoesters. In the present paper, the effects of several N-thiophosphoryl amino acids on the activity of green crab alkaline phosphatase have been studied. The results show that these derivatives of amino acids can lead to reversible inactivation. The equilibrium constants for inhibitors binding with the enzyme and/or the enzyme-substrate complexes have been determined. The obtained results show that both N-thiophosphoryl-Cys and N-thiophosphoryl-Glu were non-competitive inhibitors, while other five N-thiophosphoryl amino acids were un-competitive inhibitors. For the un-competitive inhibitors, the inhibition strength follows the order N-thiophosphoryl-Ile > -Val > -Lys > -Ala > -Tyr. Compared with respective free amino acids, it can be seen that N-thiophosphorylation of the amino acids increased their inhibition strength except the N-thiophosphoryl-Cys.

Alkaline Phosphatase↗

PubMed1976

Subcellular distribution of a gonadotropin-induced form of mouse ovarian alkaline phosphatase.

The subcellular distributions of alkaline phosphates I (the major activity of prepubertal mouse ovaries) and alkaline phosphatase Ib (a kinetically distinct isoenzyme induced in large amounts by injection of human chorionic gonadotropin or luteinizing hormone) were studied by differential rate centrifugation and discontinuous density gradient centrifugation of ovarian homogenates from control and gonadotropin-treated mice. The distributions of the two alkaline phosphatases were alike and were similar to those of nucleotidase, Mg2+ -dependent ATPase and Co2+ -stimulated naphthylamidase activities, suggesting that they were associated with plasma-membrane vesicles.

Alkaline Phosphatase↗

PubMed1972

The nature of the alkaline phosphatases of bile.

The main alkaline phosphatase isoenzymes of human bile have been purified by DEAE-cellulose chromatography. Characteristics of the isoenzymes, such as electrophoretic mobility before and after butanol extraction, Michaelis constant, and change in electrophoretic mobility following exposure to neuraminidase have been studied and compared with isoenzymes from other sources. The results show that the main alkaline phosphatase of bile is derived from the liver. It is present as a protein phosphatidylcholine complex.

Alcohols↗

PubMed1989

Intestinal-like alkaline phosphatase expressed in normal human adult kidney.

Human adult kidney was found to contain not only the 'tissue-unspecific alkaline phosphatase' but also another alkaline phosphatase isozyme. By use of monoclonal antibodies specific for human intestinal alkaline phosphatase, this kidney isozyme was purified to homogeneity by immunoaffinity chromatography. The structural and kinetic properties of the enzyme were compared with those of the other alkaline phosphatase isozymes expressed in normal human tissues, i.e. the placental, intestinal, meconial (fetal), liver and kidney isozymes. The new kidney isozyme was clearly different from both the tissue-unspecific and the adult intestinal alkaline phosphatase as regards isoelectric point, molecular mass and peptide maps after cyanogen bromide cleavage, but it was found to be identical to the meconial alkaline phosphatase. The results demonstrate simultaneous expression of two alkaline phosphatase isozymes in human kidney, one of which is normally related only to the fetal intestine.

Alkaline Phosphatase↗

PubMed1976

An ultrahistochemical study of the distribution of acid and alkaline phosphatases in placentae from normal and complicated pregnancies.

The subcellular localisation of acid and alkaline phosphatase has been studied in the trophoblast of placentae from both normal and complicated pregnancies. In placentae from uncomplicated pregnancies the number of trophoblastic acid-phosphatase-containing organelles decreases progressively as gestation proceeds whilst alkaline-phosphatase activity, although abundant at term, could not be demonstrated during the early stages of pregnancy. The acid-phosphatase-containing organelles are of two types; one is a small round body which is probably a lysosome whilst the other is a multivesicular body. The alkaline phosphatase is distributed mainly on the syncytial microvilli and plasma-membrane. It is suggested that the marked lysosomal activity during early pregnancy is related to the architectural refashioning of the placenta during this period and that there are two phosphatase-linked transfer systems in the trophoblast, one dependent upon acid-phosphatase-containing multivesicular bodies and being utilised during early pregnancy and the other reliant upon alkaline phosphatase and dominating during the second half of gestation. In placentae from prolonged pregnancies there is a further decrease in trophoblastic acid phosphatase and, usually, a continuing increase in alkaline-phosphatase activity. In placentae from babies of low birth weight this trend is sometimes reversed and alkaline-phosphatase activity either disappears or its reaction product diffuses throughout the syncytium; this is usually accompanied by a marked increase in the number of acid-phosphatase-containing multivesicular bodies. Placentae from women with pre-eclampsia show no loss of alkaline-phosphatase activity but are characterised by an increased number of lysosomal bodies.

Acid Phosphatase↗

PubMed1983

Alkaline phosphatase secretion-negative mutant of Bacillus licheniformis 749/C.

An alkaline phosphatase secretion-blocked mutant of Bacillus licheniformis 749/C was isolated. This mutant had defects in the phoP and phoR regions of the chromosome. The selection procedure was based on the rationale that N-methyl-N'-nitro-N-nitrosoguanidine can induce mutations of closely linked multiple genes. The malate gene and the phoP and phoR genes are located at the 260-min position in the Bacillus subtilis chromosome; hence, the malate gene could be used as a marker for the mutation of the phoP and phoR regions of the chromosome. In a two-step selection procedure, strains defective in malate utilization were first selected with the cephalosporin C procedure. Second, these malate-defective strains were further screened in a dye medium to select strains with defects in alkaline phosphatase secretion. One stable mutant (B. licheniformis 749/cNM 105) had a total secretion block for alkaline phosphatase and had the following additional characteristics: (i) the amount of alkaline phosphatase synthesized was comparable to that in the wild type; (ii) the alkaline phosphatase was membrane bound; (iii) the mutant strain alkaline phosphatase, in contrast to that of the wild type, could not be extracted with MgCl2, although the amounts of protein extracted from each strain were comparable; (iv) the sodium dodecyl sulfate-polyacrylamide gel pattern of MgCl2-extracted proteins from the mutant strain was different from that of the wild-type proteins; (v) the mutant, unlike the wild type, could not use malate as a sole source of carbon; and (vi) the outside surface of the wall of the mutant cells contained an additional electron-dense layer that was not present on the wild-type cell wall surface.

Alkaline Phosphatase↗

PubMed1979

[Alkaline phosphatase activity and properties in the organs of cattle and sheep].

Alkaline-phosphatase activity and the physico-chemical properties of the liver, lung, spleen, kidney, intestine, bone and placenta of 25 clinically healthy cattle and 30 clinically healthy sheep were investigated. High alkaline phosphatase activity was detected in kidneys and intestines. The alcaline phosphatase of cattle and sheep liver, spleen, kidney, lung, bone and placenta was thermo-labile and sensitive to l-arginine, l-homoarginine and imidazole, but was not sensitive to l-phenylalanine. Bone phosphatase of cattle and sheep was sensitive to urea. Intestinal phosphatase of cattle proved thermostable, sensitive to l-phenylalanine and not sensitive to l-arginine, l-homoarginine, imidasol and urea. Agarose gel electrophoresis of alkaline phosphatase indicated the presence of one fraction only and liver alkaline phosphatase proved to be the fastest. Sheep liver alkaline phosphatase had two fractions while sheep intestinal and placental alkaline phosphatase had three fractions and some of them were faster than liver alkaline phosphatase.

Alkaline Phosphatase↗

PubMed1998

Characterization of alkaline phosphatase genes expressed in seminoma by cDNA cloning.

Two members of a placental alkaline phosphatase (PLAP) family, PLAP and PLAP-like or germ cell alkaline phosphatase, are aberrantly expressed in tumors of ecotropic origin. To characterize alkaline phosphatase induced in seminoma, alkaline phosphatase cDNA clones were isolated from a cDNA library constructed from seminoma cells and characterized by nucleotide sequence determination. Thus, isolated cDNA clones were classified into two types, germ cell alkaline phosphatase (PLAP-like) and liver/bone/kidney-type alkaline phosphatase (L/B/K AP). These results suggest that other than the PLAP family members, the expression of L/B/K AP is enhanced in seminoma and can serve as a tumor marker in seminoma.

Alkaline Phosphatase↗

PubMed1989

[Phosphoethanolamine--a substrate of alkaline phosphatase isolated from rat calvaria].

Alkaline phosphatase from calvaria of 8 to 12-day-old Wistar rats was purified to electrophoretic homogeneity by a simple procedure (homogenisation, solubilisation by Triton X-100, DEAE-Sephacel ion exchange chromatography). For the holoenzyme, a Mr of about 160 kDa was determined, and it seems to consist of two identical subunits. The pH optima for the hydrolysis of phosphoethanolamine and p-nitrophenylphosphate are 10.0 and pH 9.0-10.5, respectively. The rate constants for the hydrolysis of phosphoethanolamine, p-nitrophenylphosphate and other phosphomonoesters at pH 10.0 are comparable, but the Km values differ by one to two orders of magnitude. At physiological pH (7.5) the maximum hydrolysis rate of the substrates phosphoethanolamine and p-nitrophenylphosphate was only 8% and 5%, respectively, of that determined at the pH optimum. On the basis of the kinetic data an in vivo function of alkaline phosphatase in bones as a monophosphate ester hydrolyzing enzyme seems unlikely.

Alkaline Phosphatase↗

PubMed1998

Inorganic pyrophosphate-phosphohydrolytic activity associated with rat osseous plate alkaline phosphatase.

Purified membrane-bound alkaline phosphatase from rat osseous plate hydrolyzed pyrophosphate in the presence of magnesium ions, with a specific activity of 92.7 U/mg. Optimal apparent pH for pyrophosphatase activity was 8.0 and it remained unchanged on increasing the pyrophosphate concentration. In the absence of magnesium ions the enzyme had a Km = 88 microM and V = 36.7 U/mg for pyrophosphate and no inhibition by excess substrate was observed. Pyrophosphatase activity was rapidly destroyed at temperatures above 40 degrees C, but magnesium ions apparently protected the enzyme against denaturation. Sodium metavanadate (Ki = 1.0 mM) was a competitive inhibitor of pyrophosphatase activity, while levamisole (Ki = 8.2 mM) and theophylline (Ki = 7.4 mM) were uncompetitive inhibitors. Magnesium ions (K0.5 = 1.7 microM) stimulated pyrophosphatase activity, while cobalt (Ki = 48.5 microM) and zinc (Ki = 22.0 microM) ions were non-competitive inhibitors. Manganese and calcium ions had no effect on pyrophosphatase activity. The Mw of the pyrophosphatase protein was 130 kDa by gel filtration, but a value of 65 kDa was obtained by dissociative gel electrophoresis, suggesting that it was a dimer of apparently identical subunits. These results suggested that pyrophosphatase activity stems from the membrane-bound osseous plate alkaline phosphatase and not from a different protein.

Alkaline Phosphatase↗

PubMed1987

Isolation and purification of placental type alkaline phosphatase from a seminoma.

An alkaline phosphatase (EC 3.1.3.1) of the placental type was isolated from a seminoma type of human testicular cancer tissue and was purified to homogeneity by sulfate-mediated chromatography on a column of Cibacron Blue Sepharose 4B. The purified enzyme had a specific activity of 40.6 kU per gram of protein and was obtained in a yield of 37%. The purification procedure used was simple and economical, and may be used to purify alkaline phosphatase isoenzymes from other cancer tissues. This is the first report of the purification of the enzyme in seminoma. Inhibition studies suggest that this enzyme is a Nagao variant rather than the Regan type reported in several cancer tissues.

Alkaline Phosphatase↗

PubMed1987

Purification and partial amino acid sequencing of rat bone tumor (UMR106) alkaline phosphatase.

Cultured rat osteosarcoma (UMR106) alkaline phosphatase was purified to apparent homogeneity by sequential application of polyclonal antibody affinity, DEAE-cellulose, and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of a dominant band (using silver staining) corresponding to a molecular weight of 80,000. The amino acid composition was similar to those of various alkaline phosphatases. The N-terminal amino acid sequence was determined as follows: Phe-Val-Pro-Glu-Lys-Glu-Lys- Asp-Pro-Ser-Tyr-Trp-Arg-Gln-Gln-Ala-Gln-Glu-Thr-Leu- Lys-Asn-Ala-Leu-Lys-?-Gln-Lys-?-Asn-Val-Asn-Ala-Lys.

Alkaline Phosphatase↗

PubMedDate not supplied

Low serum alkaline phosphatase activity in Wilson's disease.

Low values for serum alkaline phosphatase activity were observed early in the course of two patients with Wilson's disease presenting with the combination of severe liver disease and Coombs' negative acute hemolytic anemia. A review of other cases of Wilson's disease revealed that 11 of 12 patients presenting with hemolytic anemia had values for serum alkaline phosphatase less than their respective sex- and age-adjusted mean values; in eight, serum alkaline phosphatase activity was less than the lower value for the normal range of the test. Low values for serum alkaline phosphatase were much less common in Wilson's disease patients with more chronic forms of presentation. Copper added in high concentration to serum in vitro did not have an important effect on serum alkaline phosphatase activity. The mechanism responsible for the decrease in serum alkaline phosphatase activity in patients is uncertain.

Adolescent↗

PubMed2001

Dephosphorylation of MnDPDP and related compounds by acid and alkaline phosphatase.

The enzymatic dephosphorylation of the magnetic resonance imaging contrast agent Teslascan was studied in in vitro experiments with acid phosphatase (prostatic, from human semen) and alkaline phosphatase (from human placenta). The active component, MnDPDP (manganese (II)-N,N'-dipyridoxylethylenediamine-N,N'-diacetate-5,5'-bis(phosphate), was dephosphorylated by both enzymes to the monophosphate MnDPMP and the totally dephosphorylated compound MnPLED. The corresponding zinc compound, ZnDPDP (which is a result of in vivo metabolism), was also dephosphorylated by both enzymes to ZnDPMP and ZnPLED. In separate experiments, both enzymes dephosphorylated MnDPMP and ZnDPMP. With the same amount of enzyme units, alkaline phosphatase was almost four times more active than acid phosphatase in dephosphorylating MnDPDP and ZnDPDP with only minor differences whether the substrate contained Mn or Zn. A similar difference in enzymatic activity was seen with the monophosphates, MnDPMP and ZnDPMP. This, taken together with the approximately 50 times higher activity of alkaline phosphatase than acid phosphatase in serum shows that alkaline phosphatase is responsible for most of the dephosphorylation of MnDPDP and its metabolites in vivo.

Acid Phosphatase↗

PubMed1981

Response-surface-optimized, zinc-enhanced assay for serum alkaline phosphatase.

An optimized assay for alkaline phosphatase (EC 3.1.3.1) is reported. A discrete analyzer, the DuPont Automatic Clinical Analyzer (aca), was used for this study. The assay is based on results of response-surface experimental co-optimization techniques, and response is enhanced over the present aca assay. A key feature is the incorporation of zinc ions, both to fully optimize the assay and to reduce the sensitivity of measured activity to zinc-binding impurities in the buffer, 2-amino-2-methyl-1-propanol. In addition, a simple technique is described for measuring relative concentrations of zinc-binding impurities in this buffer. These features should be considered in the design of any assay for alkaline phosphatase that is based on p-nitrophenyl phosphate as substrate and 2-amino-2-methyl-1-propanol as buffer.

Alkaline Phosphatase↗

PubMed1989

The effects of cadmium on the p-nitrophenyl phosphatase and inorganic pyrophosphatase activities of alkaline phosphatase in developing hamster tooth germs.

p-Nitrophenyl phosphatase (p-NPP-ase) and inorganic pyrophosphatase (PPi-ase) activities originate from the same alkaline phosphatase enzyme. Only the PPi-ase site has zinc (Zn2+) as a cofactor. Cadmium (Cd2+) in concentrations from 10(-5) mol/l upwards inhibited the PPi-ase activity, but did not inhibit the p-NPP-ase activity at all. In mineralizing tooth germs Cd2+ may replace Zn2+, thereby changing the specific stereoconfiguration in the active centre needed for PPi-ase activity, but not that for p-NPP-ase activity.

4-Nitrophenylphosphatase↗

PubMed1993

Expression and nature of the alkaline phosphatase gene in cultured osteosarcoma cells.

The molecular mechanism for the differences in specific activity of alkaline phosphatase in six human osteosarcoma cell lines was investigated. Five of the lines expressed only the tissue-non-specific or liver/bone/kidney isoenzyme of alkaline phosphatase. The sixth line had the lowest levels of alkaline phosphatase and this was determined to be a mixture of liver/bone/kidney isoenzyme and at least one other form. The mRNA of liver/bone/kidney alkaline phosphatase was identified by Northern analysis in the three cell lines that expressed the largest amount of alkaline phosphatase catalytic activity. This mRNA was indistinguishable in size from that seen in control mRNA from normal kidney (2.5 kb). Southern analysis demonstrated that EcoRI or HindIII restriction fragment patterns and the intensity of the bands, of the liver/bone/kidney alkaline phosphatase gene in the osteosarcoma cell lines were identical to that of the control DNA from normal peripheral blood leukocytes. Thus, the gene coding for liver/bone/kidney alkaline phosphatase appears to be intact in all of these osteosarcoma cells and it is unlikely that rearrangement, deletion or amplification of the gene is responsible for its activation or inactivation. Slot blot analysis revealed varying amounts of the transcripts of the liver/bone/kidney isoenzyme in each of the cell lines. The best fit line of a plot of the log of the level of mRNA of alkaline phosphatase vs. the log of the specific activity of liver/bone/kidney alkaline phosphatase was constructed. This gave a Pearson correlation coefficient of 0.92 (P < 0.008), demonstrating a significant relationship between the two variables. It is likely that the regulation of alkaline phosphatase activity is at the transcriptional process rather than the translational or post-translational processes and that the specific activity of the enzyme may be controlled by the amount of steady-state mRNA of the liver/bone/kidney isoenzyme.

Alkaline Phosphatase↗

PubMed1990

Structure of alkaline phosphatases.

The crystal structure of alkaline phosphatase (AP) from Escherichia coli, which is a prototype for mammalian APs, has been refined to a crystallographic R-factor of 0.184 at 2.0 A resolution. During the course of the refinement residues 380 to 410 were retraced and 190 to 200 were shifted by one residue, and substantial changes in the active site of the enzyme were made. Based on the refined structure and the sequences of mammalian enzymes (25-30% strict homology) we have modelled the core of the three dimensional structures of the mammalian alkaline phosphatases. Considerable circumstantial evidence suggests that this is valid despite the fact that the mammalian enzymes are larger, contain carbohydrate and are membrane associated through a phosphatidylinositol moiety. The active site of the molecule is highly conserved but specific changes in the secondary ligands to bound phosphate and the Mg metal are observed.

Alkaline Phosphatase↗