Histochemical studies on the effect of thyroid hormone on alkaline and acid phosphatase activities in liver of fish and amphibia.

The Lata fishes (Ophicephalus punctatus) showed increased alkaline and acid phosphatase activities in liver after immersion for 15-30 days in thyroxine-containing medium (0.025 mug/ml). A single injection of thyroxine (1-2 mug/g of body weight) caused increased acid phosphatase activity in liver of Lata fish in comparison to the controls on the 5th day after experiment but the alkaline phosphatase activity remained unchanged. Both alkaline and acid phosphatases showed increased activities in liver of Lata fishes treated with a single injection of 4 mug of thyroxine per g of body weight on the 5th day. Immersion of Lata fishes in thiourea solution (1 mg/ml) for 15 days did not show any alteration in alkaline or acid phosphatase activities but these enzyme activities decreased after 30 days' immersion in thiourea solution in comparison to the controls. A seasonal variation of alkaline and acid phosphatase activities was observed in liver of Lata fishes. More alkaline phosphatase activity was found in liver of summer fishes than in winter fishes. The winter fishes showed more acid phosphatase activity than the summer fishes. Three consecutive injections of thyroxine (0.1 mug/g of body weight) to toads (Bufo melanostictus) caused increased alkaline and acid phosphatase activities in liver on the 5th day of the experiment, in comparison to the controls.     

Presence and activity of alkaline phosphatase in two human osteosarcoma cell lines

The presence and activity of alkaline phosphatase in SAOS-2 and TE-85 human osteosarcoma cells grown in culture were examined at the ultrastructural level. A monoclonal antibody raised against purified human bone osteosarcoma alkaline phosphatase was used to localize the enzyme in cultures of the osteosarcoma cells. Similar cultures were analyzed for alkaline phosphatase activity using an enzyme cytochemical method with cerium as the capture agent. Alkaline phosphatase was immunolocalized at the light microscopic level in an osteogenic sarcoma and ultrastructurally on the SAOS-2 cell membrane and the enclosing membrane of extracellular vesicular structures close to the cells. In contrast, the TE-85 cells were characterized by the absence of all but a few traces of immunolabeling at the cell surface. Enzyme cytochemical studies revealed strong alkaline phosphatase activity on the outer surface of the SAOS-2 cell membrane. Much lower enzyme activity was observed in the TE-85 cells. The results support biochemical data from previous studies and confirm that SAOS-2 cells have a significantly greater concentration of alkaline phosphatase at the plasma membrane.     

Sub-lethal effect of synthetic pyrethroid pesticide on metabolic enzymes and protein profile of non-target Zebra fish,Danio rerio

Extensive application of pesticide in agricultural field affects the enzymatic activity of non-target animals, including fishes. In this study, the impact of sublethal concentration of fenvalerate on marker enzymes of freshwater Zebra fish was evaluated. Pesticide-induced stress can specifically affect non target fishes, through elevated level of reactive oxygen species which is responsible for biochemical, cell metabolism and physiological activities. The oxidative stress mediated by fenvalerate at sub lethal concentrations after 28 days of exposure of Zebra fish. Following 28 days of exposure of pesticide, catalase, superoxide dismutase, aspartate amino transferases, alanine amino transferase, alkaline phosphatase and acid phosphatase were assessed. Results revealed reduction of superoxide dismutase activity after 28 days of exposure in sub lethal concentration of fenvalerate in liver and gills. In liver, catalase activity was found to be less in fenvalerate exposed fish than control fish. In liver, increase of 75.75% aspartate amino transferase and 38% increase in alanine amino transferase in gills. SGPT activity was relatively higher than SGOT suggests more contribution of phyruvalate than oxaloacetate formation. Fenvalerate induced changes in acid phosphatase and alkaline phosphatase activity in the liver and gills of Zebra fish after four weeks of exposure. Fenvalerate induced expression of various stress proteins in gill, liver, followed by muscle. Some proteins lost its intensity due to fenvalerate toxicity. Result revealed that enzyme assays and SDS-PAGE analysis for protein subunits determination is relevant tool to monitor stress in freshwater ecosystem. The findings suggest that in monitoring fenvalerate toxicity programme, enzyme activities can be potent diagnostic tool for fenvalerate induced toxicity.     

Tissue alkaline phosphatase activity in selected freshwater teleosts.

1. Kinetic studies of tissue alkaline phosphatase activity were carried out on eight species of teleost fishes taken from the Mississippi River. 2. Liver alkaline phosphatase activity expressed in mumoles of p-nitrophenol released per minute per gram of protein were as follows: Micropterus salmoides, 20.31; Stizostedion vitreum, 19.22; Pomoxis nigromaculatus, 18.76; Lepisosteus platostomus, 16.26; Esox lucius, 16.14; Hiodon tergisus, 13.83; Ictalurus punctatus, 10.87; Aplodinotus grunniens, 7.94. 3. Specific activity of the kidney alkaline phosphatase was about ten times the level of liver alkaline phosphatase for the species examined.     

The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum.

1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, before immunoaffinity purification of the liver enzyme, resulted in the same specific activity and subunit Mr. The native Mr of the sulphobetaine 14-solubilized enzyme was consistent with the enzyme being a dimer of two identical subunits and was higher than that of the butanol-extracted enzyme, presumably due to the binding of the detergent micelle. 2. Pure bone and liver alkaline phosphatase were used to raise further antibodies to the two isoenzymes. Altogether, 27 antibody-producing cell lines were cloned from 12 mice. Several of these antibodies showed a greater than 2-fold preference for bone alkaline phosphatase in the binding assay used for screening. No antibodies showing a preference for liver alkaline phosphatase were successfully cloned. None of the antibodies showed significant cross-reaction with placental or intestinal alkaline phosphatase. Epitope analysis of the 27 antibodies using liver alkaline phosphatase as antigen gave rise to six groupings, with four antibodies unclassified. The six major epitope groups were also observed using bone alkaline phosphatase as antigen. 3. Serum from patients with cholestasis contains soluble and particulate forms of alkaline phosphatase. The soluble serum enzyme had the same size and charge as butanol-extracted liver enzyme on native polyacrylamide-gel electrophoresis. Cellulose acetate electrophoresis separated the soluble and particulate serum alkaline phosphatases as slow- and fast-moving forms respectively. In the presence of sulphobetaine 14 all the serum enzyme migrated as the slow-moving form on cellulose acetate electrophoresis. Monoclonal anti-(alkaline phosphatase) immunoadsorbents did not bind the particulate form of alkaline phosphatase in cholestatic serum but bound the soluble form. In the presence of sulphobetaine 14 all the cholestatic serum alkaline phosphatase bound to the immunoadsorbents. 4. The electrophoretic and immunological data are consistent with both particulate and soluble forms of alkaline phosphatase in cholestatic serum being derived from the hepatocyte membrane.     

Cortisol modification of HeLa 65 alkaline phosphatase. Decreased phosphate content of the induced enzyme.

Alkaline phosphatase activity of HeLa cells is increased 5-20-fold during growth in medium with cortisol. The increase in enzyme activity is due to an enhanced catalytic efficiency rather than an increase in alkaline phosphatase protein in induced cells. In the present study the chemical composition of control and induced forms of alkaline phosphatase were investigated to determine the enzyme modification that may be responsible for the increased catalytic activity. HeLa alkaline phosphatase is a phosphoprotein and the induced form of the enzyme has approximately one-half of the phosphate residues associated with control enzyme. The decrease in phosphate residues of the enzyme apparently alters its catalytic activity. Other chemical components of purified alkaline phosphatase from control and induced cells are similar; these include sialic acid, hexosamine and sulfhydryl residues.     

Alkaline phosphatase in the integument of Ceratitis capitata: Developmental profile and functional properties

Electrophoretic analysis of alkaline phosphatase from the integument during development, reveals two bands of enzyme activity. One corresponding to phosphatase activity during pupation and just prior to eclosion and the other during the middle of the pupal stages. On the contrary in the haemolymph there is one band on enzyme activity through all the developmental stages. The haemolymph alkaline phosphatase band does not comigrate with any integumental enzyme band. The developmental profile of the integumental alkaline phosphatase activity has also been compared to that of the haemolymph. It was found that the pattern of activity is completely different. In the integument, two peaks of enzyme activity were found: one just prior to pupation and the other during eclosion. These two peaks do not coincide to that of haemolymph alkaline phosphatase activity. The pH optimum for both enzyme forms of third instar larvae, although broad especially for haemolymph form, was clearly in the alkaline range, with a peak at pH 8.5–9.0. The two isozymes have different affinities for the substrate tyrosine-O-phosphate. Tyrosine-O-phosphate is the preferred substrate for the integumental enzyme form with a K_m of 0.4 mM. We suggest that alkaline phosphates from the integument is specific for the hydrolysis of tyrosine-O-phosphate.     

Effect of polyenic antibiotics on the activity of alkaline phosphatase from Candida albicans]

Polyenic antibiotics (levorin, amphotericin B, nistatin) inhibit in vivo and in vitro the activity of membrane alkaline phosphatase from sensitive Candida albicans strain, and their inhibitory effect is twice lower on the enzyme from the resistant strain. A correlation is observed between the antibiotic concentration and the inhibitory effect on alkaline phosphatase activity. Nistatin is found to be the least efficient inhibitor (among the antibiotics studied) of alkaline phosphatase. The treatment of membranes with polyenic antibiotics does not result in solubilization of membrane proteins nad alkaline phosphatase. The data obtained are considered with respect to the effect of polyenic antibiotics on cell membrane structure.     

Plasma membrane localization of alkaline phosphatase in HeLa cells.

The localization of alkaline phosphatase in HeLa cells was examined by electron microscopic histochemistry and subcellular fractionation techniques. Two monophenotypic sublines of HeLa cells which respectively produced Regan and non-Regan isoenzymes of alkaline phosphatase were used for this study. The electron microscopic histochemical results showed that in both sublines the major location of alkaline phosphatase is in the plasma membrane. The enzyme reaction was occasionally observed in some of the dense body lysosomes. This result was supported by data obtained from a subcellular fractionation study which showed that the microsomal fraction rich in plasma membrane fragments had the highest activity of alkaline phosphatase. The distribution of this enzyme among the subcellular fractions closely paralleled that of the 5'-nucleotidase, a plasma membrane marker enzyme. Characterization of the alkaline phosphatase present in each subcellular fraction showed identical enzyme properties, which suggests that a single isoenzyme exists among fractions obtained from each cell line. The results, therefore, confirm the reports suggesting that plasma membrane is the major site of alkaline phosphatase localization in HeLa cells. The absence of any enzyme reaction in the perimitochondrial space in these cultured tumor cells also indicates that the mitochondrial localization of the Regan isoenzyme reported in ovarian cancer may not be a common phenomenon in Regan-producing cancer cells.     

L'influence du cadmium sur l'activite de la phosphatase alcaline dans divers tissus de Lepomis gibbosus L. (Pisces; Centrarchidae)

To study the effects of cadmium on the activity of Zn-metalloenzymes, fishes have been exposed to different concentrations of that metal. The concentrations of Zn and Cd and the activity of alkaline phosphatase were measured in some tissues. From the results it seems there is no competition between Zn and Cd for the enzymatic sites of the alkaline phosphatase.

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L'influence du cadmium sur l'activite de la phosphatase alcaline dans divers tissus de Lepomis gibbosus L. (Pisces; Centrarchidae)

     

Induction of Rat Liver Alkaline Phosphatase by Bile Duct Ligation

Bile duct ligation causes a five- to sevenfold increase in the activity of rat liver alkaline phosphatase within 12 hours after ligation and a similar rise in the activity of alkaline phosphatase in serum. The increased serum activity is due entirely to the appearance of a new isoenzyme that has the properties of rat liver alkaline phosphatase. The increase in both serum and liver alkaline phosphatase is prevented by the prior administration of cycloheximide in a dose that inhibits protein synthesis by 70%. Rat liver alkaline phosphatase was then purified to homogeneity. Antibody was raised to purified rat liver alkaline phosphatase in rabbits. The antibody was coupled to sepharose 4B and affinity columns made. ³-H-leucine was then injected into the portal veins of sham operated rats and rats with bile duct ligation four hours after ligation. One hour after injection and five hours after ligation, animals were sacrificed. Liver alkaline phosphatase was purified by means of affinity chromatography and double immunoprecipitation with rabbit antibody to rat liver alkaline phosphatase and goat anti-rabbit gamma globulin. Bile duct ligation increased the incorporation of ³-H-leucine into liver alkaline phosphatase more than threefold compared with sham operated rats, 164 CPM/mg protein vs. 49 CPM/mg protein (p < .001). The data indicate that the increased activity of rat liver alkaline phosphatase after bile duct ligation is due to enzyme induction rather than to activation of a pre-existing, relatively inactive enzyme.     

Induction of plasma membrane alkaline phosphatase in rat liver

We have determined alkaline phosphatase activity in total liver plasma membrane fractions from rats subjected to a partial hepatectomy and sham operated with or without manipulation of the liver. In all these cases, an increase of the enzyme activity was observed. Kinetic studies of alkaline phosphatase activity performed on plasma membrane fractions from rats subjected to a partial hepatectomy suggest that alkaline phosphatase increase is produced by de novo biosynthesis of enzyme molecules. Determination of alkaline phosphatase activity in purified plasma membrane subfractions corresponding to each of the three functional regions of the hepatocyte surface (blood sinusoidal, lateral and bile canalicular), indicates that the increase of the enzyme activity observed after partial hepatectomy is selectively induced in the bile canalicular domain of the hepatocyte plasma membrane.     

Phosphoester specificity of purified human liver alkaline phosphatase.

Kinetic parameters for the hydrolysis of a number of physiologically important phosphoesters by purified human liver alkaline phosphatase have been determined. The enzyme was studied at pH values of 7.0 to 10.0. The affinity of the enzyme for the compounds was determined by competition experiments and by their direct employment as substrates. Phosphodiesters and phosphonates were not hydrolysed but the latter were inhibitors. Calcium and magnesium ions inhibited the hydrolysis of ATP and PP1 and evidence is presented to show that the metal complexes of these substrates are not hydrolysed by alkaline phosphatase. A calcium-stimulated ATPase activity could not be demonstrated for the purified enzyme or the enzyme in the presence of a calcium-dependent regulator protein. Nevertheless, the influence of magnesium and calcium ions on the ATPase activity of alkaline phosphatase means that precautions must be taken when assaying for Ca2+-ATPase in the presence of alkaline phosphatase. The low substrate Km values and the hydrolysis which occurs at pH 7.4 mean that the enzyme could have a significant phosphohydrolytic role. However, liver cell phosphate concentrations, if accessible to the enzyme, are sufficient to strongly inhibit this activity.     

5-Bromo-2'-deoxyuridine induces placental alkaline phosphatase biosynthesis in cultured choriocarcinoma cells

5-Bromo-2'-deoxyuridine (BrdUrd) stimulated the biosynthesis and hence increased the activity of placental alkaline phosphatase in choriocarcinoma cells. While BrdUrd had no effect on the rate of degradation or processing of placental alkaline phosphatase, it increased the rate of phosphatase synthesis. The stimulation of enzyme activity could be completely accounted for by the increase in alkaline phosphatase protein. Both control and BrdUrd-induced cells contained polypeptides of 61,500 and 64,500 Da, identified as the precursor and fully processed forms of placental alkaline phosphatase monomer. The half-life of this enzyme monomer in both control and BrdUrd-treated cells was estimated to be 36 h. BrdUrd induced a specific increase in the placental alkaline phosphatase mRNA leading to the observed enhancement of biosynthesis. The continued rise in alkaline phosphatase biosynthesis in BrdUrd-induced cells following BrdUrd removal indicated that this analog acted by incorporation into DNA.     

An in vitro production of bone specific alkaline phosphatase

Induced alkaline phosphatase has been extracted from osteosarcoma cells grown in tissue culture medium. The extracted enzyme has been purified. Using electrophoresis, inhibition studies, and thermolability, the enzyme was categorized as alkaline phosphatase of osseous origin. Antibodies to this enzyme were reacted against alkaline phosphatase extracted from cadaveric bone, liver, intestine, kidney and fresh placenta. The antibodies were specific against alkaline phosphatase of osseous origin only. No cross-reaction occurred with the enzyme extracted from other sources. The data derived from these studies indicate that alkaline phosphatase of bone is a specific enzyme of osseous tissue. Furthermore, the enzyme has specific antigenic and other properties which distinguish it from alkaline phosphatases from other sources. A model for in vitro production of a specific alkaline phosphatase of bone is presented.     

Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase

Alkaline phosphatase prepared from mammalian cell cultures was found to have alkaline inorganic pyrophosphatase activity. Both of these activities appear to be associated with a single protein, as demonstrated by: (1) concomitant purification of alkaline phosphatase and alkaline inorganic pyrophosphatase; (2) proportional precipitation of alkaline phosphatase and inorganic pyrophosphatase activities by titrating constant amounts of an enzyme preparation with increasing concentration of antibody; (3) immune electrophoresis, which showed that precipitin bands that have alkaline phosphatase activity also have pyrophosphatase activity; (4) inhibition of pyrophosphatase activity by cysteine, an inhibitor of alkaline phosphatase activity; (5) similar subcellular localization of the two enzyme activities as demonstrated by histochemical methods; (6) hormonal and substrate induction of alkaline phosphatase activity in mammalian cell cultures, which produced a nearly parallel rise in inorganic pyrophosphatase activity.     

Inhibitory effect of dibutyryl cyclic adenosine monophosphate on the induction of alkaline phosphatase in human fetal liver cell line

When human fetal liver cells (HuL-1-317), cultured continuously in a serum-free medium, were incubated with a combination of prednisolone, butyrate and a hypertonic concentration of NaCl at 37 degrees C, alkaline phosphatase activity increased. However, the addition of dibutyryl adenosine cyclic monophosphate (Bt2cAMP) to these agents inhibited the increase in alkaline phosphatase activity in a dose-dependent manner: the inhibitory effect of Bt2cAMP was significant at 0.05 mM, but disappeared at 0.01 mM. Both cycloheximide and actinomycin D inhibited the increase in alkaline phosphatase activity with the combination described above. Western blotting showed that this enzyme activity increase was a consequence of greater biosynthesis of enzyme molecules in HuL-1-317 cells, and that Bt2cAMP regulated the synthesis of enzyme molecules. We conclude that the changes in alkaline phosphatase activity under various conditions are based on the changes in the number of enzyme molecules in HuL-1-317 cells.     

Purification and characterization of phytase from rat intestinal mucosa.

Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1.3.8 or 3.1.3.26) was purified from rat intestinal mucosa. The purified enzyme preparation exhibited two protein bands on SDS-polyacrylamide gel electrophoresis with estimated molecular masses of 70 kDa and 90 kDa. Rabbit antisera prepared against the 90K subunit cross-reacted with the 70K subunit on immunoblotting. The peptide maps of the 70K and 90K subunits were similar, and the N-terminal amino acid sequences of the two subunit proteins were almost identical. Treatments to remove sugar moieties from the proteins showed that the two subunit proteins had different oligosaccharide chains, although the difference in their molecular masses was not due to the difference in their oligosaccharide compositions. The purified enzyme also showed activity of alkaline phosphatase (orthophosphoric monoester phosphohydrolase; EC 3.1.3.1), but the properties of the two enzyme activities were different; the optimum pH for phytase activity was 7.5, while that for alkaline phosphatase was 10.4. Phytase activity did not necessarily require divalent cations, while Mg2+ was essential for alkaline phosphatase activity. Phenylalanine, a specific inhibitor of intestine-type alkaline phosphatase had no effect on the phytase activity.