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PubMed1983

Induction of hepatic alkaline phosphatase by colchicine: role of cholestasis and bile acids.

We studied the induction of hepatic alkaline phosphatase by colchicine, a drug which impairs bile flow. By analogy to bile duct obstruction, where retained bile acids induce alkaline phosphatase, we hypothesized that the mechanism of induction involves the drug's cholestatic property and subsequent intrahepatic retention of bile acids. After administering colchicine to bile-fistula rats, we monitored bile flow. In colchicine-treated rats which did not develop cholestasis, we varied the transhepatic flux of taurocholate 15-fold over the physiologic range in order to study the role of intrahepatic bile acids. Induction of alkaline phosphatase was observed when cholestasis was absent and the bile acid flux was 5% of normal. The magnitude of enzyme induction was not augmented by increasing the transhepatic flux of taurocholate. The results demonstrate that colchicine induces hepatic alkaline phosphatase by a mechanism which operates independently of cholestasis and is not modified by the level of bile acid in the liver.

Alkaline Phosphatase↗
PubMed1976

Stabilization of human serum alkaline phosphatase to histidine-induced heat inactivation by tryptic digestion.

1. Serum alkaline phosphatase [EC 3.1.3.1] was strongly inactivated by histidine during incubation at pH 8.0 and 45degrees; however, tryptic digestion of the serum strongly protected the enzyme against inactivation by histidine. In the absence of histidine, however, neither heat inactivation of the phosphatase nor the effect of trypsin [EC 3.4.21.4] was observed. Factors affecting the alkaline phosphatase inactivation were studied further. 2. The effect of trypsin on the histidine-induced heat inactivation differed considerably according to the tissue source of the enzyme, which suggests a possible method for distinguishing alkaline phosphatase isoenzymes.

Alkaline Phosphatase↗
PubMed1978

Acid- and alkaline phosphatase in amniotic fluid in normal and complicated pregnancy.

171 samples of amniotic fluid were obtained by abdominal amniocentesis from 67 women with complicated pregnancies (isoimmunization, diabetes mellitus or toxaemia). The levels of heat-labile alkaline phosphatase (HLAP), heat-stable alkaline phosphatase (HSAP) and acid phosphatase (AcP) were determined and compared to the enzyme levels in 179 samples from women with normal pregnancies of corresponding gestational ages. HLAP showed two "peaks" of activity, one in the 5th-22nd week and the other at term. HSAP and AcP showed increased activity at term. HSAP was decreased (p less than 0.01) in isoimmunization between the 36th and 40th week. 11 cases of toxaemia with placental insufficiency showed no differences in the levels of HLAP and HSAP compared with normal pregnancy. AcP showed no differences between normal and complicated pregnancy. Samples contaminated by blood showed no significant increase in the acid- and alkaline phosphatase levels. Samples contaminated by meconium showed a complex pattern. Some samples had normal enzyme levels, some had high levels of HLAP only and some had high levels of HSAP and AcP. The origin of the enzymes is not known with certainty. HSAP in amniotic fluid is most likely not of placental but intestinal origin. Determinations of acid- and alkaline phosphatase in amniotic fluid seem to be of little values in the clinical management of complicated pregnancy.

Acid Phosphatase↗
PubMedDate not supplied

Alkaline phosphatase activatable polymeric cross-linkers and their use in the stabilization of proteins.

We report the synthesis of polymeric cross-linking agents, poly(glutamic acid) poly(phosphorothioates), and their use in the cross-linking and stabilization of proteins upon treatment with alkaline phosphatase. We have shown that poly(phosphorothioates) are excellent substrates of alkaline phosphatase, yielding thiolated polymers which react covalently with electrophilic groups introduced into the proteins. Three proteins of different structure and function were cross-linked using this method: calf intestinal alkaline phosphatase, glucose oxidase (Aspergillus niger), and (R)-phycoerythrin. The cross-linking of alkaline phosphatase is self-catalyzed since this enzyme catalyzes the hydrolysis of phosphates, unmasking thiolates which react with the maleimide prederivatized alkaline phosphatase. Incubation of buffered solutions of native alkaline phosphatase at 45 degreesC for 7-14 days resulted in a 35% higher loss of enzymatic activity compared to that of cross-linked enzyme. The effect of cross-linking glucose oxidase is even more notable, ranging from 800% stabilization at 37 degrees C and pH 9.0 to 3000% at 37 degrees C and pH 7.4. (R)-Phycoerythrin cross-linked with 1-3 equiv of poly(phosphorothioates) and incubated at 45 degrees C for 45 days was 20% more fluorescent than the native (R)-phycoerythrin subjected to the same conditions. The stabilizing effect of cross-linking was confirmed by comparing the rate of loss of quaternary structure of the cross-linked (R)-phycoerythrin with that of the native protein.

Alkaline Phosphatase↗
PubMed1978

A double antibody solid phase radioimmunoassay for placental alkaline phosphatase.

A solid-phase "direct" radioimmunoassay for determination of placental alkaline phosphatase in serum was developed. The assay is highly sensitive, with a minimum detectable dose of 9 ng protein per ml. The specificity for the placental type of the enzyme, in comparison to other serum alkaline phosphatases, was found to be convincing. The choice of genetic types of either placental alkaline phosphatase or antiserm was found not to influence the radioimmunoassay. With the described technique the serum concentration of this enzyme during normal pregnancy was measured. A 25-fold increase from low levels during the first trimester up to 252 +/- 70 ng/ml (mean +/- S.D.) in gestation week 40 was observed.

Alkaline Phosphatase↗
PubMed1984

Quantitation of human intestinal and liver/bone alkaline phosphatase in serum by rocket electroimmunoassay.

The separation and quantitation of the various human serum alkaline phosphatases has been largely by indirect and/or imprecise methods. A rocket electroimmunoassay method has been developed for the quantitation of the alkaline phosphatases, which uses enzymatic activity to detect the rocket and bromochloroindolylphosphate as substrate. The assay for intestinal phosphatase requires only one-dimensional electrophoresis, since the antibody to the intestinal enzyme does not cross-react with the other phosphatases. Both liver and bone enzymes give a line of identity when tested against antibody directed against the liver phosphatases, and these phosphatases require separation first by acrylamide gel electrophoresis before quantitation by crossed immunoelectrophoresis. Liver/bone enzyme levels in serum are easily detected within the linear range of the assay without concentration of the serum. The assay is 10-fold more sensitive than quantitation by acrylamide gel electrophoresis. The inability to detect circulating intestinal alkaline phosphatase in the serum of most fasting hospitalized patients has been documented by this sensitive assay, and the predominance of the liver isoenzyme has been confirmed. The assay should prove useful for determining the tissue of origin of serum alkaline phosphatases, and in providing quantitative data for physiological studies. It is not adaptable for automation and will not prove useful in the routine clinical laboratory.

Alkaline Phosphatase↗
PubMed1988

Comparison of alkaline phosphatase activity in normal, lactating and neoplastic rat mammary tissue.

1. Alkaline phosphatase activity in NMU-induced rat mammary tumours was compared with activity in normal and lactating mammary gland. 2. Both tumour and normal mammary alkaline phosphatase were sensitive to heat inactivation and inhibition by phenylalanine. 3. Specific activity of enzyme in tumours was comparable to normal mammary tissue. 4. Mammary gland alkaline phosphatase increased markedly in late pregnancy and early lactation. 5. Bromocryptine treatment had no effect on enzyme activity in lactating mammary gland.

Alkaline Phosphatase↗
PubMed2002

Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome.

BACKGROUND: In humans, there are four alkaline phosphatases, and each form exhibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnant with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. RESULTS: To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60-80% of activity. CONCLUSION: Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome.

Alkaline Phosphatase↗
PubMed1992

Significance of bone alkaline phosphatase, CA 15-3 and CEA in the detection of bone metastases during the follow-up of patients suffering from breast carcinoma.

After the introduction (1, 2) and methodical evaluation (3, 4) of a new method for the quantitative measurement of the bone isoenzyme of alkaline phosphatase (test-combination bone alkaline phosphatase, Boehringer Mannheim), we started a retrospective clinical study for the follow-up investigations of breast cancer patients. Our aim was to establish the significance of the routinely used tumour markers, CEA and CA 15-3, in combination with bone alkaline phosphatase for the early detection of metastatic spread to the bone. We investigated 492 sera from 92 patients suffering from breast carcinoma, and we compared each date of investigation with the results of the clinical examination and with the results of medical imaging, if that had been performed. From a previous study involving skeleton scintigraphy (5) we knew that single examinations do not allow a differential diagnosis between benign and malignant disorders of the bone, so we based our calculations on differences between sequential investigations. We found that in follow-up investigations of patients with breast carcinoma the combined determination of CEA, CA 15-3 and bone alkaline phosphatase may be indicative for the localisation of metastatic disease. The determination of the bone alkaline phosphatase is easy to handle with a short assay time and good reproducibility; it can therefore be recommended.

Alkaline Phosphatase↗
PubMed1983

Characterization of acid and alkaline phosphatase activity in preparations of tubulin and microtubule-associated proteins.

Acid and alkaline phosphatase activity, determined by the hydrolysis of p-nitrophenyl phosphate, was found in preparations of microtubules purified from bovine brain by temperature-dependent assembly-disassembly and ion-exchange chromatography. Phosphocellulose-purified tubulin contained an associated acid phosphatase activity, stimulated by Mg2+ and by Zn2+. Alkaline phosphatase activity with a pH optimum of 10.4 was measured in a fraction of microtubule-associated proteins (MAPs). Kinetics and the effects of sodium fluoride, sodium tartrate, sulfhydryl-blocking agents, EDTA and Zn2+ are reported.

Acid Phosphatase↗
PubMed2006

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture.

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MW(r) of about 120 kDa and specific PNPP activity of 1200 U/mg. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 U/mg), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and beta-glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function.

Alkaline Phosphatase↗
PubMed1982

A variant alkaline phosphatase in renal cell carcinoma.

We report a case of renal cell carcinoma in which up to 32% of the abnormally increased alkaline phosphatase activity in serum was contributed by a variant alkaline phosphatase originating in the primary tumor and its secondary deposits. The variant enzyme was probably an altered form of normal renal alkaline phosphatase. The rest of the alkaline phosphatase activity in the serum was of hepatic origin, but no abnormality of the liver was discovered at autopsy.

Adenocarcinoma↗
PubMed1980

[Alkaline phosphatase properties of the olfactory lining of the bull].

Biochemical and electrophoretic characteristics of enzyme alkaline phosphatase and its localization in the bull's olfactory mucose structures have been investigated. The activity of enzymes was found to change with odour stimulation. Using electron microscope histochemistry a considerable activity of this enzyme has been demonstrated in the structures secreting olfactory mucus, and in the mucus itself, with alkaline phosphatase macromolecules being concentrated on the surface membrane of olfactory cilia, forming a layer around these. The activity of alkaline phosphatase was found in some receptor cells. The electrophoretic analysis allowed to find five molecular forms of alkaline phosphatase, having different substrate specificity. Isoenzymes II and III, splitting alpha-sodium naphthylphosphate, displayed similar properties with those of intestinal serum isoenzymes. Other three of the five isoenzymes of olfactorymucose have no analogues in the serum that could be regarded as a possible involvement in olfactory epithelium of phosphatases performing specific functions typical of this receptor organ.

Alkaline Phosphatase↗
PubMedDate not supplied

Fc site-specific labeling of immunoglobulins with calf intestinal alkaline phosphatase.

A strategy is described for the site-specific conjugation of alkaline phosphatase to the Fc region of immunoglobulins. Mild oxidation of immunoglobulins by sodium periodate followed by reductive amination with excess cystamine and sodium cyanoborohydride covalently attaches cystamine specifically to the oligosaccharide chains of the antibody's Fc region. The disulfide bonds of the conjugated cystamine moieties are then reduced to free thiols with a low concentration of dithiothreitol. The thiol-modified antibodies (typically containing three to four thiols per Fc region) are then brought into contact with maleimide-functionalized alkaline phosphatase to form antibody-alkaline phosphatase conjugates. The Fc site-specific conjugation methodology was optimized and successfully applied to a number of different monoclonal and polyclonal antibodies. The conjugates prepared show assay performances superior to those prepared by more conventional means. Evidence obtained indicates that this superior conjugate performance results from total retention of the antibody's antigen binding activity following the Fc site-specific conjugation procedure.

Alkaline Phosphatase↗
PubMed1985

Coordinate regulation of collagen and alkaline phosphatase levels in chick embryo chondrocytes.

Chick embryo tibial chondrocytes release into their extracellular matrix several species of proteochondroitin sulfate and collagen as well as matrix vesicles that are rich in Ca2+ and alkaline phosphatase and that appear to play a role in the calcification of cartilage. To determine whether there was any parallel regulation of the production of these products, the rates of collagen synthesis by cultured chick embryo tibial chondrocytes were altered, and the resulting changes in proteochondroitin sulfate synthesis and alkaline phosphatase levels in the cells were measured. As the rate of collagen synthesis was increased by adding increasing amounts of ascorbic acid to the culture medium, there was a parallel increase in the level of alkaline phosphatase. Similarly, when the rate of collagen synthesis was inhibited by adding 3,4-dehydroproline to the culture medium, the levels of alkaline phosphatase fell. The alkaline phosphatase in the culture medium was associated with vesicles which appeared to be matrix vesicles. It was recovered quantitatively by filtration through membranes with a pore size of 0.1 mu and measured by solubilizing the alkaline phosphatase from the membrane with detergent and assaying with 4-methylumbelliferyl phosphate as the substrate. When the matrix vesicles from the culture medium were analyzed for collagen types, it was found that only Type X collagen was recovered in this fraction. The implications of the association of Type X collagen and the matrix vesicles, both of which are found primarily in growth plate cartilage in the zone of hypertrophied chondrocytes which is in the process of mineralization, are discussed.

Alkaline Phosphatase↗
PubMed1983

Effect of ethanol on alkaline phosphatase activity in HeLa cells.

Incubation of HeLa cells with 10-80 mM ethanol, caused a dose-dependent decrease in alkaline phosphatase specific activity. With 80 mM ethanol, the decrease became apparent after 24-48 hr incubation. The same dose-dependent effect was observed with the nonmetabolizable alcohol t-butanol. The effect of ethanol was not due to toxicity as the cells continued to replicate for weeks in the presence of ethanol and was reversible. There were no differences in the Km values of alkaline phosphatase in crude extracts or solubilized enzyme prepared either from control or 80 mM ethanol-treated cells. Hydrocortisone and choline chloride, which by themselves increase alkaline phosphatase specific activity, altered the effect of ethanol on alkaline phosphatase. In the presence of either 1 microM hydrocortisone or 40 mM choline chloride, neither 10 nor 20 mM ethanol decreased alkaline phosphatase specific activity.

Alkaline Phosphatase↗
PubMed1989

Isolation and characterization of monoclonal antibodies against alkaline phosphatase of Pseudomonas aeruginosa.

Monoclonal antibodies against the alkaline phosphatase of Pseudomonas aeruginosa were produced from spleen cells of BALB/c mice primed with purified alkaline phosphatase of P. aeruginosa ATCC 10145 and SP20/Ag-14 myeloma cells. The eight stable clones established produced antibodies that reacted by enzyme-linked immunosorbent and indirect immunofluorescence assays with all bacterial strains of P. aeruginosa, including the 17 serotypes and two nontypable strains. Three of the clones cross-reacted only with some Pseudomonas species of the rRNA homology group I defined by N. J. Palleroni (in N. R. Krieg and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, 8th ed., p. 140-218, 1984). The other clones also interacted with other species, including Pseudomonas acidovorans and Xanthomonas maltophilia. Because other species of the genera Aeromonas and Acinetobacter and species of the family Enterobacteriaceae were not detected by these monoclonal antibodies, the antibodies could be used as reagents for routine detection of P. aeruginosa in clinical specimens. Interactions of the antibodies with other Pseudomonas species such as P. fluorescens and P. stutzeri are not important, since these species are susceptible to the same antipseudomonal agents.

Alkaline Phosphatase↗
PubMed1987

The physical characteristics and enzymatic modification of fetal intestinal alkaline phosphatase in amniotic fluid.

The predominant form of alkaline phosphatase in amniotic fluid at 16 to 18 weeks gestation has the inhibition characteristics of the isoenzymes from adult and fetal small intestine. These characteristics are shared by an alkaline phosphatase of presumed intestinal origin occasionally found in the sera of premature neonates. However, in contrast to the rapid anodal migration of the latter enzyme, amniotic fluid phosphatase is of low electrophoretic mobility in polyacrylamide gel, and it has a high molecular weight. Digestion of amniotic fluid with bromelain produces a rapidly-migrating active enzyme form, with a molecular weight of about 135,000, compared with 145,000 for the fetal intestinal phosphatase in serum. The bromelain-treated amniotic fluid phosphatase is similar in size to a Kasahara isoenzyme in the serum of a cancer patient, which is itself thought to result from re-expression of a fetal intestinal phosphatase gene.

Adult↗