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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human placental homogenates and membrane fractions were centrifuged on continuous sucrose density gradients, with or without buoyant density perturbation of plasma-membranes by digitonin, and aliquots of each gradient fraction were assayed for a range of plasma-membrane and intracellular organelle markers, and for specific binding and inactivation of radiolabelled GnRH agonist (GnRHA), Buserelin ([D-Ser(tBu)6] GnRH 1-9 ethylamide). GnRH agonist (GnRHA) binding equilibrated in the same regions of control gradients as the plasma-membrane markers, EGF-receptor and alkaline phosphatase. Moreover, binding of 125I-labelled GnRHA and 125I-labelled chicken GnRH II (ch GnRH II) was enriched in the same regions of the gradients, indicating that both bound to the same membrane fractions. Digitonin pretreatment increased the buoyant density of all three placental plasma-membrane markers to a similar degree. Intracellular organelle markers (and hCG content) equilibrated in different regions of the gradient to placental surface-membranes, and were not perturbed appreciably by digitonin. In contrast, inactivation of 125I-labelled GnRHA was associated largely with the soluble (cytosol) fraction which failed to enter the gradient, and little tracer inactivation was observed in fractions enriched in GnRHA binding activity. Similar results were obtained with fractionated rat pituitary membranes. We conclude that: (a) placental GnRH binding sites do not represent binding of radiolabelled ligand to GnRH-degrading enzymes, (b) degradation of radiolabelled ligand is associated largely with placental cytosol fractions, and (c) GnRH binding activity appears to be associated largely with placental plasma-membranes.
Placenta
PMID:Human placental gonadotrophin-releasing hormone (GnRH) binding sites: II. Comparison of binding and inactivation of 125I-labelled GnRH agonist to subcellular fractions following density gradient centrifugation. 133 45

It was found that mitochondria from human placenta exhibited an ADPase activity with the following characteristics. The enzyme responsible for this activity was associated with the inner mitochondrial membrane. It was not released by treatment of the submitochondrial particles with solutions of high ionic strength. Maximal ADP hydrolysis was reached at pH 8. Specific inhibitors for alkaline phosphatase (L-phenylalanine), myokinase (P1,P5-di(adenosine-5')pentaphosphate), or 5'-nucleotidase (concanavalin A) did not decrease ADP hydrolysis. ATP synthesis from ADP by myokinase was about 13 nmol/mg/min, whereas ADP hydrolysis reached values around 500 to 550 nmol/mg/min, indicating that a myokinase-H+ATPase combination could not account for the observed rates of ADP hydrolysis. The activity was stimulated by Mg2+, but high concentrations of this cation produced inhibition. High ADP concentrations did not inhibit ADPase activity. Kinetic measurements of the activity in the submitochondrial particles showed that the true substrate was ADP-Mg. The kinetic studies showed V(app) values of 476 and 270 nmol/mg/min, and Kmapp values of 416 and 8.7 microM.
Placenta
PMID:Subcellular localization and properties of adenosine diphosphatase in human placenta. 147 Jun 6

We describe the culture of human term placental trophoblast cells on cell-free amniotic membrane, with medium on both sides. Over the course of 2 days, the isolated cells, initially simple, mononucleated and probably cytotrophoblast, form a confluent layer of multinucleated syncytial cells with morphological and immunocytochemical properties of syncytiotrophoblast. This layer becomes polarized with respect to morphology, alkaline phosphatase distribution and hCG secretion. Contamination with amnion cells, and with other cell types that are present in placental tissue, was less than 1 per cent. A preliminary investigation of the permeability properties of the preparation showed that the trophoblast cell layer, rather than the amniotic membrane, was rate-limiting to transtrophoblast transfer, but that possible effects of the supporting membrane should be considered. The transtrophoblast transfer of D-glucose and the non-metabolisable analogue, 3-O-methyl-D-glucose (3OMG), had saturable and non-saturable/leak components in both directions, indicating that carrier-mediated processes were involved. The non-metabolisable amino acid 2-aminoisobutyrate (AIB) was both accumulated within the trophoblast cells, and transferred by saturable and non-saturable processes from the microvillous side, but no saturable accumulation or transfer was observed from the basal side, at the concentrations tried. The results suggest that this model may prove suitable for studies of transtrophoblast transfer.
Placenta
PMID:Two-sided culture of human placental trophoblast. Morphology, immunocytochemistry and permeability properties. 170 71

We have developed hybridization probes that clearly distinguish the RNA transcripts of the two closely related human heat-stable alkaline phosphatase genes. RNA from the PLAP-1 gene, encoding the term placental alkaline phosphatase, is the predominant transcript in placenta from 8 weeks to term. Transcripts from the PLAP-2 gene, encoding the germ-cell or PLAP-like enzyme, are also detectable in the placenta, but at no more than 2 per cent the level of PLAP-1 transcripts.
Placenta
PMID:Trace expression of the germ-cell alkaline phosphatase gene in human placenta. 172 15

In this report, we describe the generation of specific antibodies to rat alkaline phosphatase and the temporal and regional characteristics of alkaline phosphatase expression during maturation of the rat chorioallantoic placenta. An antipeptide antiserum was generated to the amino terminal 15 amino acids of rat alkaline phosphatase. The antiserum specifically recognized alkaline phosphatase. Alkaline phosphatase expression was monitored in the junctional and labyrinth zones of the chorioallantoic placenta by Western and Northern blot analyses. Alkaline phosphatase protein and mRNA were present in both the junctional and labyrinth zones on day 13 of gestation. As gestation advanced, alkaline phosphatase mRNA and protein expression decreased below the limits of detection in the junctional zone, while alkaline phosphatase expression increased in the labyrinth zone. Labyrinthine alkaline phosphatase migrated predominantly as a 95-kDa species, whereas rat kidney expressed exclusively the 75-kDa species. Enzymatic deglycosylation of the 75- and 95-kDa alkaline phosphatase species resulted in the generation of a 55-kDa species. In summary, alkaline phosphatase expression is a useful indicator of trophoblast differentiation.
Placenta
PMID:Expression of alkaline phosphatase in differentiated rat labyrinthine trophoblast tissue. 175 73

Cytochemistry revealed that alkaline phosphatase was localized predominantly to the maternal facing plasma membrane of syncytiotrophoblast layer II in the haemotrichorial rat placenta at term. Plasma membrane vesicles prepared from term rat placenta by homogenization, treatment with MgCl2, and differential centrifugation were enriched 14-fold in alkaline phosphatase activity as compared to homogenate. These vesicles were mainly oriented right side out, as shown by a lack of effect of saponin treatment on alkaline phosphatase activity. Na+ uptake into the vesicles under equilibrium exchange conditions was significantly stimulated (P less than 0.01) nearly threefold in the presence of an outwardly directed H+ gradient as compared to no gradient. The stimulation by a H+ gradient was abolished in the presence of 0.5 mM amiloride. Measurements with the pH-sensitive dye BCECF in the absence of Na+ showed that 95.2 +/- 0.6 per cent of a 1.2 pH unit H+ gradient was dissipated from the vesicles in 2 min, but the remaining gradient was maintained for up to 15 min. These experiments therefore provide evidence that vesicles derived mainly from the maternal facing plasma membrane of syncytiotrophoblast layer II of the rat placenta possess a Na+/H+ exchanger.
Placenta
PMID:Preparation of plasma membrane vesicles from the rat placenta at term and measurement of Na+ uptake. 208 46

Plasma pyridoxal 5'-phosphate (PLP) concentrations decrease 50% in pregnant mice and erythrocyte PLP levels increase threefold over nonpregnant levels. These studies were designed to determine whether changes in the enzymes involved in synthesis and degradation of PLP in blood are altered during pregnancy. We measured net synthesis of PLP in erythrocytes and the activity of enzymes involved in the regulation of plasma and erythrocyte PLP concentration: erythrocyte pyridoxal kinase (PLK) and neutral phosphatase, and plasma and tissue alkaline phosphatase (ALP). Net synthesis of PLP and activities of erythrocyte PLK and neutral phosphatase in erythrocytes remained unchanged during pregnancy. We were unable to detect any dephosphorylation of PLP in erythrocytes of pregnant or nonpregnant mice. Mouse erythrocytes were devoid of ALP activity; neutral phosphatase was inactive with PLP and PLP was an uncompetitive inhibitor of the enzyme. Plasma ALP activity decreased 50% in the pregnant mice; therefore, it likely does not participate in the reduction of plasma PLP levels during pregnancy. Placenta had high levels of PLP-phosphatase activity (ALP) and, if it is active as an ectoenzyme in this tissue as it is in others, it may be the most important mediator of plasma PLP levels in pregnancy.
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PMID:Vitamin B-6 metabolic enzymes in blood and placenta of pregnant mice. 215 29

Growth characteristics and the expression of trophoblast-associated markers by six cell lines generated from midgestation chorioallantoic placentas of outbred (Holtzman) and inbred (Lewis, PVG.RT Ir8) rats were evaluated. The cells comprising all cell lines were epithelioid (contained cytokeratin-type intermediate filaments), had normal (2n, 4n) DNA content, and synthesized the extracellular matrix glycoprotein laminin. Variability was observed among the lines in all other characteristics: median cell size, rate of growth, serum dependency, responses to transferrin and dibutyryl cyclic adenosine-3',5'-monophosphate, synthesis of some major proteins, alkaline phosphatase activity, and the expression of immunoreactive placental lactogen-II. In general, cell lines with smaller mean cell sizes grew rapidly and required little serum for maintenance in vitro; cell lines with larger mean sizes grew more slowly and preferred higher concentrations of serum. Some associations between mean cell size/rate of growth and other characteristics were observed. No major differences were apparent between cell lines generated from outbred and inbred rat placentas. Trophoblast cell lines expressing distinct phenotypes provide a valuable new approach for studying a wide range of trophoblast cell activities.
Placenta
PMID:Isolation of phenotypically distinct trophoblast cell lines from normal rat chorioallantoic placentas. 278 30

Using density gradient centrifugation, human trophoblastic cells were enriched from mixed cell populations of enzymatically dispersed first- and third-trimester placentae. Over 95 per cent of the cells recovered were of epithelial (i.e., trophoblastic) origin, as evidenced by their cytokeratin intermediate filament positivity and vimentin negativity, examined using indirect immunofluorescence, and also by their high content of human chorionic gonadotrophin. The activities of key enzymes involved in purine degradation and re-utilization (5'-nucleotidase; AMP-deaminase; hypoxanthine phosphoribosyltransferase (HPRT); xanthine dehydrogenase/oxidase) as well as the total activity of alkaline phosphatase were measured in the trophoblastic cells. A six-fold increase in the trophoblastic alkaline phosphatase activity was noted between the first and third trimester. A 40 per cent decrease was noted in the activity of 5'-nucleotidase, which, on the basis of kinetic properties, appears to have a dominant role in the dephosphorylation of placental nucleoside-5'-monophosphates. The trophoblastic activities of AMP-deaminase, HPRT, and xanthine dehydrogenase/oxidase did not change as a function of the gestational age. In view of the relative activities of the latter two enzymes, hypoxanthine formed in the trophoblast appears more likely to be re-utilized than degraded to uric acid.
Placenta
PMID:Activities of key enzymes of purine degradation and re-utilization in human trophoblastic cells. 283 9

A discontinuous density gradient centrifugation method, devised to isolate enriched populations of trophoblast from murine definitive placentae, is described. It is concluded that the isolated adherent cells are trophoblast on the basis of the following characteristics: they are fetally derived, as determined by their donor glucose phosphate isomerase phenotype in embryo transfer experiments; epithelial cells, as shown by the presence of cytokeratin filaments and the absence of vimentin; negative for the stage-specific embryonic antigen-I (SSEA-I); and capable of progesterone secretion. Initially, they grew as individual polygonal cells, tending to form tight confluent monolayers with poorly defined intercellular boundaries. They were mono- or binucleate and increased their nuclear size with time. After two days, giant cells appeared to be formed from binucleated cells by nuclear fusion, and multinucleated cells appeared forming syncytia. Some of these cells also seemed to form giant cells. A low percentage (1 to 10 per cent) of contaminating cells, mainly macrophage-like cells, was observed. The isolated cells were a mixture of alkaline phosphatase- (AP-)positive and AP-negative cells, with some of the latter having phagocytic capacity. All were Fc receptor-negative. The possible identity of these cells in relation to trophoblast in the intact placenta is discussed. This method of isolating and characterizing trophoblast cells from the definitive mouse placenta will be a useful tool for studying the biology and immunology of trophoblast.
Placenta
PMID:Isolation and characterization of trophoblast from murine placenta. 301 19


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