Placental alkaline phosphatase isoenzyme expression by the non-HeLa DoT cervical-carcinoma cell line.

Strain E of chloridazon-degrading bacteria, when grown on L-phenylalanine accumulates cis-2,3-dihydro-2,3-dihydroxyphenylalanine. In experiments with resting cells and during growth the bacterium converts the aromatic carboxylic acids phenylacetate, phenylpropionate, phenylbutyrate and phenyl-lactate into the corresponding cis-2,3-dihydrodiol compounds. The amino acids L-phenylalanine, N-acetyl-L-phenylalanine and t-butyloxycarbonyl-L-phenylalanine were also transformed into dihydrodiols. All seven dihydrodiols, thus obtained, were characterized both by conventional analytical techniques and by the ability to serve as substrates for a cis-dihydrodiol dehydrogenase.     

Placental alkaline phosphatase types in Germany

Summary Two children with autosomal deletion (46,XY,del(12)(p11) and 46,XY/46,XY, del(5)(p13)) and normal phenotype were found among 5049 consecutive newborn children. The mother of the proband with deletion short arm 5 had the karyotype 46,XX,9qh+, but the parents had otherwise normal chromosome constitution.

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Zusammenfassung Zwei Kinder mit autosomaler Deletion (46,XY,del(12)(p11) und 46,XY/46,XY,del(5)(p13)) bei normalem Phänotyp wurden unter 5049 auslesefrei gewonnenen Neugeborenen entdeckt. Die Mutter des Probanden mit der Deletion am kurzen Arm von Nr. 5 hatte den Karyotyp 46,XX,9qh+; sonst hatten die Eltern normale Chromosomen.

     

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.     

Placental alkaline phosphatase polymorphism in northern Greece

The placental alkaline phosphatase (Aph) polymorphism was studied in Northern Greece. A new rare placental Aph variant was described.     

Expression of plasma membrane alkaline phosphatase in normal and regenerating choriocapillaris in the rabbit.

Ultrastructural histochemistry for plasma membrane nonspecific alkaline phosphatase was performed on the normal and regenerating choriocapillaris (CC) of rabbits. In normal animals the CC endothelium expressed little or no staining, whereas in regenerating CC the endothelium exhibited staining. The staining was most intense at the unfenestrated plasma membrane. As the capillaries matured and the fenestrated plasma membrane became more extensive, the staining was reduced and eventually eliminated. Pericytes did not stain in normal or regenerating CC.     

Cell cycle variations in HeLa 65 plasma membrane alkaline phosphatase

HeLa plasma membranes from M, G₁, and S phase cells were isolated from growing synchronous cell cultures. It was found that the specific activity of plasma membrane alkaline phosphatase was over three times higher in the M phase cell than in the G₁ and S phase cell. However, sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis showed that the S phase plasma membrane contained 5.5 times more alkaline phosphatase protein than did the plasma membrane from mitotic cells, and 11.0 times more than the G₁ phase plasma membrane. This would indicate that the high specific activity in mitosis was due to modification of the alkaline phosphatase protein resulting in increased enzymatic activity.     

Placental alkaline phosphatase polymorphism in some Punjabi populations

Placental isozyme of alkaline phosphatase (orthophosphoric monoester hydrolase, EC 3.1.3.1.) has been studied in very few Indian populations with respect to its polymorphic form. The present study surveys the alleles encountered in some Punjabi groups of Northern India. The distinguishing physico-chemical properties of this isozyme, viz. heat stability, steady increase in maternal plasma during the course of pregnancy, and unique electrophoretic mobility have been used for its typing. The Punjabi groups surveyed are Jat (Sikh), Baniya and Khatri (all endogamous castes) and Lower Castes and others (two mixed caste groups). These groups were found to be in Hardy-Weinberg-equilibrium, and some of them revealed intergroup heterogeneity, when compared with each other. Further comparison with other Indian populations, for which data were available, also revealed interesting results.     

Capillary volume fraction is the principal determinant of villous membrane thickness in the normal human placenta at term.

The thickness of the villous membrane is known to be an important factor in determining the morphometric diffusing capacity of the placenta at term. As yet it is not certain how areas of the villous membrane specialised for gaseous exchange, the vasculo-syncytial membranes, differentiate. One mechanism suggested is that they arise through obtrusion of the fetal capillaries contained within the stromal core. As a result the bulk of the overlying trophoblast is displaced laterally, reducing the thickness of the villous membrane to as little as 1-2 microns at these points. To test this hypothesis the relationship between the vascularity of the villi, as determined by the villous capillary volume fraction, and the mean thickness of the villous membrane was investigated. Data were taken from a recent study in which placental villi were biopsied from normal term placentae within 1 min of caesarean delivery and at 5, 10, 15 and 20 min thereafter. Since intuitively the membrane has both a maximum and a minimum thickness a sigmoid relationship was fitted to the data using least squares regression analysis. Estimates of arithmetic and harmonic mean thicknesses were then predicted from the capillary volume fraction for a large number of placentae using data from previous studies. These all employed similar stereological techniques but were performed over a number of years by several workers in this laboratory. The predicted values were tested against the measured values using paired 't' tests, but no significant differences (P greater than 0.05) were detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Placental alkaline phosphatase has a binding site for the human immunoglobulin-G Fc portion.

Affinity chromatography of human plasma on placental-alkaline-phosphatase-Sepharose columns (placental alkaline phosphatase, PLAP) yielded consistently a pure protein which was identified as IgG on the basis of electrophoretical and immunological comparisons with authentic human IgG. SDS/PAGE of the protein revealed, under reducing conditions, two polypeptides of 55 kDa and 25 kDa. The N-terminal amino acid sequence (12 residues) of the 55-kDa subunit presented high similarity (83-100%) with known sequences of immunoglobulin gamma chains. The IgG binds by its Fc portion to a fully exposed domain in the plasma-membrane-anchored PLAP. Scatchard analysis of the interaction gave a dissociation constant of 3.68 microM, a value close to those found for haematopoietic cells and syncytiotrophoblast Fc receptors. The latter was affinity purified from human placenta as the major IgG-binding component and presented cross-immunoreactivity with anti-PLAP antibodies, indicating that PLAP and the putative placental Fc receptor could be identical molecules.     

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.     

Placental alkaline phosphatase, a GPI-anchored protein, is clustered in clathrin-coated vesicles.

Pure clathrin-coated vesicles were prepared from a fresh human placenta. The analysis of their content revealed the presence of transferrin, low density lipoproteins, IgG and placental alkaline phosphatase. Since the latter is a membrane protein anchored by a glycan-phosphatidyl inositol (GPI) moiety, its presence in coated vesicles was unexpected. Placental alkaline phosphatase is neither adsorbed to the surface of the vesicles, nor appearing due to plasma membrane contaminants, but is located in the lumen of the vesicles. The presence of alkaline phosphatase in coated vesicles strengthens its postulated physiological role in the transcytosis of IgG molecules in placenta.     

Alkaline phosphatase on activated B cells characterization of the expression of alkaline phosphatase on activated B cells. Kinetics and membrane anchor.

Recently we reported that the expression of the enzyme alkaline phosphatase (APase) is a marker for B cell activation. Enzymatic activity was found only in activated B cells and not T cells. Using flow cytometry we showed that some of the APase was found on the cell membranes (mAPase) and by functional assays, some was spontaneously released into the tissue culture medium. In the present report the expression of mAPase on activated B lymphocytes is more fully characterized. Two mAb specific for rat APase were used to measure the kinetics of the membrane expression of mAPase. Within 48 h of activation, mAPase is detected by flow cytometry and increases coordinately with both the transferrin receptor and IL-2R. Maximal membrane expression of mAPase in terms of number of positive cells and mean fluorescent intensity, is detected by day 4 to 5 of culture. Using hydroxyurea and demecolcine to block cells at G1/S and G2/M, respectively, it appeared that the initial expression of mAPase occurred as cells progressed into S phase of the cell cycle. This was confirmed using two-color flow cytometric analysis with the Hoechst DNA stain 33342 and the FITC-labeled APase-specific mAb. Finally, using phosphatidylinositol-specific phospholipase C we were able to show that 60 to 80% of the mAPase is linked to the membrane via a glycosyl-phosphatidylinositol linkage. From this we have concluded that mAPase can be added to a growing list of glycoproteins that are anchored to the membrane by the glycosyl-phosphatidylinositol linkage and are expressed on differentiating B cells. This list now includes Thy-1, BLAST-1, Jlld, and mAPase.     

Placental alkaline phosphatase is efficiently targeted to rafts in supported lipid bilayers

Evidence is growing that biological membranes contain lipid microdomains or "rafts" that may be involved in processes such as cellular signaling and protein trafficking. In this study, we have used atomic force microscopy to examine the behavior of rafts in supported lipid bilayers. We show that bilayers composed of equimolar dioleoylphosphatidylcholine and sphingomyelin spontaneously form rafts, which are detectable as raised features. A comparison of the extents of protrusion of the rafts in monolayers and bilayers indicates that the rafts in the two leaflets of the bilayer coincide. The rafts were observed both in the absence and presence of cholesterol (33 mol %). Cholesterol reduced raft protrusion presumably by increasing the thickness of the non-raft bilayer. PLAP (glycosylphosphatidylinositol-anchored protein placental alkaline phosphatase) was purified and shown to exist as a dimer. Following its incorporation into supported lipid bilayers, PLAP was found to be targeted efficiently to rafts, both in the absence and presence of cholesterol. We suggest that atomic force microscopy provides a powerful tool for the study of raft structure and properties.     

Tetrameric alkaline phosphatase in human liver plasma membranes

Molecular weights of native membrane-bound alkaline phosphatase released by butanol and by nonionic detergents were more than twice that of the purified dimeric enzyme. Alkaline phosphatase released by phosphatidylinositol-specific phospholipase-C was of both high and low molecular weight: the former was a protomer of a single protein of the same molecular size as monomeric alkaline phosphatase. We conclude that the membrane-bound enzyme is probably a tetramer.     

Stimulation by ATP of alkaline phosphatase in placental plasma membranes.

1. ATP stimulated the p-nitrophenyl phosphatase activity of placental plasma membranes, with an increase in activity of approximately 100% at 5 mM ATP. The stimulation was not dependent on the presence of Mg-2-+. 2. The K-m for p-nitrophenyl phosphate was not changed by the presence of 5 mM ATP. 3. ATP hydrolysis by the plasma membrane preparation under the same assay conditions as for alkaline phosphatase was not influenced by the presence of 5 mM p-nitrophenyl phosphate. 4. Extraction of the plasma membrane preparation with n-butanol abolished the stimulatory effect of ATP, as well as Ca-2-+-activated ATPase activity.     

Synthesis and secretion of alkaline phosphatase in vitro from first-trimester and term human placentas.

The synthesis and secretion of alkaline phosphatases in vitro by human placental tissue incubated in organ culture were studied. First-trimester placenta synthesizes and secretes two different alkaline phosphatase isoenzymes (heat-labile and heat-stable), whereas in term placenta nearly all the alkaline phosphatase synthesized and secreted is heat-stable. The specific activities of alkaline phosphatases in first-trimester and term placental tissue remain constant throughout the time course of incubation. In the media, specific activities increase with time. Hence, alkaline phosphatase synthesis seems to be the driving force for its own secretion. The rates of synthesis de novo and of alkaline phosphatases were measured. The specific radioactivities of the secreted alkaline phosphatases were higher than the corresponding specific radioactivities in the tissue throughout the entire incubation period. The intracellular distribution of the alkaline phosphatase isoenzymes was compared.