Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.
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PMID:Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase. 751 12

To determine which factors may regulate the DNA binding and transcriptional properties of retinoic acid receptors (RARs and RXRs), we investigated the sensitivity of reporter genes bearing various retinoic acid response elements (RAREs) to protein phosphatases (PPases) inhibition. PPases inhibition by okadaic acid led to an increase of the reporter genes activity in a RARE-dependent and ligand-independent manner and was dependent on the type of response element used. Overexpression of protein phosphatases 2A and 1 (PP2A and PP1) decreased the inducibility of the reporter genes tested. Nuclear extracts from okadaic acid-treated COS cells displayed an 2-5-fold increased level of receptor binding to RAREs in vitro, suggesting that PPases inhibition increased the DNA binding activity of retinoid receptors. Treatment of receptors extracted from COS cells by alkaline phosphatase and partially purified PP1 and PP2A decreased their DNA binding activity, but heterodimers bound to DNA were not sensitive to phosphatase treatment. Reconstitution experiments showed that phosphorylation of both receptors increased the DNA binding activity of RXR/RAR heterodimers. Taken together, these data show that the modulation of the phosphorylation state of RARs and RXRs represents an other level of regulation of the retinoid signaling pathway.
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PMID:Protein phosphatases 1 and 2A regulate the transcriptional and DNA binding activities of retinoic acid receptors. 773 17

Two cDNAs encode rat intestinal alkaline phosphatases having completely different carboxyl-terminal peptides; one is hydrophobic and fulfills the consensus requirements for glycan phosphatidylinositol linkage, and the other is neither hydrophobic nor hydrophilic, but contains a small amino acid domain (-NSASS-) just distal to a region of 17 threonine residues. Constructs were created using 80% of the amino-terminal portion of one alkaline phosphatase and the carboxyl-terminal portions of each of the isoforms. Both of the carboxyl-terminal peptides supported glycan phosphatidylinositol linkage as demonstrated by the following criteria: 1) plasma membrane targeting in transfected COS-1 cells, 2) release of transfected alkaline phosphatase by phosphatidylinositol-specific phospholipase C, 3) appearance of the trypanosome variable glycoprotein cross-reacting determinant after phospholipase C treatment, 4) ethanolamine incorporation into newly synthesized enzyme, 5) loss of phospholipase C release after mutation of the omega and omega + 2 positions in the putative linkage site, -NSA-, and 6) evidence of surface membrane localization by immunofluorescence using antibody against rat intestinal alkaline phosphatase. These data demonstrate that a predicted hydrophobic carboxyl-terminal sequence is not essential for glycan phosphatidylinositol linkage. Moreover, because both isomers are membrane-bound, the origin of soluble enzyme in the serum is likely to arise from the action of serum phosphatidylinositol-specific phospholipase C.
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PMID:Two rat intestinal alkaline phosphatase isoforms with different carboxyl-terminal peptides are both membrane-bound by a glycan phosphatidylinositol linkage. 774 44

Treatment of acute promyelocytic leukemia (APL) blasts with cyclic adenosine monophosphate (cAMP) analogs, in combination with all-trans retinoic acid (ATRA), results in the upregulation of the expression of leukocyte alkaline phosphatase (LAP), a marker for the differentiation of the granulocyte. The synergistic interaction between the cyclic nucleotide analogs and the retinoid is not unique to APL cells, as it is observed also in the peripheral granulocytes of chronic myelogenous leukemia (CML) patients. The molecular mechanisms underlying LAP induction were studied in NB4, an immortalized APL cell line. Induction of LAP enzymatic activity is dependent on the time of exposure and on the concentrations of dibutyryl-cAMP or 8-bromo-cAMP and ATRA, two factors that influence the kinetics of appearance of detectable levels of the enzyme. Augmentation of LAP levels by ATRA and cAMP is the result of both transcriptional and early posttranscriptional events and requires de novo protein synthesis. LAP induction correlates with augmentation in the levels of the type I catalytic subunit of cAMP-dependent protein kinase transcript and with granulocytic differentiation. The transcriptional component of the process leading to increased LAP gene expression was reproduced in its main features by transient transfection experiments performed in COS-7 cells using the normal retinoic acid receptor type alpha (RAR-alpha) or the APL-specific aberrant form (PML-RAR) and the upstream promoter of the liver/bone/kidney (L/B/K)-type alkaline phosphatase gene. The promoter is upregulated by treatment with ATRA, and this upregulation is further increased by cAMP analogs.
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PMID:All-trans retinoic acid and cyclic adenosine monophosphate cooperate in the expression of leukocyte alkaline phosphatase in acute promyelocytic leukemia cells. 778 Jan 46

Microsomal-type cytochrome P450s are integral membrane proteins bound to the membrane through their N-terminal transmembrane hydrophobic segment, the signal anchor sequence. To elucidate the determinants that enable the P450s to be located in the ER, we constructed cDNAs encoding chimeric proteins in which a secretory form of carboxyesterase, carboxyesterase Sec, was connected to the N-terminus of the full-length or truncated forms of a microsomal-type P450, P450(M1), and the constructed plasmids were expressed in COS cells. Since carboxyesterase Sec is an N-glycosylated secretory protein, endo H treatment could be used to determine whether these chimeric proteins were located in the ER or not. Carboxyesterase Sec with the N-terminal 20 amino acids, containing the transmembrane region, of P450(M1), was located in the ER, as determined from the endo H sensitivity of the expressed protein and immunofluorescence staining of the cells. As the expressed protein exhibited carboxyesterase activity, it was not retained in the ER through the BiP-dependent quality control system recognizing unfolded proteins. Another chimeric protein construct in which carboxyesterase Sec was connected to the C-terminal region of rat UDP-glucuronosyltransferase (UDP-GT), that contained a double-lysin ER retention motif, was also located in the ER, as determined from the endo H sensitivity and immunofluorescence staining. On the other hand, the sugar moiety of the carboxyesterase Sec connected to the transmembrane segment of UDP-GT, Sec/GTd, was partially resistant to the endo H treatment. From the results of immunofluorescent staining and cell fractionation, it was concluded that the Sec/GTd product was located in the Golgi apparatus. These observations indicated that the N-terminal hydrophobic segment of P450(M1) is sufficient for the ER membrane retention, whereas the transmembrane segment of UDP-GT is not. To determine whether microsomal P450s are recycled between the ER and Golgi compartments or not, a DNA construct encoding cathepsin D connected to the N-terminus of P450(M1) was prepared and expressed in COS cells. The fusion protein was phosphorylated, but the phosphorylation was sensitive to alkaline phosphatase. As a control, authentic cathepsin D was subjected to phosphorylation of its oligosaccharide chain that was resistant to the alkaline phosphatase treatment. Since GlcNAc-P-transferase, which forms the alkaline phosphatase-resistant phosphodiester in the sugar chains of lysosome-targeting proteins, is located in the Golgi apparatus, it was concluded that the oligosaccharide chain of the cathepsin D portion of the fusion protein was not phosphorylated, and that the chimeric protein did not go to the Golgi apparatus.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The transmembrane region of microsomal cytochrome P450 identified as the endoplasmic reticulum retention signal. 779 74

We have transiently expressed the rat Kv2.1 K+ channel polypeptide (pKv2.1) at high levels by transfection of mammalian COS-1 cells. Kv2.1-transfected cells express a molecular mass of 108 kDa pKv2.1, larger than the size of the core polypeptide (95 kDa) predicted from the deduced primary sequence and of pKv2.1 synthesized in cell free or Xenopus oocyte translation systems. The increased size of pKv2.1 in COS-1 cells is due to a posttranslational modification that occurs early (t1/2 = 5 min) in the biosynthetic transport through the endomembrane system, presumably while the protein resides in the endoplasmic reticulum. The increased size is entirely due to phosphorylation, based on in vivo 32P-labeling and sensitivity to alkaline phosphatase digestion. Immunofluorescent localization of pKv2.1 shows intense surface labeling; no intracellular pools of retained protein are apparent. Immunogold electron microscopy confirms that the expressed polypeptide is found on the cell surface in small clusters or patches of 10-15 gold particles. Cells expressing pKv2.1 exhibit large, voltage-dependent outward currents. The pharmacological properties of the expressed Kv2.1 currents are virtually indistinguishable from those described previously in Xenopus oocytes microinjected with Kv2.1 cRNA, but differences in voltage-dependent properties were observed. High level of expression of functional pKv2.1 in these cells points to the utility of this system for the rapid biochemical, cell biological and electrophysiological analysis of altered forms of pKv2.1, and other members of the K+ channel gene family.
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PMID:Properties of Kv2.1 K+ channels expressed in transfected mammalian cells. 808 26

It has recently been shown that in the rat, dihydrotachysterol (DHT) is extensively metabolized in the side-chain in vivo along pathways similar to those of vitamin D. In addition 25-hydroxy-DHT2 [25OHDHT2] is hydroxylated at C1, producing both 1 alpha- and 1 beta- hydroxy compounds. An in vivo study in 1988 demonstrated that in normal adult subjects receiving oral DHT2, plasma 1 alpha,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations fell, but with unchanged plasma PTH levels. Down-regulation of 1,25-(OH)2D3 production by 25-(OH)DHT2 or some other unknown metabolite was also suggested as an explanation for these observations. To investigate whether either of the newly characterized 1 alpha,25- or 1 beta,25-(OH)2DHT2 was formed in vivo in normal man, DHT2 (approximately 1 mg/day, orally) was administered to healthy volunteers (three males and one female). Plasma was analyzed by high performance liquid chromatography and gas chromatography-mass spectrometry, demonstrating the formation of both 1 alpha,25- and 1 beta,25-(OH)2DHT2 in vivo in normal human subjects. Plasma levels of 1,25-(OH)2D3, PTH, ionized and total calcium, inorganic phosphate, and alkaline phosphatase were monitored. The plasma concentrations of DHT2, 25OHDHT2, and 1 alpha,25- and 1 beta,25-(OH)2DHT2 were measured by gas chromatography-mass spectrometry. In all volunteers, plasma ionized calcium increased slightly during DHT2 administration; 1,25-(OH)2D3 and PTH concentrations fell. Plasma levels of DHT2 and its metabolites rose over the same period. The average fall in the level of plasma 1,25-(OH)2D (60-70 pmol/L) was mirrored by a rise in the concentration of 1 alpha,25-(OH)2DHT2 (550 pmol/L). This ratio is appropriate, because it has previously been shown that in a reconstituted COS cell, 1 alpha,25-(OH)2DHT3 has roughly one tenth the potency of 1,25-(OH)2D3. At maximum concentration, the ratios of DHT2/25OHDHT2/1 beta,25-(OH)2DHT2/1 alpha,25-(OH)2DHT2 were approximately 10:1:2:0.1. The concentration of 1 beta,25-(OH)2DHT2 was greater than that of 25OHDHT2, and the ratio of 1 alpha,25- to 1 beta,25-(OH)2DHT2 (1:20) was substantially lower than that in rat plasma (3:10). The data presented here suggest that the active DHT2 metabolite in man is 1 alpha,25-(OH)2DHT2 and that the fall in plasma 1,25-(OH)2D seen during DHT therapy may be partly the result of suppressed PTH secretion.
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PMID:In vivo dihydrotachysterol2 metabolism in normal man: 1 alpha- and 1 beta-hydroxylation of 25-hydroxydihydrotachysterol2 and effects on plasma parathyroid hormone and 1 alpha,25-dihydroxyvitamin D3 concentrations. 820 Sep 53

All-trans retinoic acid (ATRA) is successfully used in the cyto-differentiating treatment of acute promyelocytic leukemia (APL). Paradoxically, APL cells express PML-RAR, an aberrant form of the retinoic acid receptor type alpha (RAR alpha) derived from the leukemia-specific t(15;17) chromosomal translocation. We show here that AM580, a stable retinobenzoic derivative originally synthesized as a RAR alpha agonist, is a powerful inducer of granulocytic maturation in NB4, an APL-derived cell line, and in freshly isolated APL blasts. After treatment of APL cells with AM580 either alone or in combination with granulocyte colony-stimulating factor (G-CSF), the compound induces granulocytic maturation, as assessed by determination of the levels of leukocyte alkaline phosphatase, CD11b, CD33, and G-CSF receptor mRNA, at concentrations that are 10- to 100-fold lower than those of ATRA necessary to produce similar effects. By contrast, AM580 is not effective as ATRA in modulating the expression of these differentiation markers in the HL-60 cell line and in freshly isolated granulocytes obtained from the peripheral blood of chronic myelogenous leukemia patients during the stable phase of the disease. In NB4 cells, two other synthetic nonselective RAR ligands are capable of inducing LAP as much as AM580, whereas RAR beta- or RAR gamma-specific ligands are totally ineffective. These results show that AM580 is more powerful than ATRA in modulating the expression of differentiation antigens only in cells in which PML-RAR is present. Binding experiments, using COS-7 cells transiently transfected with PML-RAR and the normal RAR alpha, show that AM580 has a lower affinity than ATRA for both receptors. However, in the presence of PML-RAR, the synthetic retinoid is a much better transactivator of retinoic acid-responsive element-containing promoters than the natural retinoid, whereas, in the presence of RAR alpha, AM580 and ATRA have similar activity. This may explain the strong cyto-differentiating potential of AM580 in PML-RAR-containing leukemic cells.
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PMID:AM580, a stable benzoic derivative of retinoic acid, has powerful and selective cyto-differentiating effects on acute promyelocytic leukemia cells. 860 43

Growth/differentiation factor-5 (GDF-5) is a member of the bone morphogenetic protein (BMP) family, which plays an important role in bone development in vivo. Mutations in the GDF-5 gene result in brachypodism in mice and Hunter-Thompson type chondrodysplasia in human. BMPs transduce their effects through binding to two different types of serine/threonine kinase receptors, type I and type II. However, binding abilities appear to be different among the members of the BMP family. BMP-4 binds to two different type I receptors, BMP receptors type IA (BMPR-IA) and type IB (BMPR-IB), and a type II receptor, BMP receptor type II (BMPR-II). In addition to these receptors, osteogenic protein-1 (OP-1, also known as BMP-7) binds to activin type I receptor (ActR-I) as well as activin type II receptors (ActR-II and ActR-IIB). Here we investigate the binding and signaling properties of GDF-5 through type I and type II receptors. GDF-5 induced alkaline phosphatase activity in a rat osteoprogenitor-like cell line, ROB-C26. 125I-GDF-5 bound to BMPR-IB and BMPR-II but not to BMPR-IA in ROB-C26 cells and other nontransfected cell lines. Analysis using COS-1 cells transfected with the receptor cDNAs revealed that GDF-5 bound to BMPR-IB but not to the other type I receptors when expressed alone. When COS-1 cells were transfected with type II receptor cDNAs, GDF-5 bound to ActR-II, ActR-IIB, and BMPR-II but not to transforming growth factor-beta type II receptor. In the presence of type II receptors, GDF-5 bound to different sets of type I receptors, but the binding was most efficient to BMPR-IB compared with the other type I receptors. Moreover, a transcriptional activation signal was efficiently transduced by BMPR-IB in the presence of BMPR-II or ActR-II after stimulation by GDF-5. These results suggest that BMPR-IB mediates certain signals for GDF-5 after forming the heteromeric complex with BMPR-II or ActR-II.
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PMID:Identification of type I and type II serine/threonine kinase receptors for growth/differentiation factor-5. 870 14

Knowledge of the number and kinds of differentiation steps characterizing cells of the osteoblast lineage is inadequate. To analyze further osteoblast differentiation, a number of labs have generated monoclonal antibodies to osteogenic cells, derived from both normal bone and osteosarcomas. A variety of immunolabelling patterns on primary cell cultures, cell lines, and tissue sections has been reported, including cell surface, cytoplasmic, and extracellular matrix-associated patterns. Most of the antibodies selected recognize predominantly the mature osteoblast and osteocyte; in addition, however, antibodies have been generated that recognize pre-osteoblasts. Some recognize cells of both the osteoblast and chondroblast lineages and may contribute to a better understanding of the lineage and phenotypic relationships between these two cell types. In addition to recognition in vivo of cell subpopulations of discrete maturational stages, changes in the immunolabelling patterns in vitro have also documented a differentiation sequence in cells undergoing osteogenesis in cell and tissue cultures. In at least two cases, the antibodies have been used to isolate subpopulations of cells from bone, including relatively pure populations of osteocytes. With the exception of several antibodies that are against alkaline phosphatase or known matrix proteins including osteocalcin, the nature of the macromolecular species recognized by most of the antibodies generated to date are unknown. Recently, however, one antibody was used to clone the cDNA for the beta-galactoside-binding lectin, galectin 3 or epsilon binding protein (epsilon BP; IgE-binding protein; Mac-2), from a lambda gt11 osteoblast expression library; another was used to clone from an ROS 17/2.8-COS cell expression library the cDNA for OTS-8, a putative target gene of early response genes stimulated in response to phorbol esters in MC3T3-E1 cells. Neither of these macromolecules had previously been identified in bone cells, but the recent molecular and cellular analyses have shown them to be developmentally and/or hormonally regulated in osteoblastic cells. These antibodies extend the available markers and support earlier observations that a variety of molecules are differentially expressed by cells at different stages of the osteoblast lineage. This chapter will not be an exhaustive survey of all immunocytochemical and immunohistochemical analyses of osteogenic cells and tissues but will focus on the approach of eliciting novel monoclonal antibodies by the injection of osteogenic cells or crude bone extracts and its potential for establishing new markers of the osteoblast lineage. We have not included a large number of studies documenting the use of antibodies raised against several known bone matrix proteins; while these have been crucial in developing our current understanding of osteogenic differentiation, we sought rather to highlight the potential of the "random" injection approach.
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PMID:Monoclonal antibodies as tools for studying the osteoblast lineage. 884 13


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