Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone synthesis. StAR is thought to increase the delivery of cholesterol to the inner mitochondrial membrane where P450scc resides. Tropic hormones acting through the intermediacy of cAMP rapidly increase pregnenolone synthesis, and this rapid steroidogenic response is believed to be due to StAR's action. The StAR protein contains two consensus sequences for phosphorylation catalyzed by protein kinase A that are conserved across all species in which the amino acid sequence of the StAR protein has been determined. We demonstrated that human StAR expressed in COS-1 cells exists in at least four species detectable by two-dimensional gel electrophoresis followed by Western blotting. The two more acidic species disappeared after treatment of the cell extracts with alkaline phosphatase. 32P was incorporated into StAR protein immunoprecipitated from COS-1 cell extracts, and a 10-min treatment with 8-bromo-cAMP increased 32P incorporation into the StAR preprotein. StAR protein generated by in vitro transcription/translation was phosphorylated by the protein kinase A catalytic subunit in the presence of [gamma-32P]ATP. Mutation of potential sites for protein kinase A-mediated phosphorylation at serine 57 and serine 195 to alanines, individually, reduced 32P incorporation from labeled ATP into StAR preprotein produced by in vitro transcription/translation when incubated with protein kinase A catalytic subunit. 32P labeling of StAR protein expressed in COS-1 cells was also reduced when serine 57 or serine 195 were mutated to alanines. A double mutant in which both serine 57 and serine 195 were changed to alanines displayed markedly reduced 32P incorporation. To determine the functional significance of StAR phosphorylation, we tested the steroidogenic activity of the wild-type StAR and mutated StAR proteins in COS-1 cells expressing the human cholesterol side chain cleavage enzyme system. Mutation of the conserved protein kinase A phosphorylation site at serine 57 had no effect on pregnenolone synthesis. However, mutation of the serine residue at 195 resulted in an approximately 50% reduction in pregnenolone production. The S195A mutant construct did not yield the more acidic species of StAR detected in two-dimensional Western blots, indicating that the mutation affected the ability of the protein to be post-translationally modified. Mutation of the corresponding serine residues in murine StAR (Ser56 and Ser194) to alanines yielded results that were similar to those obtained with human StAR; the S56A mutant displayed a modest reduction in steroidogenic activity, whereas the S194A mutant had approximately 40% of the activity of murine wild-type StAR. In contrast to the human S195A mutation, conversion of serine 195 to an aspartic acid residue had no effect on steroidogenic activity, consistent with the idea that a negative charge at this site modulates StAR function. Our observations suggest that phosphorylation of serine 194/195 increases the biological activity of StAR and that this post- or co-translational event accounts, in part, for the immediate effects of cAMP on steroid production.
 ...
PMID:Phosphorylation of steroidogenic acute regulatory protein (StAR) modulates its steroidogenic activity. 940 83

Osteoblasts and adipocytes originate from common mesenchymal precursors. With aging, there is a decrease in osteoprogenitor cells that parallels an increase of adipocytes in bone marrow. We observed that rabbit serum (RS) induces adipocyte-like differentiation in human osteosarcoma SaOS-2/B10 and MG-63 cell lines, in rat ROS17/2.8 cells, and in mouse calvaria-derived osteoblastic MB1.8 cells, as evidenced by the accumulation of Oil Red O positive lipid vesicles and the decrease in alkaline phosphatase expression. Both SaOS-2/B10 and MG-63 cells, but not ROS17/2.8 nor MB1.8 cells, express significant levels of PPARgamma mRNA, a member of the peroxisome proliferator activated receptor (PPAR) family that has been implicated in the control of adipocyte differentiation. However, both ROS17/2.8 and MG-63 cells express significant levels of the adipocyte selective marker, aP2 fatty acid binding mRNA, which can be further increased by RS. These cell types express PPARdelta/NUC-1 but not PPARalpha, indicating that cells that do not express either PPARgamma or PPARalpha are capable of differentiating into adipocyte-like cells. Transfection experiments in COS cells showed that compared with fetal bovine serum (FBS), RS is rich in agents that stimulate PPAR-dependent transcription. The stimulatory activity was ethyl acetate extractable and was 35-fold more abundant in RS than in FBS. Purification and analysis revealed that the major components of this extract are free fatty acids. Furthermore, the same fatty acids, a mixture of palmitic, oleic, and linoleic acids, activate the PPARs and induce adipocyte-like differentiation of both ROS17/2.8 and SaOS-2/B10 cells. These findings suggest that fatty acids or their metabolites can initiate the switch from osteoblasts to adipocyte-like cells.
 ...
PMID:High fatty acid content in rabbit serum is responsible for the differentiation of osteoblasts into adipocyte-like cells. 944 95

We have studied the biosynthesis and intracellular transport of tissue-nonspecific alkaline phosphatase (TNSALP) transiently expressed in COS-1 cells. Mutations were introduced into TNSALP to examine the effects of a single amino acid substitution on the activity and biosynthesis of TNSALP. The cells expressing wild-type TNSALP exhibited more than 200-fold higher alkaline phosphatase activity than untransfected ones. Pulse-chase experiments showed that TNSALP was synthesized as a 66-kDa endoglucosaminidase H (Endo H)-sensitive form and converted to EndoH-resistant forms with heterogenous molecular masses ( approximately 80 kDa), which finally appeared on the cell surface as judged by digestion with phosphatidylinositol-specific phospholipase C (PI-PLC). In contrast, a TNSALP with a Glu218-->Gly mutation exhibited no phosphatase activity at all and the 66-kDa Endo H-sensitive form was the only molecular species throughout the chase in the transfected cells. In accordance with this finding, digestion with PI-PLC and immunofluorescence observation confirmed that this mutant was never expressed on the cell surface. Another mutant with a Ala162-->Thr substitution, which naturally occurs in association with a lethal hypophosphatasia, exhibited a low activity and only a small fraction of the 66-kDa form acquired Endo-H resistance and reached the cell surface. Since the wild-type and the mutant TNSALPs were labeled with [3H]ethanolamine, a component of glycosylphosphatidylinositol (GPI), it is unlikely that the impaired intracellular transport of the two mutants is due to a failure in their modification by GPI. Interestingly, the 66-kDa Endo H-sensitive form of the TNSALP mutants but not that of the wild-type, was found to form an interchain disulfide-bonded high-molecular-mass aggregate within the cells. These results suggest that impaired intracellular transport of the TNSALP (Ala162-->Thr) molecule caused by its aggregation is the molecular basis for the lethal hypophosphatasia carrying this mutation.
 ...
PMID:Defective intracellular transport of tissue-nonspecific alkaline phosphatase with an Ala162-->Thr mutation associated with lethal hypophosphatasia. 956 33

One point mutation which converts glycine-317 to aspartate of tissue-nonspecific alkaline phosphatase (TNSALP) was reported to be associated with lethal hypophosphatasia (Greenberg, C. R., et al. Genomics 17, 215-217, 1993). In order to define the molecular defect of TNSALP underlying the pathogenesis of hypophosphatasia, we have examined the biosynthesis of TNSALP with a Gly317-->Asp substitution. When expressed in COS-1 cells, the mutant did not exhibit alkaline phosphatase activity at all, indicating that the replacement of glycine-317 with aspartate abolishes the catalytic activity of TNSALP. Pulse-chase experiments showed that the newly synthesized mutant failed to acquire Endo H-resistance and to reach the cell surface. Interestingly, this TNSALP mutant was found to form a disulfide-bonded high-molecular-mass aggregate and was rapidly degraded within the cell, though the mutant protein was modified by glycosylphosphatidylinositol (GPI). Lactacystin, an inhibitor of the proteasome, obstructed the degradation of the mutant protein, suggesting the involvement of proteasome as a part of quality control of TNSALP.
 ...
PMID:Intracellular retention and degradation of tissue-nonspecific alkaline phosphatase with a Gly317-->Asp substitution associated with lethal hypophosphatasia. 961 60

In a screening for new inhibitors of NF-KB and AP-1 mediated signal transduction pathways in COS-7 cells using secreted alkaline phosphatase (SEAP) as a reporter gene three novel compounds, cycloepoxydon (1), 1-hydroxy-2-hydroxymethyl-3-pent-1-enylbenzene (2) and 1-hydroxymethyl-3-pent-1,3-dienylbenzene (3) were isolated from fermentations of the deuteromycete strain 45-93. Cycloepoxydon inhibits the TPA-induced NF-KB and AP-1 mediated SEAP expression with an IC50 of 1-2 micrograms/ml (4.2-8.4 microns) and 3-5 micrograms/ml (12.6-21 microns) respectively. 1-Hydroxy-2-hydroxymethyl-3-pent-1-enylbenzene (2) inhibits the TPA-induced NF-KB and AP-1 mediated SEAP expression with an IC50 of 7 micrograms/ml (36.4 microns) and 5 micrograms/ml (26 microns). 3 showed only a weak inhibition of the AP-1 and no influence on NF-KB dependent reporter gene expression. In COS-7 and HeLa S3 cells electrophoretic mobility shift assays showed that cycloepoxydon strongly reduced the TPA and TNF- alpha mediated binding of NF-KB to a high affinity consensus sequence which was due to the inhibition of phosphorylation of the protein IKB.
 ...
PMID:Cycloepoxydon, 1-hydroxy-2-hydroxymethyl-3-pent-1-enylbenzene and 1-hydroxy-2-hydroxymethyl-3-pent-1,3-dienylbenzene, new inhibitors of eukaryotic signal transduction. 966 73

A murine monoclonal anti-dsDNA antibody was found to penetrate living cells and localize in the nucleus without pathologic effects. A single mutation in VH markedly enhanced cellular penetration. The mutant antibody was produced as recombinant Fab and single chain antibody fragments to investigate its use as a delivery system to target the cell nucleus. Complexes were made containing Fab fragments and alkaline phosphatase conjugated goat antibodies to mouse |gk chains. Fab fragments transported 305 kDa goat antibody-enzyme complexes into the nucleus in COS-7 and CHO cells. A single chain antibody cDNA was constructed by splice overlap extension PCR and expressed in COS-7 cells. Binding of the single chain antibody to dsDNA was shown by ELISA, and cellular penetration and nuclear localization were demonstrated in COS-7 and CHO cells. The single chain antibody cDNA was ligated into the expression vector, pEGFP, to produce a fusion protein with green fluorescent protein. The fusion protein penetrated COS-7 cells and localized in the cell nucleus. The single chain antibody produced during sustained expression in CHO cells re-entered antibody-producing cells and localized in the nucleus without affecting cell viability. Our results demonstrate the potential use of a modified autoantibody as a delivery system to target the cell nucleus.
 ...
PMID:An autoantibody is modified for use as a delivery system to target the cell nucleus: therapeutic implications. 980 41

The universal quantitation of the DNA hybridization reaction has been a goal sought by many researchers. Part of this search has been the need to develop a rapid, sensitive, easy-to-perform, and quantitative method to measure the abundance of specific mRNAs directly within cells. Conventionally mRNA detection can be done by advanced quantitative in situ hybridization (ISH) using either image analysis or fluorescence in situ hybridization (FISH), or indirectly by extraction of mRNA from cells or tissue and using Northern blot or quantitative polymerase chain reaction (PCR). We examined the quantitative nature of probe binding to intracellular mRNA in a sensitive and easy-to-use nonisotopic method of ISH previously developed in our laboratories. The method is applicable to isolated primary cells or cells in culture. The procedural details are very simple, with cells being centrifuged into 96-well microplates, fixed with formalin, and pretreated with Triton X-100 and Nonidet P-40 before photobiotin-labeled cDNA probes are applied. Biotin from the hybridization of probe to target is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and visualized by the p-nitrophenyl phosphate conversion method. The quantitative parameters of the ISH procedure were determined by measuring the levels of expression of erythropoietin (EPO) mRNA and its translated protein in transfected COS-7 cells. There is a log-linear relationship between the levels of signal obtained in the ISH reaction in 96-well microplates and the EPO protein levels measured by enzyme-linked immunosorbent assay (ELISA). This demonstrated relationship is important in the standardization and use of these procedures to measure quantitatively mRNAs within cells.
 ...
PMID:Quantitative aspects of an in situ hybridization procedure for detecting mRNAs in cells using 96-well microplates. 981 11

Several lines of evidence implicate zinc finger proteins of the Gli family in the final steps of Hedgehog signaling in normal development and disease. C-terminally truncated mutant GLI3 proteins are also associated with human syndromes, but it is not clear whether these C-terminally truncated Gli proteins fulfil the same function as full-length ones. Here, structure-function analyses of Gli proteins have been performed using floor plate and neuronal induction assays in frog embryos, as well as induction of alkaline phosphatase (AP) in SHH-responsive mouse C3H10T1/2 (10T1/2) cells. These assays show that C-terminal sequences are required for positive inducing activity and cytoplasmic localization, whereas N-terminal sequences determine dominant negative function and nuclear localization. Analyses of nuclear targeted Gli1 and Gli2 proteins suggest that both activator and dominant negative proteins are modified forms. In embryos and COS cells, tagged Gli cDNAs yield C-terminally deleted forms similar to that of Ci. These results thus provide a molecular basis for the human Polydactyly type A and Pallister-Hall Syndrome phenotypes, derived from the deregulated production of C-terminally truncated GLI3 proteins. Analyses of full-length Gli function in 10T1/2 cells suggest that nuclear localization of activating forms is a regulated event and show that only Gli1 mimics SHH in inducing AP activity. Moreover, full-length Gli3 and all C-terminally truncated forms act antagonistically whereas Gli2 is inactive in this assay. In 10T1/2 cells, protein kinase A (PKA), a known inhibitor of Hh signaling, promotes Gli3 repressor formation and inhibits Gli1 function. Together, these findings suggest a context-dependent functional divergence of Gli protein function, in which a cell represses Gli3 and activates Gli1/2 prevents the formation of repressor Gli forms to respond to Shh. Interpretation of Hh signals by Gli proteins therefore appears to involve a fine balance of divergent functions within each and among different Gli proteins, the misregulation of which has profound biological consequences.
 ...
PMID:Gli proteins encode context-dependent positive and negative functions: implications for development and disease. 1037 10

Three FLAG epitopes have been incorporated into the mammalian expression vector pCMV-5 to create a transient expression vector, p3XFLAG-CMV-7. The vector was designed to express FLAG fusion proteins that can be detected at tenfold lower expression levels than the current FLAG fusion protein expression system. The usefulness of this expression and detection system was demonstrated by expression of bacterial alkaline phosphatase in COS-7 cells. In addition, 3XFLAG bacterial alkaline phosphatase was expressed in Escherichia coli, purified on anti-FLAG M2 affinity gel, and detection of 500 pg of purified protein by Western blot analysis is demonstrated.
 ...
PMID:Multiple epitope tagging of expressed proteins for enhanced detection. 1076 59

In the liver, malonyl-CoA is central to many cellular processes, including both fatty acid biosynthesis and oxidation. Malonyl-CoA decarboxylase (MCD) is involved in the control of cellular malonyl-CoA levels, and functions to decarboxylate malonyl-CoA to acetyl-CoA. MCD may play an essential role in regulating energy utilization in the liver by regulating malonyl-CoA levels in response to various nutritional or pathological states. The purpose of the present study was to investigate the role of liver MCD in the regulation of fatty acid oxidation in situations where lipid metabolism is altered. A single MCD enzyme of molecular mass 50.7 kDa was purified from rat liver using a sequential column chromatography procedure and the cDNA was subsequently cloned and sequenced. The liver MCD cDNA was identical to rat pancreatic beta-cell MCD cDNA, and contained two potential translational start sites, producing proteins of 50.7 kDa and 54.7 kDa. Western blot analysis using polyclonal antibodies generated against rat liver MCD showed that the 50.7 kDa isoform of MCD is most abundant in heart and liver, and of relatively low abundance in skeletal muscle (despite elevated MCD transcript levels in skeletal muscle). Tissue distribution experiments demonstrated that the pancreas is the only rat tissue so far identified that contains both the 50.7 kDa and 54. 7 kDa isoforms of MCD. In addition, transfection of the full-length rat liver MCD cDNA into COS cells produced two isoforms of MCD. This indicated either that both initiating methionines are functionally active, generating two proteins, or that the 54.7 kDa isoform is the only MCD protein translated and removal of the putative mitochondrial targeting pre-sequence generates a protein of approx. 50.7 kDa in size. To address this, we transiently transfected a mutated MCD expression plasmid (second ATG to GCG) into COS-7 cells and performed Western blot analysis using our anti-MCD antibody. Western blot analysis revealed that two isoforms of MCD were still present, demonstrating that the second ATG may not be responsible for translation of the 50.7 kDa isoform of MCD. These data also suggest that the smaller isoform of MCD may originate from intracellular processing. To ascertain the functional role of the 50. 7 kDa isoform of rat liver MCD, we measured liver MCD activity and expression in rats subjected to conditions which are known to alter fatty acid metabolism. The activity of MCD was significantly elevated under conditions in which hepatic fatty acid oxidation is known to increase, such as streptozotocin-induced diabetes or following a 48 h fast. A 2-fold increase in expression was observed in the streptozotocin-diabetic rats compared with control rats. In addition, MCD activity was shown to be enhanced by alkaline phosphatase treatment, suggesting phosphorylation-related control of the enzyme. Taken together, our data demonstrate that rat liver expresses a 50.7 kDa form of MCD which does not originate from the second methionine of the cDNA sequence. This MCD is regulated by at least two mechanisms (only one of which is phosphorylation), and its activity and expression are increased under conditions where fatty acid oxidation increases.
 ...
PMID:Characterization of rat liver malonyl-CoA decarboxylase and the study of its role in regulating fatty acid metabolism. 1094 76


<< Previous 1 2 3 4 5 6 7 8 9 Next >>