Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter. When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI. In particular, the BPTI produced by E. coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea. This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed.
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PMID:Production of native, correctly folded bovine pancreatic trypsin inhibitor by Escherichia coli. 242 15

A gene encoding bovine prochymosin (PC) was fused to the coding sequence (phoA) for the Escherichia coli alkaline phosphatase (AP) signal peptide and expressed in E. coli under the control of the phoA promoter. Upon induction, an AP-PC fusion protein was produced which was neither processed nor exported into the periplasm. We investigated this lack of secretion by constructing a series of gene fusions in which different regions of the PC gene were inserted between the coding regions of the AP leader and mature protein. Analysis of the cellular location of the proteins encoded by these fusions revealed that a region of PC (between amino acids 6 and 29) prevented processing and secretion of an AP-PC fusion when inserted near to the AP signal peptide. In contrast, when this 'blocking sequence' was inserted elsewhere in AP the hybrid proteins were efficiently processed and translocation was initiated.
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PMID:A short N-proximal region of prochymosin inhibits the secretion of hybrid proteins from Escherichia coli. 251 Oct 82

The 33 kDa extrinsic polypeptide of photosystem II, also known as the manganese-stabilizing polypeptide (MSP), is located on the lumen side of the thylakoid and is involved in water oxidation. The gene for MSP, designated woxA, has been cloned from the nitrogen-fixing filamentous cyanobacterium Anabaena and sequenced. The woxA open reading frame was found to be 819 bp. The deduced amino acid sequence was 63% and 59% homologous with that of Synechococcus and Synechocystis, respectively, and 44% conserved when compared to the MSP of spinach or pea. Two cysteine residues at positions 48 and 73 were found to be conserved in cyanobacteria and plants. The first 29 amino acids are hydrophobic and may represent the transit peptide. woxA: :phoA translational fusion products, in which the body of Escherichia coli alkaline phosphatase was fused to the amino terminal portion of woxA between residues 35 and 130, yielded active alkaline phosphatase in E. coli. Thus the transit peptide of woxA functions in E. coli to transport phosphatase across the cytoplasmic membrane. S1 mapping and primer extension experiments showed that the woxA transcription initiation site is located 220 bp upstream from the translational start. The woxA promoter has some resemblance to the E. coli consensus and other known Anabaena vegetative cell promoters.
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PMID:Nucleotide sequence of the gene encoding the 33 kDa water oxidizing polypeptide in Anabaena sp. strain PCC 7120 and its expression in Escherichia coli. 251 33

During nitrogen-limited growth, Escherichia coli expresses a specific ammonium or methylammonium ion transport system (Amt). Strains carrying defects in Amt have been isolated following Tn10 transposon mutagenesis. These mutants have less than 10% of the transport activity of the parental strain. Glutamate, glutamine, arginine, or high levels (20 mM) of ammonium will serve as the sole nitrogen source for growth of these strains, and glutamine synthetase is normally expressed and repressed by the nitrogen regulatory (Ntr) system. When transformed with plasmid pGln84, containing lacZ fused to an Ntr promoter (glnLp), the Amt mutants expressed a normal level of beta-galactosidase. Furthermore, P1 bacteriophage transduction of the amt mutation into an Ntr mutant, normally constitutive for Amt, gave Amt- transductants. Therefore, the mutations are unlikely to lie within genes affecting Ntr elements. Following transformation with plasmid libraries of E. coli genomic DNA constructed in pUC9, two plasmids conferring the Amt+ phenotype on the amt mutants were isolated. These plasmids were unable to complement the Amt- phenotype of Ntr- mutants. Restriction digestion of these plasmids revealed common fragments, and Southern blot analyses indicated that the Amt-complementing sequence and the site of Tn10 insertion in the genome occur in the same 3.4-kilobase HindIII-SalI fragment. Insertion of TnphoA into this fragment produced amt::phoA fusions which gave high levels of alkaline phosphatase under nitrogen-limiting conditions but low levels during ammonia excess. This suggests that the amt product contains domains which are exported to the periplasm.
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PMID:Isolation of an ammonium or methylammonium ion transport mutant of Escherichia coli and complementation by the cloned gene. 253 89

Ultrastructural, enzyme histochemical and immunohistochemical studies were performed on tissue obtained from eight cases of malignant fibrous histiocytoma (MFH) and five cases of sacral decubitus ulcer. The MFH was composed of two major tumour cell types: fibroblast-like and histiocyte-like cells. Both cell types demonstrated abundant branching, fragmented rough endoplasmic reticulum (rER), many free ribosomes, occasional small mitochondria, an oval, elliptical or irregularly shaped nucleus with one or two prominent nucleoli and often a few dense bodies. However, pseudopodial projections, multivesicular bodies and phagosomes, common histiocyte organelles, were not seen. With little difference between cases or selection sites, the MFH cells reacted to acid phosphatase (AcP) and alpha-naphtyl butyrate esterase (ANBE) by enzyme histochemistry and with ferritin (Fer), alpha 1-antitrypsin (AT), alpha 1-antichymotrypsin (ACT), fibronectin (FN), HLA-DR, HLA-DP, Leu 10 and OKT 9 in immunohistochemical studies. MFH tumour cells did not immunostain with monocyte/macrophage markers (Leu M1, Leu M3, Mo 1, Mo 2 and Macrophage) although non-neoplastic histiocytes did react to these markers. In addition, granulation tissue, such as that found in sacral decubitus ulcers, was examined and the existence of a specific cell type called the "fibrohistiocytoid (FH) cell" was documented. The FH cell was short, spindle shaped and elliptical. Ultrastructurally, it had fragmented rER distributed in a branching pattern, dispersed free ribosomes, small mitochondria and a few dense bodies, but lacked diverse fused lysosomes and distinct pseudopodial cytoplasmic extensions. The FH cells reacted with AcP, alkaline phosphatase and ANBE but not with peroxidase using enzyme histochemistry and with Fer, AT, ACT, FN, HLA-DR, HLA-DP, Leu 10 and OKT 9 but not with monocyte/macrophage markers, C3d receptor, C3bi receptor in immunohistochemical studies. The FH cells had morphological, enzyme histochemical and immunohistochemical characteristics intermediate between fibroblasts and histiocytes. Similarities between MFH cells and the FH cells seen in chronic inflammation are discussed.
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PMID:Malignant fibrous histiocytoma: similarities to the "fibrohistiocytoid cells" in chronic inflammation. 254 May 88

The nucleotide sequence of the Vibrio alginolyticus alkaline serine exoprotease A (ProA) gene cloned in Escherichia coli was determined. The exoprotease A gene (proA) consisted of 1602 bp which encoded a protein of 534 amino acids (aa) with an Mr of 55,900. The region upstream from the gene was characterized by a putative promoter consensus region (-10 -35), a ribosome-binding site and ATG start codon. The proA gene encodes a typical 21-aa N-terminal signal sequence which, when fused to alkaline phosphatase by means of transposon TnphoA, was able to mediate transport of the alkaline phosphatase to the periplasm in E. coli. Deletions of up to 106 aa from the C terminus of ProA did not result in the loss of extracellular protease activity. Additional V. alginolyticus genes were not involved in the secretion into the medium of the cloned ProA in E. coli. The amino acid sequence of ProA showed low overall homology to a Serratia marcescens serine exoprotease but significant homology was detected with other subtilisin family exoproteases. The fungal proteinase K, another sodium dodecyl sulfate-resistant protease, had 44% aa homology with ProA.
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PMID:Nucleotide sequence of the Vibrio alginolyticus calcium-dependent, detergent-resistant alkaline serine exoprotease A. 254 61

We demonstrated the utility of Escherichia coli alkaline phosphatase, encoded by phoA, as a reporter molecule for genetic fusions in Rhodobacter sphaeroides. A portion of the R. sphaeroides cycA gene was fused to phoA, yielding a fusion protein comprising the putative signal sequence and first 10 amino acids of the cytochrome c2 apoprotein joined to the sixth amino acid of alkaline phosphatase. The fusion protein was efficiently transported to the periplasm of R. sphaeroides as determined by enzyme activity, Western immunoblot analysis, and immunogold electron microscopy. We also documented the ability of an R. sphaeroides mutant, RS104, with gross defects in photosynthetic membrane morphology to efficiently recognize and translocate the fusion protein to the periplasmic compartment. The inclusion of 500 base pairs of R. sphaeroides DNA in cis to the cycA structural gene resulted in a 2.5-fold increase in alkaline phosphatase activity in photosynthetically grown cells compared with the activity in aerobically grown cells, demonstrating that the fusion protein is regulated in a manner similar to that of cytochrome c2 regulation. We also constructed two pUC19-based plasmids suitable for the construction of translational fusions to phoA. In these plasmids, translational fusions of phoA to the gene under consideration can be made in all three reading frames, thus facilitating construction and expression of fusion protein systems utilizing phoA.
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PMID:Construction, expression, and localization of a CycA::PhoA fusion protein in Rhodobacter sphaeroides and Escherichia coli. 255 61

A library of fepA::phoA gene fusions was generated in order to study the structure and secretion of the Escherichia coli K-12 ferric enterobactin receptor, FepA. All of the fusion proteins contained various lengths of the amino-terminal portion of FepA fused in frame to the catalytic portion of bacterial alkaline phosphatase. Localization of FepA::PhoA fusion proteins in the cell envelope was dependent on the number of residues of mature FepA present at the amino terminus. Hybrids containing up to one-third of the amino-terminal portion of FepA fractionated with their periplasm, while those containing longer sequences of mature FepA were exported to the outer membrane. Outer membrane-localized fusion proteins expressed FepA sequences on the external face of the outer membrane and alkaline phosphatase moieties in the periplasmic space. From sequence determinations of the fepA::phoA fusion joints, residues within FepA which may be exposed on the periplasmic side of the outer membrane were identified.
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PMID:Export of FepA::PhoA fusion proteins to the outer membrane of Escherichia coli K-12. 255 64

Manoil and Beckwith (1985) have constructed a transposon, TnphoA, that permits the generation of hybrid proteins composed of alkaline phosphatase (AP) lacking its signal peptide fused to amino-terminal sequences of other proteins. This transposon has been used to localize export signals and analyze membrane topology of bacterial proteins. We have applied this approach to the membrane fusion protein (F) of respiratory syncytial virus (RSV). The transposon TnphoA and a plasmid directing bacterial expression of the F gene were used to construct F-AP hybrids. These hybrids yielded AP activity, indicating the presence of viral sequences that promoted protein transport through the cytoplasmic membrane. Sequence analysis showed that TnphoA was inserted at four different positions within the F1 subunit. Deletion of the hydrophobic F1 amino-terminus (fusion-related domain) resulted in AP transport to the periplasm, suggesting that the hydrophobic amino-terminus of the F2 subunit is sufficient to promote protein export. Some hybrids were apparently cleaved at or near the F2/F1 junction. The periplasmic localization of an uncleaved hybrid strongly suggested that the fusion-related domain of the F protein, when in the uncleaved F0 precursor, can be moved across the bacterial cytoplasmic membrane. Although these results apply to the recombinant F protein, they agree with the presumed signal sequence and membrane topology of the native F glycoprotein. Thus, this method may be useful in determining membrane topology and in localizing important domains of viral proteins.
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PMID:Alkaline phosphatase fusions to the respiratory syncytial virus F protein as an approach to analyze its membrane topology. 255 67

Colchicine- and vinblastine-induced depolymerization of microtubules (MTs) in the intestinal epithelium of rats and mice resulted in significant delivery of three apical membrane proteins (alkaline phosphatase, sucrase-isomaltase, and aminopeptidase N) to the basolateral membrane domain. In addition, typical brush borders (BBs) occurred at the basolateral cell surface, consisting of numerous microvilli that contained the four major components of the cytoskeleton of apical microvilli (actin, villin, fimbrin, and the 110-kD protein). Formation of basolateral microvilli required polymerization of actin and proceeded at glycocalyx-studded plaques that resembled the dense plaques located at the tips of apical microvilli. BBs from the basolateral membrane became internalized into BB-containing vacuoles which served as recipient organelles for newly synthesized apical membrane proteins. The BB vacuoles fused with each other and finally were inserted into the apical BB. Polarized distribution of Na+,K+-ATPase, a basolateral membrane protein, was not affected by drug-induced depolymerization of MTs. These observations indicate that Golgi-derived carrier vesicles (CVs) containing apical membrane proteins are vectorially guided to the apical cell surface by a retrograde transport along MTs. MTs are uniformly oriented towards a narrow space underneath the apical terminal web (termed subterminal space) that contains MT-organizing properties and controls polarized alignment of MTs. In contrast to apical CVs, targeting of basolateral CVs appears to be independent of MTs but demands a barrier at the apical membrane domain that prevents basolateral CVs from apical fusion (transport barrier hypothesis).
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PMID:Role of microtubules in polarized delivery of apical membrane proteins to the brush border of the intestinal epithelium. 256 63


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