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)

Cells containing a protein fusion consisting of the Rhizobium leguminosarum bv. viciae nodulation protein, NodT, fused to PhoA, produced alkaline phosphatase activity, indicating that the N terminus of NodT could translocate PhoA across the inner membrane. Cellular fractionation suggested that the NodT::PhoA fusion is targetted to the outer membrane. NodT resembles a family of bacterial outer membrane proteins including TolC, PrtF, CyaE and AprF, which are involved in secretion. By analogy, NodT (together with the inner membrane putative transport proteins NodI and NodJ) is proposed to be involved in the secretion of nodulation factors.
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PMID:Rhizobium leguminosarum NodT is related to a family of outer-membrane transport proteins that includes TolC, PrtF, CyaE and AprF. 764 32

In wild-type strains of Escherichia coli, alkaline phosphatase (AP), either when present as a soluble protein or when fused to a membrane protein, is only active after translocation to the periplasm. In thioredoxin reductase (trxB) mutants, however, cytoplasmically localized AP can form disulphide bonds and can reach an active conformation. Once it has folded in the cytoplasm, it can no longer be translocated. On the other hand, when AP is fused to periplasmic domains of a membrane protein, translocation can be more rapid than folding. Thus, expressing hybrids of AP and integral membrane proteins in a trxB mutant generates competition between folding of AP in the cytoplasm and its translocation to the periplasm. The cellular localization of AP can be monitored in phosphoserine phosphatase (serB) mutants causing auxotrophy for L-serine. Cytoplasmically but not periplasmically localized AP can compensate for the lack of SerB, leading to growth on indicator plates. As expected, when AP was fused to cytoplasmic domains of membrane proteins, serB-mediated auxotrophy was abolished. Surprisingly, AP fusions to periplasmic domains exhibited a non-uniform response pattern. Fusions that translocate AP rapidly did not complement the SerB defect, while those that export AP only slowly could do so. The usefulness of these strains for studying a variety of aspects related to membrane protein biogenesis is discussed.
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PMID:Synthetic competition between cytoplasmic folding and translocation of a soluble membrane protein domain. 765 6

Linker insertions in the pullulanase structural gene (pulA) were examined for their effects on pullulanase activity and cell surface localization in Escherichia coli carrying the cognate secretion genes from Klebsiella oxytoca. Of the 23 insertions, 11 abolished pullulanase activity but none were found to prevent secretion. To see whether more drastic changes affected secretion, we fused up to five reporter proteins (E. coli periplasmic alkaline phosphatase, E. coli periplasmic maltose-binding protein, periplasmic TEM beta-lactamase, Erwinia chrysanthemi extracellular endoglucanase Z, and Bacillus subtilis extracellular levansucrase) to three different positions in the pullulanase polypeptide: close to the N terminus of the mature protein, at the C terminus of the protein, or at the C terminus of a truncated pullulanase variant lacking the last 256 amino acids. Only 3 of the 13 different hybrids were efficiently secreted: 2 in which beta-lactamase was fused to the C terminus of full-length or truncated pullulanase and 1 in which maltose-binding protein was fused close to the N terminus of pullulanase. Affinity-purified endoglucanase-pullulanase and pullulanase-endoglucanase hybrids exhibited apparently normal levels of pullulanase activity, indicating that the conformation of the pullulanase segment of the hybrid had not been dramatically altered by the presence of the reporter. However, pullulanase-endoglucanase hybrids were secreted efficiently if the endoglucanase component comprised only the 60-amino-acid, C-terminal cellulose-binding domain, suggesting that at least one factor limiting hybrid protein secretion might be the size of the reporter.
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PMID:Extracellular secretion of pullulanase is unaffected by minor sequence changes but is usually prevented by adding reporter proteins to its N- or C-terminal end. 766 12

The fusion of lysosomes and other granules with phagocytic vesicles (endo-fusion) and cell membrane (exocytosis) was simultaneously examined in non-phagocytosing guinea-pig polymorphonuclear leukocytes (PMNs). beta-Glucuronidase as a typical lysosomal enzyme, acid phosphatase as another lysosomal enzyme, and alkaline phosphatase as a specific granule enzyme were assayed. PMNs released these three enzymes in the presence of serum but the extents of exocytosis differed considerably: release of beta-glucuronidase, alkaline phosphatase and acid phosphatase was 6.9, 4.3 and 3.3%, respectively. Acid phosphatase was released even in the absence of serum, whereas the other two enzymes were not. These three enzymes showed also different responses in endofusion: the preformed phagocytic vesicles fused with the granules containing beta-glucuronidase and acid phosphatase, but scarcely fused with those containing alkaline phosphatase, although all these enzymes were recovered in increasing amounts in the phagolysosome fraction when the cells were allowed to phagocytose continuously. These results suggest that fusion of these three types of granules with phagocytic vesicles (endo-fusion) and plasma membrane (exocytosis) is heterogeneous and regulated by different mechanisms in guinea-pig PMNs.
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PMID:Heterogeneity in lysosomal fusion with phagocytic vesicles and cell membrane in non-phagocytosing guinea-pig polymorphonuclear leukocyte. 771 92

A fusion protein, Sta-CBDCex, which comprises streptavidin with a cellulose-binding domain (CBDCex) fused to its C terminus, was produced in the cytoplasm of Escherichia coli, where it formed inclusion bodies. Renatured Sta-CBDCex, recovered from the inclusion bodies, adsorbed to Avicel, a microcrystalline cellulose. The cellulose-bound Sta-CBDCex in turn bound biotinylated alkaline phosphatase or biotinylated beta-glucosidase. The immobilized beta-glucosidase remained fully active during 2 weeks of continuous column operation at 50 degrees C.
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PMID:A streptavidin-cellulose-binding domain fusion protein that binds biotinylated proteins to cellulose. 776 88

We have investigated the transcriptional control elements of the human interferon (IFN)-gamma-induced tryptophanyl-tRNA synthetase (hWRS) gene and characterized the transcripts. Transcription leads to a series of mRNAs with different combinations of the first exons. The full-length mRNA codes for a 55-kDa protein (hWRS), but a mRNA lacking exon II is present in almost as high amounts as the full-length transcript. This alternatively spliced mRNA is probably translated into a 48-kDa protein starting from Met48 in exon III. The predicted 48-kDa protein corresponds exactly to an IFN-gamma-inducible protein previously detected by two-dimensional gel electrophoresis. By isolation of genomic clones and construction of plasmids containing hWRS promoter fragments fused to the secreted alkaline phosphatase reporter gene we have mapped a promoter region essential for IFN-mediated gene activation. This region contains IFN-stimulated response elements (ISRE) as well as a Y-box and a gamma-activated sequence (GAS) element. IFN-gamma inducibility of hWRS depends on ongoing protein synthesis, suggesting that so far undescribed transcription factors apart from the latent GAS-binding protein p91 contribute to gene activation. This could be interferon-regulatory factor-1, which binds ISRE elements.
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PMID:Transcriptional regulation of the interferon-gamma-inducible tryptophanyl-tRNA synthetase includes alternative splicing. 781

The 1.3 S biotinylatable subunit of Proprionibacterium shermanii transcarboxylase complex was fused to the C-terminus of the human neurokinin 1 receptor gene and introduced into the Semliki Forest virus expression vector pSFV1. RNA transcribed from pSFV1-NK1-biot and pSFV-Helper2 was coelectroporated into BHK cells permitting in vivo packaging of recombinant virus. Infection of BHK and CHO cells with SFV-NK1-biot virus yielded high level of the fusion receptor as detected by metabolic labeling, immunoblotting with streptavidin alkaline phosphatase and binding to substance P. Like native receptor, the biotinylated receptor fusion was able to stimulate Ca2+ mobilization in infected CHO cells, indicating functional coupling to guanine-nucleotide-binding proteins.
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PMID:Functional activity of a biotinylated human neurokinin 1 receptor fusion expressed in the Semliki Forest virus system. 788 38

The iron starvation-induced, 2,042-amino-acid protein HMWP2 of Yersinia enterocolitica has two internal hydrophobic segments which might promote its export and association with the cytoplasmic membrane. To determine whether part of HMWP2 could be exported beyond the periplasmic face of the cytoplasmic membrane, we used TnphoA mutagenesis to construct 10 hybrid proteins in which periplasmic alkaline phosphatase (PhoA) was fused to the end of C-terminally truncated HMWP1 (at amino acid positions 1751 and 1753 two independent isolates]) had high alkaline phosphate activity (close to that of the native enzyme), both in Escherichia coli and in Y. pseudotuberculosis, indicating that the PhoA segment of the hybrid reached the periplasm. Deletion studies showed that the export signal resides in the second hydrophobic segment of HMWP2. This result would be compatible with the topology of the protein in the cytoplasmic membrane predicted from the distribution of charged amino acids at either end of the two hydrophobic segments. However, two hybrids in which the junction was even further toward the C terminus of HMMWP2 (at positions 1793 and 1999) had only weak alkaline phosphatase activity, suggesting that the predicted topology is incorrect. The location of HMWP2 was therefore determined by subcellular fractionation. The results indicate that HMPW2 is mainly cytoplasmic, consistent with its presumed role in the ATP-dependent, nonribosomal synthesis of an unknown peptide. We propose that the high alkaline phosphatase activity associated with some of the HMWP-2-PhoA hybrids results from the unmasking of the cryptic export signal activity in the second hydrophobic segment of HMPW2.
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PMID:Yersinia spp. HMWP2, a cytosolic protein with a cryptic internal signal sequence which can promote alkaline phosphatase export. 789 1

The cDNA coding for human big endothelin 1 (bigET-1), preceded by an optimized collagenase recognition sequence and followed by a stop codon, was fused in frame to the C-terminal region of alkaline phosphatase (AP). The fusion protein (AP-bigET), expressed in Escherichia coli K12 upon the lowering of organic phosphate concentrations, consisted of alkaline phosphatase (1-447), the collagenase cleavage site (Gly-Pro-Ala)4, and glycylprolyl-bigET-1. AP-bigET accumulated intracellularly in the form of inclusion bodies that were extensively washed and finally extracted by 8 M urea to yield highly enriched AP-bigET. Upon digestion of the fusion protein with collagenase, two disulfide conformeres of glycylprolyl-bigET-1 (bigET-1A and bigET-1B) could be purified by reverse-phase FPLC. Upon treatment with dipeptidylpeptidase IV to remove the N-terminal glycylprolyl-dipeptide, the later-eluting form of bigET-1 (bigET-1B) coeluted with authentic human bigET-1 on reverse-phase HPLC. BigET-1A and bigET-1B were formed at a ratio of 1:3. After reduction and S-pyridylethylation, both conformers coeluted with authentic but reduced bigET-1. Their amino acid sequences were identical. Both forms were converted by digestion with pepsin to the respective ET-1 conformeres (ET-1A and ET-1B) that were purified. In vasoconstriction assays, ET-1B but not ET-1A, at 10(-8) M, evoked a maximal response indistinguishable from that of authentic ET-1.
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PMID:Purification of human big endothelin 1 derived through cleavage with collagenase and dipeptidylpeptidase IV from a fusion protein expressed in Escherichia coli. 790 63

An Escherichia coli expression-export vector was constructed (pCGV1, 6.3 kb) containing the alkaline phosphatase structural gene (phoA) located downstream from the phage lambda pR and pL promoters positioned in tandem and the cIts857 gene encoding lambda thermosensitive repressor. The phoA gene is fused to DNA encoding a hybrid signal sequence that contains the N-terminal portion of the beta-lactamase (Bla) signal sequence and the C-terminal region of the PhoA signal sequence. Within the DNA encoding hybrid signal sequence, a unique NheI restriction site is present where polymerase chain reaction (PCR)-amplified genes may be cloned. The 5' PCR primers reconstitute the C-terminal portion of either the PhoA or Bla signal sequences to restore an intact signal peptide. Recombinant phoA- clones are selected on indicator plates containing 5-bromo-4-chloro-3-indolyl phosphate.
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PMID:A novel Escherichia coli expression-export vector containing alkaline phosphatase as an insertional inactivation screening system. 792 33


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