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)

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.
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PMID:Placental alkaline phosphatase, a GPI-anchored protein, is clustered in clathrin-coated vesicles. 155 May 85

Escherichia coli lac permease is a polytopic integral membrane protein with six translocated (periplasmic) domains. Individual N-terminal cytoplasmic regions and membrane-spanning segments adjacent to each of the periplasmic domains acted as export signals for an attached sensor protein (alkaline phosphatase). However, the export activity of one of the spanning segments was considerably lower than that of the others, and was limited by the presence of a positively charged residue (Arg302). These observations are compatible with models of membrane protein insertion in which hydrophilic domains are translocated independently. However, the results suggest that efficient translocation may sometimes require interaction between individual spanning segments.
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PMID:Membrane protein spanning segments as export signals. 156 45

We have investigated the role of clathrin in vacuolar protein sorting using yeast strains harboring a temperature-sensitive allele of clathrin heavy chain (chc1-ts). After a 5 min incubation at the non-permissive temperature (37 degrees C), the chc1-ts strains displayed a severe defect in the sorting of lumenal vacuolar proteins. Sorting of a vacuolar membrane protein, alkaline phosphatase, and transport to the surface of a cell wall protein, was not affected at 37 degrees C. In chc1-ts cells incubated at 37 degrees C, secretion of the missorted lumenal vacuolar protein carboxypeptidase Y (CPY) was blocked by the sec1 mutation which prevents fusion of secretory vesicles to the plasma membrane. Unexpectedly, chc1-ts cells incubated for extended periods at 37 degrees C regained the ability to sort CPY. Cells carrying deletions of the CHC1 gene (chc1 delta) also sorted CPY to the vacuole even when subjected to temperature shifts. Vacuolar delivery of CPY in chc1 delta cells was not blocked by sec1 suggesting that transport does not occur by secretion and endocytosis. These results provide in vivo evidence that clathrin plays a role in the Golgi complex in sorting of vacuolar proteins from the secretory pathway. With time, however, yeast cells lacking functional clathrin heavy chains are able to adapt in a way that allows restoration of vacuolar protein sorting in the Golgi complex. These conclusions clarify previous studies of chc1 delta cells which raised the possibility that clathrin is not involved in vacuolar protein sorting.
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PMID:A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast. 163 56

The topology of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides has been examined by generating gene fusions with alkaline phosphatase. Gene fusions were generated at random locations within the fbcB gene encoding the cytochrome b subunit. These fusion products were expressed in Escherichia coli and were screened for alkaline phosphatase activity on chromogenic plates. 33 in-frame fusions which showed activity were further characterized. The fusion junctions of all those fusions which had a high specific activity were clustered in three regions of the cytochrome b polypeptide, and thus these regions were tentatively assigned as being near the periplasmic surface. The data are consistent with a model containing eight transmembrane helices. In order to explore the validity of the gene fusion approach for a protein not normally expressed in E. coli, the topology of the L-subunit of the photosynthetic reaction center from R. sphaeroides was also explored using phoA gene fusions. A similar protocol was used as with the cytochrome b subunit. The gene fusions with high specific activity were shown to be in regions of the L-subunit polypeptide known to be at or near the periplasmic surface, as defined by the high resolution structure determined by X-ray crystallography. These data demonstrate the utility of this approach for determining membrane protein topology and extend potential applications to include at least some proteins not normally expressed in E. coli.
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PMID:The use of gene fusions to examine the membrane topology of the L-subunit of the photosynthetic reaction center and of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides. 164 18

The expression of many of the known virulence determinants of Bordetella pertussis is coordinately regulated by the vir regulatory locus and reduced in response to environmental signals called modulators. We have previously identified eight TnphoA gene fusions in B. pertussis in which the expression of alkaline phosphatase was maximal in the absence of the modulators nicotinic acid and MgSO4. We have termed the genes identified by these fusions vir-activated genes. Here we report the characterization of these TnphoA mutant strains. Four fusion strains were defective in known virulence determinants. For one of these, fusion strain SK39, Southern blot hybridization demonstrated that TnphoA was inserted in the S1 subunit gene of pertussis toxin. Hemagglutination assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblots identified three fusions strains, SK16, SK75, and SK91, that were defective in filamentous hemagglutinin. Whereas all three filamentous hemagglutinin-defective mutants showed either normal or enhanced colonization, the pertussis toxin-defective mutant showed a marked defect in pulmonary persistence. Of the four other fusion strains, two were deficient in outer membrane proteins. One of these, strain SK8, was defective in a major outer membrane protein of 95 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This strain colonized mouse lungs less well and did not induce lymphocytosis after aerosol challenge. The other strain, SK34, was defective in four outer membrane proteins, three of which were detectable only on a Western blot with polyclonal sera against B. pertussis. Two of our gene fusion strains did not show any defect in identifiable vir-regulated proteins.
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PMID:Characterization of vir-activated TnphoA gene fusions in Bordetella pertussis. 165 62

Yersinia pestis, the etiologic agent of bubonic plague, contains a 75-kb virulence plasmid, called pCD1 in Y. pestis KIM. The low-Ca(2+)-response genes of Y. pestis regulate both bacterial growth and the expression of pCD1-encoded virulence determinants in response to temperature and the presence of Ca2+ or nucleotides. This study characterizes the nucleotide sequence and protein product of the lcrD locus. An lcrD mutant, in contrast to the parent Y. pestis, did not undergo growth restriction or induce strong expression of the V antigen when grown under conditions (37 degrees C, no Ca2+) expected to elicit maximal expression of pCD1 genes. DNA sequence analysis of the cloned lcrD locus showed a single open reading frame that could encode a protein with a molecular weight of 77,804 and a pI of 4.88. LcrD was identified as a 70-kDa inner membrane protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. LcrD membrane topology was investigated by using lcrD-phoA translational fusions generated with the transposon TnphoA. The alkaline phosphatase activities of the resultant hybrid proteins were consistent with a model predicting eight amino-terminal transmembrane segments that anchor a large cytoplasmic carboxyl-terminal domain to the inner membrane.
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PMID:LcrD, a membrane-bound regulator of the Yersinia pestis low-calcium response. 165 87

A method for the simultaneous preparation of microvillous and basal membrane vesicles from human placental syncytiotrophoblast is described. Mg2(+)-aggregated basal membranes are separated from microvillous membranes by low-speed centrifugation after initial homogenization and centrifugation steps. Microvillous membranes (MVM) are obtained from the low speed supernatant while basal membranes (BM) contained in the Mg2(+)-aggregated material are resuspended and further purified on a sucrose step gradient. MVM and BM prepared by this method were enriched 20-fold and 11-fold as determined by the membrane marker enzymes, alkaline phosphatase (MVM) and adenylate cyclase (BM). There was minimal cross-contamination of the two isolated plasma membrane fractions and the yields obtained were 26% (MVM) and 21% (BM) compared to the initial homogenate. The MVM and BM fractions were free from contamination by mitochondrial or lysosomal membranes and showed only minor contamination by microsomal membranes. The two membrane fractions were also tested for the presence of non-syncytial plasma membranes by electrophoretic immunoblotting. Contamination of both MVM and BM by fibroblast, endothelial, macrophage and cytotrophoblast plasma membranes amounted to less than 15% of the total membrane protein as determined by immunoblotting. Vesicle orientation, determined from the latency of specific concanavalin A binding, was 88 +/- 4% right-side out for MVM and 73 +/- 12% right-side out for BM. This simple preparative procedure produces a high yield of both MVM and BM from human placenta. The analytical data demonstrates that 'paired' MVM and BM fractions derived from the same placental tissue have a high purity in terms not only of contamination by intracellular membranes, but also in terms of contamination by non-syncytial plasma membranes.
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PMID:Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta. 170 Sep 23

A 7.2 kb Bg/II restriction fragment, which increases the production of several extracellular enzymes, including alkaline phosphatase, amylase, protease, lipase and beta-galactosidase, was cloned in Streptomyces lividans from the DNA of S. griseus ATCC 10137. This gene (named saf) showed a positive gene dosage effect on production of extracellular enzymes. When the saf gene was introduced into cells in high copy numbers it delayed the formation of pigments and spores in S. lividans and also retarded actinorhodin production in Streptomyces coelicolor. The saf gene hybridized with specific bands in the DNA of several Streptomyces strains tested. A 1 kb fragment containing the saf gene was sequenced and contains an open reading frame (ORF) of 306 nucleotides which encodes a polypeptide of Mr 10,500. This ORF is contained within a fragment of 432 bp which retained activity in Streptomyces. A fragment with promoter activity is present upstream of the saf reading frame. The predicted Saf polypeptide has a strong positive charge, and does not show a typical amino acid composition for a membrane protein, and contains a DNA-binding domain similar to those found in several regulatory proteins.
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PMID:Cloning and characterization of a gene of Streptomyces griseus that increases production of extracellular enzymes in several species of Streptomyces. 170 69

Placental alkaline phosphatase (PLAP) is anchored in the plasma membrane by a phosphatidylinositol-glycan moiety (PI-glycan). PI-glycan is added posttranslationally to the nascent peptide chain after the removal of 29 amino acids from the COOH-terminus. The contribution of selected COOH-terminal amino acids to the signal for PI-glycan addition was tested by creating a fusion protein with the COOH-terminus of PLAP and a secreted protein and by mutagenesis of specific PLAP COOH-terminal amino acids. The cDNA encoding the COOH-terminus of PLAP was fused in frame to the cDNA for human clotting Factor X and expressed in transfected COS-1 cells. Fusion proteins containing 32 amino acids of the PLAP COOH-terminus were modified by PI-glycan addition. Thus, the signal for PI-glycan modification must reside in these amino acids. Next, the region between the hydrophobic domain and the cleavage site was examined for additional determinants. Mutations of the hydrophilic residues in the spacer region demonstrated that these amino acids do not contribute to the signal for PI-glycan addition. Deletion of amino acids in the spacer region prevented the addition of PI-glycan suggesting that the length of the spacer domain or the amino acids around the cleavage site are important determinants. Finally, we demonstrated that interruption of the hydrophobic domain by a charged residue prevents PI-glycan addition and results in a protein that is secreted into the medium. The finding that a single Leu to Arg substitution in the hydrophobic domain converts a PI-glycan anchored, membrane protein to a secreted protein suggests that an essential signal for the correct sorting of PI-glycan anchored proteins versus secreted proteins resides in the hydrophobic domain. Substitution of a charged amino acid for a hydrophobic amino acid may be a mechanism for producing membrane bound and secreted forms of the same protein.
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PMID:Site-specific mutations in the COOH-terminus of placental alkaline phosphatase: a single amino acid change converts a phosphatidylinositol-glycan-anchored protein to a secreted protein. 173 Jul 77

The yeast VPS15 gene encodes a novel protein kinase homolog that is required for the sorting of soluble hydrolases to the yeast vacuole. In this study, we extend our previous mutational analysis of the VPS15 gene and show that alterations of specific Gps15p residues, that are highly conserved among all protein kinase molecules, result in the biological inactivation of Vps15p. Furthermore, we demonstrate here that short C-terminal deletions of Vps15p result in a temperature-conditional defect in vacuolar protein sorting. Immediately following the temperature shift, soluble vacuolar hydrolases, such as carboxypeptidase Y and proteinase A, accumulate as Golgi-modified precursors within a saturable intracellular compartment distinct from the vacuole. This vacuolar protein sorting block is efficiently reversed when mutant cells are shifted back to the permissive temperature; the accumulated precursors are rapidly processed to their mature forms indicating that they have been delivered to the vacuole. This rapid and efficient reversal suggests that the accumulated vacuolar protein precursors were present within a normal transport intermediate in the vacuolar protein sorting pathway. In addition, this protein delivery block shows specificity for soluble vacuolar enzymes as the membrane protein, alkaline phosphatase, is efficiently delivered to the vacuole at the non-permissive temperature. Interestingly, the C-terminal Vps15p truncations are not phosphorylated in vivo suggesting that the phosphorylation of Vps15p may be critical for its biological activity at elevated temperatures. The rapid onset and high degree of specificity of the vacuolar protein delivery block in these mutants suggests that the primary role of Vps15p is to regulate the sorting of soluble hydrolases to the yeast vacuolar compartment.
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PMID:A genetic and structural analysis of the yeast Vps15 protein kinase: evidence for a direct role of Vps15p in vacuolar protein delivery. 175 16


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