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)

When alkaline phosphatase is fused to the periplasmic domain of a cytoplasmic membrane protein, it is efficiently exported to the periplasm. Such a hybrid protein exhibits high alkaline phosphatase enzymatic activity. When alkaline phosphatase is fused to the cytoplasmic domain of a membrane protein, it remains, for the most part, in the cytoplasm. Such fusions exhibit low enzymatic activity. However, stable retention of alkaline phosphatase in the cytoplasm requires the presence in the fusion protein of the cytoplasmic loop ordinarily present in that position in the native, unfused protein. Using oligonucleotide-directed mutagenesis, we have shown that positively charged amino acids are required for the stable cytoplasmic localization of the fused alkaline phosphatase. We propose that, in addition to hydrophobic transmembrane segments, positively charged amino acids in the hydrophilic cytoplasmic domains of a membrane protein are determinants of the protein's topology.
 ...
PMID:Positively charged amino acid residues can act as topogenic determinants in membrane proteins. 259 79

Placental alkaline phosphatase (PLAP) is normally anchored to the plasma membrane of cells by a phosphatidylinositol-glycan anchor after removal of a carboxyl-terminal peptide from the nascent enzyme. To investigate the signals required for this processing we constructed a chimeric cDNA. The latter was designed to code for a truncated precursor form of PLAP, containing the phosphatidylinositol-glycan attachment site but incapable of any form of membrane attachment, fused to a carboxyl-terminal peptide of vesicular stomatis virus glycoprotein. Expression of the PLAP-vesicular stomatis virus glycoprotein chimeric cDNA in transfected COS cells produced an enzymatically active protein that was attached to the plasma membrane, with the PLAP domain on the outer surface. Assays for the presence of phosphatidylinositol-glycan attachment proved negative, whereas an antibody assay confirmed the presence of the vesicular stomatis virus glycoprotein carboxyl-terminal peptide, leading to the conclusion that the truncated PLAP is attached to the cells by the membrane-spanning domain of the vesicular stomatis virus glycoprotein. In light of previous findings on carboxyl-terminal requirements of PLAP these studies suggest that an essential signal for correct sorting between transmembrane insertion and phosphatidylinositol-glycan attachment resides in the cytoplasmic domain.
 ...
PMID:Conversion of placental alkaline phosphatase from a phosphatidylinositol-glycan-anchored protein to an integral transmembrane protein. 264 36

A recombinant plasmid carrying a bovine growth hormone gene fused with the regulatory and signal regions of the alkaline phosphatase gene of E. coli was constructed. The bovine growth hormone gene expression as well as protein partial processing and secretion into the periplasm have been shown to take place under phosphate starvation, i.e. conditions of alkaline phosphatase derepression.
 ...
PMID:[Biosynthesis and secretion of bovine growth hormone in Escherichia coli under the control of the secretory vector containing a promoter and signal region of the alkaline phosphatase gene]. 267 71

The Tsr protein of Escherichia coli is a chemosensory transducer that mediates taxis toward serine and away from certain repellents. Like other bacterial transducers, Tsr spans the cytoplasmic membrane twice, forming a periplasmic domain of about 150 amino acids and a cytoplasmic domain of about 300 amino acids. The 32 N-terminal amino acids of Tsr resemble the consensus signal sequence of secreted proteins, but they are not removed from the mature protein. To investigate the function of this N-terminal sequence in the assembly process, we isolated translational fusions between tsr and the phoA and lacZ genes, which code for the periplasmic enzyme alkaline phosphatase and the cytoplasmic enzyme beta-galactosidase, respectively. All tsr-phoA fusions isolated code for proteins whose fusion joints are within the periplasmic loop of Tsr, and all of these hybrid proteins have high alkaline phosphatase activity. The most N-terminal fusion joint is at amino acid 19 of Tsr. Tsr-lacZ fusions were found throughout the tsr gene. The beta-galactosidase activity of the LacZ-fusion proteins varies greatly, depending on the location of the fusion joint. Fusions with low activity have fusion joints within the periplasmic loop of Tsr. The expression of these fusions is most likely reduced at the level of translation. In addition, one of these fusions markedly reduces the export and processing of the periplasmic maltose-binding protein and the outer membrane protein OmpA, but not of intact PhoA or of the outer membrane protein LamB. A temperature-sensitive secA mutation, causing defective protein secretion, stops expression of new alkaline phosphatase activity coded by a tsr-phoA fusion upon shifting to the nonpermissive temperature. The same secA mutation, even at the permissive temperature, increases the activity and the level of expression of LacZ fused to the periplasmic loop of Tsr relative to a secA+ strain. We conclude that the assembly of Tsr into the cytoplasmic membrane is mediated by the machinery responsible for the secretion of a subset of periplasmic and outer membrane proteins. Moreover, assembly of the Tsr protein seems to be closely coupled to its synthesis.
 ...
PMID:The Tsr chemosensory transducer of Escherichia coli assembles into the cytoplasmic membrane via a SecA-dependent process. 284 45

A transposon Tn917 insertion between gerE and ilvB has identified a new developmental locus, gerM, in Bacillus subtilis. gerM96::Tn917 affects both sporulation and germination. DNA on either side of the transposon has been cloned and includes the previously cloned sdhC and gerE loci. gerE terminates 2.1 kb from the end of the transposon. The gerM96::Tn917 mutant is oligosporogenous, yielding approximately 1% of the number of wild-type heat resistant spores in liquid medium and 10% on solid medium. Six hours after the onset of sporulation alkaline phosphatase and glucose dehydrogenase levels were 90% and 7%, respectively, of those of the wild-type. At this time 50% of the mutant cells were still dividing. The occurrence of multiple polar septa and 'pygmy' cells suggested a block at stage II of sporulation. Following addition of germinants, mutant spores prepared on nutrient agar lost heat resistance normally but released slightly less dipicolinic acid than wild-type spores. They also showed only partial loss of optical density, associated with a phase-grey appearance and striations in the cortex suggesting partial degradation. Expression of the gerM gene was monitored by production of beta-galactosidase encoded by a promotorless lacZ gene fused to the gerM96::Tn917 insertion. It occurred 1.5-4 h after commencement of sporulation. Transcription was directed from a promoter on the gerE side of gerM and was unaffected by a mutation in the gerE gene.
 ...
PMID:Genetical and molecular studies on gerM, a new developmental locus of Bacillus subtilis. 284 48

The ompF gene codes for a major outer membrane protein of Escherichia coli. A plasmid was constructed in which the structural gene for human beta-endorphin is preceded by the upstream region of the ompF gene consisting of the promoter region and the coding regions for the signal peptide and the N terminus of the OmpF protein. When the plasmid was introduced into E. coli N99, and OmpF-beta-endorphin fused peptide was synthesized and secreted into the culture medium through both the cytoplasmic and outer membranes. The OmpF signal peptide was cleaved correctly during the secretion, indicating that the export of the fused protein across the cytoplasmic membrane was dependent on the signal peptide. The secretion into the culture medium was apparently selective. Neither beta-lactamase nor alkaline phosphatase (both are periplasmic proteins) appeared in the culture medium in significant amounts. The mode of passage of the fused peptide across the outer membrane is discussed.
 ...
PMID:Secretion into the culture medium of a foreign gene product from Escherichia coli: use of the ompF gene for secretion of human beta-endorphin. 293 2

We constructed a derivative of transposon Tn5 that permits the generation of hybrid proteins composed of alkaline phosphatase (EC 3.1.3.1) lacking its signal peptide fused to amino-terminal sequences of other proteins. Such a hybrid gives alkaline phosphatase activity if the protein fused to alkaline phosphatase contributes sequences that promote export and thus compensate for the missing alkaline phosphatase signal peptide. Fusions to both a secreted periplasmic protein and a complex cytoplasmic membrane protein led to alkaline phosphatase activity. TnphoA fusions should help localize export signals within the structure of a protein, such as a transmembrane protein, as well as identify new chromosomal genes for secreted and transmembrane proteins.
 ...
PMID:TnphoA: a transposon probe for protein export signals. 299 94

The structural gene (appA) for the periplasmic acid phosphatase (optimum pH 2.5) of Escherichia coli was cloned into a plasmid by using a combination of in vivo and in vitro techniques. The position and orientation of the appA gene within the cloned DNA fragment were identified by using fusions to the alkaline phosphatase gene (phoA) generated by Tn5 IS50L::phoA (TnphoA) insertions. For TnphoA-generated hybrid proteins to have high enzymatic activity, it appears that the phoA gene must be fused to a target gene coding for a signal which promotes protein export. The approach used to identify the appA gene thus appears to provide a simple general means of selectively identifying genes encoding membrane and secreted proteins.
 ...
PMID:Use of TnphoA to detect genes for exported proteins in Escherichia coli: identification of the plasmid-encoded gene for a periplasmic acid phosphatase. 303 Oct 17

A synthetic chimeric gene, coding for the human epidermal growth factor fused to the signal peptide of Escherichia coli alkaline phosphatase, was cloned into E. coli under the transcriptional control of the trp-lac (tac) promoter. Following induction with isopropylthiogalactoside, the secretion of the correctly processed protein product into the bacterial periplasm was detected and quantitated by its specific binding to the epidermal growth factor receptor. The purified protein was identical to authentic human epidermal growth factor in size, amino acid composition, primary sequence, receptor binding, and stimulation of receptor protein-tyrosine kinase activity. Based on interspecies homologies, structural considerations, and reported studies with peptide fragments, structure-function analysis was initiated with alterations of targeted amino acid residues by oligonucleotide-directed mutagenesis. The receptor binding affinity of each mutant, relative to the wild type, was measured by both radioreceptor competition and receptor tyrosine kinase stimulation assays. In general, the values obtained by the two methods were in agreement for each species of epidermal growth factor and followed the order: wild type greater than Glu24----Gly greater than Asp27----Gly much greater than Pro7----Thr greater than Tyr29----Gly greater than Leu47----His. The relatively low values obtained with the last two mutants suggest that Tyr29 and Leu47 may be important for the biological activity of human epidermal growth factor.
 ...
PMID:Cloning of authentic human epidermal growth factor as a bacterial secretory protein and its initial structure-function analysis by site-directed mutagenesis. 304 17

The gene for Staphylococcal protein A was fused to the coding sequence of bacterial beta-galactosidase, alkaline phosphatase and human insulin-like growth factor I (IGF-I). The fusion proteins, expressed in bacteria, were purified by affinity chromatography on IgG-Sepharose and antibodies were raised in rabbits. All three fusion proteins elicited specific antibodies against both the inserted protein sequences and the protein A moiety. In the case of IGF-I, the protein A moiety in the fusion protein may act as an adjuvant since native IGF-I alone is a poor immunogen. The results suggest that the protein A fusion system can be used for efficient antibody production against peptides or proteins expressed from cloned or synthetic genes. To facilitate such gene fusions a set of optimized vectors have been constructed.
 ...
PMID:Production of specific antibodies against protein A fusion proteins. 309 19


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>