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

---

Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 47-kDa lipoprotein is an abundant integral membrane protein and dominant immunogen of Treponema pallidum subsp. pallidum. Previous DNA sequencing of the 47-kDa-lipoprotein gene did not reveal consensus features representative of other bacterial lipoprotein genes; this prompted further analyses with emphasis on elucidation of the N terminus of the molecule. To assist in localizing start signals for the protein, the transcription initiation site for the 47-kDa-antigen gene was determined. RNA isolated from both T. pallidum and recombinant Escherichia coli expressing the 47-kDa antigen was used as a template in reverse transcriptase primer extension. Upon analysis of cDNA products, transcription initiation was localized to one nucleotide in T. pallidum and to two adjacent nucleotides in E. coli. When various primers were used in DNA sequencing reactions for these analyses, a previously undetected nucleotide (G) was found in the purported 5' untranslated region; this altered the upstream reading frame to reveal plausible sites for ribosome binding (GGAGG), translation initiation (GTG start codon), and signal peptidase II processing (Val-Val-Gly-Cys). Discounting acylation, the molecular weight of the mature polypeptide is 45,756 (approximately 46,600 with acylation). To derive nonacylated 47-kDa antigen for further structure-function studies, the 47-kDa-antigen gene was subcloned without acylation signals as a genetic construct encoding a glutathione S-transferase fusion protein; following cleavage with thrombin, the nonacylated 47-kDa protein was hydrophilic rather than amphiphilic but retained its antigenicity when tested against 116 human serum samples from patients with various stages of syphilis.
...
PMID:Analysis of the N-terminal region of the 47-kilodalton integral membrane lipoprotein of Treponema pallidum. 137 97

Crude adult worm antigen of Dictyocaulus viviparus was examined for specific antigens by SDS-PAGE and immunoblotting using sera from cattle experimentally infected with D. viviparus, vaccinated with a normal or a reduced dosage of the commercial lungworm vaccine, and helminth-free cattle. A D. viviparus-specific region M(r) 18,000 was identified and isolated. A lambda ZAP II cDNA expression library consisting of 4.4 x 10(5) recombinant clones (88% of the total number of clones) was constructed from D. viviparus adult worm mRNA. Rabbit antiserum to the M(r) 18,000 antigen was used to screen the cDNA library and eight positive clones were picked and allocated to the same antigenic family by sibling analysis. All clones were subcloned into the plasmid pGEX-2T, and the clone with highest expression yields was expressed as a glutathione S-transferase fusion protein (DvGST3-14) or, after cleavage with thrombin, as pure recombinant parasite protein (Dv3-14). The native parasite antigen encoded by the clone was identified. The immunodiagnostic potential of the recombinant proteins was assessed by immunoblotting.
...
PMID:Dictyocaulus viviparus: isolation and characterization of a recombinant antigen with potential for immunodiagnosis. 145 87

Four cDNA fragments encoding different portions of the alpha-subunit of human H,K-adenosine triphosphatase (ATPase) were amplified by means of the polymerase chain reaction technique, ligated into the plasmid pGEX-2T, and expressed as glutathione S-transferase fusion proteins in Escherichia coli. The fragments A (residues 163-313), Ba (residues 360-797), Bb (residues 526-797), and C (residues 822-1031) together encompass 77% of the alpha-subunit and cover most of its cytosolic part. The reactivities of autoantibodies in the sera from patients with pernicious anaemia with the recombinant fusion proteins were analysed by immunoblotting. One autoantigenic epitope was found in the NH2-terminal part of the Ba fragment--that is, between residues 360 and 525. No epitope was detected in the other fragments. The Ba fragment was cleaved off from the glutathione S-transferase fusion protein by the action of thrombin and was then further purified. By means of enzyme-linked immunosorbent assay, 28 of 42 sera (67%) from patients with pernicious anaemia were positive against the purified Ba fragment. The present results provide a final proof that the human H,K-ATPase alpha-subunit is a major autoantigen in the parietal cell and that the major epitope is located between residues 360 to 525 on the cytosolic side of the secretory membrane.
...
PMID:Localization of a pernicious anaemia autoantibody epitope on the alpha-subunit of human H,K-adenosine triphosphatase. 751 38

The cDNA encoding QPc-9.5 kDa (subunit VII) of bovine heart mitochondrial ubiquinol-cytochrome c reductase was cloned and sequenced. This cDNA is 665 base pairs long with an open reading frame of 246 base pairs that encodes an 81-amino acid mature QPc-9.5 kDa. The insert contains 395 base pairs of a 3'-noncoding sequence with a poly(A) tail. The amino acid sequence of QPc-9.5 kDa deduced from this nucleotide sequence is the same as that obtained by protein sequencing except that residue 61 is tryptophan instead of cysteine. The QPc-9.5 kDa was overexpressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-QPc) using the expression vector, pGEX/QPc. The yield of soluble active recombinant GST-QPc fusion protein depends on the induction growth time, temperature, and medium. Maximum yield of recombinant fusion protein was obtained from cells harvested 3 h postinduction of growth at 27 degrees C on LB medium containing betaine and sorbitol. QPc-9.5 kDa was released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPc-9.5 kDa showed one protein band in SDS-polyacrylamide gel electrophroesis corresponding to subunit VII of mitochondrial ubiquinol-cytochrome c reductase. Although the isolated recombinant QPc-9.5 kDa is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1 million. Addition of detergent deaggreates the isolated protein to the monomeric state, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution. The recombinant QPc-9.5 kDa binds ubiquinone and shows a spectral blue shift. Upon titration of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that the recombinant protein may be in the functionally active state.
...
PMID:Cloning, gene sequencing, and expression of the small molecular mass ubiquinone-binding protein of mitochondrial ubiquinol-cytochrome c reductase. 759 38

Osteopontin (OPN), a secreted phosphoprotein, has been implicated in various biological phenomena (e.g. bone development, sepsis, tumor progression, and metastasis). Its role in any context is poorly understood. OPN contains a conserved Gly-Arg-Gly-Asp-Ser (GRGDS) sequence, and binds to cells via integrin-mediated mechanisms. Using recombinant human osteopontin-glutathione S-transferase fusion protein and our improved hybridoma fusion partner (Sp2/mIL6), we raised murine monoclonal antibodies against osteopontin. We characterized two antibodies that recognize not only recombinant but also native human osteopontin. These antibodies do not cross-react with mouse osteopontin (recombinant protein or that secreted by ras-transformed NIH 3T3 cells), or bovine bone osteopontin, suggesting that they recognize epitopes unique to human OPN. One antibody specifically inhibited adhesion of MDA-MB-435 human breast cancer cells and ras-transformed NIH 3T3 cells to human osteopontin. This antibody failed to recognize osteopontin cleaved by thrombin, which cleaves adjacent to the cell binding domain. We previously showed that thrombin cleavage reduces osteopontin cell binding activity. Thus we postulate that this monoclonal antibody recognizes and interferes with the function of the RGD/thrombin cleavage region of human OPN.
...
PMID:Inhibition of Arg-Gly-Asp (RGD)-mediated cell adhesion to osteopontin by a monoclonal antibody against osteopontin. 808 34

The aim of this work was to construct short analogues of the repetitive water-binding domain of the Pseudomonas syringae ice nucleation protein, InaZ. Structural analysis of these analogues might provide data pertaining to the protein-water contacts that underlie ice nucleation. An artificial gene coding for a 48-mer repeat sequence from InaZ was synthesized from four oligodeoxyribonucleotides and ligated into the expression vector, pGEX2T. The recombinant vector was cloned in Escherichia coli and a glutathione S-transferase fusion protein obtained. This fusion protein displayed a low level of ice-nucleating activity when tested by a droplet freezing assay. The fusion protein could be cleaved with thrombin, providing a means for future recovery of the 48-mer peptide in amounts suitable for structural analysis by nuclear magnetic resonance spectroscopy.
...
PMID:Synthesis and expression of a gene encoding a 48-residue repeat in the Pseudomonas syringae ice nucleation protein. 818 60

Calcineurin, a protein phosphatase found in eukaryotic cells, presents a challenging problem in heterologous protein expression because it is both heterodimeric and posttranslationally modified. In this paper, we describe the cloning of both subunits (catalytic A and regulatory B) of calcineurin from a human cDNA library and their expression at high levels in Escherichia coli. The calcineurin A subunit is expressed as an insoluble glutathione S-transferase fusion protein, while the calcineurin B subunit is soluble upon direct expression. Catalytically active holoenzyme is derived from the separately expressed subunits using a three-step refolding protocol. First, the fusion protein is solubilized, then it is cleaved at the fusion junction with thrombin, and, finally, a catalytically competent calcineurin A:calcineurin B:calmodulin complex is reconstituted by cofolding the separately purified components. In addition, we show that a similar refolding protocol can be applied to a C-terminally truncated form of calcineurin A, which lacks an autoinhibitory and calmodulin-binding domain.
...
PMID:Reconstitution of active human calcineurin from recombinant subunits expressed in bacteria. 853 59

Subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex was over-expressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-RSIV) using the expression vector, pGEX/RSIV. Maximum yield of soluble active recombinant fusion protein was obtained from cells harvested 3 h after induction of growth at 37 degrees C in LB medium. Subunit IV was released from the fusion protein by proteolytic cleavage with thrombin. When subjected to SDS-polyacrylamide gel electrophoresis, isolated recombinant subunit IV of R. sphaeroides cytochrome b-c1 complex. Although the isolated recombinant subunit IV is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1000 kDa. The addition of detergent deaggregates the isolated protein, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution. When the three-subunit core cytochrome b-c1 complex, purified from RS delta IV-adapted chromatophores containing a fraction of the wild type cytochrome b-c1 complex activity, was reacted with varying amounts of recombinant subunit IV, the activity increased as the subunit IV concentration increased. Maximum activity restoration was reached when 1 mol of subunit IV/mol of three-subunit core complex was used. The reconstituted cytochrome b-c1 complex is similar to the wild-type complex in molecular size, apparent Km for Q2H2, and inhibitor sensitivity, indicating that recombinant subunit IV is properly assembled into the active cytochrome b-c1 complex. A tryptophan residue in subunit IV was found to be involved in the interaction with the three-subunit core complex.
...
PMID:Functional expression of subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex and reconstitution of recombinant protein with three-subunit core complex. 856 59

Cyclic guanosine 5'-monophosphate (cGMP) phosphodiesterase (PDE) regulates the level of cGMP on transduction of a visual signal in vertebrate photoreceptor cells. Two identical inhibitory PDE gamma subunits (Pgammas) block catalytic activity of PDE-alpha and -beta subunits (Palphabeta) in the dark. The primary regions of Pgamma involved in the interaction with Palphabeta are a central polycationic region, Pgamma-24-45, and a C-terminal region of Pgamma. Recently, we have shown that the C-terminal region of Pgamma, which is the major Pgamma inhibitory domain, blocks PDE activity by binding to the catalytic site of PDE (Artemyev, N. O., Natochin, M., Busman, M., Schey, K. L., and Hamm, H. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5407-5412). Here, we localize the site on the rod cGMP PDE alpha subunit that binds to the central polycationic domain of Pgamma. This site is located within a region that links a second noncatalytic cGMP binding site with the catalytic domain of PDE. A polypeptide coresponding to this region, Palpha-461-553, expressed as a glutathione S-transferase fusion protein in Escherichia coli and isolated after cleavage of the fusion protein with thrombin, blocks inhibition of PDE activity by Pgamma. In addition, Palpha-461-553 binds to the Pgamma-24-45 region (Kd, 7 microM), as measured by a fluorescent increase in a Pgamma-24-45Cys peptide labeled with 3-(bromoacetyl)-7-diethylaminocoumarin. The Palpha-461-553 region was further characterized by using a set of synthetic peptides. A peptide corresponding to residues 517-541 of Palpha (Palpha-517-541) effectively suppressed inhibition of PDE activity by Pgamma and bound to Pgamma-24-45Cys labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (Kd, 22 microM). Palpha-517-541 also competes with the activated rod G-protein alpha-subunit for binding to Pgamma labeled with lucifer yellow vinyl sulfone. This suggests that light activation of rod PDE by the G-protein transducin involves competition between transducin alpha-guanosine 5'-triphosphate and Palpha-517-541 for binding to the Pgamma-24-45 region. Based on the results, we propose a linear model of interactions between catalytic and inhibitory PDE subunits.
...
PMID:An interface of interaction between photoreceptor cGMP phosphodiesterase catalytic subunits and inhibitory gamma subunits. 870 12

In the search for the essential functional domains of the large mechanosensitive ion channel (MscL) of E. coli, we have cloned several mutants of the mscL gene into a glutathione S-transferase fusion protein expression system. The resulting mutated MscL proteins had either amino acid additions, substitutions or deletions in the amphipathic N-terminal region, and/or deletions in the amphipathic central or hydrophilic C-terminal regions. Proteolytic digestion of the isolated fusion proteins by thrombin yielded virtually pure recombinant MscL proteins that were reconstituted into artificial liposomes and examined for function by the patch-clamp technique. The addition of amino acid residues to the N-terminus of the MscL did not affect channel activity, whereas N-terminal deletions or changes to the N-terminal amino acid sequence were poorly tolerated and resulted in channels exhibiting altered pressure sensitivity and gating. Deletion of 27 amino acids from the C-terminus resulted in MscL protein that formed channels similar to the wild-type, while deletion of 33 C-terminal amino acids extinguished channel activity. Similarly, deletion of the internal amphipathic region of the MscL abolished activity. In accordance with a recently proposed spatial model of the MscL, our results suggest that (i) the N-terminal portion participates in the channel activation by pressure, and (ii) the essential channel functions are associated with both, the putative central amphipathic alpha-helical portion of the protein and the six C-terminal residues RKKEEP forming a charge cluster following the putative M2 membrane spanning alpha-helix.
...
PMID:Molecular dissection of the large mechanosensitive ion channel (MscL) of E. coli: mutants with altered channel gating and pressure sensitivity. 914 55


1 2 3 Next >>

---