Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The superfamily of olfactory receptor genes, whose products are thought to be activated by odorant ligands, is critical for odor recognition. Two olfactory receptors, olp4 from rat and OR17-4 from human, were overexpressed in Sf9 insect cells. The presence of the proteins in cell membranes was monitored by immunoblotting with peptide-specific polyclonal antibodies directed against the C-terminal sequences of these receptors and with a mAb against an N-terminal octapeptide epitope tag. A DNA sequence that codes for a His6 tag, which binds tightly to a Ni2+-chelate-affinity column, was incorporated into the N-termini of both genes. The expressed olfactory receptors were found mainly in the cell-membrane fraction. The proteins were difficult to solubilize by many detergents and only lysophosphatidylcholine was found to be both suitable for efficient solubilization of the overexpressed olfactory receptors and compatible with the purification system used. After solubilization, the olfactory receptors were purified to near homogeneity by affinity chromatography on nickel nitrilotriacetic acid resin and by cation-exchange chromatography. Electrophoresis of the purified proteins and visualization with Coomassie Blue staining or by immunoblotting with specific antibodies, revealed bands of 32, 69 and 94 kDa, which were identified as the monomeric, dimeric and trimeric forms of the receptor proteins. The oligomeric forms were resistant to reduction and alkylation, and are therefore thought to be held together by non-covalent hydrophobic interactions that are resistant to SDS. This finding is similar to previous observations for other guanine-nucleotide-binding-regulatory-protein-coupled receptors. Reconstitution in phospholipid vesicles showed that the purified olfactory receptors insert specifically into the lipid bilayer. This provides a means to study functional reconstitution with putative transduction components such as olfactory guanine-nucleotide-binding-regulatory protein.
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PMID:Overexpression, solubilization and purification of rat and human olfactory receptors. 866 47

DNA encoding the coat protein (P3) of a Scottish isolate of potato leafroll virus (PLRV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) such that the coat protein was expressed either in an unmodified form or with the addition of the amino acid sequence MHHHHHHGDDDDKDAMG at the N terminus (P3-6H). Insect cells infected with these recombinant baculoviruses accumulated substantial amounts of P3 and P3-6H. P3 could not be recovered from cell extracts unless it was denatured in SDS but a proportion of the P3-6H was recoverable in a soluble form in non-denaturing conditions. Immunogold labelling of sections of infected cells showed that P3 accumulated in nuclei in large amorphous bodies. In contrast, although much of the P3-6H also accumulated in nuclei, it formed virus-like particles (VLP) which were often grouped in close-packed, almost cystalline arrays. When electron microscope grids coated with antibodies to PLRV were floated on cell extracts containing P3-6H, VLP were trapped which were indistinguishable from PLRV particles trapped from extracts of PLRV-infected plants. The VLP co- sedimented in sucrose gradients with PLRV particles which suggests that the VLP contained RNA. VLP collected from sucrose density gradient fractions contained protein which reacted with nickel chelated to nitrilotriacetic acid, a histidine-specific reagent. Cells infected with either recombinant baculovirus also synthesized a protein, with an Mr of about 17000, which was shown to be the translation product of the P4 gene which is in the +1 reading frame within the coat protein gene. This protein was also found in the nuclear fraction of infected cells but was more readily soluble than was P3.
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PMID:Assembly of virus-like particles in insect cells infected with a baculovirus containing a modified coat protein gene of potato leafroll luteovirus. 875 74

The proteinase previously described as an unidentified component of E. coli A2 extracts which hydrolyses actin at a new cleavage site (Khaitlina et al. (1991) FEBS Lett. 279, 49) was isolated and further characterized. A chromatographic method of proteinase purification was developed by which a purity of more than 80% was attained. The enzyme was identified as a single, 32 kDa polypeptide (ECP 32) by SDS-PAGE and non-denaturing electrophoresis as well as by ion-exchange chromatography and gel filtration. The N-terminal sequence of ECP 32 was determined to be: AKTSSAGVVIRDIFL. The activity of ECP 32 is inhibited by o-phenanthroline, EDTA, EGTA and zincone. The EDTA-inactivated enzyme can be reactivated by cobalt, nickel and zinc ions. Based on these properties ECP 32 was classified as a metalloproteinase (EC 3.4.24). Limited proteolysis of skeletal muscle actin between Gly-42 and Val-43 was observed at enzyme substrate mass ratios of 1:25 to 1:3000. Two more sites between Ala-29 and Val-30, and between Ser-33 and Ile-34 were cleaved by ECP 32 in heat- or EDTA-inactivated actin. Besides actin, only histones and DNA-binding protein HU were found to be substrates of the proteinase, confirming its high substrate specificity. Its molecular mass, N-terminal sequence and enzymatic properties distinguish ECP 32 from any known metalloproteinases of E. coli, and we therefore conclude that it is a new enzyme.
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PMID:Purification and characterization of the proteinase ECP 32 from Escherichia coli A2 strain. 876 29

A novel enzyme, trehalose synthase, was purified from a cell-free extract of Pimelobacter sp. R48 to an electrophoretically homogeneous state by successive chromatographies on DEAE-Toyopearl 650, Butyl-Toyopearl 650, and Mono Q HR5/5 columns. The molecular weight of the enzyme was estimated to be 62,000 by SDS-polyacrylamide gel electrophoresis, and the enzyme had a pI of 4.6 by gel isoelectrofocusing. The enzyme catalyzed the conversion of maltose into trehalose by intramolecular transglucosylation. The enzyme also converted into maltose but was inactive on other saccharides. The N-terminal amino acid of the enzyme was serine. The optimum pH and temperature were pH7.5 and 20 degrees C, respectively. The enzyme was stable in the range of pH 6.0-9.0 and up to 30 degrees C for 60 min. The rate of conversion of maltose into trehalose was independent of the maltose concentration. The maximum yield of trehalose from maltose were 81.8%, 80.9%, and 76.7% at 5, 15, and 25 degrees C, respectively. The activity was inhibited by Cu2+, Hg2+, Ni2+, Zn2+, and Tris.
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PMID:Purification and properties of a novel enzyme, trehalose synthase, from Pimelobacter sp. R48. 882 31

Stable microtubules (MTs) are well known to contain acetylated alpha-tubulin. Ni2+-induced MT bundling may be accompanied with such tubulin post-translational modification. To explore this possibility as a mechanism of Ni2+-induced cytoskeletal injury, we have examined acetylated alpha-tubulin levels in cultured 3T3 cells by both immunoprecipitation assays and fluorescent staining of MTs using a monoclonal antibody (clone 6-11B-1) specific for acetylated artubulin. Cell extracts prepared from [35S]methionine labeled cultures in the presence or absence of Ni2+ were immunoprecipitated and analyzed by SDS-PAGE followed by autoradiography. A predominant protein band (molecular mass 55 kDa), representing acetylated alpha-tubulin, appeared in Ni2+-treated cells in a dose and time-dependent manner whereas the corresponding labeled protein band was only barely detectable in control cells. Consistent with the immunoblot findings, MTs in control 3T3 cells in the absence of Ni2+ were not labeled by the 6-11B-1 antibody except for some short, discontinuous segments localized in the cell center or the perinuclear MT organizing center area. In contrast, treatment of cells with NiCl2 (2 mM for 20 hr) resulted in, as expected, the formation of MT bundles that were intensely stained by the 6-11B-1 monoclonal antibody specific for acetylated alpha-tubulin. Furthermore, MTs containing acetylated alpha-tubulin in Ni2+-treated cells were resistant to disassembly induced by nocodazole, and at least partially resistant to cold temperature (0 degrees C), which also depolymerizes MTs. Since acetylated alpha-tubulin serves as a marker for the presence of stable MTs, the marked enhancement of alpha-tubulin acetylation in Ni2+-treated cells indicates that stabilization of MTs may be an important mechanism by which Ni2+ induces cell injury since stabilized MTs in turn should favor MT bundling, an unusual form of cytoskeletal perturbation in response to Ni2+ exposure.
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PMID:Nickel (Ni2+) enhancement of alpha-tubulin acetylation in cultured 3T3 cells. 888 64

The activation of the human transforming growth factor (TGF-beta) system begins with the cytokine-induced association of the extracellular domains of two structurally related receptor subunits. To study the protein-protein interactions between TGF-beta and the ligand-specific receptor subunit, the extracellular domain of the human TGF-beta receptor type II (T beta R-II) has been expressed as an intracellular protein in insect cells using the baculovirus expression system. The cDNA construct was engineered to encode amino acids 24-159 (the signal sequence 1-23 was lacking) preceded by one initiator methionine residue and six histidine residues added at the carboxy terminus. The soluble receptor accumulated in the cytoplasm of infected cells and was purified by one-step nickel-chelate affinity chromatography. The purified protein was not glycosylated; it migrated as a single band of apparent mass 19.5 kDa in SDS/polyacrylamide gels, and had a homogeneous N-terminal sequence. We have established a solid-phase binding assay using radioiodinated TGF-beta 3 and capture antibodies to immobilize the soluble receptor. In this assay, the apparent dissociation constant of the TGF-beta type-II receptor ectodomain for TGF-beta 3 was approximately 150 nM (this value is approximately 1000-fold higher than that of the cell-membrane receptor complex of living cells). The affinity of TGF-beta 3 for the unglycosylated ectodomain of T beta R-II from insect cells was lower than the affinity for the recombinant glycosylated ectodomain T beta R-II from mouse cells. The novel assay has been used to characterize affinities and specificities of TGF-beta 3, TGF-beta 2, corresponding mutants and hybrid proteins, as well as a related protein, BMP-2. The assay could also be used to search for inhibitors.
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PMID:The unglycosylated extracellular domain of type-II receptor for transforming growth factor-beta. A novel assay for characterizing ligand affinity and specificity. 891 30

Type 1 plasminogen activator inhibitor (PAI-1) is a key regulator of the fibrinolytic cascade that is stored in a rapidly releasable form within platelet alpha-granules. To identify proteins that may participate in the targeting or storage of this potent inhibitor, this report investigates the applicability of utilizing filamentous bacteriophages to display proteins expressed by cells containing a regulated secretory pathway and their enrichment based upon an interaction with PAI-1. For this purpose, RNA was extracted from AtT-20 cells (i.e. a classical model cell system for intracellular protein sorting), reverse transcribed, amplified using polymerase chain reaction primers containing internal restriction sites, and cloned into the phagemid pCOMB3H for expression as fusion constructs with the bacteriophage gene III protein. Escherichia coli was transformed with the phagemids and infected with VCSM13 helper phage, and the resulting AtT-20 cDNA-bacteriophage library was enriched by panning against solid- and solution-phase PAI-1. The enriched cDNA library was subcloned into a prokaryotic expression vector system that replaces the gene III protein with a decapeptide tag for immunologic quantitation. One novel cDNA clone (i.e. A-61), which preferentially recognized solution-phase PAI-1 and reacted positively with antibodies derived from a rabbit immunized with alpha-granules, was subcloned into the prokaryotic expression vector pTrcHis to create a construct containing an N-terminal six-histidine purification tag. This construct was expressed in E. coli, purified by nickel-chelate chromatography followed by preparative SDS-polyacrylamide gel electrophoresis, and utilized for the generation of polyclonal antibodies. Immunoblotting analysis employing antibodies against the purified A-61 construct revealed a 23-kDa protein present in the regulated secretory pathway of AtT-20 cells. The 23-kDa molecule was purified from media conditioned by AtT-20 cells by ion exchange chromatography on DEAE-Sephacel, molecular sieve chromatography on Sephacryl S-100, chromatofocusing on Polybuffer exchanger 94, and affinity chromatography on PAI-1-Sepharose. N-terminal amino acid sequencing of a 16-kDa Lys-C proteolytic fragment of the 23-kDa storage granule protein was employed to confirm its identity with the cDNA sequence of clone A-61. These data indicate that phage display of cDNA libraries fused to the C-terminal region of the gene III protein and their enrichment via an interaction with a target molecule can be utilized to define other proteins present within a particular cellular pathway.
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PMID:Purification of storage granule protein-23. A novel protein identified by phage display technology and interaction with type I plasminogen activator inhibitor. 893 62

We describe the expression, purification, and characterization of human interleukin-1 beta converting enzyme (ICE) containing an affinity tag and modified to resist autoproteolysis. The point mutation Asp381 to Glu was added to eliminate the major site of autolytic degradation while maintaining catalytic activity, and an N-terminal polyhistidine tag was added in place of the ICE pro-region to facilitate purification. N-His (D381E) ICE was expressed in Escherichia coli and purified by nickel-chelating Sepharose and size-exclusion chromatography (SEC). The enzyme was stabilized greater than 80-fold against autolytic degradation relative to wild-type N-His ICE. SDS-PAGE analysis with silver-staining revealed no impurities, and 85% of the protein was catalytically active as determined by titration with a novel titrant, PD 163594 (3-[2-(2-benzyloxycarbonylamino-3-methylbutyrylamino)prop ionylamino]-4- oxo-5-(2-oxo-2H-chromen-7-yloxypentanoic acid). An oxidized adduct of ICE with glutathione, formed by disulfide rearrangement with oxidized glutathione to inhibit and stabilize the enzyme during purification, was rapidly reduced upon exposure to 5 mM DTT. One mole of glutathione was released per mole of active enzyme. Of the nine cysteines in ICE, eight were present in their reduced form in the glutathione adduct. N-His (D381E) ICE cleaved Ac-YVAD-Amc with the Michaelis-Menten parameters K(M) = 14 microM and Kcat = 0.7 s-1, values essentially identical to those reported for enzyme from natural sources.
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PMID:Preparation of an autolysis-resistant interleukin-1 beta converting enzyme mutant. 894 55

The voltage-gated K+ channel of T-lymphocytes, Kv1.3, was heterologously expressed in African Green Monkey kidney cells (CV-1) using a vaccinia virus/T7 hybrid expression system; each infected cell exhibited 10(4) to 5 x 10(5) functional channels on the cell surface. The protein, solubilized with detergent (3-[cholamidopropyl)dimethylammonio]-1-propanesulfonic acid or cholate), was purified to near-homogeneity by a single nickel-chelate chromatography step. The Kv1.3 protein expressed in vaccinia virus-infected cells and its purified counterpart are both modified by a approximately 2-kDa core-sugar moiety, most likely at a conserved N-glycosylation site in the external S1-S2 loop; absence of the sugar does not alter the biophysical properties of the channel nor does it affect expression levels. Purified Kv1.3 has an estimated size of approximately 64 kDa in denaturing SDS-polyacrylamide electrophoresis gels, consistent with its predicted size based on the amino acid sequence. By sucrose gradient sedimentation, purified Kv1.3 is seen primarily as a single peak with an approximate mass of 270 kDa, compatible with its being a homotetrameric complex of the approximately 64-kDa subunits. When reconstituted in the presence of lipid and visualized by negative-staining electron microscopy, the purified Kv1.3 protein forms small crystalline domains consisting of tetramers with dimensions of approximately 65 x 65 A. The center of each tetramer contains a stained depression which may represent the ion conduction pathway. Functional reconstitution of the Kv1.3 protein into lipid bilayers produces voltage-dependent K+-selective currents that can be blocked by two high affinity peptide antagonists of Kv1.3, margatoxin and stichodactylatoxin.
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PMID:Purification, visualization, and biophysical characterization of Kv1.3 tetramers. 899 50

A thermostable pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41) has been purified to homogeneity from Thermus caldophilus GK-24 by chromatographic methods, including gel-filtration and ion-exchange chromatography. The specific activity of the enzyme was increased 431-fold with a recovery of 13.2%. The purified enzyme was a monomer, M(r) = 65 kDa as estimated by SDS-PAGE and gel filtration. The pI was 6.1. The enzyme was most active at pH 5.5. The activity was maximal at 75 degrees C and stable up to 95 degrees C for 30 min at pH 5.5. The enzyme was stable to incubation from pH 3.5 to pH 8.0 at 4 degrees C for 24 h. The activity of the enzyme was stimulated by Mn2+ and Mg2+ ions. Ni2+, Ca2+, Co2+ ions and EDTA did not inhibit the enzyme activity. The enzyme hydrolyzed the alpha-1,6 linkages of amylopectin, glycogens, alpha, beta-limited dextrin, and pullulan. The enzyme caused the complete hydrolysis of pullulan to maltotriose. The activity was inhibited by alpha-, beta-, or gamma-cyclodextrins. The N-terminal sequence [(AIa-Pro-Gln-(Asp or Tyr)- Asn-Leu-Leu-Xaa-ILe-Gly-Ala(Ser)] showed some similarity to those of bacterial pullulanases.
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PMID:Purification and biochemical characterization of pullulanase type I from Thermus caldophilus GK-24. 902 41


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