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)

Using low-speed sedimentation equilibrium we have established that vinculin binds to alpha-actinin with a Kd of 1.3 x 10(-5) M. Electron microscopy of negatively stained preparations of vinculin revealed spherical particles (diameter 11.2 nm; S.D. 1.7 nm, n = 21), whereas alpha-actinin appeared as a rod-shaped particle (length 33 nm; S.D. 3.3 nm, n = 23). Mixtures of the two proteins contained both 'lollipop'- and 'dumbell'-shaped particles which we interpret as either one or two spherical vinculin molecules associated with the ends of the alpha-actinin rod. We have further defined the vinculin-binding site in alpha-actinin using 125I-vinculin and a gel-blot assay in which proteolytic fragments of alpha-actinin and fragments of alpha-actinin expressed in Escherichia coli were resolved by SDS/PAGE and blotted to nitrocellulose. 125I-vinculin bound to polypeptides derived from the spectrin-like repeat region of alpha-actinin, but did not bind to the actin-binding domain. Binding was inhibited by a 100-fold molar excess of unlabelled vinculin. Using a series of glutathione S-transferase fusion proteins we have mapped the vinculin-binding site to a region toward the C-terminal end of the molecule (alpha-actinin residues 713-749). 125I-vinculin also bound to fusion proteins containing this sequence which had been immobilized on glutathione-agarose beads. The vinculin-binding site is localized in a highly conserved region of the molecule close to the first of two EF-hand calcium-binding motifs.
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PMID:Identification of the vinculin-binding site in the cytoskeletal protein alpha-actinin. 803 76

Polyclonal antibodies were raised to partial and full-length synthetic peptides of human papillomavirus type 16 (HPV-16) E5. Antisera specificity for HPV-16 E5 was demonstrated by their ability to recognize not only their peptide immunogens but also full-length peptide and a glutathione S-transferase-E5 fusion protein. The most reactive antiserum, PE-6, raised to a full-length peptide, was used in Western blot analysis to identify HPV-16 E5 protein from exfoliated cervical cells. A strong, single band at approximately 20K was detected in two of six HPV-16-positive samples from women with a history of low-grade cervical intraepithelial neoplasia. The apparent M(r) by SDS-PAGE suggests that HPV-16 E5 forms homodimers in vivo, but not through cysteine linkage.
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PMID:Detection of E5 oncoprotein in human papillomavirus type 16-positive cervical scrapes using antibodies raised to synthetic peptides. 807 47

The activities of the conjugative enzymes, glutathione S-transferase and UDP-glucuronyl-transferase, have been measured in vitro in the livers of camels, guinea-pigs and rats. Some sex differences were observed in the levels of these conjugative enzymes. In rats and guinea-pigs, females had higher UDP-glucuronyltransferase activity than males. In camels, females had higher glutathione S-transferase activity than males. In these species, the cytochrome P-450 isozymes observed between the 50,000 and 60,000 mol. wt regions have been separated and characterized by SDS-polyacrylamide gel electrophoresis. Camels showed lower levels of all types of cytochrome P-450 isozymes, while guinea-pigs showed higher levels of most of these isozymes. In general, camels seemed to have the lowest drug-metabolizing enzyme activity when compared to rats and guinea-pigs.
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PMID:Comparison of cytochrome P-450 content and conjugative enzymes in livers of camels (Camelus dromedarius), guinea-pigs (Cavia porcellus) and rats (Rattus norvegicus). 808 58

Muscarinic acetylcholine receptors (mAChR, human m2 subtype) expressed in Sf9 (Spodoptera frugiperda) cells using the baculovirus system were purified and subjected to phosphorylation by a mAChR kinase, which was partially purified from porcine cerebrum. Two bands with apparent molecular masses of 59 kDa and 39 kDa as determined by SDS/PAGE were found to be phosphorylated in an agonist-dependent manner. Both bands were labeled by the irreversible muscarinic ligand [3H]propylbenzilylcholine mustard. Molecular masses of the [32P]phosphorylated or [3H]propylbenzilylcholine-mustard-labeled bands decreased following treatment with N-glycanase. The 59-kDa and 39-kDa bands were converted to 52-kDa and 32-kDa bands, respectively, indicating that both the 59-kDa and 39-kDa bands contain the amino-terminal region where glycosylation sites are present. The ratio of incorporated [32P]phosphate and bound [3H]propylbenzilylcholine mustard was essentially the same for the 59-kDa and 39-kDa bands, indicating that all the phosphorylation sites reside in the sequence of 39 kDa from the amino-terminal region. The amounts of incorporated [32P]phosphate were estimated to be 10-11/receptor, with 7-8 serine and 3-4 threonine, but no phosphorylated tyrosine residues. Further treatment of [32P]phosphorylated or [3H]propylbenzilylcholine-mustard-labeled receptors with V8 protease indicated that the phosphorylation sites were not present in 30-kDa amino-terminal segment. These results indicate that the phosphorylation sites are localized in the range 30-39 kDa from the amino terminus, which consists of primarily the central part of the third intracellular loop. Consistent with this conclusion, a fusion protein containing glutathione S-transferase linked to a peptide corresponding to residues 227-324 of the central part of the third intracellular loop was found to be phosphorylated by the mAChR kinase in a heparin-sensitive manner.
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PMID:Location of agonist-dependent-phosphorylation sites in the third intracellular loop of muscarinic acetylcholine receptors (m2 subtype). 811 96

We have isolated a variant [PC3(R)] of the human prostate PC3 tumor cell line which showed resistance to several anticancer drugs. Studies to evaluate the mechanisms of resistance to anticancer drugs in the PC3(R) cell line indicated that mdr1 was not overexpressed. Studies also indicated that activities of topo I and topo II were not different in these cell lines, nor was there any difference in the formation of drug-induced KCl-SDS precipitable complexes, indicating that topoisomerases were not involved in the development of resistance in PC3(R) cells. While the activity of glutathione S-transferase and total glutathione levels were also similar in these cell lines, the glutathione peroxidase activity in PC3(R) cells was 5-fold lower than in PC3 cells. Furthermore, proto-oncogene expression for c-jun, c-myc, and H-ras was significantly higher in resistant cells than in sensitive cells, indicating that the amplification of early response genes may play a role in the emergence of de novo resistance in PC3(R) cells.
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PMID:Oncogene overexpression and de novo drug-resistance in human prostate cancer cells. 815 44

A method for the examination of the glutathione S-transferase isoenzyme profiles in human liver using a new HPLC affinity support is described. Liver cytosol was injected directly onto an HPLC column (5 x 0.46 cm) containing a support with a covalently bound affinity ligand (S-octylglutathione) specific for the isoenzymes. Contaminating cytosolic proteins were removed in a washing step. The isoenzymes were eluted with a linear gradient of a different affinity ligand in the mobile phase. Coinciding with the affinity ligand gradient, a salt gradient (0-200 mM sodium chloride) was applied. In this manner the isoenzymes were fractionated into the enzymatically active homodimers and heterodimers. The classes of the affinity fractionated isoenzymes were determined by SDS-PAGE and ELISA while the subunit content was determined by reversed-phase chromatography. For one liver three Alpha class isoenzyme subunits, forming three heterodimers and two homodimers, were detected. Five livers were examined, and the homodimer A1-1 was found to be the predominant glutathione S-transferase isoenzyme. Minor amounts of Pi and Mu class isoenzymes were also detected. This non-denaturing high-performance affinity chromatography method reduced analysis time by a factor of ten when compared to other affinity analysis methods for the glutathione S-transferases.
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PMID:Examination of glutathione S-transferase isoenzyme profiles in human liver using high-performance affinity chromatography. 818 Jun 56

The tertiary structure of erythropoietin (EPO) remains to be elucidated by X-ray crystallography. Although the amino acid sequence of EPO is known, the specific features that confer its biological activity are not well understood. In order to study the structure-function relationships of EPO by in vitro mutagenesis, we have used the vector pGEX-2T to express human and murine EPO fused to the carboxyl terminus of glutathione S-transferase (GST) in E. coli. The fusion proteins were the predicted size (46 kDa) by SDS-PAGE. GST-huEPO eluted from glutathione-agarose using reduced glutathione (GSH) was tested by radioimmunoassay and in a mouse spleen cell assay (MSCA). Dose-response curves parallel to recombinant human EPO (rHuEPO) were obtained in both assays. The ratio of immuno- to bioactivity was 4.7:1. Thus the presence of the 26 kDa GST protein at the end terminus of EPO does not abrogate biological activity. GST-mEPO also gave dose-response curves parallel to rHuEPO in the MSCA but not in the RIA. The wild-type murine and three mutant GST-EPO fusion proteins (166 Des-Arg, Glu 159-->Val, and Arg 163-->Glu) were tested in the MSCA and assayed for GST activity. The ratio of bioactivity to enzyme activity for the Arg 163-->Glu mutant was approximately one third of the value obtained for each of the other fusion proteins, indicating that arginine at 163 is functionally important for EPO activity. The availability of these human and murine gene constructs in pGEX should facilitate site-directed mutagenesis and permit detailed studies of the structure-function relationships for the two erythropoietins.
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PMID:Structure-function relationships of the erythropoietin molecule. 818 27

A baculovirus vector system that expresses cloned DNA sequences as glutathione S-transferase fusion proteins was developed. This system was used to express and purify the lymphocyte-specific tyrosine kinase p56lck. This recombinant p56lck was purified to homogeneity in a single chromatography step using glutathione resin. By SDS gel analysis recombinant p56lck was found to migrate as two species with molecular masses of approximately 56,000 Da. p56lck purified in this manner retained a high level of activity, phosphorylated an exogenous substrate on tyrosine residues, and underwent autophosphorylation on tyrosine residues. The Km (approximately 0.33 mmol) and Vmax (approximately 83 pmol min-1 mg-1) values were also determined by using enolase as a substrate.
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PMID:Analysis of the tyrosine protein kinase p56lck expressed as a glutathione S-transferase fusion protein in Spodoptera frugiperda cells. 825 50

1. Glutathione S-transferase isoenzymes from Mytilus edulis and M. galloprovincialis have been partially purified by glutathione-sepharose affinity chromatography followed by Mono Q anion exchange fast protein liquid chromatography (f.p.l.c.). 2. The tissue distribution of glutathione S-transferase in M. edulis has been studied. Using 1-chloro-2,4-dinitrobenzene as substrate, highest specific activity is observed in the gill, the main feeding organ. Affinity-purified extracts of this organ give a characteristic f.p.l.c. profile. A similar profile is obtained with affinity-purified extracts of the digestive gland of M. galloprovincialis. 3. The subunit structure of the purified isoenzymes has been studied by SDS polyacrylamide gel electrophoresis and reversed-phase h.p.l.c. The subunits have similar molecular weights and h.p.l.c. retention times to rat glutathione S-transferases.
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PMID:Separation of multiple forms of glutathione S-transferase from the blue mussel, Mytilus edulis. 828 41

We selected three kinds of plasmids for expression of C3a as fusion proteins. The proteins were purified by affinity chromatography using the respective specific resins, and their activities were measured by guinea pig platelet aggregation. We showed that polyhistidine (polyHis)-C3a fusion protein was able to exhibit 30% of the activity of natural C3a. However, glutathione S-transferase (GST)-C3a fusion protein exhibited only 10% of such activity, and no activity was measured for maltose binding protein (MBP)-C3a fusion protein. The purified polyHis-C3a fusion protein was attached to the Ni-NTA agarose column in an attempt to isolate the C3a receptor from guinea pig platelets. The C3a binding protein isolated from digitonin-solubilized guinea pig platelet membrane was approximately 50 kDa on SDS-polyacrylamide gel. This is the first report of C3a fusion protein production with biological activity.
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PMID:Expression of biologically active C3a as fusion proteins. 829 43


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