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)

Galanin receptors were solubilized from rat brain using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Binding of 125I-galanin to the soluble fraction was time- and temperature-dependent, saturable, and reversible. Scatchard analysis of binding data indicated that the soluble extract contained a single class of galanin binding sites with a Kd of 0.8 nM and a Bmax of 26 fmol/mg of protein. Unlabeled galanin and its fragments galanin(2-29) and galanin(1-15) antagonized the binding of 125I-galanin to CHAPS-solubilized extracts with relative potencies similar to those observed with membrane receptors. Galanin(3-29) was found inactive. Binding of 125I-galanin to CHAPS extracts was inhibited by guanine nucleotides with the following rank order of potency: GMP-P-(NH)P greater than GTP greater than GDP. Molecular analysis of the soluble galanin receptor by covalent cross-linking of 125I-galanin to CHAPS extracts using disuccinimidyl tartrate and further identification on SDS-PAGE indicated that the soluble galanin binding site behaves as a protein of Mr 54,000. After incubation of CHAPS extracts with 125I-galanin, gel filtration on Sephacryl S-300 followed by ultracentrifugation on sucrose density gradient revealed a binding component with the following hydrodynamic parameters: Stokes radius, 5 nm; s20,w, 4.5 S; Mr, 98,000; frictional ratio, 1.6. GMP-P(NH)P treatment of CHAPS extracts gave rise to a molecular form with the following characteristics: Stokes radius, 4 nm; s20,w, 3.3 S; Mr, 57,000; frictional ratio, 1.4.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Solubilization and molecular characterization of active galanin receptors from rat brain. 137 97

Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residue in the active site of tonoplast H(+)-ATPase from etiolated mung-bean (Vigna radiata L.) seedlings. FITC caused marked inactivation of the enzyme activities of both membrane-bound and soluble ATPase and its associated H+ translocation. The SDS/PAGE pattern revealed that the FITC-binding site was in the large (A) subunit of ATPase. Inhibition could be substantially prevented by its physiological substrate ATP, pyrophosphate and nucleotides in the decreasing order: ATP greater than pyrophosphate greater than ADP greater than AMP greater than GTP greater than CTP greater than UTP. The mode of inhibition by FITC was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first-order kinetics with a Ki of 0.33 mM, a minimum inactivation half-time of 110 s, and a first-order rate constant of 0.244 s-1. A double-logarithmic plot of apparent rate constant versus FITC concentration gave a slope of 0.913, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labelling studies indicated that the incorporation of approx. 1 mol of FITC/mol of ATPase is sufficient to inhibit ATPase completely. The enhancement and blue shift of emission maxima of FITC after modification of ATPase indicated that the labelled lysine residue was located in a relatively hydrophobic domain.
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PMID:Inhibition of tonoplast ATPase from etiolated mung bean seedlings by fluorescein 5'-isothiocyanate. 138 33

Highly purified GTP-cyclohydrolase was obtained by fractionation of cell extracts with ammonium sulfate, ion-exchange and hydrophobic chromatography. The N-terminal amino acid sequence and amino acid composition of the protein were determined. According to SDS-PAGE data, the molecular weight of the enzyme is 45 kDa. The active enzyme has several isoforms separable by native electrophoresis. The maximal enzyme activity is determined at 1.5 mM Mn2+; 70% of enzymatic activity is detected with Mg2+. The enzyme is inhibited by heavy metal ions and chelators and is inactive in the absence of thiol-reducing agents. The enzyme activity is detected in a broad range of pH with a maximum at pH 8.2. The pyrimidine product of the GTP-cyclohydrolase reaction. 2.5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate was purified and identified. Another product of this reaction is pyrophosphate.
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PMID:[Purification and properties of GTP-cyclohydrolase from Bacillus subtilis]. 139 Dec 11

A series of in vivo and in vitro experiments were conducted to determine the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) administered on the expression of c-ras. Differences in c-ras expression between control and TCDD treated groups were determined by immunoassay of p21ras protein, or indirectly measured by the specific binding of 3H-GTP to hepatic plasma membrane preparations. Intraperitoneal injection of sublethal doses of TCDD significantly elevated (P less than 0.05, Student t test) levels of hepatic p21ras protein in Sprague-Dawley rats and TCDD sensitive C57BL/6J mice. Such an increase occurred at an early stage of poisoning in the C57BL/6J mice. The earliest increase was detectable 6 hr after dosing, and the difference became statistically significant by 12 and 24 hr after dosing. In contrast, TCDD tolerant DBA/2J mice had only a marginal increase in hepatic p21ras protein which did not become statistically significant even at 24 hr host-dosing. TCDD evoked increases in hepatic p21ras protein of C57BL/6J mice were accompanied by the increase in the specific binding of GTP to hepatic plasma membranes. Column chromatography of solubilized rat hepatic membrane proteins on sephadex G-50 showed TCDD administration increased levels of a 3H-GTP binding protein with MW of approximately 21 Kd. 3H-GTP binding in total hepatic membranes was also elevated (P less than 0.05, Fisher PLSD multiple comparison test) 6 hr and 24 hr after dosing of C57BL/6J mice, but as expected the effect of TCDD was not as conspicuous as that found in the plasma membrane. TCDD treatment increased levels of a 21 Kd protein found in the in vitro translation products of RNA purified from guinea pig liver. This protein was identified as a c-ras protein based upon its ability to bind GTP, precipitation by a polyclonal antibody against the rasHa and Ki proteins and subsequent SDS-PAGE which showed a single protein band of approximately 21 Kd.
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PMID:TCDD causes stimulation of c-ras expression in the hepatic plasma membranes in vivo and in vitro. 140 41

Incubation of 80S ribosomes with a substoichiometric amount of [alpha-32P]GTP and with eEF-2 resulted in the specific labeling of one ribosomal protein which migrated very close to the position of the acidic phosphoprotein P2 from the 60S subunit in two-dimensional isofocusing-SDS gel electrophoresis. Localization of protein P2 in this electrophoretic system was ascertained by correlation with its position in the standard two-dimensional acidic-SDS gel electrophoresis after its specific phosphorylation by casein kinase II. Labeling of the ribosomal protein was dependent on the presence of eEF-2, and could be attributed to [alpha-32P]GDP binding from the results of chase experiments and HPLC identification, this binding being very likely responsible for the slight shift in the electrophoretical position of the protein. Incubation of ribosomes with tRNA(Phe) in the absence of mRNA induced the release of the bound GDP.
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PMID:Binding of GDP to a ribosomal protein after elongation factor-2 dependent GTP hydrolysis. 142 Mar 8

ODC was purified to homogeneity from E. coli K12 MG1655 strain transformed with a pBR322 plasmid carrying the ODC gene. This preparation was homogeneous as it was analyzed by SDS-polyacrylamide gel electrophoresis. From this preparation the amino-terminal sequence analysis was obtained. The native ODC of E. coli is activated by ATP, GTP, CTP and UTP at 10(-3) M concentration to around 170-300%. Our results indicate that the recombinant ODC is activated only by GTP and UTP at 10(-3) M 370% and 300%, respectively. When the recombinant ODC was incubated with calf intestine alkaline phosphatase, this inactive ODC can be reversibly activated allosterically only by GTP or UTP at a concentration of 10(-6) or 10(-5) M. That GTP or UTP can allosterically convert the inactive form of ODC to an active form suggests that these analogues may be the in vivo physiological regulators of ODC.
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PMID:Allosteric activation by nucleotides of the inactive by phosphatase ornithine decarboxylase of Escherichia coli. 144 81

Several methods for the in vitro assembly of microtubules from postmortem human brain were compared for the purpose of obtaining microtubule preparations that best retained their microtubule-associated proteins. The polymerized microtubules from the preparations were examined by negative staining and electron microscopy and shown to consist of well-formed microtubules with varying amounts of abnormal assembly products that differed between methods. The microtubule protein was analyzed by SDS-polyacrylamide gel electrophoresis, quantitative densitometry, as well as trans-blotted onto membranes which were reacted with monoclonal antibodies to tubulin subunits and microtubule-associated proteins. All the preparations were found to contain both the alpha- and beta-tubulin subunits with quantitative differences, but they varied most, both quantitatively and qualitatively, in their content of microtubule-associated proteins. The optimal method for the assembly of soluble tubulin from postmortem human brain cytosol into intact microtubules which specifically retained most of their MAPs, especially tau, employed 4 M glycerol assembly buffer in the presence of 10 microM taxol and 1 mM GTP. The isolation methods were used to compare young and aged brains, and there were fewer microtubule-associated proteins, especially tau, associated with the microtubules in advanced age, in all preparations.
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PMID:Comparison of methods for the in vitro assembly of postmortem human brain microtubules that retain the microtubule-associated protein tau. 149 96

Small G-proteins encoded by ras-like genes are ubiquitous in eukaryotic cells. These G-proteins are believed to play a role in central processes, such as signal transduction, cell differentiation and membrane vesicle transport. By screening genomic and cDNA libraries of the colonial alga, Volvox carteri f. nagariensis, with ypt DNA probes from Zea mays, we have identified the first member of a ypt gene family, yptV1, within a green alga. The 1538-bp yptV1 gene of V. carteri consists of nine exons and eight introns and has three potential polyadenylation sites 210, 420 and 500 bp downstream from the UGA stop codon. The derived 203-amino-acid polypeptide, YptV1, exhibits 81% similarity with Ypt1 from mouse, with the corresponding genes sharing four identical intron positions. Recombinant YptV1 (reYptV1) produced in Escherichia coli retains the ability to bind GTP after SDS-PAGE and immobilization on nitrocellulose. Immunological studies using polyclonal antibodies against reYptV1 indicate that the protein is present in the membrane fraction of a V. carteri extract and is expressed throughout the whole life-cycle of the alga. Similar to other Ras-like proteins, YptV1 contains two conserved C-terminal cysteine residues suggesting post-translational modification(s), such as isoprenylation or palmitoylation, required for membrane anchoring. The presumptive role of YptV1 in cytoplasmic vesicle transport is briefly discussed.
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PMID:The yptV1 gene encodes a small G-protein in the green alga Volvox carteri: gene structure and properties of the gene product. 151 89

Excimer-forming cysteines in tubulin are detected by the presence of excimer fluorescence in N-(1-pyrenyl)maleimide-labeled tubulin. The ratio of excimer/monomer fluorescence of labeled protein remained unchanged upon its dilution. These results indicating that both partner of each pair(s) of cysteine are located in the same subunit. The excimer fluorescence is insensitive to prior treatment of tubulin with either colchicine or GTP, indicating that pairs of cysteines protected by those drugs are not involved in excimer formation. This excimer fluorescence of N-(1-pyrenyl)maleimide-labeled tubulin disappeared upon treatment with SDS, guanidinium chloride (GdmCl) and urea. Studies with GdmCl induced unfolding of N-(1-pyrenyl)maleimide-labeled tubulin showed that the loss of excimer fluorescence precedes subunit dissociation. The loss of both colchicine-binding activity and the excimer fluorescence with increasing temperature indicates a major conformational change of the tubulin molecule at elevated temperatures.
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PMID:Excimer fluorescence of pyrene-maleimide-labeled tubulin. 154 Dec 90

Vpu as a human-immunodeficiency-virus-type-1-encoded 81-amino-acid integral-membrane protein was expressed in Escherichia coli using the inducible ptrc promoter of an ATG fusion vector. Recombinant Vpu is associated with membranes of E. coli and could be partially solubilized by detergents. Recombinant Vpu was phosphorylated in vitro with purified porcine casein kinase II (CKII) as well as with a CKII-related protein kinase found in cytoplasmic extracts of human and hamster cells. Recombinant Vpu associated with E. coli membranes has turned out to be the best substrate for in vitro phosphorylation with CKII. This reaction can be inhibited by heparin and the ATP analogue 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB), both known to be potent inhibitors of CKII. Radiolabelled gamma ATP and gamma GTP were used as phosphate donors in vitro phosphorylation of recombinant Vpu. In vivo phosphorylation of Vpu in HIV-1-infected H9 cells was also inhibited by DRB. We concluded therefrom that the Vpu protein is phosphorylated by the ubiquitous CKII in HIV-1-infected human host cells. Two seryl residues in the sequence of Vpu (position 52 and 56) correspond to the consensus S/TXXD/E for CKII. These potential phosphorylation sites are located within a well-conserved dodecapeptide of Vpu (residues 47-58), which is found in different HIV-1 strains as well as in a Vpu-like protein of SIVCPZ. Monoclonal and polyclonal antibodies directed against two different epitopes of Vpu were used for immunoprecipitation of Vpu from HIV-1-infected cells and for detection of Vpu in Western blot analyses. Vpu from HIV-1-infected cells as well as recombinant Vpu expressed in E. coli were determined by SDS/PAGE using 6 M urea to be 9 kDa, which corresponds to the calculated molecular mass of Vpu.
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PMID:Human-immunodeficiency-virus-type-1-encoded Vpu protein is phosphorylated by casein kinase II. 154 Dec 98


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