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1.
Geoffrey R. Banks David G. Barker 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1985,826(4):180-185
Yeast DNA ligase is radioactively labelled in vitro by incubating a crude cell extract with [α-32P]ATP. The product of this reaction is the stable covalent ligase-AMP adduct, which can be characterized by its reactivity with either pyrophosphate or nicked DNA and visualized by gel electrophoresis and autoradiography. The Saccharomyces cerevisiae DNA ligase was identified as an 89 kDa polypeptide by exploiting the fact that transformants with multiple copies of the plasmid-encoded DNA ligase (CDC9) gene overproduce the enzyme by two orders of magnitude. A similar strategy has been used to identify the Schizosaccharomyces pombe DNA ligase as an 87 kDa polypeptide. Both values agree well with the coding capacities of the respective cloned gene sequences. When the S. cerevisiae ligase is greatly overproduced with respect to wild-type levels, a second polypeptide of 78.5 kDa is also labelled and has the same properties as the 89 kDa adduct. We suggest that this polypeptide is generated by proteolysis. 相似文献
2.
Electrostatic calculations and model-building suggest that DNA bound to CAP is sharply bent 总被引:20,自引:0,他引:20
Two observations suggest that DNA, upon binding to E. coli catabolite gene activator protein (CAP), is sharply bent by a total angle of at least 100-150 degrees: (1) The electrostatic potential field of CAP shows regions of positive potential that form a ramp on 3 sides of the protein. (2) The DNA binding site size as determined by DNA ethylation interference with binding, (Majors: "Control of the E. coli Lac Operon at the Molecular Level." Ph.D. Thesis, Harvard University, Cambridge, 1977) and by relative affinities of DNA fragments of various lengths (Liu-Johnson et al.: Cell 47:995-1005, 1986) requires severe bending of the DNA to maintain its favorable electrostatic contact with the protein. 相似文献
3.
R. D. Myers T. H. Privette R. L. Hornsby H. S. Swartzwelder 《Neurochemical research》1988,13(10):989-995
The kinetics of45Ca2+ efflux were examined at circumscribed sites in the perfused hippocampus of the freely moving rat with either one of two tetrahydroisoquinoline (TIQ) products, tetrahydropapaveroline (THP) or salsolinol. Guide tubes for unilateral push-pull perfusion were implanted stereotaxically to rest just above sites within the dorsal hippocampus. Upon recovery from surgery, a tissue site in the hippocampus was prelabeled with 1.0 l of45Ca2+ (2.0 Ci) injected through the indwelling guide tube. After 16–20 hr had elapsed, successive push-pull perfusions of the site were carried out with an artificial cerebrospinal fluid (CSF), at 5.0 min intervals and at a rate of 20 l/min, in order to obtain a control washout curve of declining radioactivity. On the fifth of a series of 5.0 min perfusions, either THP or salsolinol was added to the perfusion medium in a concentration of 10 or 100 ng/l. Then the hippocampal site was perfused again with control CSF for the collection of an additional three samples. Although THP in both of the test concentrations generally augmented the efflux of45Ca2+, the temporal course and magnitude of the enhancement depended on the anatomical site of the perfusion. In the more rostral hippocampal planes of AP 3.0 and AP 4.0, THP caused a delayed efflux of the cation, after the perfusion of THP had been discontinued, in nearly half of the loci reactive to the TIQ. Similarly, salsolinol enhanced significantly the efflux of45Ca2+ in a concentration-dependent manner during the interval of its perfusion within the hippocampal plane of AP 3.0. These results suggest that both THP and salsolinol can affect differentially the kinetics of45Ca2+ efflux and that these differences are contingent on the circumscribed anatomical site of push-pull perfusion. It is envisaged that a part of the neurochemical effects of the two TIQs when acting centrally are mediated by membrane mechanisms involving Ca2+ transport, metabolism or other cellular activity of the cation. 相似文献
4.
The -carboxyl groups of Glu-43() and Glu-22() of hemoglobin-S (HbS), two intermolecular contact residues of deoxy protein, are activated by carbodiimide atp H 6.0. The selectivity of the modification by the two nucleophiles, glycine ethyl ester (GEE) and glucosamine, is distinct. Influence ofN-hydroxysulfosuccinimide, a reagent that rescues carbodiimide-activated carboxyl (O-acyl isourea) as sulfo-NHS ester, on the overall selectivity and efficiency of the coupling of Glu-22() and Glu-43() with nucleophiles has been investigated. Sulfo-NHS increases the extent of coupling of nucleophiles to HbS. The rescuing efficiency of sulfo-NHS(increase in modification) with GEE and galactosamine as nucleophiles is 2.0 and 2.8, respectively. In the presence of sulfo-NHS, the extent of modification of a carboxyl group is a direct reflection of the extent to which it is activated (i.e., the protonation state of the carboxyl group). The modification reaction exhibits very high selectivity for Glu-43() with GEE and galactosamine (GA) in the presence of sulfo-NHS. From the studies of the kinetics of amidation of oxy-HbS at its Glu-43() (i.e., chemical reactivity) as a function of thepH in the region of 5.5–7.5, the apparentpKa of its -carboxyl group has been calculated to be 6.35. Deoxygenation of HbS, nearly doubles the chemical reactivity of Glu-43() of HbS atpH 7.0. It is suggested that the increased hydrophobicity of the microenvironment of Glu-43(), which occurs on deoxygenation of the protein, is reflected as the increased chemical reactivity of the -carboxyl group and could be one of the crucial preludes to the polymerization process. 相似文献
5.
E D Gundelfinger 《FEBS letters》1983,157(1):133-138
The interaction between the three Drosophila DNA-dependent RNA polymerases (EC 2.7.7.6) and the DNA template or the RNA product was investigated by photochemical cross-linking and binding studies, using RNA polymerase subunits immobilized on nitro-cellulose filters. It can be shown that the two largest subunits are responsible for the binding of the enzymes to both template and newly-synthesized RNA. 相似文献
6.
7.
Summary Insitu perfusion of rat liver was performed with a medium containing glucose-cysteine adduct [2-(D-gluco-pentahydroxypentyl) thiazolidine-4-carboxylic acid, glc-cys] and its effect on glutathione (GSH) and ATP levels and bile production was examined. The GSH content in the liver was maintained at the original level during perfusion with 1 mM glc-cys for 2h, while it decreased significantly in the absence of glc-cys. After 4h of perfusion without glc-cys, ATP content and bile production decreased significantly besides the decrease in GSH content, but they were maintained at the original levels with glc-cys. When the perfusion was performed with the liver of rats injected with diethyl maleate (DEM), the GSH level, which was decreased to 6.0% of the control by DEM injection, was restored to 22.6% of the original level by perfusion with 2mM glc-cys for 30 min. Data indicate that glccys is a cysteine prodrug with protective action on the liver. 相似文献
8.
Summary Effect of intraperitoneal administration (5 mmol/kg of body weight) of glucose- cysteine adduct (glc-cys) as a cysteine prodrug in rat tissues was studied. Cysteine levels in liver and kidney increased to 1.08 and 1.98mol per g or ml, respectively, at 2h after the administration. GSH levels did not change substantially. However, when glc-cys was injected to rats treated with diethyl maleate, a GSH-depleting agent, the decreased GSH levels were restored rapidly. The recoveries in liver and kidney were 72% at 1h and 66% at 2h, respectively, after glc-cys administration. Metabolism of glc-cys was assessed by urinary excretion of glc-cys, sulfate and taurine. Average excretion of glc-cys was 2.86mmol/kg/24h after glc-cys administration. Increased excretions of sulfate and taurine were 0.77 and 0.14mmol/kg/24h, respectively. Data show that, although glc-cys excretion was relatively rapid, glc-cys was effectively utilized for GSH synthesis in GSH-depleted tissues. 相似文献
9.
10.
R.M. Maiorino A.J. Gandolfi K. Brendel J.R. Mac Donald I.G. Sipes 《Chemico-biological interactions》1982,38(2):175-188
Numerous xenobiotics are known to be bioactivated and to covalently bind to proteins, but the resulting amino acid adducts (AAAs) are unknown. In this study the AAAs of twelve 14C-labeled aliphatic halides were examined after formation in an in vitro microsomal system. After exhaustive solvent extraction of the precipitated microsomal protein, the AAAs were isolated by Pronase digestion, followed by filtration through a 500 mol. wt. exclusion membrane. The liberated AAAs were applied to a constant flow DC-4A cation exchange column, resolved by stepwise buffer elution, collected and counted for radioactivity. Column recovery for applied radioactivity was 100 ± 4%. Generally, 1–4 different AAAs (defined by eluting radioactivity) were resolved, with each organohalogen displaying a characteristic elution profile. Methyl iodide, trichloroethylene and 1,2-dichloroethylene had a single major AAA while bromotrichloromethane, 1,2-dibromoethane, 1,1,1-trichloroethane, 1,2-dichloroethane, 1,1,2-trichloroethane, 2-bromo-2-chloro-1,1,1-trifluoroethane, chloroform and carbon tetrachloride had up to 4 AAAs or more, indicating combinations of binding site(s) and reactive intermediate(s). The single AAA formed following incubation of methyl iodide with the microsomes was identified as S-methylcysteine. Thus, this method appears capable of resolving binding sites and is the initial isolation step for identifying specific adducts to proteins. 相似文献