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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

rad5 (rev2) mutants of Saccharomyces cerevisiae are sensitive to UV light and other DNA-damaging agents, and RAD5 is in the RAD6 epistasis group of DNA repair genes. To unambiguously define the function of RAD5, we have cloned the RAD5 gene, determined the effects of the rad5 deletion mutation on DNA repair, DNA damage-induced mutagenesis, and other cellular processes, and analyzed the sequence of RAD5-encoded protein. Our genetic studies indicate that RAD5 functions primarily with RAD18 in error-free postreplication repair. We also show that RAD5 affects the rate of instability of poly(GT) repeat sequences. Genomic poly(GT) sequences normally change length at a rate of about 10(-4); this rate is approximately 10-fold lower in the rad5 deletion mutant than in the corresponding isogenic wild-type strain. RAD5 encodes a protein of 1,169 amino acids of M(r) 134,000, and it contains several interesting sequence motifs. All seven conserved domains found associated with DNA helicases are present in RAD5. RAD5 also contains a cysteine-rich sequence motif that resembles the corresponding sequences found in 11 other proteins, including those encoded by the DNA repair gene RAD18 and the RAG1 gene required for immunoglobin gene arrangement. A leucine zipper motif preceded by a basic region is also present in RAD5. The cysteine-rich region may coordinate the binding of zinc; this region and the basic segment might constitute distinct DNA-binding domains in RAD5. Possible roles of RAD5 putative ATPase/DNA helicase activity in DNA repair and in the maintenance of wild-type rates of instability of simple repetitive sequences are discussed.
Mol Cell Biol 1992 Sep
PMID:Saccharomyces cerevisiae RAD5-encoded DNA repair protein contains DNA helicase and zinc-binding sequence motifs and affects the stability of simple repetitive sequences in the genome. 132 6

Neuronal cell lines provide a source of pure populations of neurons and allow the properties of many neurotransmitter receptors to be studied. However, none of these cells have been reported to express functional gamma-aminobutyric acid (GABA)A receptors. Indeed, there have been no reports of cell lines expressing functional amino acid receptors. Using biochemical and electrophysiological techniques, we have identified a neuronal cell line expressing functional GABAA receptors. Membranes from immortalized hypothalamic (GT1-7) neurons bound [3H]muscimol but not [3H]flunitrazepam. GABA-activated chloride currents, recorded from GT1-7 cells, were blocked by bicuculline and Zn2+ but were insensitive to diazepam. These results suggest that GABAA receptors on GT1-7 cells lack gamma subunits. The neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one and pentobarbital both modulated GABAA receptors in these cells. Polymerase chain reaction analysis of the cells revealed the presence of mRNAs encoding alpha 1, beta 1, and beta 3 polypeptides. GT1-7 cells provide a useful model system for studying the regulation of GABAA receptor polypeptide expression.
Mol Pharmacol 1992 Aug
PMID:Immortalized hypothalamic GT1-7 neurons express functional gamma-aminobutyric acid type A receptors. 132 30

In the preceding paper it was shown that an isoform of serum albumin (ASA; active serum albumin) causes a rapid retraction of neurites and increases intracellular content of Ins1,4,5P3 in PC12 cells. Here we examined whether ASA's effects in nerve growth factor-differentiated PC12 cells were mediated through the Ins1,4,5P3/Ca2+ second messenger pathway by monitoring intracellular Ca2+ (Ca2+i) with Fura2. It was found that ASA caused a dose-dependent increase in Ca2+i. In Ca(2+)-free medium, the increase in Ca2+i elicited by ASA was smaller, but the rise in Ins1,4,5P3 content was not appreciably changed. The small Ca2+i increase seen in Ca(2+)-free medium was probably due to the release of Ca2+ from Ins1,4,5P3-sensitive intracellular stores. In Ca(2+)-containing medium the Ca2+ transient induced by ASA was not affected by organic Ca2+ channel blockers, but decreased when Co2+, Mn2+ or Zn2+ were present in the extracellular medium. The effect of other ligands, such as carbachol and bradykinin, whose receptors are coupled to the phosphoinositide system was also investigated. Carbachol at concentrations from 2 to 200 microM, and bradykinin at a concentration of 2 microM did not cause neurite retraction, whereas 200 microM bradykinin caused an approximately 40% decrease in neurite length. Thapsigargin, a Ca(2+)-ATPase inhibitor, caused a sustained elevation of Ca2+i and retraction of neurites, whereas depolarization of the cells by high K+ gave only a transient elevation of Ca2+i, and no neurite retraction. Therefore, a sustained elevation in Ca2+i might be a sufficient trigger to induce neurite retraction in differentiated PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Brain Res Mol Brain Res 1992 Aug
PMID:The effect of active serum albumin on PC12 cells: II. Intracellular Ca2+ transients and their role in neurite retraction. 132 93

Exposure of mouse colliculi neurons to selective 5-hydroxytryptamine (5-HT)4 agonists was accompanied by a rapid desensitization of the receptor-stimulated adenylyl cyclase response. Half-maximal desensitization occurred after 2 min. Only exposure of neurons to selective 5-HT4 agonists led to a potent desensitization of the 5-HT4-mediated response. Neurons exposed to other agents, like isoproterenol, vasoactive intestinal peptide, or forskolin, that increase cAMP levels did not undergo any desensitization of 5-HT4 receptors. Activation of protein kinase A with either 8-bromo-cAMP or dibutyryl-cAMP or application of inhibitors of protein kinase A-dependent phosphorylation did not change the rate of 5-HT4-induced desensitization. No shift to lower potency of 5-HT4 agonists in the concentration-response curve was observed. These results suggest that 5-HT4 receptor agonists induced homologous but not cAMP-mediated heterologous desensitization. A good correlation was found between the affinities of nine 5-HT4 agonists and their abilities to desensitize the adenylyl cyclase response. This may indicate that homologous desensitization is a function of the mean occupancy time of the receptors by agonists. When permeabilized neurons were loaded with heparin, an inhibitor of the beta-adrenergic receptor kinase (beta ARK), 5-HT4 receptor desensitization was reduced by 30-40%. Interestingly, Zn2+, an other inhibitor of beta ARK, totally prevented 5-HT4-induced desensitization. Pretreatment of neurons with concanavalin A, reported to inhibit sequestration of beta-adrenergic receptors from the cell surface, reduced the desensitization process by 70%. These data suggest that both sequestration and phosphorylation by beta ARK, or another specific agonist-dependent receptor kinase, are involved in homologous desensitization of 5-HT4 receptors coupled to adenylyl cyclase.
Mol Pharmacol 1992 Nov
PMID:Characterization of homologous 5-hydroxytryptamine4 receptor desensitization in colliculi neurons. 133 63

The rate of the redox reaction between porcine MbO2 and ferri-Cyt c at different ionic strengths in the pH range 5-8 has been studied. At low ionic strength (I = 0-0.1) the pH dependence curve was found to have a sigmoid shape with pKeff approximately 5.7, implying the effect of ionization of His-119(GH1) at the "active site" of myoglobin on the kinetics of the process. In this range of ionic strengths the rate of the reaction decreases sharply. The slope of the curve in the coordinates of IgKexp versus square root of I/1 + square root of I varies depending on pH. It is greater at pH less than or equal to 6 and smaller at pH 7.5, which is due to deprotonation of His(GH1). At high ionic strength (I greater than 0.1) the rate of electron transfer is negligible, independent of pH and does not practically change as I increases from 0.1 to 1. It is shown that the local electrostatic interactions play a decisive role in the formation of an efficient electron-transfer complex between Mb and Cyt c. The binding of the zinc ion to His(GH1) was found to inhibit the electron transfer at I = 0.01, similarly to what occurs at a high ionic strength, though the "reactive" charges of the proteins are not screened and the positive charge at His(GH1) is retained. This suggests that His(GH1) is directly involved in the mechanism of electron transfer from Mb to Cyt c. The data obtained are compared with earlier data on the effect of pH, ionic strength and zinc ions on the reaction between MbO2 from sperm whale and Cyt c. To explain the higher efficiency of pig MbO2 as electron donor, the electrostatic and steric properties of both myoglobins have been analyzed.
Mol Biol (Mosk)
PMID:[Study of electron transport in heme proteins. X. Effect of pH, ionic strength, and zinc ions and the rate of ferricytochrome c reduction by oxymyoglobin from swine heart]. 133 70

A set of mutants of transcription factor IIIA (TFIIIA) have been prepared in which successive zinc-finger domains have been deleted from the carboxyl terminus of the protein. These have been analyzed by hydroxyl radical footprinting to map the location of contacts to DNA by individual zinc-finger domains of TFIIIA. The results suggest that the nine zinc fingers of TFIIIA are organized into three DNA-binding domains of three fingers each. The spatial relationship between zinc-finger contacts to 5S DNA suggests that the two domains which interact with either end of the DNA-binding site of TFIIIA (fingers 1-3 and 7-9) have a compact conformation, similar to that exemplified by the zif268 cocrystal structure [Pavletich, N.P., & Pabo, C.O. (1991) Science 252, 809-817]. However, the central domain (fingers 4-6) has a much more extended conformation, following a path nearly parallel to the helix axis and contacting over 20 base pairs of DNA in the center of the binding site of TFIIIA. These results strongly support two recently proposed and radically new models for the TFIIIA/5S DNA complex [Hayes, J.J., & Tullius, T.D. (1992) J. Mol. Biol. 227, 407-417; Clemens, K.R., Liao, X., Wolf, V., Wright, P.E., & Gottesfeld, J.M. (1992) Proc. Natl. Acad. Sci. U.S.A. (in press)].
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PMID:Locations of contacts between individual zinc fingers of Xenopus laevis transcription factor IIIA and the internal control region of a 5S RNA gene. 133 65

There are at least three isozymes (C alpha, C beta, and C gamma) of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase (PKA) (Beebe, S., Oyen, O., Sandberg, M., Froysa, A., Hansson, V., and Jahnsen, T. (1990) Mol. Endocrinol. 4, 465-475). To compare the C gamma and C alpha isozymes, the respective cDNAs were expressed in permanently transformed Kin-8 PKA-deficient Y1 adrenal cells using the mouse metallothionein promoter. The recombinant C subunits were characterized as immunoreactive, zinc-inducible, cAMP-dependent kinase activities. In contrast to C alpha, histone was a better substrate than Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) for C gamma. Furthermore, C gamma histone kinase activity was not inhibited by the protein kinase inhibitor peptide (5-24 amide), which has been widely used as a PKA-specific inhibitor. The major C gamma peak (type I) eluted from DEAE-Sepharose at a higher NaCl concentration (120 mM) than the C alpha type I eluted (70 mM). C gamma and C alpha type II eluted between 220 and 240 mM NaCl. C gamma required higher concentrations of cAMP than C alpha did for dissociation from the mutant type I holoenzyme. These differences provided a basis for the separation of the mutant RI-associated isozymes on DEAE-Sepharose. Both C alpha (41-42 kDa) and C gamma (39-40 kDa) were identified by a C subunit antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. Zinc induced the PKA-mediated rounding phenotype in C gamma and C alpha clones, thereby restoring the cells to the parent Y1 adrenal cell phenotype. Collectively, these data indicate that C gamma is an active PKA C subunit but suggest that C gamma and C alpha have different protein and peptide recognition determinants.
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PMID:The C gamma subunit is a unique isozyme of the cAMP-dependent protein kinase. 133 96

Analysis of a T-cell antigen receptor (TCR) alpha promoter from a variable gene segment (V) revealed a critical GT box element which is also found in upstream regions of several V alpha genes, TCR enhancer, and regulatory elements of other genes. This element is necessary for TCR gene expression and binds several proteins. These GT box-binding proteins were identified as members of a novel Sp1 multigene family. Two of them, which we term Sp2 and Sp3, were cloned. Sp2 and Sp3 contain zinc fingers and transactivation domains similar to those of Sp1. Like Sp1, Sp2 and Sp3 are expressed ubiquitously, and their in vitro-translated products bind to the GT box in TCR V alpha promoters. Sp3, in particular, also binds to the Sp1 consensus sequence GC box and has binding activity similar to that of Sp1. As the GT box has also previously been shown to play a role in gene regulation of other genes, these newly isolated Sp2 and Sp3 proteins might regulate expression not only of the TCR gene but of other genes as well.
Mol Cell Biol 1992 Oct
PMID:Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression. 134

The assembly of the insulin hexamer brings the six B13 glutamate side-chains at the centre into close proximity. Their mutual repulsion is unfavourable and zinc co-ordination to B10 histidine is necessary to stabilize the well known zinc-containing hexamers. Since B13 is always a carboxylic acid in all known sequences of hexamer forming insulins, it is likely to be important in the hormone's biology. The mutation of B13 Glu-->Gln leads to a stable zinc-free hexamer with somewhat reduced potency. The structures of the zinc-free B13 Gln hexamer and the 2Zn B13 insulin hexamer have been determined by X-ray analysis and refined with 2.5 A and 2.0 A diffraction data, respectively. Comparisons show that in 2Zn B13 Gln insulin, the hexamer structure (T6) is very like that of the native hormone. On the other hand, the zinc-free hexamer assumes a quaternary structure (T3/R3) seen in the native 4Zn insulin hexamer, and normally associated only with high chloride ion concentrations in the medium. The crystal structures show the B13 Gln side-chains only contact water in contrast to the B13 glutamate in 2Zn insulin. The solvation of the B13 Gln may be associated with this residue favouring helix at B1 to B8. The low potency of the B13 Gln insulin also suggests the residue influences the hormone's conformation.
J Mol Biol 1992 Dec 20
PMID:Role of B13 Glu in insulin assembly. The hexamer structure of recombinant mutant (B13 Glu-->Gln) insulin. 136 49

[3H]Dextrorphan recognition sites were characterized in rat brain membranes. The pharmacological profile and regional distribution of [3H]dextrorphan binding sites appear to distinguish these sites from those labeled either by [3H]dextromethorphan or by putative sigma receptor radioligands. Data from thoroughly washed forebrain membranes suggest that [3H]dextrorphan predominantly labels a high affinity site defined by the activated state of the N-methyl-D-aspartate (NMDA) receptor-channel complex. Regulation of [3H]dextrorphan binding by specific modulators of NMDA receptor function suggests that [3H]dextrorphan binding is predominantly localized to a domain of the receptor-channel complex also recognized by the prototypical noncompetitive antagonist radioligands (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) and [3H]1-[1-(2-thienyl)cyclohexyl]piperidine (TCP). The critical relationship between [3H]dextrorphan binding and activation of the NMDA receptor-complex is suggested by the profound dependence of [3H]dextrorphan binding on glutamate in well washed membranes. Basal specific [3H]dextrorphan binding is nearly totally suppressed by the specific competitive NMDA antagonist D(-)-2-amino-5-phosphonopentanoic acid (D-AP5), in a glutamate- but not glycine-surmountable manner. Glutamate and glycine each stimulate [3H]dextrorphan binding in a concentration-dependent manner, effecting maximal increases from control of up to 30- and 14-fold, respectively. The NMDA receptor specificity of the modulation of [3H]dextrorphan binding by glutamate and glycine is indicated by the sensitivity of their effects to competitive antagonism by D-AP5 and 3-amino-1-hydroxy-2-pyrrolidone (HA-966), respectively, and by the accordant rank orders of potency of glycine analogs as modulators of [3H]dextrorphan binding and as ligands at the strychnine-insensitive glycine site. The divalent cations Mg2+ and Zn2+ and the polyamines spermine and spermidine regulate [3H]dextrorphan binding in a manner consistent with radioligand interaction at the noncompetitive NMDA antagonist domain. Mg2+ and spermidine regulate [3H]dextrorphan binding biphasically in well washed forebrain membranes, whereas Zn2+ monotonically inhibits [3H]dextrorphan binding. Mg2+ and spermidine regulate [3H]dextrorphan binding with qualitative similarity and in a contrasting fashion to their regulation of [3H]MK-801 and [3H]TCP binding. First, spermidine and Mg2+ are significantly more potent modulators of [3H]dextrorphan binding than of [3H]MK-801 and [3H]TCP binding in well washed membranes; second, whereas the potencies of spermidine and Mg2+ as modulators of [3H]MK-801 and [3H]TCP binding are significantly increased by glutamate and glycine in well washed membranes, their potencies as regulators of [3H]dextrorphan binding appear to be unaffected by glutamate and glycine.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1992 Jan
PMID:High affinity [3H]dextrorphan binding in rat brain is localized to a noncompetitive antagonist site of the activated N-methyl-D-aspartate receptor-cation channel. 137 Jul 4


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