scholarly journals Chicken MAR-binding protein ARBP is homologous to rat methyl-CpG-binding protein MeCP2.

1997 ◽  
Vol 17 (9) ◽  
pp. 5656-5666 ◽  
Author(s):  
J M Weitzel ◽  
H Buhrmester ◽  
W H Strätling
Keyword(s):  
Satellite Dna ◽  
Binding Protein ◽  
Repetitive Sequences ◽  
Band Shift ◽  
High Affinity ◽  
Vitro Binding ◽  
Mouse Satellite Dna ◽  
Chicken Lysozyme ◽  
Protein Mecp2

Here, we describe the cloning and further characterization of chicken ARBP, an abundant nuclear protein with a high affinity for MAR/SARs. Surprisingly, ARBP was found to be homologous to the rat protein MeCP2, previously identified as a methyl-CpG-binding protein. A region spanning 125 amino acids in the N-terminal halves is 96.8% identical between chicken ARBP and rat MeCP2. A deletion mutation analysis using Southwestern and band shift assays identified this highly conserved region as the MAR DNA binding domain. Alignment of chicken ARBP with rat and human MeCP2 proteins revealed six trinucleotide amplifications generating up to 34-fold repetitions of a single amino acid. Because MeCP2 was previously localized to pericentromeric heterochromatin in mouse chromosomes, we analyzed the in vitro binding of ARBP to various repetitive sequences. In band shift experiments, ARBP binds to two chicken repetitive sequences as well as to mouse satellite DNA with high affinity similar to that of its binding to chicken lysozyme MAR fragments. In mouse satellite DNA, use of several footprinting techniques characterized two high-affinity binding sites, whose sequences are related to the ARBP binding site consensus in the chicken lysozyme MAR (5'-GGTGT-3'). Band shift experiments indicated that methylation increased in vitro binding of ARBP to mouse satellite DNA two- to fivefold. Our results suggest that ARBP/MeCP2 is a multifunctional protein with roles in loop domain organization of chromatin, the structure of pericentromeric heterochromatin, and DNA methylation.

scholarly journals A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability.

1987 ◽  
Vol 7 (9) ◽  
pp. 3268-3276 ◽  
Author(s):  
A B Sachs ◽  
R W Davis ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Association Constant ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
High Affinity ◽  
Terminal Domain ◽  
Necessary And Sufficient

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

Substrate specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa

1993 ◽  
Vol 39 (7) ◽  
pp. 722-725 ◽  
Author(s):  
John L. Wylie ◽  
Elizabeth A. Worobec
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Glucose Transport ◽  
Transport System ◽  
Binding Protein ◽  
High Affinity ◽  
Glucose Binding ◽  
Glucose Transport System ◽  
Glucose Binding Protein

Specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa was examined. At a concentration of [14C]glucose near the Vmax of the system, inhibition by maltose, galactose, and xylose was detected. This inhibition is similar to that detected in earlier in vivo studies and correlates with the known specificity of OprB, a glucose-specific porin of P. aeruginosa. At a level of [14C]glucose 100 times lower, only unlabelled glucose inhibited uptake to any extent. This matches the known in vitro specificity of the periplasmic glucose binding protein. These findings were used to explain the discrepancy between earlier in vivo and in vitro results reported in the literature.Key words: Pseudomonas aeruginosa, glucose transport, OprB, glucose binding protein.

scholarly journals Znu Is the Predominant Zinc Importer in Yersinia pestis during In Vitro Growth but Is Not Essential for Virulence

2010 ◽  
Vol 78 (12) ◽  
pp. 5163-5177 ◽  
Author(s):  
Daniel C. Desrosiers ◽  
Scott W. Bearden ◽  
Ildefonso Mier ◽  
Jennifer Abney ◽  
James T. Paulley ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Binding Protein ◽  
Bacterial Pathogens ◽  
Titration Calorimetry ◽  
Growth Defect ◽  
Uptake System ◽  
High Affinity ◽  
Zn Uptake ◽  
Low Concentrations

ABSTRACT Little is known about Zn homeostasis in Yersinia pestis, the plague bacillus. The Znu ABC transporter is essential for zinc (Zn) uptake and virulence in a number of bacterial pathogens. Bioinformatics analysis identified ZnuABC as the only apparent high-affinity Zn uptake system in Y. pestis. Mutation of znuACB caused a growth defect in Chelex-100-treated PMH2 growth medium, which was alleviated by supplementation with submicromolar concentrations of Zn. Use of transcriptional reporters confirmed that Zur mediated Zn-dependent repression and that it can repress gene expression in response to Zn even in the absence of Znu. Virulence testing in mouse models of bubonic and pneumonic plague found only a modest increase in survival in low-dose infections by the znuACB mutant. Previous studies of cluster 9 (C9) transporters suggested that Yfe, a well-characterized C9 importer for manganese (Mn) and iron in Y. pestis, might function as a second, high-affinity Zn uptake system. Isothermal titration calorimetry revealed that YfeA, the solute-binding protein component of Yfe, binds Mn and Zn with comparably high affinities (dissociation constants of 17.8 ± 4.4 nM and 6.6 ± 1.2 nM, respectively), although the complete Yfe transporter could not compensate for the loss of Znu in in vitro growth studies. Unexpectedly, overexpression of Yfe interfered with the znu mutant's ability to grow in low concentrations of Zn, while excess Zn interfered with the ability of Yfe to import iron at low concentrations; these results suggest that YfeA can bind Zn in the bacterial cell but that Yfe is incompetent for transport of the metal. In addition to Yfe, we have now eliminated MntH, FetMP, Efe, Feo, a substrate-binding protein, and a putative nickel transporter as the unidentified, secondary Zn transporter in Y. pestis. Unlike other bacterial pathogens, Y. pestis does not require Znu for high-level infectivity and virulence; instead, it appears to possess a novel class of transporter, which can satisfy the bacterium's Zn requirements under in vivo metal-limiting conditions. Our studies also underscore the need for bacterial cells to balance binding and transporter specificities within the periplasm in order to maintain transition metal homeostasis.

scholarly journals The neuronal RNA binding protein Nova-1 recognizes specific RNA targets in vitro and in vivo.

1997 ◽  
Vol 17 (6) ◽  
pp. 3194-3201 ◽  
Author(s):  
R J Buckanovich ◽  
R B Darnell
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Motor Dysfunction ◽  
High Affinity ◽  
Loop Region ◽  
Nuclear Rna ◽  
Rna Ligands

Nova-1, an autoantigen in paraneoplastic opsoclonus myoclonus ataxia (POMA), a disorder associated with breast cancer and motor dysfunction, is a neuron-specific nuclear RNA binding protein. We have identified in vivo Nova-1 RNA ligands by combining affinity-elution-based RNA selection with protein-RNA immunoprecipitation. Starting with a pool of approximately 10(15) random 52-mer RNAs, we identified long stem-loop RNA ligands that bind to Nova-1 with high affinity (Kd of approximately 2 nM). The loop region of these RNAs harbors a approximately 15-bp pyrimidine-rich element [UCAU(N)(0-2)]3 which is essential for Nova-1 binding. Mutagenesis studies defined the third KH domain of Nova-1 and the [UCAU(N)(0-2)]3 element as necessary for in vitro binding. Consensus [UCAU (N)(0-2)], elements were identified in two neuronal pre-mRNAs, one encoding the inhibitory glycine receptor alpha2 (GlyR alpha2) and a second encoding Nova-1 itself. Nova-1 protein binds these RNAs with high affinity and specificity in vitro, and this binding can be blocked by POMA antisera. Moreover, both Nova-1 and GlyR alpha2 pre-mRNAs specifically coimmunoprecipitated with Nova-1 protein from brain extracts. Thus, Nova-1 functions as a sequence-specific nuclear RNA binding protein in vivo; disruption of the specific interaction between Nova-1 and GlyR alpha2 pre-mRNA may underlie the motor dysfunction seen in POMA.

A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability

1987 ◽  
Vol 7 (9) ◽  
pp. 3268-3276 ◽  
Author(s):  
A B Sachs ◽  
R W Davis ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Association Constant ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
High Affinity ◽  
Terminal Domain ◽  
Necessary And Sufficient

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

Androgen and estrogen binding in rat skeletal and perineal muscles

1976 ◽  
Vol 54 (1) ◽  
pp. 50-55 ◽  
Author(s):  
Jean Y. Dubé ◽  
Renée Lesage ◽  
Roland R. Tremblay
Keyword(s):  
Gel Filtration ◽  
Levator Ani ◽  
Female Rats ◽  
High Affinity ◽  
Male And Female Rats ◽  
Vitro Binding ◽  
Bulbocavernosus Muscle ◽  
Muscle Complex ◽  
Estrogen Binding

Specific in vitro binding of [3H]testosterone (T), 5α-[3H]dihydrotestosterone (DHT), and [3H]estradiol (E2) was demonstrated in the 30 000 × g supernatant (cytosol) of thigh muscles (TM) and of the levator ani – bulbocavernosus muscle complex (LA–BC) by gel filtration through Sephadex G-25 columns. In TM cytosol, T and E2 [are bound with high affinity (Ka = 1.1 × 109 M−1 and 2.3 × 109 M−1, respectively) whereas DHT binding is of lower affinity (Ka = 5.0 × 107 M−1).] In LA–BC cytosol, T, E2, and DHT are bound with high affinity (Ka = 1.9 × 109 M−1, 0.3 × 109 M−1 and 0.5 × 109 M−1, respectively). Competition experiments suggest that the binding of the three hormones (T, E2, and DHT) is due to different proteins. In addition to TM and LA–BC, T and E2 binding was found in other muscles of male and female rats, including gastrocnemius, the pectoralis, diaphragm, and heart.

Role of high-affinity folate-binding protein in the plasma distribution of tetrahydrofolate in pigs

1996 ◽  
Vol 270 (1) ◽  
pp. R105-R110 ◽  
Author(s):  
K. Sasaki ◽  
M. Natsuhori ◽  
M. Shimoda ◽  
Y. Saima ◽  
E. Kokue
Keyword(s):  
Protein Binding ◽  
Binding Capacity ◽  
Binding Protein ◽  
Folate Binding Protein ◽  
High Affinity ◽  
Plasma Distribution ◽  
The Stability ◽  
Plasma Ultrafiltrate

Stability and protein-binding properties of tetrahydrofolate (THF) in pig plasma were studied in vitro. THF in plasma was stable for more than 120 min when it existed in a bound form, whereas THF both in plasma ultrafiltrate and in plasma ultrafiltrate plus porcine albumin was degraded rapidly and disappeared soon after its addition. These results suggest that high-affinity folate-binding protein (HFBP) is related to the stability of THF. THF-protein binding kinetic analysis showed that porcine plasma had HFBP and low-affinity binding protein (albumin) for THF. Dissociation constant and maximal binding capacity of HFBP were calculated to be 0.4 and 70 nM, respectively, indicating that > 98% of endogenous plasma THF existed in bound form with HFBP. Porcine albumin was not essentially a protein that binds and protects endogenous THF from degradation. We conclude that most endogenous THF binds to HFBP and only the unbound form of THF is rapidly degraded in pig plasma. HFBP protects THF from degradation and allows THF to exist stably in pig plasma. In addition, HFBP may govern the species specificity of plasma folate distribution in pigs.

scholarly journals An RNA-binding protein specifically interacts with a functionally important domain of the downstream element of the simian virus 40 late polyadenylation signal.

1991 ◽  
Vol 11 (10) ◽  
pp. 5312-5320 ◽  
Author(s):  
Z W Qian ◽  
J Wilusz
Keyword(s):  
Binding Site ◽  
Rna Binding ◽  
Specific Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Band Shift

We have identified an RNA-binding protein which interacts with the downstream element of the simian virus 40 late polyadenylation signal in a sequence-specific manner. A partially purified 50-kDa protein, which we have named DSEF-1, retains RNA-binding specificity as assayed by band shift and UV cross-linking analyses. RNA footprinting assays, using end-labeled RNA ladder fragments in conjunction with native gel electrophoresis, have identified the DSEF-1 binding site as 5'-GGGGGAGGUGUGGG-3'. This 14-base sequence serves as an efficient DSEF-1 binding site when placed within a GEM4 polylinker-derived RNA. Finally, the DSEF-1 binding site restored efficient in vitro 3' end processing to derivatives of the simian virus 40 late polyadenylation signal in which it substituted for the entire downstream region. DSEF-1, therefore, may be a sequence-specific binding factor which regulates the efficiency of polyadenylation site usage.

Synthesis and biological activity of histogranin and related peptides

1995 ◽  
Vol 73 (2) ◽  
pp. 209-214 ◽  
Author(s):  
Jyoti A. Prasad ◽  
Vijay K. Shukla ◽  
Simon Lemaire
Keyword(s):  
Nmda Receptor Antagonist ◽  
The Other ◽  
Peptide Fragments ◽  
Antagonist Activity ◽  
Brain Membrane ◽  
High Affinity ◽  
Aspartate Receptor ◽  
Vitro Binding

Histogranin (HN) was first isolated from bovine adrenal medulla and shown to be a pentadecapeptide displaying N-methyl-D-aspartate (NMDA) receptor antagonist activity. To determine the active pharmacophore of HN, fragments of the peptide were synthesized and their structure–activity relationships studied by measuring their ability to displace the binding of [125I][Ser1]HN to rat brain membrane preparations and to block NMDA-induced convulsions in mice. In the binding assay, only the full length peptide HN and HN(1–10) displayed a high affinity (Ki of 72 and 162 nM, respectively). All other tested fragments with deletions at the N- and (or) C-terminals of the molecule showed large (16- to 2500-fold) decreases in potency. The least active peptide fragment tested was HN(6–10) (Ki of 164 μM). In vivo, HN and HN(2–15) (100 nmol; i.c.v.) produced 94 and 40% protection against NMDA-induced convulsions in mice, respectively. None of the other peptide fragments displayed significant anticonvulsant activity. The protective activity of HN (60 and 100 nmol) was markedly antagonized by coadministration of HN(1–10) (100 nmol). The results indicate that the in vivo anti-NMDA and in vitro binding activities of HN and related peptides, with the exception of HN(1–10), depend upon the integrity of the molecule. On the other hand, the high affinity of HN(1–10) for HN binding sites correlates well with its antagonist effects towards the activity of the parent peptide.Key words: histogranin, peptide receptor, N-methyl-D-aspartate receptor, anticonvulsant.

Sign in / Sign up

Export Citation Format

Share Document