No Functional Difference of the Two Iron-Binding Sites of Human Transferrin in Vitro and in Vivo

1. According to the Fletcher-Huehns hypothesis there exists a functional difference between the two iron-binding sites of transferrin. 2. The aim of the study presented was to evaluate this hypothesis in a homogeneous system, with human bone marrow cells and pure human monoferric transferrins A and B. 3. For this reason normal human bone marrow cells were incubated with human monoferric transferrin. The monoferric transferrins A and B were obtained by selective labelling at different pH of apotransferrin followed by preparative isoelectric focusing in granulated gels. The uptake of iron by the cell suspensions from monoferric transferrins A and B was equal. 4. In a heterogeneous but more active system for the removal of iron from human transferrin in vitro the two human monoferric transferrins did not show any significant functional differences. 5. No support for the Fletcher-Huehns hypothesis could be obtained.

Download Full-text

The behavior of transferrin iron in the rat

Blood ◽

10.1182/blood.v57.2.218.218 ◽

1981 ◽

Vol 57 (2) ◽

pp. 218-228 ◽

Cited By ~ 16

Author(s):

H Huebers ◽

W Bauer ◽

E Huebers ◽

E Csiba ◽

C Finch

Keyword(s):

Binding Sites ◽

Specific Binding ◽

Ferrous Ammonium Sulfate ◽

Iron Binding ◽

Body Tissues ◽

Iron Deficient ◽

Diferric Transferrin ◽

Iron Exchange

Abstract The behavior of rat transferrin has been investigated employing acrylamide gel electrophoresis and isoelectric focusing. In vitro trace labeling with iron chelates at 30 min was 93%-98% effective, whereas binding by simple ferric salts was reduced to 71%-76%. Complete and specific binding of 59FeSO4 by the iron binding sites of transferrin was demonstrated after in vitro or in vivo addition of ferrous ammonium sulfate in pH 2 saline up to the point of iron saturation. In vitro the radioriron transferrin complex in plasma was stable and its iron had a negligible exchange with other transferrin binding sites over several hours. The distribution of radioiron added in vitro or through absorption was shown to be random between the binding sites of slow and fast transferrin molecule. Iron distribution among body tissues was similar for mono- and diferric transferrin iron and was not affected by the site distribution of iron on the transferrin molecule. The only important aspect of transferrin iron binding was the more rapid tissue uptake of iron in the diferric form was compared to monoferric transferrin. Additional in vivo effects on internal iron exchange were produced by changes in the iron balance of the animal. In the iron loaded animal, monoferric transferrin injected into the plasma was rapidly loaded by iron from tissue and thereby converted to diferric transferrin. Injection of diferric transferrin in the iron deficient animal was associated with a rapid disappearance from circulation of the original complex and a subsequent appearance of monoferric transferrin as a result of iron returning from tissues. These observations support the concept that plasma iron behaves as a single pool except that diferric iron exchange occurs at a more rapid rate than dose monoferric iron exchange.

Download Full-text

The behavior of transferrin iron in the rat

Blood ◽

10.1182/blood.v57.2.218.bloodjournal572218 ◽

1981 ◽

Vol 57 (2) ◽

pp. 218-228

Author(s):

H Huebers ◽

W Bauer ◽

E Huebers ◽

E Csiba ◽

C Finch

Keyword(s):

Binding Sites ◽

Specific Binding ◽

Ferrous Ammonium Sulfate ◽

Iron Binding ◽

Body Tissues ◽

Iron Deficient ◽

Diferric Transferrin ◽

Iron Exchange

The behavior of rat transferrin has been investigated employing acrylamide gel electrophoresis and isoelectric focusing. In vitro trace labeling with iron chelates at 30 min was 93%-98% effective, whereas binding by simple ferric salts was reduced to 71%-76%. Complete and specific binding of 59FeSO4 by the iron binding sites of transferrin was demonstrated after in vitro or in vivo addition of ferrous ammonium sulfate in pH 2 saline up to the point of iron saturation. In vitro the radioriron transferrin complex in plasma was stable and its iron had a negligible exchange with other transferrin binding sites over several hours. The distribution of radioiron added in vitro or through absorption was shown to be random between the binding sites of slow and fast transferrin molecule. Iron distribution among body tissues was similar for mono- and diferric transferrin iron and was not affected by the site distribution of iron on the transferrin molecule. The only important aspect of transferrin iron binding was the more rapid tissue uptake of iron in the diferric form was compared to monoferric transferrin. Additional in vivo effects on internal iron exchange were produced by changes in the iron balance of the animal. In the iron loaded animal, monoferric transferrin injected into the plasma was rapidly loaded by iron from tissue and thereby converted to diferric transferrin. Injection of diferric transferrin in the iron deficient animal was associated with a rapid disappearance from circulation of the original complex and a subsequent appearance of monoferric transferrin as a result of iron returning from tissues. These observations support the concept that plasma iron behaves as a single pool except that diferric iron exchange occurs at a more rapid rate than dose monoferric iron exchange.

Download Full-text

GAGA Factor Isoforms Have Distinct but Overlapping Functions In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.21.24.8565-8574.2001 ◽

2001 ◽

Vol 21 (24) ◽

pp. 8565-8574 ◽

Cited By ~ 20

Author(s):

Anthony J. Greenberg ◽

Paul Schedl

Keyword(s):

Fusion Protein ◽

Transcriptional Activation ◽

Functional Difference ◽

Wild Type ◽

Gaga Factor ◽

Phenotypic Analysis ◽

Alternatively Spliced

ABSTRACT The Drosophila melanogaster GAGA factor (encoded by the Trithorax-like [Trl] gene) is required for correct chromatin architecture at diverse chromosomal sites. The Trl gene encodes two alternatively spliced isoforms of the GAGA factor (GAGA-519 and GAGA-581) that are identical except for the length and sequence of the C-terminal glutamine-rich (Q) domain. In vitro and tissue culture experiments failed to find any functional difference between the two isoforms. We made a set of transgenes that constitutively express cDNAs coding for either of the isoforms with the goal of elucidating their roles in vivo. Phenotypic analysis of the transgenes in Trl mutant background led us to the conclusion that GAGA-519 and GAGA-581 perform different, albeit largely overlapping, functions. We also expressed a fusion protein with LacZ disrupting the Q domain of GAGA-519. This LacZ fusion protein compensated for the loss of wild-type GAGA factor to a surprisingly large extent. This suggests that the Q domain either is not required for the essential functions performed by the GAGA protein or is exclusively used for tetramer formation. These results are inconsistent with a major role of the Q domain in chromatin remodeling or transcriptional activation. We also found that GAGA-LacZ was able to associate with sites not normally occupied by the GAGA factor, pointing to a role of the Q domain in binding site choice in vivo.

Download Full-text

Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2091-2103.1993 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2091-2103

Author(s):

S Türkel ◽

P J Farabaugh

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Activation ◽

Binding Sites ◽

Transcriptional Repression ◽

Physiological Control ◽

Reading Frame ◽

Negative Region ◽

Activation Site

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

Download Full-text

Progastrin1–80stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00351.2002 ◽

2003 ◽

Vol 284 (2) ◽

pp. G328-G339 ◽

Cited By ~ 41

Author(s):

P. Singh ◽

X. Lu ◽

S. Cobb ◽

B. T. Miller ◽

N. Tarasova ◽

...

Keyword(s):

Epithelial Cells ◽

Binding Sites ◽

Intestinal Epithelial Cells ◽

Full Length ◽

Intestinal Epithelial ◽

Growth Effects ◽

High Affinity ◽

Significant Difference

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG1–80) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1–1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK1and CCK2receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK1-R and CCK2-R on IEC cells. High-affinity ( Kd= 0.5–1.0 nM) binding sites for125I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG ≥ COOH-terminally extended G17 ≥ G-Gly > G17 > *CCK-8 (* significant difference; P< 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

Download Full-text

Temperature-dependence of the DnaA–DNA interaction and its effect on the autoregulation of dnaA expression

Biochemical Journal ◽

10.1042/bj20120876 ◽

2012 ◽

Vol 449 (2) ◽

pp. 333-341 ◽

Cited By ~ 6

Author(s):

Chiara Saggioro ◽

Anne Olliver ◽

Bianca Sclavi

Keyword(s):

Temperature Dependence ◽

Binding Sites ◽

Dna Interaction ◽

Bacterial Dna ◽

Dnaa Protein ◽

High Affinity ◽

Key Factor

The DnaA protein is a key factor for the regulation of the timing and synchrony of initiation of bacterial DNA replication. The transcription of the dnaA gene in Escherichia coli is regulated by two promoters, dnaAP1 and dnaAP2. The region between these two promoters contains several DnaA-binding sites that have been shown to play an important role in the negative auto-regulation of dnaA expression. The results obtained in the present study using an in vitro and in vivo quantitative analysis of the effect of mutations to the high-affinity DnaA sites reveal an additional effect of positive autoregulation. We investigated the role of transcription autoregulation in the change of dnaA expression as a function of temperature. While negative auto-regulation is lost at dnaAP1, the effects of both positive and negative autoregulation are maintained at the dnaAP2 promoter upon lowering the growth temperature. These observations can be explained by the results obtained in vitro showing a difference in the temperature-dependence of DnaA–ATP binding to its high- and low-affinity sites, resulting in a decrease in DnaA–ATP oligomerization at lower temperatures. The results of the present study underline the importance of the role for autoregulation of gene expression in the cellular adaptation to different growth temperatures.

Download Full-text

SSMART: Sequence-structure motif identification for RNA-binding proteins

10.1101/287953 ◽

2018 ◽

Cited By ~ 2

Author(s):

Alina Munteanu ◽

Neelanjan Mukherjee ◽

Uwe Ohler

Keyword(s):

Binding Sites ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Sequence Motif ◽

Primary Sequence ◽

Sequence Structure ◽

Rna Targets

AbstractMotivationRNA-binding proteins (RBPs) regulate every aspect of RNA metabolism and function. There are hundreds of RBPs encoded in the eukaryotic genomes, and each recognize its RNA targets through a specific mixture of RNA sequence and structure properties. For most RBPs, however, only a primary sequence motif has been determined, while the structure of the binding sites is uncharacterized.ResultsWe developed SSMART, an RNA motif finder that simultaneously models the primary sequence and the structural properties of the RNA targets sites. The sequence-structure motifs are represented as consensus strings over a degenerate alphabet, extending the IUPAC codes for nucleotides to account for secondary structure preferences. Evaluation on synthetic data showed that SSMART is able to recover both sequence and structure motifs implanted into 3‘UTR-like sequences, for various degrees of structured/unstructured binding sites. In addition, we successfully used SSMART on high-throughput in vivo and in vitro data, showing that we not only recover the known sequence motif, but also gain insight into the structural preferences of the RBP.AvailabilitySSMART is freely available at https://ohlerlab.mdc-berlin.de/software/SSMART_137/[email protected]

Download Full-text

The closely related Drosophila sry beta and sry delta zinc finger proteins show differential embryonic expression and distinct patterns of binding sites on polytene chromosomes

Development ◽

10.1242/dev.110.1.141 ◽

1990 ◽

Vol 110 (1) ◽

pp. 141-149 ◽

Cited By ~ 1

Author(s):

F. Payre ◽

S. Noselli ◽

V. Lefrere ◽

A. Vincent

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Polytene Chromosomes ◽

Duplication Event ◽

Amino Acid Sequences ◽

Evolutionary Divergence ◽

Coding Region ◽

Protein Coding

Serendipity (sry) beta (beta) and delta (delta) are two finger protein genes resulting from a duplication event. Comparison of their respective protein products shows interspersed blocks of conserved and divergent amino-acid sequences. The most extensively conserved region corresponds to the predicted DNA-binding domain which includes 6 contiguous fingers; no significant sequence conservation is found upstream and downstream of the protein-coding region. We have analysed the evolutionary divergence of the sry beta and delta proteins on two separate levels, their embryonic pattern of expression and their DNA-binding properties in vitro and in vivo. By using specific antibodies and transformant lines containing beta-galactosidase fusion genes, we show that the sry beta and sry delta proteins are maternally inherited and present in embryonic nuclei at the onset of zygotic transcription, suggesting that they are transcription factors involved in this process. Zygotic synthesis of the sry beta protein starts during nuclear division cycles 12–13, prior to cellularisation of the blastoderm, while the zygotic sry delta protein is not detectable before germ band extension (stage 10 embryos). Contrary to sry delta, the zygotic sry beta protein constitutes only a minor fraction of the total embryonic protein. The sry beta and delta proteins made in E. coli bind to DNA, with partly overlapping specificities. Their in vivo patterns of binding to DNA, visualised by immunostaining polytene chromosomes, differ both in the number and position of their binding sites. Thus changes in expression pattern and DNA-binding specificity have contributed to the evolution of the sry beta and delta genes.

Download Full-text

Growth of Neisseria gonorrhoeae in the Female Mouse Genital Tract Does Not Require the Gonococcal Transferrin or Hemoglobin Receptors and May Be Enhanced by Commensal Lactobacilli

Infection and Immunity ◽

10.1128/iai.70.5.2549-2558.2002 ◽

2002 ◽

Vol 70 (5) ◽

pp. 2549-2558 ◽

Cited By ~ 39

Author(s):

Ann E. Jerse ◽

Emily T. Crow ◽

Amy N. Bordner ◽

Ishrat Rahman ◽

Cynthia Nau Cornelissen ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Genital Tract ◽

Female Genital Tract ◽

Iron Chelator ◽

Iron Store ◽

Human Transferrin ◽

Mucosal Surfaces ◽

Phase Variants

ABSTRACT Neisseria gonorrhoeae is capable of utilizing a variety of iron sources in vitro, including human transferrin, human lactoferrin, hemoglobin, hemoglobin-haptoglobin complexes, heme, and heterologous siderophores. Transferrin has been implicated as a critical iron store for N. gonorrhoeae in the human male urethra. The demonstration that gonococci can infect the lower genital tracts of estradiol-treated BALB/c mice in the absence of human transferrin, however, suggests that other usable iron sources are present in the murine genital tract. Here we demonstrate that gonococcal transferrin and hemoglobin receptor mutants are not attenuated in mice, thereby ruling out transferrin and hemoglobin as essential for murine infection. An increased frequency of phase variants with the hemoglobin receptor “on” (Hg+) occurred in ca. 50% of infected mice; this increase was temporally associated with an influx of neutrophils and detectable levels of hemoglobin in the vagina, suggesting that the presence of hemoglobin in inflammatory exudates selects for Hg+ phase variants during infection. We also demonstrate that commensal lactobacilli support the growth of N. gonorrhoeae in vitro unless an iron chelator is added to the medium. We hypothesize that commensal lactobacilli may enhance growth of gonococci in vivo by promoting the solubilization of iron on mucosal surfaces through the production of metabolic intermediates. Finally, transferrin-binding lipoprotein (TbpB) was detected on gonococci in vaginal smears, suggesting that although gonococci replicate within the genital tracts of mice, they may be sufficiently iron-stressed to express iron-repressible proteins. In summary, these studies support the potential role of nontransferrin, nonhemoglobin iron sources during gonococcal infection of the female genital tract.

Download Full-text