scholarly journals A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

2020 ◽  
Vol 48 (16) ◽  
pp. 8914-8926
Author(s):  
Erin E Cutts ◽  
J Barry Egan ◽  
Ian B Dodd ◽  
Keith E Shearwin
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Attachment Site ◽  
Binding Energies ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Binding Model ◽  
Specific Binding Sites

Abstract The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

scholarly journals A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

2019 ◽  
Author(s):  
Erin Cutts ◽  
J. Barry Egan ◽  
Ian Dodd ◽  
Keith Shearwin
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Attachment Site ◽  
Binding Energies ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Binding Model ◽  
Specific Binding Sites

AbstractThe Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA, and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl’s repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

Atrial natriuretic factor binding sites in the jejunum

1989 ◽  
Vol 256 (2) ◽  
pp. G436-G441 ◽  
Author(s):  
C. Bianchi ◽  
G. Thibault ◽  
A. De Lean ◽  
J. Genest ◽  
M. Cantin
Keyword(s):  
Binding Sites ◽  
Atrial Natriuretic Factor ◽  
Specific Binding ◽  
Rat Tissues ◽  
Rat Jejunum ◽  
Specific Binding Sites ◽  
Natriuretic Factor ◽  
Atrial Natriuretic

We have studied the localization and the characterization of atrial natriuretic factor (ANF) binding sites by radioautographic techniques. Quantitative in vitro radioautography with a computerized microdensitometer demonstrated the presence of high-affinity, low-capacity 125I-ANF-(99-126) binding sites (Kd, 48 pM; Bmax, 63 fmol/mg protein) mainly in the villi of 20-microns slide-mounted transverse sections of the rat jejunum. Competition curves showed 50% inhibitory concentrations of 55 and 1,560 pM for ANF-(99-126) and ANF-(103-123), respectively. In vivo electron microscope radioautography showed that 80% of the silver grains were localized on the lamina propria fibroblast-like cells, 18% on mature enterocytes, and 2% on capillaries. Bradykinin and adrenocorticotropin did not compete with ANF binding. These results demonstrate that ANF binding sites in the rat jejunum possess the pharmacological characteristics of functional ANF receptors encountered in other rat tissues, and ultrastructural radioautographs show their cellular distribution. Taken together, these results demonstrate the presence and the localization of specific binding sites for ANF in the jejunal villi of the rat small intestine.

Autoradiographic detection of kinin receptors in the human medulla of control, hypertensive, and diabetic donors

2002 ◽  
Vol 80 (4) ◽  
pp. 249-257 ◽  
Author(s):  
Hudson de Sousa Buck ◽  
Brice Ongali ◽  
Gaétan Thibault ◽  
Charles J Lindsey ◽  
Réjean Couture
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Specific Binding ◽  
Inferior Olivary Nucleus ◽  
Binding Studies ◽  
Specific Binding Sites ◽  
Kinin Receptors ◽  
Medullary Nuclei ◽  
Inferior Olivary

Kinins have been elected to the status of central neuromediators. Their effects are mediated through the activation of two G-protein-coupled receptors, denoted B1 and B2. Functional and binding studies suggested that B1 and B2 receptors are upregulated in the medulla and spinal cord of hypertensive and diabetic rats. The aim of this study was to localize and quantify kinin receptors in post-mortem human medulla obtained from normotensive, hypertensive, and diabetic subjects, using in vitro receptor autoradiography with the radioligands [125I]HPP-HOE140 (B2 receptor) and [125I]HPP[des-Arg10]-HOE140 (B1 receptor). Data showed specific binding sites for B2 receptor (0.4–1.5 fmol/mg tissue) in 11 medullary nuclei from 4 control specimens (paratrigeminal > ambiguus > cuneate, gelatinous layer of the caudal spinal trigeminal nucleus > caudal and interpolar spinal trigeminal, external cuneate, solitary tract > hypoglossal > gracile > inferior olivary nuclei). Increased density of B2 receptor binding sites was observed in seven medullary nuclei of four hypertensive specimens (paratrigeminal > external cuneate > interpolar and caudal spinal trigeminal, gracile, inferior olivary > hypoglossal nuclei). B2 receptor binding sites were seemingly increased in the same medullary nuclei of two diabetic specimens. Specific binding sites for B1 receptor (1.05 and 1.36 fmol/mg tissue) were seen only in the inferior olivary nucleus in two out of the ten studied specimens. The present results support a putative role for kinins in the regulation of autonomic, nociceptive, and motor functions at the level of the human medulla. Evidence is also provided that B2 receptors are upregulated in medullary cardiovascular centers of subjects afflicted of cardiovascular diseases.Key words: bradykinin, hypertension, diabetes, human brain.

scholarly journals Contribution of gangliosides to thyroxin binding with plasma membranes

1995 ◽  
Vol 41 (2) ◽  
pp. 28-30
Author(s):  
T. S. Saatov ◽  
F. Ya. Gulyamova ◽  
G. U. Usmanova
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Specific Binding ◽  
Brain Cell ◽  
Cell Recognition ◽  
High Affinity ◽  
Specific Binding Sites

Besides intracellular receptors of thyroid hormones, specific binding sites for T3 and T4 were detected on plasma membranes (PM) of some cells and a relationship between membrane reception .and lipid composition of membranes shown. The parameters of 125I-T4 binding to highly purified PM of hepatic and cerebral cells of rats were studied. The hepatic and cerebral cellular membranes were found to contain two sites of hormone binding each, one of these sites being characterized by a high affinity and low capacity, and the other by low affinity and a higher binding capacity. The association constant of highly affine site of hepatocyte membranes was found to be higher than that of brain cell membranes. T4 membranous receptors may be significant in the process of cell “recognition" by the hormone. In vivo and in vitro experiments with 125I-T4 and 14C-labeled thyroxin in ganglioside fractions showed appreciable binding of the hormone to Gm3 fraction, this evidently pointing to participation of this, ganglioside in T4 interaction with membrane receptor. It is possible that gangliosides situated on membranous surface are components of or function as receptors.

Autoradiographic determination of dexamethasone binding sites along the rabbit nephron

1983 ◽  
Vol 244 (3) ◽  
pp. F325-F334 ◽  
Author(s):  
N. Farman ◽  
A. Vandewalle ◽  
J. P. Bonvalet
Keyword(s):  
Proximal Tubule ◽  
Binding Sites ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Nuclear Binding ◽  
Specific Binding Sites ◽  
In Vitro Incubation ◽  
Collecting Tubules

Specific binding sites of tritiated dexamethasone ([3H]dex) along the tubule of rabbit kidney were investigated using an autoradiographic method (dry film) on isolated tubular segments. After in vitro incubation of kidney pyramids with [3H]dex (0.15-53 nM) in the presence or absence of an excess (X200) of unlabeled dexamethasone, tubular segments were microdissected and processed for autoradiography. A quantitative analysis of specific labeling over cytoplasm and nuclei was performed. Specific nuclear binding was observed in all tubular segments beyond the pars recta. This binding was dose dependent and reached much higher values than those reported for aldosterone. In the proximal tubule, the specific labeling was also high but remained mostly cytoplasmic. The meaning of these drastically different intracellular localizations is still open to interpretation. Autoradiography was performed after in vivo injection of [3H]dex and [3H]aldosterone. The results were not different from those described here for dexamethasone and from those previously reported for aldosterone after in vitro incubation. We conclude that specific nuclear binding sites for dexamethasone range over the nephron except for proximal tubule, with no great difference among segments, in contrast to specific sites for aldosterone, which are restricted to distal and cortical collecting tubules. The exact significance of the proximal cytoplasmic specific binding of [3H]dex remains to be determined.

Brain Specific Benzodiazepine Receptors

1978 ◽  
Vol 133 (3) ◽  
pp. 249-260 ◽  
Author(s):  
Claus Bræstrup ◽  
Richard F. Squires
Keyword(s):  
Binding Sites ◽  
Neuronal Degeneration ◽  
Specific Binding ◽  
Benzodiazepine Receptors ◽  
Clinical Effects ◽  
Single Class ◽  
Specific Binding Sites ◽  
Benzodiazepine Binding Sites

SummaryBrain membranes from rat and human contain a single class of brain specific binding sites for pharmacologically and clinically active benzodiazepines. There is good correlation between the pharmacological effects of benzodiazepines and the affinity for the 3H-diazepam binding site.Benzodiazepine binding sites are not present on glial cells. Selective neuronal degeneration experiments in rats indicate a neuronal localization. 3H-Flunitrazepam is a very suitable ligand for affinity binding and it binds to the same class of binding sites as 3H-diazepam.Our results indicate that the in vitro3H-diazepam and 3H-flunitrazepam binding sites are the receptors which in vivo mediate various pharmacological and clinical effects of benzodiazepines.

scholarly journals Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative.

1995 ◽  
Vol 15 (3) ◽  
pp. 1405-1421 ◽  
Author(s):  
C C Adams ◽  
J L Workman
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Sites ◽  
Specific Binding ◽  
General Mechanism ◽  
Nf Kappa B ◽  
Nucleosomal Dna ◽  
Nucleosome Binding

To investigate mechanisms by which multiple transcription factors access complex promoters and enhancers within cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores. We used a purified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kappa B (KBF1), to reconstituted nucleosome cores containing different combinations of binding sites. Here we show that binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative. Thus, the binuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kappa B all participated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particular DNA-binding domain. Simultaneous binding by two factors increased the affinity of individual factors for nucleosomal DNA by up to 2 orders of magnitude. Importantly, cooperative binding resulted in efficient nucleosome binding by factors (USF and NF-kappa B) which independently possess little nucleosome-binding ability. The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-binding and dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was responsible for the increased affinity of additional factors. Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of the orientation of the binding sites on the nucleosome. These results indicate that cooperative nucleosome binding is a general mechanism that may play a significant role in loading complex enhancer and promoter elements with multiple diverse factors in chromatin and contribute to the generation of threshold responses and transcriptional synergy by multiple activator sites in vivo.

scholarly journals The behavior of transferrin iron in the rat

Blood ◽  
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

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.

scholarly journals A yeast ARS-binding protein activates transcription synergistically in combination with other weak activating factors.

1990 ◽  
Vol 10 (3) ◽  
pp. 887-897 ◽  
Author(s):  
A R Buchman ◽  
R D Kornberg
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Essential Gene ◽  
Specific Binding ◽  
Binding Protein ◽  
Point Mutations ◽  
Yeast Genes

ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

The binding of [3H]mepyramine to histamine H1 receptors in monkey brain

1985 ◽  
Vol 63 (6) ◽  
pp. 756-759 ◽  
Author(s):  
B. Bielkiewicz ◽  
D. A. Cook
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Histamine Receptor ◽  
Detailed Examination ◽  
Mammalian Brain ◽  
Binding Studies ◽  
Peripheral Tissues ◽  
Monkey Brain ◽  
Specific Binding Sites ◽  
Histamine H1 Receptors

Several laboratories have reported ligand binding studies using radioactive histamine H1 antagonists to label the H1 receptors in mammalian brain. We have extended these studies to a detailed examination of the binding of [3H]mepyramine to monkey brain and have shown that the distribution is similar to that in man, with specific binding sites being concentrated in the frontal cortex with relatively low binding to the pons and basal ganglia. The binding shows a single saturable component with a KD of about 1 nM and a Hill plot slope close to unity. These observations are the same for all structures tested. Comparison with data from other laboratories suggests that in this species, the histamine receptor is the same as that in peripheral tissues. From Ki values for various ligands and comparison of KD estimates in other species, the receptor seems to be essentially identical to the H1 receptor in central and peripheral tissues of the guinea pig and also to that in human brain. The rat and possibly the dog have minor differences from the monkey in terms of KD values for [3H]mepyramine binding.

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